Article

Endogenous nitric oxide mechanisms mediate stretch-dependency of Ca2+-release in cardiomyocytes

November 2001
Nature Cell Biology 3(10):867-73

November 2001
3(10):867-73

DOI:10.1038/ncb1001-867

Source
PubMed

Authors:

Martin Vila Petroff

Independent Researcher

Chantal Dessy

Université Catholique de Louvain - UCLouvain

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Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K-Akt-endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.

Тhe link of T(–786)C аnd G894T polymorphisms of the endothelial NO-synthase gene with endothelial vasodilatory function and long-term clinical prognosis in patients with chronic heart failure and reduced left ventricular ejection fraction

Article

Full-text available

Jul 2022

The aim – сhronic heart failure (CHF) is associated with endothelial dysfunction. The pivotal role of nitric oxide in the maintenance of endothelial function (EF) is well-known. But it is unknown whether endothelial nitric oxide synthase (eNOS) gene polymorphismis associated with both EF and clinical outcomes in systolic CHF.Materials and methods. 116 stable (NYHA II–III) ischemic CHF patients with left ventricular ejection fraction (LVEF)≤ 45 % were examined. Flow-mediated vasodilation (FMVD) of a. brachialis was carried out by standard cuff test. Patients were followed-up for a median of twenty months to determine long-term outcomes. The frequency of T(–786)C genotypes was: TT – 40.5 % (n=47), TC – 43.1 % (n=50), CC – 16.4 % (n=19); the frequency of G894T genotypes was: GG 56.0 % (n=65), GT 33.6 % (n=39), ТТ 10.4 % (n=12).Results and discussion. FMVD in patients with TT genotype of T(–786)C polymorphisms was 7.2 [4.7; 8.3] %, in patients with TC – 6.6 [4.4; 9.1] %, where as FMVD in patients with genotype CC was 4.7 [2.8; 6.0] %, p=0.034 for TT vs. CC; p=0.046 for TC vs. CC. FMVD in patients with GG genotype of G894T polymorphisms was 7.1 [4.3; 9.4] %, in patients with GT – 6.2 [5.1; 8.1] %, in patients with genotype TT was 4.2 [2.5; 5.3] %. The difference between FMVD was significant only TT vs. CC – p=0.030. The patients with CC genotype demonstrated a significantly higher heart failure hospitalization rate (log-rank 5.304, p=0.021) and higher cardiovascular (CV) mortality rate (log-rank 4.011, p=0.045) as compared with the TT homozygote group. LVEF, FMVD, and CC genotype were the predictors of CV mortality in univariate Cox regression analysis, and only LVEF and FMVD in multivariate Cox model. Long-term outcomes were similar in patients with GG, GT and TT genotypes of G894T polymorphisms.Conclusion. In stable ischemic systolic CHF CC T(–786)C eNOS genotype is associated with worse FMVD response and worse long-term outcome versus TT T(–786)C eNOS genotype. TT G(984)T eNOS genotype is associated with worse FMVD response only, but not with long-term outcomes versus GG G(894)T eNOS genotype.

The role of activation of two different sGC binding sites by NO‐dependent and NO‐independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes

Article

Full-text available

Apr 2022

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd³⁺‐sensitive nonselective cation current, an analog of ISAC, while in a stretched cell it eliminates ISAC. The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC, this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation. ODQ eliminates ISAC, but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC. 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

Electrophysiological and Molecular Mechanisms of Sinoatrial Node Mechanosensitivity

Article

Full-text available

Aug 2021

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.

Nitric Oxide and Mechano-Electrical Transduction in Cardiomyocytes

Article

Full-text available

Dec 2020

The ability§ of the heart to adapt to changes in the mechanical environment is critical for normal cardiac physiology. The role of nitric oxide is increasingly recognized as a mediator of mechanical signaling. Produced in the heart by nitric oxide synthases, nitric oxide affects almost all mechano-transduction pathways within the cardiomyocyte, with roles mediating mechano-sensing, mechano-electric feedback (via modulation of ion channel activity), and calcium handling. As more precise experimental techniques for applying mechanical stresses to cells are developed, the role of these forces in cardiomyocyte function can be further understood. Furthermore, specific inhibitors of different nitric oxide synthase isoforms are now available to elucidate the role of these enzymes in mediating mechano-electrical signaling. Understanding of the links between nitric oxide production and mechano-electrical signaling is incomplete, particularly whether mechanically sensitive ion channels are regulated by nitric oxide, and how this affects the cardiac action potential. This is of particular relevance to conditions such as atrial fibrillation and heart failure, in which nitric oxide production is reduced. Dysfunction of the nitric oxide/mechano-electrical signaling pathways are likely to be a feature of cardiac pathology (e.g., atrial fibrillation, cardiomyopathy, and heart failure) and a better understanding of the importance of nitric oxide signaling and its links to mechanical regulation of heart function may advance our understanding of these conditions.

The Slow Force Response to Stretch: Controversy and Contradictions

Article

Jan 2019

When exposed to an abrupt stretch, cardiac muscle exhibits biphasic active force enhancement. The initial, instantaneous, force enhancement is well explained by the Frank‐Starling mechanism. However, the cellular mechanisms associated with the second, slower, phase remain contentious. This review explores hypotheses regarding this ‘slow force response’ with the intention of clarifying some apparent contradictions in the literature. The review is partitioned into three sections. The first section considers pathways that modify the intracellular calcium handling to address the role of the sarcoplasmic reticulum in the mechanism underlying the slow force response. The second section focuses on extracellular calcium fluxes and explores the identity and contribution of the stretch‐activated, non‐specific, cation channels as well as signalling cascades associated with G‐protein coupled receptors. The final section introduces promising candidates for the mechanosensor(s) responsible for detecting the stretch perturbation. This article is protected by copyright. All rights reserved.

Regulation of cardiac calcium by mechanotransduction: Role of mitochondria

Article

Sep 2018

Arrhythmogenic Mechanisms in Heart Failure: Linking Beta-Adrenergic Stimulation, Stretch and Calcium

Article

Full-text available

Sep 2018

Heart failure (HF) is associated with elevated sympathetic tone and mechanical load. Both systems activate signalling transduction pathways that increase cardiac output, but eventually become part of the disease process itself leading to further worsening of cardiac function. These alterations can adversely contribute to electrical instability, at least in part due to the modulation of Ca2+ handling at the level of the single cardiac myocyte. The major aim of this review is to provide a definitive overview of the links and cross talk between beta-adrenergic stimulation, mechanical load and arrhythmogenesis in the setting of HF. We will initially review the role of Ca2+ in the induction of both early and delayed afterdepolarizations, the role that beta-adrenergic stimulation plays in the initiation of these and how the propensity for these may be altered in HF. We will then go onto reviewing the current data with regards to the link between mechanical load and afterdepolarizations, the associated mechano-sensitivity of the ryanodine receptor and other stretch activated channels that may be associated with HF-associated arrhythmias. Furthermore, we will discuss how alterations in local Ca2+ microdomains during the remodelling process associated the HF may contribute to the increased disposition for beta-adrenergic or stretch induced arrhythmogenic triggers. Finally, the potential mechanisms linking beta-adrenergic stimulation and mechanical stretch will be clarified, with the aim of finding common modalities of arrhythmogenesis that could be targeted by novel therapeutic agents in the setting of HF.

Effects of S-Nitrosoglutathione on Electrophysiological Manifestations of Mechanoelectric Feedback

Article

Full-text available

Dec 2018

Electromechanical coupling studies have described the intervention of nitric oxide and S-nitrosylation processes in Ca²⁺ release induced by stretch, with heterogeneous findings. On the other hand, ion channel function activated by stretch is influenced by nitric oxide, and concentration-dependent biphasic effects upon several cellular functions have been described. The present study uses isolated and perfused rabbit hearts to investigate the changes in mechanoelectric feedback produced by two different concentrations of the nitric oxide carrier S-nitrosoglutathione. Epicardial multielectrodes were used to record myocardial activation at baseline and during and after left ventricular free wall stretch using an intraventricular device. Three experimental series were studied: (a) control (n = 10); (b) S-nitrosoglutathione 10 µM (n = 11); and (c) S-nitrosoglutathione 50 µM (n = 11). The changes in ventricular fibrillation (VF) pattern induced by stretch were analyzed and compared. S-nitrosoglutathione 10 µM did not modify VF at baseline, but attenuated acceleration of the arrhythmia (15.6 ± 1.7 vs. 21.3 ± 3.8 Hz; p < 0.0001) and reduction of percentile 5 of the activation intervals (42 ± 3 vs. 38 ± 4 ms; p < 0.05) induced by stretch. In contrast, at baseline using the 50 µM concentration, percentile 5 was shortened (38 ± 6 vs. 52 ± 10 ms; p < 0.005) and the complexity index increased (1.77 ± 0.18 vs. 1.27 ± 0.13; p < 0.0001). The greatest complexity indices (1.84 ± 0.17; p < 0.05) were obtained during stretch in this series. S-nitrosoglutathione 10 µM attenuates the effects of mechanoelectric feedback, while at a concentration of 50 µM the drug alters the baseline VF pattern and accentuates the increase in complexity of the arrhythmia induced by myocardial stretch.

Modulations of Cardiac Functions and Pathogenesis by Reactive Oxygen Species and Natural Antioxidants

Article

Full-text available

May 2021

Homeostasis in the level of reactive oxygen species (ROS) in cardiac myocytes plays a critical role in regulating their physiological functions. Disturbance of balance between generation and removal of ROS is a major cause of cardiac myocyte remodeling, dysfunction, and failure. Cardiac myocytes possess several ROS-producing pathways, such as mitochondrial electron transport chain, NADPH oxidases, and nitric oxide synthases, and have endogenous antioxidation mechanisms. Cardiac Ca2+-signaling toolkit proteins, as well as mitochondrial functions, are largely modulated by ROS under physiological and pathological conditions, thereby producing alterations in contraction, membrane conductivity, cell metabolism and cell growth and death. Mechanical stresses under hypertension, post-myocardial infarction, heart failure, and valve diseases are the main causes for stress-induced cardiac remodeling and functional failure, which are associated with ROS-induced pathogenesis. Experimental evidence demonstrates that many cardioprotective natural antioxidants, enriched in foods or herbs, exert beneficial effects on cardiac functions (Ca2+ signal, contractility and rhythm), myocytes remodeling, inflammation and death in pathological hearts. The review may provide knowledge and insight into the modulation of cardiac pathogenesis by ROS and natural antioxidants.

Mechano‐electric and mechano‐chemo‐transduction in cardiomyocytes

Article

Full-text available

Feb 2020
J PHYSIOL-LONDON

Cardiac excitation‐contraction (E‐C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano‐electro‐transduction (commonly referred to as mechano‐electric coupling, MEC) and mechano‐chemo‐transduction (MCT) mechanisms at cell and molecular levels which couple to Ca²⁺‐electro and E‐C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano‐transduction to the Ca²⁺ homeodynamics and to the electrical excitation are essential for understanding the E‐C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca²⁺ dynamics). Of course, such separation is artificial since Ca²⁺ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca²⁺ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium. image

Quantitative tests reveal that microtubules tune the healthy heart but underlie arrhythmias in pathology

Article

Full-text available

Jan 2019
J PHYSIOL-LONDON

Key points Our group previously discovered and characterized the microtubule mechanotransduction pathway linking diastolic stretch to NADPH oxidase 2‐derived reactive oxygen species signals that regulate calcium sparks and calcium influx pathways. Here we used focused experimental tests to constrain and expand our existing computational models of calcium signalling in heart. Mechanistic and quantitative modelling revealed new insights in disease including: changes in microtubule network density and properties, elevated NOX2 expression, altered calcium release dynamics, how NADPH oxidase 2 is activated by and responds to stretch, and finally the degree to which normalizing mechano‐activated reactive oxygen species signals can prevent calcium‐dependent arrhythmias. Abstract Microtubule (MT) mechanotransduction links diastolic stretch to generation of NADPH oxidase 2 (NOX2)‐dependent reactive oxygen species (ROS), signals we term X‐ROS. While stretch‐elicited X‐ROS primes intracellular calcium (Ca²⁺) channels for synchronized activation in the healthy heart, the dysregulated excess in this pathway underscores asynchronous Ca²⁺ release and arrhythmia. Here, we expanded our existing computational models of Ca²⁺ signalling in heart to include MT‐dependent mechanotransduction through X‐ROS. Informed by new focused experimental tests to properly constrain our model, we quantify the role of X‐ROS on excitation–contraction coupling in healthy and pathological conditions. This approach allowed for a mechanistic investigation that revealed new insights into X‐ROS signalling in disease including changes in MT network density and post‐translational modifications (PTMs), elevated NOX2 expression, altered Ca²⁺ release dynamics (i.e. Ca²⁺ sparks and Ca²⁺ waves), how NOX2 is activated by and responds to stretch, and finally the degree to which normalizing X‐ROS can prevent Ca²⁺‐dependent arrhythmias.

Oxidative Stress in Cardiac Tissue of Patients Undergoing Coronary Artery Bypass Graft Surgery: The Effects of Overweight and Obesity

Article

Full-text available

Dec 2018
OXID MED CELL LONGEV

Background: Obesity is one of the major cardiovascular risk factors and is associated with oxidative stress and myocardial dysfunction. We hypothesized that obesity affects cardiac function and morbidity by causing alterations in enzymatic redox patterns. Methods: Sixty-one patients undergoing coronary artery bypass grafting (CABG) were included in the study. Excessive right atrial myocardial tissue emerging from the operative connection to the extracorporeal circulation was harvested. Patients were assigned to control (n = 19, body mass index (BMI): <25 kg/m2), overweight (n = 25, 25 kg/m2 < BMI < 30 kg/m2), or obese (n = 17, BMI: >30 kg/m2) groups. Oxidative enzyme systems were studied directly in the cardiac muscles of patients undergoing CABG who were grouped according to BMI. Molecular biological methods and high-performance liquid chromatography were used to detect the expression and activity of oxidative enzymes and the formation of reactive oxygen species (ROS). Results: We found increased levels of ROS and increased expression of ROS-producing enzymes (i.e., p47phox, xanthine oxidase) and decreased antioxidant defense mechanisms (mitochondrial aldehyde dehydrogenase, heme oxygenase-1, and eNOS) in line with elevated inflammatory markers (vascular cell adhesion molecule-1) in the right atrial myocardial tissue and by trend also in serum (sVCAM-1 and CCL5/RANTES). Conclusion: Increasing BMI in patients undergoing CABG is related to altered myocardial redox patterns, which indicates increased oxidative stress with inadequate antioxidant compensation. These changes suggest that the myocardium of obese patients suffering from coronary artery disease is more susceptible to cardiomyopathy and possible damage by ischemia and reperfusion, for example, during cardiac surgery.

Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction

Article

Full-text available

Sep 2018
PFLUG ARCH EUR J PHY

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca²⁺, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2′,7′-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca²⁺ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1 mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10 μmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca²⁺ waves, and the number of beats after electrical stimulation (0.2 μmol/L isoproterenol), while 3 μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them. A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca²⁺ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca²⁺ waves.

Circulation Research Arrhythmogenic Manifestations of Chagas Disease: Perspectives From the Bench to Bedside

Article

Full-text available

May 2024
CIRC RES

Chagas cardiomyopathy caused by infection with the intracellular parasite Trypanosoma cruzi is the most common and severe expression of human Chagas disease. Heart failure, systemic and pulmonary thromboembolism, arrhythmia, and sudden cardiac death are the principal clinical manifestations of Chagas cardiomyopathy. Ventricular arrhythmias contribute significantly to morbidity and mortality and are the major cause of sudden cardiac death. Significant gaps still exist in the understanding of the pathogenesis mechanisms underlying the arrhythmogenic manifestations of Chagas cardiomyopathy. This article will review the data from experimental studies and translate those findings to draw hypotheses about clinical observations. Human-and animal-based studies at molecular, cellular, tissue, and organ levels suggest 5 main pillars of remodeling caused by the interaction of host and parasite: immunologic, electrical, autonomic, microvascular, and contractile. Integrating these 5 remodeling processes will bring insights into the current knowledge in the field, highlighting some key features for future management of this arrhythmogenic disease.

Ryanodine receptor dysfunction causes senescence and fibrosis in Duchenne dilated cardiomyopathy

Article

Full-text available

Jan 2024

Background Duchenne muscular dystrophy (DMD) is an X‐linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD ( mdx ) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca ²⁺ ) cycling related to early‐stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age‐dependent DCM. Methods Here, we used hiPSC‐CMs from DMD patients selected by Speckle‐tracking echocardiography and canine DMD cardiac biopsies to assess key early‐stage Duchenne DCM features. Results Dystrophin deficiency was associated with RyR2 remodelling and SR Ca ²⁺ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early‐stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC‐CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC‐CMs. We observed cardiac hypocontractility in DMD hiPSC‐CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 μm for DMD vs. 2.26 ± 0.08 μm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis. Conclusions We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca ²⁺ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD‐associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2‐mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD‐induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.

Modulating NO–GC Pathway in Pulmonary Arterial Hypertension

Article

Full-text available

Dec 2023
INT J MOL SCI

The pathogenesis of complex diseases such as pulmonary arterial hypertension (PAH) is entirely rooted in changes in the expression of some vasoactive factors. These play a significant role in the onset and progression of the disease. Indeed, PAH has been associated with pathophysiologic alterations in vascular function. These are often dictated by increased oxidative stress and impaired modulation of the nitric oxide (NO) pathway. NO reduces the uncontrolled proliferation of vascular smooth muscle cells that leads to occlusion of vessels and an increase in pulmonary vascular resistances, which is the mainstay of PAH development. To date, two classes of NO-pathway modulating drugs are approved for the treatment of PAH: the phosphodiesterase-5 inhibitors (PD5i), sildenafil and tadalafil, and the soluble guanylate cyclase activator (sGC), riociguat. Both drugs provide considerable improvement in exercise capacity and pulmonary hemodynamics. PD5i are the recommended drugs for first-line PAH treatment, whereas sGCs are also the only drug approved for the treatment of resistant or inoperable chronic thromboembolic pulmonary hypertension. In this review, we will focus on the current information regarding the nitric oxide pathway and its modulation in PAH.

The Slow Force Response and Simultaneous Changes in Ca2+ Transient in Healthy and Failing Rat Atrial and Ventricular Myocardium

Article

Full-text available

Feb 2023

The Slow Force Response (SFR) is an important mechanism of adaptation of myocardial contractility to the mechanical load. The discrepancies between the SFR in atrial and ventricular myocardium are poorly characterized; moreover, the correlation between the mechanical response and underlying changes in Ca2+ transient are unknown. We measured simultaneous slow changes in force and Ca2+ transient developed in right atrial and ventricular (RA and RV) muscles of healthy control rats (CONT) and rats with monocrotaline-induced pulmonary heart failure (MCT) as a response to rapid muscle stretch. The SFR was positive in RA muscles but negative in RV muscles in CONT and MCT groups. The extent of SFR was significantly higher in the failing myocardium. The slow changes in peak active force showed little correlation with rate- and time-related characteristics of isometric force. The relative changes in Ca2+ transient diastolic level, amplitude, time-to-peak and time to decay from peak to 50% amplitude at the end of SFR were significantly smaller compared to the extent of SFR. Plotting the extent of SFR as peak active force vs the extent of change in Ca2+ transient diastolic level or amplitude revealed that only the latter followed nearly linear behavior while the former had no substantial correlation. We conclude that the minor changes in Ca2+ transient characteristics during SFR underlie the much greater changes observed in the peak active force. The atrioventricular discrepancies in the SFR indicate different adaptive responses of the contractility of these chambers to the external mechanical loading.

Absence of Nitrergic Modulation of Starling Response in Haemoglobin-Less Antarctic Fish Chionodraco hamatus

Article

Full-text available

Nov 2022

The Frank-Starling response is an intrinsic heart property that is particularly evident in the fish heart because piscine cardiomyocytes are extremely sensitive to stretch. Several mechanisms and compounds influence the Frank-Starling response, including the free radical nitric oxide produced by nitric oxide synthases in the vascular endothelium and cardiomyocytes of all vertebrates. Besides its role in scavenging nitric oxide, hemoglobin may act as a source and transporter. In this context, the hemoglobin-less Antarctic teleost Chionodraco hamatus (icefish) represents a unique opportunity to investigate the involvement of nitric oxide in the Frank-Starling response. Using an isolated perfused heart preparation, weverified a basal nitrergic tone that is not implicated in the Frank-Starling response. In addition, by comparing nitric oxide synthases expression and activation in C. hamatus and the red-blooded Antarctic teleost Trematomus bernacchii, we found the endothelial isoform of nitric oxide synthase (the primary generator of nitric oxide during shear stress) to be less expressed and activated in the former.

Nitrosylation of cardiac CaMKII at Cys290 mediates mechanical afterload‐induced increases in Ca transient and Ca sparks

Article

Full-text available

Oct 2022
J PHYSIOL-LONDON

Cardiac mechanical afterload induces an intrinsic autoregulatory increase in myocyte Ca²⁺ dynamics and contractility to enhance contraction (known as the Anrep effect or slow force response). Our prior work has implicated both nitric oxide (NO) produced by NO synthase 1 (NOS1) and calcium/calmodulin‐dependent protein kinase II (CaMKII) activity as required mediators of this form of mechano‐chemo‐transduction. To test whether a single S‐nitrosylation site on CaMKIIδ (Cys290) mediates enhanced sarcoplasmic reticulum Ca²⁺ leak and afterload‐induced increases in sarcoplasmic reticulum (SR) Ca²⁺ uptake and release, we created a novel CRISPR‐based CaMKIIδ knock‐in (KI) mouse with a Cys to Ala mutation at C290. These CaMKIIδ‐C290A‐KI mice exhibited normal cardiac morphometry and function, as well as basal myocyte Ca²⁺ transients (CaTs) and β‐adrenergic responses. However, the NO donor S‐nitrosoglutathione caused an acute increased Ca²⁺ spark frequency in wild‐type (WT) myocytes that was absent in the CaMKIIδ‐C290A‐KI myocytes. Using our cell‐in‐gel system to exert multiaxial three‐dimensional mechanical afterload on myocytes during contraction, we found that WT myocytes exhibited an afterload‐induced increase in Ca²⁺ sparks and Ca²⁺ transient amplitude and rate of decline. These afterload‐induced effects were prevented in both cardiac‐specific CaMKIIδ knockout and point mutant CaMKIIδ‐C290A‐KI myocytes. We conclude that CaMKIIδ activation by S‐nitrosylation at the C290 site is essential in mediating the intrinsic afterload‐induced enhancement of myocyte SR Ca²⁺ uptake, release and Ca²⁺ transient amplitude (the Anrep effect). The data also indicate that NOS1 activation is upstream of S‐nitrosylation at C290 of CaMKII, and that this molecular mechano‐chemo‐transduction pathway is beneficial in allowing the heart to increase contractility to limit the reduction in stroke volume when aortic pressure (afterload) is elevated. image Key points A novel CRISPR‐based CaMKIIδ knock‐in mouse was created in which kinase activation by S‐nitrosylation at Cys290 (C290A) is prevented. How afterload affects Ca²⁺ signalling was measured in cardiac myocytes that were embedded in a hydrogel that imposes a three‐dimensional afterload. This mechanical afterload induced an increase in Ca²⁺ transient amplitude and decay in wild‐type myocytes, but not in cardiac‐specific CaMKIIδ knockout or C290A knock‐in myocytes. The CaMKIIδ‐C290 S‐nitrosylation site is essential for the afterload‐induced enhancement of Ca²⁺ transient amplitude and Ca²⁺ sparks.

Neurogranin regulates calcium-dependent cardiac hypertrophy

Article

Jul 2022
Exp Mol Pathol

Intracellular Ca²⁺-calmodulin (CaM) signaling plays an important role in Ca²⁺-CaM-dependent kinase (CaMKII) and calcineurin (CaN)-mediated cardiac biology. While neurogranin (Ng) is known as a major Ca²⁺-CaM modulator in the brain, its pathophysiological role in cardiac hypertrophy has never been studied before. In the present study, we report that Ng is expressed in the heart and depletion of Ng dysregulates Ca²⁺ homeostasis and promotes cardiac failure in mice. 10-month-old Ng null mice demonstrate significantly increased heart to body weight ratios compared to wild-type. Using histological approaches, we identified that depletion of Ng increases cardiac hypertrophy, fibrosis, and collagen deposition near perivascular areas in the heart tissue of Ng null mice. Ca²⁺ spark experiments revealed that cardiac myocytes isolated from Ng null mice have decreased spark frequency and width, while the duration of sparks are significantly increased. We also identified that lack of Ng increases CaMKIIδ signaling and periostin protein expression in these mouse hearts. Overall, we are the first study to explore how Ng expression in the heart plays an important role in Ca²⁺ homeostasis in cardiac myocytes as well as the pathophysiology of cardiac hypertrophy and fibrosis.

Potential Mechanism of Dingji Fumai Decoction Against Atrial Fibrillation Based on Network Pharmacology, Molecular Docking, and Experimental Verification Integration Strategy

Article

Full-text available

Nov 2021

Background: Dingji Fumai Decoction (DFD), a traditional herbal mixture, has been widely used to treat arrhythmia in clinical practice in China. However, the exploration of the active components and underlying mechanism of DFD in treating atrial fibrillation (AF) is still scarce. Methods: Compounds of DFD were collected from TCMSP, ETCM, and literature. The targets of active compounds were explored using SwissTargetPrediction. Meanwhile, targets of AF were collected from DrugBank, TTD, MalaCards, TCMSP, DisGeNET, and OMIM. Then, the H-C-T-D and PPI networks were constructed using STRING and analyzed using CytoNCA. Meanwhile, VarElect was utilized to detect the correlation between targets and diseases. Next, Metascape was employed for systematic analysis of the mechanism of potential targets and protein complexes in treating AF. AutoDock Vina, Pymol, and Discovery Studio were applied for molecular docking. Finally, the main findings were validated through molecular biology experiments. Results: A total of 168 active compounds and 1,093 targets of DFD were collected, and there were 89 shared targets between DFD and AF. H-C-T-D network showed the relationships among DFD, active compounds, targets, and AF. Three functional protein complexes of DFD were extracted from the PPI network. Further systematic analysis revealed that the regulation of cardiac oxidative stress, cardiac inflammation, and cardiac ion channels were the potential mechanism of DFD in treating AF. Addtionally, molecular docking verified the interactions between active compounds and targets. Finally, we found that DFD significantly increased the level of SIRT1 and reduced the levels of ACE, VCAM-1, and IL-6. Conclusions: DFD could be utilized in treating AF through a complicated mechanism, including interactions between related active compounds and targets, promoting the explanation and understanding of the molecular biological mechanism of DFD in the treatment of AF.

S-nitrosoglutathione reductase (GSNOR) deficiency accelerates cardiomyocyte differentiation of induced pluripotent stem cells

Article

Full-text available

Sep 2021

Introduction: Induced pluripotent stem cells (iPSCs) provide a model of cardiomyocyte (CM) maturation. Nitric oxide signaling promotes CM differentiation and maturation, although the mechanisms remain controversial. Aim: The study tested the hypothesis that in the absence of S-nitrosoglutathione reductase (GSNOR), a denitrosylase regulating protein S-nitrosylation, the resultant increased S-nitrosylation accelerates the differentiation and maturation of iPSC-derived cardiomyocytes (CMs). Methods and results: iPSCs derived from mice lacking GSNOR (iPSCGSNOR-/-) matured faster than wildtype iPSCs (iPSCWT) and demonstrated transient increases in expression of murine Snail Family Transcriptional Repressor 1 gene (Snail), murine Snail Family Transcriptional Repressor 2 gene (Slug) and murine Twist Family BHLH Transcription Factor 1 gene (Twist), transcription factors that promote epithelial-to-mesenchymal transition (EMT) and that are regulated by Glycogen Synthase Kinase 3 Beta (GSK3β). Murine Glycogen Synthase Kinase 3 Beta (Gsk3β) gene exhibited much greater S-nitrosylation, but lower expression in iPSCGSNOR-/-. S-nitrosoglutathione (GSNO)-treated iPSCWT and human (h)iPSCs also demonstrated reduced expression of GSK3β. Nkx2.5 expression, a CM marker, was increased in iPSCGSNOR-/- upon directed differentiation toward CMs on Day 4, whereas murine Brachyury (t), Isl1, and GATA Binding Protein (Gata4) mRNA were decreased, compared to iPSCWT, suggesting that GSNOR deficiency promotes CM differentiation beginning immediately following cell adherence to the culture dish-transitioning from mesoderm to cardiac progenitor. Conclusion: Together these findings suggest that increased S-nitrosylation of Gsk3β promotes CM differentiation and maturation from iPSCs. Manipulating the post-translational modification of GSK3β may provide an important translational target and offers new insight into understanding of CM differentiation from pluripotent stem cells. One sentence summary: Deficiency of GSNOR or addition of GSNO accelerates early differentiation and maturation of iPSC-cardiomyocytes.

Physical Exercise and Cardiac Repair: The Potential Role of Nitric Oxide in Boosting Stem Cell Regenerative Biology

Article

Full-text available

Jun 2021

Over the years strong evidence has been accumulated showing that aerobic physical exercise exerts beneficial effects on the prevention and reduction of cardiovascular risk. Exercise in healthy subjects fosters physiological remodeling of the adult heart. Concurrently, physical training can significantly slow-down or even reverse the maladaptive pathologic cardiac remodeling in cardiac diseases, improving heart function. The underlying cellular and molecular mechanisms of the beneficial effects of physical exercise on the heart are still a subject of intensive study. Aerobic activity increases cardiovascular nitric oxide (NO) released mainly through nitric oxidase synthase 3 activity, promoting endothelium-dependent vasodilation, reducing vascular resistance, and lowering blood pressure. On the reverse, an imbalance between increasing free radical production and decreased NO generation characterizes pathologic remodeling, which has been termed the “nitroso-redox imbalance”. Besides these classical evidence on the role of NO in cardiac physiology and pathology, accumulating data show that NO regulate different aspects of stem cell biology, including survival, proliferation, migration, differentiation, and secretion of pro-regenerative factors. Concurrently, it has been shown that physical exercise generates physiological remodeling while antagonizes pathologic remodeling also by fostering cardiac regeneration, including new cardiomyocyte formation. This review is therefore focused on the possible link between physical exercise, NO, and stem cell biology in the cardiac regenerative/reparative response to physiological or pathological load. Cellular and molecular mechanisms that generate an exercise-induced cardioprotective phenotype are discussed in regards with myocardial repair and regeneration. Aerobic training can benefit cells implicated in cardiovascular homeostasis and response to damage by NO-mediated pathways that protect stem cells in the hostile environment, enhance their activation and differentiation and, in turn, translate to more efficient myocardial tissue regeneration. Moreover, stem cell preconditioning by and/or local potentiation of NO signaling can be envisioned as promising approaches to improve the post-transplantation stem cell survival and the efficacy of cardiac stem cell therapy.

Molecular Basis of Atrial Fibrillation Initiation and Maintenance

Article

Full-text available

Mar 2021

Atrial fibrillation (AF) is the most common cardiac arrhythmia, largely associated to morbidity and mortality. Over the past decades, research in appearance and progression of this arrhythmia have turned into significant advances in its management. However, the incidence of AF continues to increase with the aging of the population and many important fundamental and translational underlaying mechanisms remain elusive. Here, we review recent advances in molecular and cellular basis for AF initiation, maintenance and progression. We first provide an overview of the basic molecular and electrophysiological mechanisms that lead and characterize AF. Next, we discuss the upstream regulatory factors conducting the underlying mechanisms which drive electrical and structural AF-associated remodeling, including genetic factors (risk variants associated to AF as transcriptional regulators and genetic changes associated to AF), neurohormonal regulation (i.e., cAMP) and oxidative stress imbalance (cGMP and mitochondrial dysfunction). Finally, we discuss the potential therapeutic implications of those findings, the knowledge gaps and consider future approaches to improve clinical management.

Mechanotranduction Pathways in the Regulation of Mitochondrial Homeostasis in Cardiomyocytes

Article

Full-text available

Jan 2021

Mitochondria are one of the most important organelles in cardiomyocytes. Mitochondrial homeostasis is necessary for the maintenance of normal heart function. Mitochondria perform four major biological processes in cardiomyocytes: mitochondrial dynamics, metabolic regulation, Ca²⁺ handling, and redox generation. Additionally, the cardiovascular system is quite sensitive in responding to changes in mechanical stress from internal and external environments. Several mechanotransduction pathways are involved in regulating the physiological and pathophysiological status of cardiomyocytes. Typically, the extracellular matrix generates a stress-loading gradient, which can be sensed by sensors located in cellular membranes, including biophysical and biochemical sensors. In subsequent stages, stress stimulation would regulate the transcription of mitochondrial related genes through intracellular transduction pathways. Emerging evidence reveals that mechanotransduction pathways have greatly impacted the regulation of mitochondrial homeostasis. Excessive mechanical stress loading contributes to impairing mitochondrial function, leading to cardiac disorder. Therefore, the concept of restoring mitochondrial function by shutting down the excessive mechanotransduction pathways is a promising therapeutic strategy for cardiovascular diseases. Recently, viral and non-viral protocols have shown potentials in application of gene therapy. This review examines the biological process of mechanotransduction pathways in regulating mitochondrial function in response to mechanical stress during the development of cardiomyopathy and heart failure. We also summarize gene therapy delivery protocols to explore treatments based on mechanical stress–induced mitochondrial dysfunction, to provide new integrative insights into cardiovascular diseases.

3D mechanical analysis of a self-contractile cell with stress fibers reorganization

Article

Apr 2021
APPL MATH MODEL

Contractile stress generated by a cell itself is crucial in sensing its surrounding microenvironment and regulating cell adhesion, differentiation and cytokinesis. However, the precise mechanisms underlying mechanotransduction remain unknown. In this paper, based on the Eshelby inclusion problem, we develop a theoretical model to characterize quantitatively the mechanical environment around a self-contractile cell experiencing stress fibers reorganization. We divide the contractile stress into two parts: the constant contractile stress and the perturbed contractile stress due to stress fibers reorganization, for internal stress fibers have enough time to reorganize actively during long-term deformation, leading to changes of contractile stress in both magnitude and direction. Obtained results suggest that stress fibers reorganization may cause significant changes in the mechanical environment of the cell, helpful for exploring the mechanisms behind cell mechanotransduction.

Mitochondrial Ca2+, redox environment and ROS emission in heart failure: Two sides of the same coin?

Article

Feb 2021

Heart failure (HF) is a progressive, debilitating condition characterized, in part, by altered ionic equilibria, increased ROS production and impaired cellular energy metabolism, contributing to variable profiles of systolic and diastolic dysfunction with significant functional limitations and risk of premature death. We summarize current knowledge concerning changes of intracellular Na⁺ and Ca²⁺ control mechanisms during the disease progression and their consequences on mitochondrial Ca²⁺ homeostasis and the shift in redox balance. Absent existing biological data, our computational modeling studies advance a new ‘in silico’ analysis to reconcile existing opposing views, based on different experimental HF models, regarding variations in mitochondrial Ca²⁺ concentration that participate in triggering and perpetuating oxidative stress in the failing heart and their impact on cardiac energetics. In agreement with our hypothesis and the literature, model simulations demonstrate the possibility that the heart’s redox status together with cytoplasmic Na⁺ concentrations act as regulators of mitochondrial Ca²⁺ levels in HF and of the bioenergetics response that will ultimately drive ATP supply and oxidative stress. The resulting model predictions propose future directions to study the evolution of HF as well as other types of heart disease, and to develop novel testable mechanistic hypotheses that may lead to improved therapeutics.

Integrative computational modeling of calcium handling and cardiac arrhythmias

Thesis

Full-text available

Jan 2021

Henry Sutanto

[CLICK DOI ABOVE TO GET THE FREE FULLTEXT] Cardiomyocyte calcium handling is a major determinant of excitation-contraction coupling. Alterations in one or more calcium-handling proteins may induce arrhythmias through the formation of ectopic activity, direct and indirect ion-channel regulation, and structural remodeling. Due to the complex and tight interactions between calcium and other molecules within a cardiomyocyte, it remains experimentally challenging to study the exact contributions of calcium-handling abnormalities to arrhythmogenesis. Multiscale computational studies performed in close collaboration with laboratory experiments create new opportunities to unravel the mechanisms of arrhythmogenesis. This thesis describes the roles of integrative computational modeling in unraveling the arrhythmogenic consequences of calcium- handling abnormalities.

Mechanoelectrical feedback in pulmonary vein arrhythmogenesis: Clinical challenges and therapeutic opportunities

Article

Full-text available

Jun 2020

Mechanoelectrical feedback is an important factor in the pathophysiology of atrial fibrillation (AF). Ectopic electrical activity originating from pulmonary vein (PV) myocardial sleeves has been found to trigger and maintain paroxysmal AF. Dilated PVs by high stretching force may activate mechanoelectrical feedback, which induces calcium overload and produces afterdepolarization. These results, in turn, increase PV arrhythmogenesis and contribute to initiation of AF. Paracrine factors, effectors of the renin‐angiotensin system, membranous channels, or cytoskeleton of PV myocytes may modulate PV arrhythmogenesis directly through mechanoelectrical feedback or indirectly through endocardial/myocardial cross‐talk. The purpose of this review is to present laboratory and translational relevance of mechanoelectrical feedback in PV arrhythmogenesis. Targeting mechanoelectrical feedback in PV arrhythmogenesis may shed light on potential opportunities and clinical concerns of AF treatment.

Cardiac Mechano-Electric Coupling: Acute Effects of Mechanical Stimulation on Heart Rate and Rhythm

Article

May 2020

The heart is vital for biological function in almost all chordates, including human. It beats continually throughout our life, supplying the body with oxygen and nutrients while removing waste products. If it stops - so does life. The heartbeat involves precise coordination of the activity of billions of individual cells, as well as their swift and well-coordinated adaption to changes in physiological demand. Much of the vital control of cardiac function occurs at the level of individual cardiac muscle cells, including acute beat-by-beat feedback from the local mechanical environment to electrical activity (as opposed to longer-term changes in gene expression, and functional or structural remodelling). This process is known as Mechano-Electric Coupling (MEC). In the current review, we: present evidence for, and implications of, MEC in health and disease in human; summarise our understanding of MEC effects gained from whole animal, organ, tissue, and cell studies; identify potential molecular mediators of MEC responses; and demonstrate the power of computational modelling in developing a more comprehensive understanding of 'what makes the heart tick'.

Cardiomyocyte calcium handling in health and disease: Insights from in vitro and in silico studies

Article

Full-text available

Mar 2020
PROG BIOPHYS MOL BIO

Calcium (Ca2+) plays a central role in cardiomyocyte excitation-contraction coupling. To ensure an optimal electrical impulse propagation and cardiac contraction, Ca2+ levels are regulated by a variety of Ca2+-handling proteins. In turn, Ca2+ modulates numerous electrophysiological processes. Accordingly, Ca2+-handling abnormalities can promote cardiac arrhythmias via various mechanisms, including the promotion of afterdepolarizations, ion-channel modulation and structural remodeling. In the last 30 years, significant improvements have been made in the computational modeling of cardiomyocyte Ca2+ handling under physiological and pathological conditions. However, numerous questions involving the Ca2+-dependent regulation of different macromolecular complexes, cross-talk between Ca2+-dependent regulatory pathways operating over a wide range of time scales, and bidirectional interactions between electrophysiology and mechanics remain to be addressed by in vitro and in silico studies. A better understanding of disease-specific Ca2+-dependent proarrhythmic mechanisms may facilitate the development of improved therapeutic strategies. In this review, we describe the fundamental mechanisms of cardiomyocyte Ca2+ handling in health and disease, and provide an overview of currently available computational models for cardiomyocyte Ca2+ handling. Finally, we discuss important uncertainties and open questions about cardiomyocyte Ca2+ handling and highlight how synergy between in vitro and in silico studies may help to answer several of these issues.

Stretch modulation of cardiac contractility: importance of myocyte calcium during the slow force response

Article

Full-text available

Jan 2020

The mechanical response of the heart to myocardial stretch has been understood since the work of muscle physiologists more than 100 years ago, whereby an increase in ventricular chamber filling during diastole increases the subsequent force of contraction. The stretch-induced increase in contraction is biphasic. There is an abrupt increase in the force that coincides with the stretch (the rapid response), which is then followed by a slower response that develops over several minutes (the slow force response, or SFR). The SFR is associated with a progressive increase in the magnitude of the Ca²⁺ transient, the event that initiates myocyte cross-bridge cycling and force development. However, the mechanisms underlying the stretch-dependent increase in the Ca²⁺ transient are still debated. This review outlines recent literature on the SFR and summarizes the different stretch-activated Ca²⁺ entry pathways. The SFR might result from a combination of several different cellular mechanisms initiated in response to activation of different cellular stretch sensors.

An Update on Hydrogen Sulfide and Nitric Oxide Interactions in the Cardiovascular System

Article

Full-text available

Sep 2018
OXID MED CELL LONGEV

Hydrogen sulfide (H 2 S) and nitric oxide (NO) are now recognized as important regulators in the cardiovascular system, although they were historically considered as toxic gases. As gaseous transmitters, H 2 S and NO share a wide range of physical properties and physiological functions: they penetrate into the membrane freely; they are endogenously produced by special enzymes, they stimulate endothelial cell angiogenesis, they regulate vascular tone, they protect against heart injury, and they regulate target protein activity via posttranslational modification. Growing evidence has determined that these two gases are not independent regulators but have substantial overlapping pathophysiological functions and signaling transduction pathways. H 2 S and NO not only affect each other’s biosynthesis but also produce novel species through chemical interaction. They play a regulatory role in the cardiovascular system involving similar signaling mechanisms or molecular targets. However, the natural precise mechanism of the interactions between H 2 S and NO remains unclear. In this review, we discuss the current understanding of individual and interactive regulatory functions of H 2 S and NO in biosynthesis, angiogenesis, vascular one, cardioprotection, and posttranslational modification, indicating the importance of their cross-talk in the cardiovascular system.

Functional heterogeneity of CPVT-mutant human cardiac ryanodine receptors: evaluation of the influence of S2031 and S2808 phosphorylation sites

Thesis

Jun 2018

Shanna Hamilton

Mechanisms of stretch-induced electro-anatomical remodeling and atrial arrhythmogenesis

Article

May 2024
J MOL CELL CARDIOL

Identification of RNA reads encoding different channels in isolated rat ventricular myocytes and the effect of cell stretching on L-type Ca 2+current

Article

Full-text available

Oct 2023
BIOL DIRECT

Background The study aimed to identify transcripts of specific ion channels in rat ventricular cardiomyocytes and determine their potential role in the regulation of ionic currents in response to mechanical stimulation. The gene expression levels of various ion channels in freshly isolated rat ventricular cardiomyocytes were investigated using the RNA-seq technique. We also measured changes in current through CaV1.2 channels under cell stretching using the whole-cell patch-clamp method. Results Among channels that showed mechanosensitivity, significant amounts of TRPM7, TRPC1, and TRPM4 transcripts were found. We suppose that the recorded L-type Ca²⁺ current is probably expressed through CaV1.2. Furthermore, stretching cells by 6, 8, and 10 μm, which increases ISAC through the TRPM7, TRPC1, and TRPM4 channels, also decreased ICa,L through the CaV1.2 channels in K⁺ in/K⁺ out, Cs⁺ in/K⁺ out, K⁺ in/Cs⁺ out, and Cs⁺ in/Cs⁺ out solutions. The application of a nonspecific ISAC blocker, Gd³⁺, during cell stretching eliminated ISAC through nonselective cation channels and ICa,L through CaV1.2 channels. Since the response to Gd³⁺ was maintained in Cs⁺ in/Cs⁺ out solutions, we suggest that voltage-gated CaV1.2 channels in the ventricular myocytes of adult rats also exhibit mechanosensitive properties. Conclusions Our findings suggest that TRPM7, TRPC1, and TRPM4 channels represent stretch-activated nonselective cation channels in rat ventricular myocytes. Probably the CaV1.2 channels in these cells exhibit mechanosensitive properties. Our results provide insight into the molecular mechanisms underlying stretch-induced responses in rat ventricular myocytes, which may have implications for understanding cardiac physiology and pathophysiology.

Nitric Oxide: Physiological Functions, Delivery, and Biomedical Applications

Article

Full-text available

Aug 2023

Nitric oxide (NO) is a gaseous molecule that has a central role in signaling pathways involved in numerous physiological processes (e.g., vasodilation, neurotransmission, inflammation, apoptosis, and tumor growth). Due to its gaseous form, NO has a short half-life, and its physiology role is concentration dependent, often restricting its function to a target site. Providing NO from an external source is beneficial in promoting cellular functions and treatment of different pathological conditions. Hence, the multifaceted role of NO in physiology and pathology has garnered massive interest in developing strategies to deliver exogenous NO for the treatment of various regenerative and biomedical complexities. NO-releasing platforms or donors capable of delivering NO in a controlled and sustained manner to target tissues or organs have advanced in the past few decades. This review article discusses in detail the generation of NO via the enzymatic functions of NO synthase as well as from NO donors and the multiple biological and pathological processes that are modulated by NO. The methods for incorporation of NO donors into diverse biomaterials including physical, chemical, or supramolecular techniques are summarized. Then, these NO-releasing platforms are highlighted in terms of advancing treatment strategies for various medical problems.

Nitric Oxide and Cardiovascular Diseases: Cardioprotection, Complications and Therapeutics

Chapter

Mar 2023

Perpetually increasing cardiovascular complications significantly contribute to economic slow-down in developing nations. Indeed, adverse cardiovascular events are among the world’s greatest mortality factors. The underlying cause behind these events is hypertension, which in advance stages, manifests with the development of multifactorial outcomes ultimately leading to organ damage and subsequent death of the individual. One of the major reasons behind the onset of hypertension is endothelial dysfunction, a physiological and clinical situation where normal functions of vascular endothelium are altered. This alteration results in a lack of proper production as well as the distribution of nitric oxide, which is a potent vasorelaxant. Efforts to maintain adequate NO signaling are always in practice. One of such approaches is targeting cytochrome b5 reductase3 at the myoendothelial junction, an anatomical location between endothelial cells and vascular smooth muscle cells. This chapter highlights the production and distribution of NO by nitric oxide synthases and cytochrome b5 reductase3, respectively, its contribution in various cascades of vascular homeostasis and its established role in cardiovascular disorders followed by different strategies and a glimpse of the clinical studies considered to improve NO signaling in vivo.

The Multidisciplinary Team (MDT) meeting and the role of pathology

Chapter

Apr 2007

Progress in Pathology reviews many aspects of pathology, describing issues of everyday diagnostic relevance and the mechanisms underlying some of these processes. Each volume in the series reviews a wide range of topics and recent advances in pathology of relevance to daily practice, keeping consultants, trainees, laboratory staff and researchers abreast of developments as well as providing candidates for the MRCPath and other examinations with answers to some of the questions they will encounter. Highly illustrated in full colour, topics covered in this volume include: Immunohistochemistry as a diagnostic aid in gynaecological pathology, Drug induced liver injury, Childhood lymphoma, Immune responses to tumours, Post-mortem imaging, Understanding the Human Tissue Act 2004 and much more. Volume 7 of Progress in Pathology will be an essential addition to the shelves and laboratory benches of every practising pathologist.

Drug induced liver injury

Chapter

Apr 2007

Understanding the Human Tissue Act 2004

Chapter

Apr 2007

Myocardial performance

Chapter

Aug 2013

In recent years our understanding of molecular mechanisms of drug action and interindividual variability in drug response has grown enormously. Meanwhile, the practice of anesthesiology has expanded to the preoperative environment and numerous locations outside the OR. Anesthetic Pharmacology: Basic Principles and Clinical Practice, 2nd edition, is an outstanding therapeutic resource in anesthesia and critical care: Section 1 introduces the principles of drug action, Section 2 presents the molecular, cellular and integrated physiology of the target organ/functional system and Section 3 reviews the pharmacology and toxicology of anesthetic drugs. The new Section 4, Therapeutics of Clinical Practice, provides integrated and comparative pharmacology and the practical application of drugs in daily clinical practice. Edited by three highly acclaimed academic anesthetic pharmacologists, with contributions from an international team of experts, and illustrated in full colour, this is a sophisticated, user-friendly resource for all practitioners providing care in the perioperative period.

The NO signalling pathway in aortic aneurysm and dissection

Article

Full-text available

Nov 2021
BRIT J PHARMACOL

Recent studies have shown that NO is a central mediator in diseases associated with thoracic aortic aneurysm, such as Marfan syndrome. The progressive dilation of the aorta in thoracic aortic aneurysm ultimately leads to aortic dissection. Unfortunately, current medical treatments have neither halt aortic enlargement nor prevented rupture, leaving surgical repair as the only effective treatment. There is therefore a pressing need for effective therapies to delay or even avoid the need for surgical repair in thoracic aortic aneurysm patients. Here, we summarize the mechanisms through which NO signalling dysregulation causes thoracic aortic aneurysm, particularly in Marfan syndrome. We discuss recent advances based on the identification of new Marfan syndrome mediators related to pathway overactivation that represent potential disease biomarkers. Likewise, we propose iNOS, sGC and PRKG1, whose pharmacological inhibition reverses aortopathy in Marfan syndrome mice, as targets for therapeutic intervention in thoracic aortic aneurysm and are candidates for clinical trials.

Expanding the Arsenal: Development of G-Protein Receptor Kinase 5 Inhibitors Utilizing a Covalent Strategy

Thesis

Jan 2020

Rachel A. Rowlands

Many cellular events are regulated by G-protein coupled receptors (GPCRs). In order to maintain homeostasis, GPCR kinases (GRKs) selectively recognize and phosphorylate activated GPCRs for internalization. There is growing evidence that both GRK2 and GRK5 have roles in pathological heart failure. Previous work has demonstrated that targeting GRK2 can reduce the amount of receptor desensitization that occurs in cardiomyocytes and increase cardiac output. However, the close homologue GRK5 is equally well expressed in cardiac tissue. Herein, we describe our efforts toward developing selective GRK5 inhibitors using covalent engagement of a non-conserved cysteine to elucidate the role of GRK5 in cardiovascular diseases. Initially, we had identified a lead compound with a pyrrolopyrimidine scaffold (GSK2163632) from the GSK Published Kinase Inhibitor Set that had modest potency for GRK5 and GRK2. Due to the similarity of the hinge region and binding pockets of GRK2 and GRK5, an approach towards building out GRK2 activity had to be based upon non-conserved residues such as Cys474. We used this approach to build covalent, and thereby selective, GRK5 inhibitors. Our initial designs allowed us to identify one strongly covalent inhibitor based on the pyrrolopyrimidine scaffold (CCG-265328) which features a weak electrophile and has 90-fold selectivity for GRK5 over GRK2. Previously, engagement of Cys474 was confirmed through tandem MS/MS, suggesting that this covalent interaction is driving selectivity. Structure-activity relationships (SAR) also revealed that linker length and degrees of freedom had less of an effect on covalent engagement than the reactivity of the electrophilic warhead. Additionally, SAR revealed that without a warhead, the modified pyrrolopyrimidine scaffold (CCG-264561) had only modest potency. We tried to improve potency through exploration of two separate scaffolds mined from a virtual screen and the literature respectively. From a virtual screen an aminopyridine compound (Chembl-1607632A) was independently synthesized and a few analogues were created. We discovered that this scaffold was unviable due to both poor potency (> 1 µM) and untransferable SAR. However, the indolinone scaffold derived from the literature proved a much more tractable option, with low nanomolar potency for the independently synthesized reference compound (CCG-271421). Interestingly, the warhead SAR developed in the pyrrolopyrimidine scaffold proved to be intractable. Indeed, the most reactive warheads, the haloketones (CCG-273220, CCG-273463) were considerably more potent and selective than the pyrrolopyrimidines (CCG-265328). In fact, the haloketones (CCG-273441) proved to be the most potent and selective covalent GRK5 inhibitors (IC50 = 3.8 nM, 1300-fold selective over GRK2) to date. Thus, we have demonstrated through development of the pyrrolopyrimidine series (CCG-265328) that Cys474 can serve as a covalent handle to convey high levels of selectivity through covalent engagement with a warhead and that this covalent strategy is transferable to different scaffolds known in kinase drug discovery, provided these scaffolds have sufficient inherent potency.

Towards precision medicine in heart failure

Article

Jun 2021

The number of therapies for heart failure (HF) with reduced ejection fraction has nearly doubled in the past decade. In addition, new therapies for HF caused by hypertrophic and infiltrative disease are emerging rapidly. Indeed, we are on the verge of a new era in HF in which insights into the biology of myocardial disease can be matched to an understanding of the genetic predisposition in an individual patient to inform precision approaches to therapy. In this Review, we summarize the biology of HF, emphasizing the causal relationships between genetic contributors and traditional structure-based remodelling outcomes, and highlight the mechanisms of action of traditional and novel therapeutics. We discuss the latest advances in our understanding of both the Mendelian genetics of cardiomyopathy and the complex genetics of the clinical syndrome presenting as HF. In the phenotypic domain, we discuss applications of machine learning for the subcategorization of HF in ways that might inform rational prescribing of medications. We aim to bridge the gap between the biology of the failing heart, its diverse clinical presentations and the range of medications that we can now use to treat it. We present a roadmap for the future of precision medicine in HF.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice

Article

Aug 2020

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates post-exercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically-necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers, or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in DMD patients.

Effect of (nitric oxide) NO donors on cardiac function

Thesis

Jan 2003

David A. Sarkar

Previous reports have demonstrated a range of negative and latterly positive inotropic responses in cardiac preparations exposed to NO. We set out to investigate the effect of NO donors on isolated myocytes and to elucidate the underlying mechanism and experimental factors governing the observed response. In isolated guinea-pig ventricular cardiomyocytes newer classes of NO donors including nitrosothiols (GSNO and SNAP) and NONOates (DEANO) induced a positive inotropic response. SNP and GTN showed no positive inotropy. The response was enhanced by co-administration of isoprenaline and reversibly abolished by the free NO scavenger oxyhaemoglobin. ODQ (soluble guanyl cyclase inhibitor) and Rp-cAMPS (protein kinase A inhibitor) did not abolish the effect. Measurement of myocyte cyclic nucleotides demonstrated a rise in cGMP, but not cAMP. Microelectrode recordings of the action potential and steady state ICa during exposure to DEANO (10?M) found no change in the action potential, though the ICa was increased with preservation of the current-voltage relationship. A faster rate of NO donor decomposition was associated with positive inotropy. Breakdown of nitrosothiols was enhanced by the presence of myocytes. Functionally the fast NO releaser DEANO was more likely to induce an increase in cell shortening compared with the slow releaser detanonoate. Positive inotropy was demonstrated in rabbit and human myocytes (from failing and non-failing hearts) but not in rat. In multi- cellular preparations the inotropic effect was reduced or absent. Myocytes from guinea-pigs treated with lipopolysaccharide demonstrated a depressed response to -adrenoceptor stimulation, which was not reversed by the nitric oxide synthase (NOS) inhibitor L-NAME. The positive inotropic response to DEANO (10 M) was unaffected. In conclusion the experiments demonstrated a positive inotropic effect of certain NO donors with variation in the response related to NO donor kinetics, animal species and preparation complexity. The effect was independent of cyclic nucleotides and mediated via the L-type calcium channel.

Focus on Cardiac Mechano-Electric Feedback

Article

Sep 2002

Peter Kohl

3D Printed Micro-Scale Force Gauge Arrays to Improve Human Cardiac Tissue Maturation and Enable High Throughput Drug Testing

Article

Dec 2018
ACTA BIOMATER

Human induced pluripotent stem cell – derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications. Statement of Significance The application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.

Inducible Nitric Oxide Synthase (iNOS) and Heart Failure

Chapter

Dec 2003

Over the last 15 years, the nitric oxide (NO) literature has experienced exponential growth (from 7 papers on endogenous NO in 1987 to over 45,000 in 2002). The link between inducible nitric oxide synthase (iNOS) and heart failure is accepted as an important association and is opening potential therapeutic avenues for the treatment of heart failure. Most literature suggests a deleterious effect of iNOS on heart function in dilated cardiomyopathy, ischemic cardiomyopathy, septic shock, cardiac allograft rejection, and viral and autoimmune myocarditis, although some controversial arguments continue.

Control of Cardiac Muscle Cell Function by an Endogenous Nitric Oxide Signaling System

Article

Full-text available

Feb 1993

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.

Calcium Sparks: Elementary Events Underlying Excitation-Contraction Coupling in Heart Muscle

Article

Full-text available

Nov 1993

Spontaneous local increases in the concentration of intracellular calcium, called "calcium sparks," were detected in quiescent rat heart cells with a laser scanning confocal microscope and the fluorescent calcium indicator fluo-3. Estimates of calcium flux associated with the sparks suggest that calcium sparks result from spontaneous openings of single sarcoplasmic reticulum (SR) calcium-release channels, a finding supported by ryanodine-dependent changes of spark kinetics. At resting intracellular calcium concentrations, these SR calcium-release channels had a low rate of opening (approximately 0.0001 per second). An increase in the calcium content of the SR, however, was associated with a fourfold increase in opening rate and resulted in some sparks triggering propagating waves of increased intracellular calcium concentration. The calcium spark is the consequence of elementary events underlying excitation-contraction coupling and provides an explanation for both spontaneous and triggered changes in the intracellular calcium concentration in the mammalian heart.

Endothelin-1 Is Involved in Mechanical Stress-induced Cardiomyocyte Hypertrophy

Article

Full-text available

Mar 1996

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10(-8) approximately 10(-7) M) maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an approximately 3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.

Endothelial nitric oxide synthase targeting to caveolae - Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells

Article

Full-text available

Oct 1996

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.

Differential regulation of nicotinic acid–adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP

Article

Full-text available

Jun 1998

The sea urchin egg has been used as a system to study calcium-release mechanisms induced by inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR), and more recently, nicotinic acid-adenine dinucleotide phosphate (NAADP). In order that cADPR and NAADP may be established as endogenous messengers for calcium release, the existence of intracellular enzymes capable of metabolizing these molecules must be demonstrated. In addition, intracellular levels of cADPR and NAADP should be under the control of extracellular stimuli. It has been shown that cGMP stimulates the synthesis of cADPR in the sea urchin egg. The present study shows that the sea urchin egg is capable of synthesizing and degrading NAADP. cADPR and NAADP synthetic activities appear to be separate, with different cellular localizations, pH and temperature optima. We suggest that in the sea urchin egg, cADPR and NAADP production may be differentially regulated by receptor-coupled second messengers, with cADPR production being regulated by cGMP and NAADP production modulated by cAMP.

Nitric Oxide Inhibits Caspase-3 by S-Nitrosationin Vivo

Article

Full-text available

Apr 1999

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 byS-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody.S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO byN-methyl-d-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely preventedS-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 μm) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with β-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-l-cysteine (50 μm). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity viaS-nitrosation of the Cys-163 residue of caspase-3.

Fas-Induced Caspase Denitrosylation

Article

Full-text available

May 1999

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Alvarez, B. V., Perez, N. G., Ennis, I. L., Camilion de Hurtado, M. C. & Cingolani, H. E. Mechanisms underlying the increase in force and Ca2+ transient that follow stretch of cardiac muscle: a possible explanation of the Anrep effect. Circ. Res. 85, 716-722

Article

Full-text available

Nov 1999
CIRC RES

Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca(2+)](i). Rat ventricular trabeculae were stretched from approximately 88% to approximately 98% of L(max), and the second force phase was analyzed. Intracellular pH, [Na(+)](i), and Ca(2+) transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94+/-0.2 g/mm(2) (P<0.01, n = 4), with the second phase accounting for 28+/-2% of the total increase (P<0.001, n = 4). During this phase, SBFI(340/380) ratio increased from 0.73+/-0.01 to 0.76+/-0.01 (P<0.05, n = 5) with an estimated [Na(+)](i) rise of approximately 6 mmol/L. [Ca(2+)](i) transient, expressed as fura-2(340/380) ratio, increased by 9.2+/-3.6% (P<0.05, n = 5). The increase in [Na(+)](i) was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na(+)](i) and [Ca(2+)](i) transient were blunted by AT(1) or ET(A) blockade. Our data indicate that the second force phase and the increase in [Ca(2+)](i) transient after stretch result from activation of the Na(+)/H(+) exchanger (NHE) increasing [Na(+)](i) and leading to a secondary increase in [Ca(2+)](i) transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET(A) receptors.

Gbeta gamma -dependent Phosphoinositide 3-Kinase Activation in Hearts with in Vivo Pressure Overload Hypertrophy

Article

Full-text available

Mar 2000

Activation of phosphoinositide 3-kinases is coupled to both phosphotyrosine/growth factor and G protein-coupled receptors. We explored the role of phosphoinositide 3-kinase activation in myocardium during in vivo pressure overload hypertrophy in mice. Cytosolic extracts from wild type hypertrophied hearts showed a selective increase in the phosphoinositide 3-kinase γ isoform. To address the role of G protein-coupled receptor-mediated activation of phosphoinositide 3-kinase, we used transgenic mice with cardiac-specific overexpression of a Gβγ sequestering peptide. Extracts from hypertrophied transgenic hearts showed complete loss of phosphoinositide 3-kinase activation, indicating a Gβγ-dependent process. To determine the class of G proteins that contribute Gβγ dimers for in vivophosphoinositide 3-kinase activation, two strategies were used: 1) transgenic mice with cardiac-specific overexpression of a Gq inhibitor peptide and 2) pertussis toxin treatment prior to pressure overload in wild type mice. Pressure overloaded Gq inhibitor transgenic mice showed a complete absence of phosphoinositide 3-kinase activation, whereas pretreatment with pertussis toxin showed robust phosphoinositide 3-kinase activation. Taken together, these data demonstrate that activation of the phosphoinositide 3-kinase during in vivo pressure overload hypertrophy is Gβγ-dependent and the Gβγ dimers arise from stimulation of Gq-coupled receptors.

A Fundamental Role for the Nitric Oxide-G-Kinase Signaling Pathway in Mediating Intercellular Ca 2+ Waves in Glia

Article

Full-text available

Apr 2000
J NEUROSCI

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Reactive oxygen species (ROS)-induced ROS release: A new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes

Article

Full-text available

Nov 2000

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

Chapter 36. Role of Nitric Oxide in Myocardial Function

Chapter

Dec 2000

Over the past two decades, the role of nitric oxide (NO) and related congeners in cardiovascular pharmacology have largely been elucidated. This chapter focuses on the extravascular production of NO in the control of cardiac contractile function. Cardiac muscle cells express the NO synthase isoform originally described in endothelial cells (eNOS), which is coupled to both β- adrenergic and cholinergic receptors in cardiac myocytes, including specialized pacemaker and conduction cells within the heart. The molecular pharmacology of NO generation in control of myocardial contractile function by the autonomic nervous system is described in detail, as is the targeting of eNOS to caveolar microdomains and its interactions with caveolin-3. Evidence pointing to the presence of another NOS isoform in the sarcoplasmic reticulum of cardiac myocytes is briefly reviewed. Response to systemic sepsis or localized infection is described.

Xu, L., Eu, J.P., Meissner, G. & Stamler, J.S. Activation of the cardiac calcium release channel (Ryanodine receptor) by Poly-S-Nitrosylation. Science 279, 234-237

Article

Jan 1998

L. J. Xu

Several ion channels are reportedly redox responsive, but the molecular basis for the changes in activity is not known. The mechanism of nitric oxide action on the cardiac calcium release channel (ryanodine receptor) (CRC) in canines was explored. This tetrameric channel contains ∼84 free thiols and is S-nitrosylated in vivo. S-Nitrosylation of up to 12 sites (3 per CRC subunit) led to progressive channel activation that was reversed by denitrosylation. In contrast, oxidation of 20 to 24 thiols per CRC (5 or 6 per subunit) had no effect on channel function. Oxidation of additional thiols (or of another class of thiols) produced irreversible activation. The CRC thus appears to be regulated by poly-S-nitrosylation (multiple covalent attachments), whereas oxidation can lead to loss of control. These results reveal that ion channels can differentiate nitrosative from oxidative signals and indicate that the CRC is regulated by posttranslational chemical modification(s) of sulfurs.

Mechanisms Underlying the Increase in Force and Ca2+ Transient That Follow Stretch of Cardiac Muscle : A Possible Explanation of the Anrep Effect

Article

Oct 1999

The skeletal muscle Ca2+ release channel: coupled O2 sensor and NO signaling functions

Article

Sep 2000

Ion channels have been studied extensively in ambient O2 tension (pO2), whereas tissue PO2 is much lower. The skeletal muscle calcium release channel/ryanodine receptor (RyR1) is one prominent example. Here we report that PO2 dynamically controls the redox state of 6-8 out of 50 thiols in each RyR1 subunit and thereby tunes the response to NO. At physiological pO2, nanomolar NO activates the channel by S-nitrosylating a single cysteine residue. Among sarcoplasmic reticulum proteins, S-nitrosylation is specific to RyR1 and its effect on the channel is calmodulin dependent. Neither activation nor S-nitrosylation of the channel occurs at ambient PO2. The demonstration that channel cysteine residues subserve coupled O2 sensor and NO regulatory functions and that these operate through the prototypic allosteric effector calmodulin may have general implications for the regulation of redox-related systems.

S-nitrosylation: Spectrum and specificity

Article

Feb 2001

Nitric oxide regulates a broad functional spectrum of proteins by S-nitrosylation. Specificity is conferred by acid–base and hydrophobic motifs that target critical cysteine residues and by protein–protein interactions that confine the signals in space.

Shah, A. M. & MacCarthy, P. A. Paracrine and autocrine effects of nitric oxide on myocardial function. Pharmacol. Ther. 86, 49-86

Article

May 2000
PHARMACOL THERAPEUT

Complex paracrine interactions exist between endothelial cells and cardiac myocytes in the heart. Cardiac endothelial cells release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1, angiotensin II, adenylpurines) that exert direct effects on myocyte function, independent of changes in coronary flow. Some of these mediators are also generated by cardiac myocytes, often under pathological conditions. This review focuses on the role of NO in this paracrine/autocrine pathway. NO modulates several aspects of “physiological” myocardial function (e.g., excitation-contraction coupling; myocardial relaxation; diastolic function; the Frank-Starling response; heart rate; β-adrenergic inotropic response; and myocardial energetics and substrate metabolism). The effects of NO are influenced by its cellular and enzymatic source, the amount generated, the presence of reactive oxygen species, interactions with neurohumoral and other stimuli, and the relative activation of cyclic GMP-dependent and -independent signal transduction pathways. The relative physiological importance of endothelium- and myocyte-derived NO remains to be established. In pathological situations (e.g., ischemia-reperfusion, left ventricular hypertrophy, heart failure, transplant vasculopathy and rejection, myocarditis), NO can potentially exert beneficial or deleterious effects. Beneficial effects of NO can result from endothelial-type nitric oxide synthase-derived NO or from spatially and temporally restricted expression of the inducible isoform, inducible-type nitric oxide synthase. Deleterious effects may result from (1) deficiency of NO or (2) excessive production, often inducible-type nitric oxide synthase-derived and usually with concurrent reactive oxygen species production and peroxynitrite formation. The balance between beneficial and deleterious effects of NO is of key importance with respect to its pathophysiological role.

Role of Ion Channels and Exchangers in Mechanical Stretch–Induced Cardiomyocyte Hypertrophy

Article

Mar 1998

We have previously reported that stretching of cardiomyocytes activates the phosphorylation cascade of protein kinases, including Raf-1 kinase and mitogen-activated protein (MAP) kinases, followed by an increase in protein synthesis partly through enhanced secretion of angiotensin II and endothelin-1. Membrane proteins, such as ion channels and exchangers, have been postulated to first receive extracellular stimuli and evoke intracellular signals. The present study was performed to determine whether mechanosensitive ion channels and exchangers are involved in stretch-induced hypertrophic responses. Neonatal rat cardiomyocytes cultured on expandable silicone dishes were stretched after pretreatment with a specific inhibitor of stretch-sensitive cation channels (gadolinium and streptomycin), of ATP-sensitive K+ channels (glibenclamide), of hyperpolarization-activated inward channels (CsCl), or of the Na+-H+ exchanger (HOE 694). Pretreatment with gadolinium, streptomycin, glibenclamide, and CsCl did not show any inhibitory effects on MAP kinase activation by mechanical stretch. HOE 694, however, markedly attenuated stretch-induced activation of Raf-1 kinase and MAP kinases by approximately 50% and 60%, respectively, and attenuated stretch-induced increase in phenylalanine incorporation into proteins. In contrast, HOE 694 did not inhibit angiotensin II-and endothelin-1-induced Raf-1 kinase and MAP kinase activation. These results suggest that among many mechanosensitive ion channels and exchangers, the Na+-H+ exchanger plays a critical role in mechanical stress-induced cardiomyocyte hypertrophy.

Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes

Article

Nov 1989

1. We measured intracellular Ca2+ transients during rapid cooling contractures (RCCs) in guinea-pig ventricular myocytes using the fluorescent Ca2+ indicator, Indo-1. 2. Rapid cooling of myocytes from 22 to 0-1 degrees C induced a rapid increase in [Ca2+]i which preceded the peak of the contraction and was sometimes large enough to saturate Indo-1. This indicates that [Ca2+]i may reach greater than 10 microM during an RCC. 3. The [Ca2+]i during the RCC slowly declined from its peak value and most of this decline in [Ca2+]i can be attributed to slow reaccumulation of Ca2+ by the sarcoplasmic reticulum (SR) in the cold. RCCs induced in the absence of Cao2+, were not different from control, supporting previous conclusions that RCCs depend exclusively on intracellular Ca2+ stores. 4. RCCs are depressed by long rest periods (rest decay) or by exposure to ryanodine or caffeine, which supports conclusions that RCCs are due to Ca2+ release from the SR. The rest decay of RCCs can be almost completely prevented by applying Nao(+)-free solution during the rest period. This implies that the loss of SR Ca2+ during rest depends on the sarcolemmal Na(+)-Ca2+ exchange (and not the sarcolemmal Ca2(+)-ATPase pump). 5. Rapid rewarming during an RCC normally leads to an additional transient contraction (or rewarming spike), without any increase in [Ca2+]i. Thus, the rewarming spike might be attributable to an increase in myofilament Ca2+ sensitivity induced by rewarming. 6. A second RCC is used to assess the fraction of Ca2+ which is re-sequestered by the SR during relaxation from the first RCC. In control solution progressive RCCs decline in amplitude, but in Na(+)-free, Ca2(+)-free solution they are of constant amplitude. We conclude that the SR Ca2+ pump and Na(+)-Ca2+ exchange are responsible for relaxation and that the latter may account for 20-50% of relaxation. 7. These results support the use of RCCs as a useful means of assessing SR Ca2+ content in intact cardiac muscle cells.

Length-dependent changes in myocardial contractile state

Article

Jun 1973
Am J Physiol

The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle

Article

Jul 1982

1. The calcium-sensitive photoprotein aequorin was micro-injected into cells of rat and cat ventricular muscles. The resulting light emission is a function of intracellular free calcium concentration ([Ca2+]i). The transient increases in [Ca2+]i that accompany contraction were monitored. 2. After an increase in muscle length, the developed tension increased immediately and then showed a slow increase over a period of minutes. The peak [Ca2+]i in each contraction was initially unchanged after an increase in muscle length but then showed a slow increase with a time course similar to that of the slow tension change. 3. As a consequence of these slow changes, the shape of the tension-length relation depends on the procedure used to determine it and this change in shape can be attributed to changes in activation. 4. Immediately after an increase in muscle length the calcium transient was abbreviated. 5. When a quick release was performed during a contraction, a short-lived increase in the [Ca2+]i was observed following the release. 6. The two previous observations can both be explained if the binding constant of troponin for calcium is a function of developed tension.

Quantitative studies on plasmalemmal folds and caveolae of rabbit ventricular myocardial cells

Article

Mar 1980

Plasmalemmal folds and caveolae were investigated by qualitative and quantitative analysis of electron micrographs obtained by freeze fracture and transmission electron microscopy (TEM) of rabbit right ventricular papillary muscles whose mean sarcomere lengths ranged from 1.64 to 2.28 micron. In passively extended muscles, folds were observed at sarcomere lengths of 2.3 micron and could be shown by extrapolation to become completely extended at a maximum sarcomere length of 2.8 micron. It was concluded that the plasmalemma does not contribute to resting tension in the physiological range of sarcomere lengths. Caveolae are present in both the external plasmalemmal envelope and T-tubular plasmalemma. They show no preferential distribution with respect to underlying myofibrillar striations or membrane folds and are nearly devoid of membrane particles in freeze-fractured material. The surface density of caveolar necks (4.0/micron2 apparent plasmalemmal area) is only 16-20% of that reported for frog skeletal muscle. Caveolae augment plasmalemmal area by 21-32%, assuming two or three caveolae per neck, respectively. Caveolar membrane does not serve as a reservoir of membrane to be recruited into external plasmalemma, at least over the physiological range of sarcomere lengths. In heart muscle they do not account for the T-tubular access resistance, and their function in this tissue remains unknown.

Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes

Article

Jun 1995
Am J Physiol

The release of sarcoplasmic reticulum (SR) Ca in cardiac muscle during excitation-contraction coupling is known to be graded by the amount of activating Ca outside the SR (i.e., Ca-induced Ca release). However, little is known about how intra-SR Ca affects the release process. In this study we assessed how the fractional SR Ca release as described by Bassani et al. [Am. J. Physiol. 265 (Cell Physiol. 34): C533-C540, 1993] is affected by alteration of trigger Ca and of SR Ca content. Experiments were done with isolated ferret ventricular myocytes using indo 1 to measure Ca concentration, perforated patch to measure Ca current (ICa), caffeine application to release SR Ca, and thapsigargin to completely block SR Ca uptake. For what we consider a Normal SR Ca load and trigger Ca [action potential at 0.5 Hz with 2 mM extracellular Ca concentration ([Ca]o)], 35 +/- 3% of the SR Ca content was released at a twitch. Changing trigger Ca by altering [Ca]o (to 0.5 and 8 mM) at a test twitch changed this fractional SR Ca release to 10 +/- 2 and 59 +/- 6%, with the same SR Ca load (and peak ICa changed in a parallel manner in separate voltage-clamp experiments). Three different levels of SR Ca load were studied (Low, Normal, and High; by action potential stimulation at different frequencies from 0.05 to 0.8 Hz) using the same standard test trigger Ca (2 mM). Surprisingly, the High-load condition only increased SR Ca content by approximately 4% but appeared to be very close to the limiting SR Ca capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ load of guinea-pig ventricular myocytes determines efficacy of brief Ca2+ currents as trigger for Ca2+ release

Article

Dec 1994

1. In guinea-pig ventricular cells, the concentration of ionized cytosolic calcium ([Ca2+]o) was estimated from the fluorescence of 100 microM K5-indo-1. At 36 degrees C and 2 mM [Ca2+]o, the Ca2+ load of the cells was varied by 1 Hz trains of conditioning clamp pulses to -30 mV (low Ca2+ load), 0 mV (intermediate Ca2+ load) and paired pulses (high Ca2+ load). After seven pulses potentiation was steady and short test pulses to 0 mV were tested for their efficacy in triggering [Ca2+]c transients. The influx of trigger Ca2+ was graded by varying the test-pulse duration between 1 and 180 ms. 2. After a 3 min rest period, [Ca2+]c was 100 +/- 20 nM (mean +/- S.E.M.) and 2 ms test pulses were unable to induce [Ca2+]c transients. Test pulses of 2 ms duration, however, induced [Ca2+]c transients after potentiation with single or paired pulses. 3. At high cellular Ca2+ load, the amplitude of the [Ca2+]c transients (delta[Ca2+]c) gradually increased with pulse durations up to 8 ms. Pulse durations between 8 and 160 ms, however, did not further increase delta[Ca2+]c as if the largest part of the [Ca2+]c transient was due to regenerative contribution of Ca(2+)-induced Ca2+ release. 4. Pulses of 160 ms duration induced 'saturating' responses whose amplitudes delta[Ca2+]c, t = infinity decreased from 938 +/- 120 nM at high Ca2+ load, to 610 +/- 90 and 350 +/- 120 nM at intermediate and low Ca2+ loads, respectively. 5. Delta[Ca2+]c was more sensitive to the duration of Ca2+ influx at low or intermediate Ca2+ loads than at high Ca2+ load. When delta[Ca2+]c was plotted against the test-pulse duration, 50% of delta[Ca2+]c, t = infinity was found to be at 9 +/- 2 ms (low), 4.6 +/- 1 ms (intermediate) or 1.8 +/- 0.5 ms pulses (high Ca2+ load). Correspondingly, the efficacy of 2 ms test pulses in triggering [Ca2+]c transients increased with the Ca2+ load. 6. At high Ca2+ load, [Ca2+]c peaked nearly independently of pulse duration at 19 +/- 3 ms. At intermediate or low Ca2+ load, time to peak increased with pulse duration. 7. The results confirm the theory that sarcoplasmic reticulum (SR) Ca2+ release contributes an amount to the [Ca2+]c transient that increases with the cellular Ca2+ load. The results are compatible with the hypothesis that SR Ca2+ release can be activated by both Ca2+ influx and by SR Ca2+ release and that the latter mechanism constitutes a positive feedback, the amplification of which increases with the amount of releasable Ca2+.

Novel method to alter length and load in isolated mammalian cardiac myocytes

Article

Nov 1994
Am J Physiol

We have devised a novel technique enabling reversible gradations in the resting and contraction length of intact mammalian ventricular myocytes of up to 15-18% over slack length. Enzymatically isolated single cells are embedded in a transparent, elastic, cross-linked fibrin matrix, contained within a narrow elastic tube. Reversible gradations in cell length are produced via fibrin matrix stretch, produced by stretching the tube. Simultaneous measurement of cell length, edge motion, and indo 1 fluorescence during auxotonic contractions permits characterization of cell contractile function. Although force cannot be directly measured, the time integral of contractile force (i.e., relative contractile impulse, a contractile index that is independent of shortening constraints) is derived combining myocyte shortening and matrix loading. Relatively small degrees of myocyte stretch produce a lightly afterloaded model dominated by variations in preload in which there is parallel augmentation of shortening and contractile impulse (force) development. At higher degrees of stretch, significant afterloading is introduced, resulting in the development of an inverse relationship between shortening and impulse (approaching isometric conditions). Length-dependent Ca2+ myofilament activation and load-dependent relaxation are readily demonstrated in intact isolated mammalian ventricular myocytes.

Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro

Article

Jan 1994
CELL

Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle in response to mechanical load. How muscle cells convert mechanical stimuli into growth signals has been a long-standing question. Using an in vitro model of load (stretch)-induced cardiac hypertrophy, we demonstrate that mechanical stretch causes release of angiotensin II (Ang II) from cardiac myocytes and that Ang II acts as an initial mediator of the stretch-induced hypertrophic response. The results not only provide direct evidence for the autocrine mechanism in load-induced growth of cardiac muscle cells, but also define the pathophysiological role of the local (cardiac) renin-angiotensin system.

Lakatta, E. G. Cardiovascular regulatory mechanisms in advanced age. Physiol. Rev. 73, 413-467

Article

May 1993

E G Lakatta

Ca2+ uptake in GH3 cells during hypotonic swelling: The sensory role of stretch-activated ion channels

Article

Jul 1996
Am J Physiol

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

Changes in [Ca2+]i, [Na+]i and Ca2+ current in isolated rat ventricular myocytes following an increase in cell length

Article

Apr 1996

Isolated rat ventricular myocytes were stretched using carbon fibres to investigate the mechanisms underlying the increase in contraction following stretch. 2. [Ca2+]i and [Na+]i were monitored using the fluorescent indicators fura-2 and sodium-binding benzofuran isophthalate, respectively. The L-type Ca2+ current was recorded simultaneously with contraction using the perforated patch-clamp technique. 3. Mechanical stretch caused an immediate increase in contraction, followed by a slow increase. Contraction was prolonged immediately after the stretch, but did not change during the slow phase. 4. The Ca2+ transient did not change immediately after the stretch. The slow increase in contraction was accompanied by an increase in the amplitude of the Ca2+ transient. However, diastolic [Ca2+]i did not change significantly following stretch. 5. [Na+]i did not change significantly either immediately, or during the slow increase in contraction, after the stretch. 6. The L-type Ca2+ current was not significantly altered either by mechanical loading of the cell with carbon fibres or by stretching the cell. 7. These results suggest that: (1) the rapid increase in contraction following a stretch is due to an increase in myofilament Ca2+ sensitivity rather than to changes in the L-type Ca2+ current or [Na+]i; and (2) a slow increase in the Ca2+ transient underlies the slow increase in contraction in isolated myocytes, but is not caused by either an increase in diastolic [Ca2+]i or a change in [Na+]i (and hence Ca2+ influx via Na(+)-Ca2+ exchange) or a change in myofilament Ca2+ sensitivity.

Nitric oxide activates skeletal and cardiac ryanodine receptors

Article

Feb 1997
CELL CALCIUM

The endothelial-derived relaxing factor, nitric oxide (NO.) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca2+ reuptake by ATP-driven Ca2+ pumps. NO did not directly alter Ca(2+)-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca2+ release induced by nitric oxide free radicals (NO.) was potentiated by cysteine providing compelling evidence that NO. in the presence of O2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.

Defective Excitation-Contraction Coupling in Experimental Cardiac Hypertrophy and Heart Failure

Article

Jun 1997

Cardiac hypertrophy and heart failure caused by high blood pressure were studied in single myocytes taken from hypertensive rats (Dahl SS/Jr) and SH-HF rats in heart failure. Confocal microscopy and patch-clamp methods were used to examine excitation-contraction (EC) coupling, and the relation between the plasma membrane calcium current (ICa) and evoked calcium release from the sarcoplasmic reticulum (SR), which was visualized as "calcium sparks." The ability of ICa to trigger calcium release from the SR in both hypertrophied and failing hearts was reduced. Because ICa density and SR calcium-release channels were normal, the defect appears to reside in a change in the relation between SR calcium-release channels and sarcolemmal calcium channels. beta-Adrenergic stimulation largely overcame the defect in hypertrophic but not failing heart cells. Thus, the same defect in EC coupling that develops during hypertrophy may contribute to heart failure when compensatory mechanisms fail.

Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes

Article

Mar 1997
Am J Physiol

In heart, spontaneous local increases in cytosolic Ca2+ concentration ([Ca2+]i) called "Ca2+ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca2+ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca2+ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca2+ sparks and SR Ca2+ content gradually decreased in rabbit. When rabbit SR Ca2+ content was decreased by reduction of stimulation rate. the initial resting spark frequency was also decreased, even though resting [Ca2+]i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na+/Ca2+ exchange (which also prevents SR Ca2+ depletion during rest). These results suggest that elevation of SR Ca2+ content can increase Ca2+ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca2+ content was constant and Ca2+ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca2+]i or SR Ca2+. This effect was also apparent in rabbit cells when SR Ca2+ depletion was prevented by blocking Na+/Ca2+ exchange. Stimulation of Ca2+ extrusion via Na+/Ca2+ exchange in the rat (by Ca2+-free superfusion, which slowly depletes SR Ca2+ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca2+ sparks increased with increasing SR Ca2+ content. In the Ca2+-overloaded state, fusion of sparks or long-lasting localized increases of [Ca2+]i were observed with increased spark frequency. We conclude that the resting frequency of Ca2+ sparks can be independently affected by changes in SR Ca2+ content, [Ca2+]i, or rest period. The latter may reflect recovery of the SR Ca2+ release channels from inactivation or adaptation.

Angiotensin II Activates Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells

Article

Sep 1997

Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein-coupled receptors. The potential involvement of PI3K in G protein-linked angiotensin II (Ang II)-dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10(-5) to 10(-8) mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT1) receptor. Immunofluorescent microscopy revealed that Ang II (10(-6) mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10(-6) mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.

Mechanical Transduction of Nitric Oxide Synthesis in the Beating Heart

Article

Oct 1997

NO alters contractile and relaxant properties of the heart. However, it is not known whether changes in ventricular loading conditions affect cardiac NO synthesis. To understand this potential contractile-relaxant autoregulatory mechanism, production of cardiac NO in response to mechanical stimuli was measured in vivo using a porphyrinic sensor placed in the left ventricular myocardium. The beating rabbit heart exhibited cyclic changes in [NO], peaking at 2.7+/-0.1 micromol/L near the endocardium and 0.93+/-0.20 micromol/L in the midventricular myocardium (concentrations were 15+/-4% lower in the rat heart). In the present study, we demonstrate for the first time that increasing or decreasing ventricular preload in vivo is followed by parallel changes in [NO], which may represent a novel autoregulatory mechanism to adjust cardiac performance or perfusion on a beat-to-beat basis. To quantify the relationship between applied force and NO synthesis, intermittent compressive or distending forces applied to ex vivo nonbeating hearts were shown to cause bursts of NO synthesis, with peak [NO] linearly related to ventricular transmural pressure. Experiments in which denuding cardiac endothelial and endocardial cells abrogated the NO signal indicate that these cells transduce mechanical stimulation into NO production in the heart. Taken together, these studies may help explain load-dependent relaxation, cardiac memory for mechanical events of preceding beats, diseases associated with myocardial distension, autoregulation of myocardial perfusion, and protection from thrombosis in the turbulent flow environment within the beating heart.

Basal Release of Nitric Oxide Augments the Frank-Starling Response in the Isolated Heart

Article

Sep 1997

The Frank-Starling response contributes to the regulation of cardiac output. The major underlying subcellular mechanism is a length-dependent change in myofilament responsiveness to Ca2+. Recent studies indicate that nitric oxide decreases myofilament responsiveness to Ca2+ and modulates myocardial relaxation and left ventricular (LV) diastolic function. We therefore investigated the interaction between nitric oxide and the Frank-Starling response. Isolated ejecting guinea pig hearts (constant afterload and heart rate) were studied before and after interventions. Elevation of filling pressure from 10 to 20 cm H2O increased cardiac output, LV end-diastolic pressure (LVEDP), and peak LV pressure (LVPmax). In the presence of N(G)-monomethyl-L-arginine (L-NMMA, 10 micromol/L; n=10) or free hemoglobin (1 micromol/L; n=8), preload-induced increases in cardiac output were significantly attenuated but baseline cardiac output was unaffected. The effects of L-NMMA were inhibited in the presence of excess L-arginine (100 micromol/L; n=6). These changes were not attributable to alterations in coronary flow. Prostaglandin F2alpha (0.01 micromol/L; n=6), which reduced coronary flow, failed to alter the cardiac output response to preload elevation. The exogenous nitric oxide donor sodium nitroprusside (1 micromol/L; n=6) reduced cardiac output at the lowest preload but not at higher preloads. LVEDP was elevated after L-NMMA and hemoglobin but reduced after sodium nitroprusside. Basal intracardiac production of nitric oxide significantly augments preload-induced rises in cardiac output in the isolated ejecting guinea pig heart. The mechanism appears to be unrelated to changes in coronary flow and may involve direct effects of nitric oxide on myocardial diastolic and/or systolic function.

Marte BM, Downward BMPKB/Akt-connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem Sci 22:355-358

Article

Oct 1997
TRENDS BIOCHEM SCI

PKB/Akt is a serine/threonine kinase that contains a pleckstrin-homology (PH) domain and is activated in response to growth-factor treatment of cells by a mechanism involving phosphoinositide 3-OH kinase. PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, perhaps explaining its discovery as a retroviral oncogene and its amplification in many human tumours.

Activation of the cardiac calcium release channel (Ryanodoine receptor) by poly-S-nitrosylation

Article

Feb 1998

Several ion channels are reportedly redox responsive, but the molecular basis for the changes in activity is not known. The mechanism of nitric oxide action on the cardiac calcium release channel (ryanodine receptor) (CRC) in canines was explored. This tetrameric channel contains approximately 84 free thiols and is S-nitrosylated in vivo. S-Nitrosylation of up to 12 sites (3 per CRC subunit) led to progressive channel activation that was reversed by denitrosylation. In contrast, oxidation of 20 to 24 thiols per CRC (5 or 6 per subunit) had no effect on channel function. Oxidation of additional thiols (or of another class of thiols) produced irreversible activation. The CRC thus appears to be regulated by poly-S-nitrosylation (multiple covalent attachments), whereas oxidation can lead to loss of control. These results reveal that ion channels can differentiate nitrosative from oxidative signals and indicate that the CRC is regulated by posttranslational chemical modification(s) of sulfurs.

Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts

Article

Mar 1998

This study tested the hypothesis that nitric oxide (NO) and atrial natriuretic peptide (ANP) can attenuate the effects of adrenergic agonists on the growth of cardiac myocytes and fibroblasts. In ventricular cells cultured from neonatal rat heart, ANP and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) caused concentration-dependent decreases in the norepinephrine (NE)-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts. In myocytes, the NO synthase inhibitor NG-monomethyl-L-arginine potentiated NE-stimulated [3H]leucine incorporation. In both cell types, ANP and SNAP increased intracellular cGMP levels, and their growth-suppressing effects were mimicked by the cGMP analogue 8-bromo-cGMP. Furthermore, in myocytes, 8-bromo-cGMP attenuated the alpha1-adrenergic receptor-stimulated increases in c-fos. Likewise, ANP and 8-bromo-cGMP attenuated the alpha1-adrenergic receptor- stimulated increase in prepro-ANP mRNA and the alpha1-adrenergic receptor-stimulated decrease in sarcoplasmic reticulum calcium ATPase mRNA. The L-type Ca2+ channel blockers verapamil and nifedipine inhibited NE-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts, and these effects were not additive with those of ANP, SNAP, or 8-bromo-cGMP. In myocytes, the Ca2+ channel agonist BAY K8644 caused an increase in [3H]leucine incorporation which was inhibited by ANP. These findings indicate that NO and ANP can attenuate the effects of NE on the growth of cardiac myocytes and fibroblasts, most likely by a cGMP-mediated inhibition of NE-stimulated Ca2+ influx.

Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins

Article

Aug 1998

Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.

Amplitude Distribution of Calcium Sparks in Confocal Images: Theory and Studies with an Automatic Detection Method

Article

Mar 1999

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.

Activation of Distinct cAMP-Dependent and cGMP-Dependent Pathways by Nitric Oxide in Cardiac Myocytes

Article

Jun 1999

Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.

Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells

Article

Jul 1999
Am J Physiol

Involvement of Akt/Protein kinase B (PKB), a serine/threonine kinase with a pleckstrin-homology domain, in angiotensin II (ANG II)-induced signal transduction was investigated in cultured vascular smooth muscle cells (VSMC). Stimulation of the cells with ANG II led to a marked increase in the kinase activity of Akt/PKB, which coincided with Ser-473 phosphorylation. ANG II-stimulated Akt/PKB activation was rapid, concentration dependent, and inhibited by the AT1-receptor antagonist CV-11974, but not by pertussis toxin. Akt/PKB activity was stimulated by the Ca2+ ionophore ionomycin, suggesting the possible involvement of Ca2+ in ANG II-stimulated Akt/PKB activation. However, blockade of Ca2+ mobilization by BAPTA-AM only partially inhibited ANG II-stimulated Akt/PKB activation. ANG II-stimulated Akt/PKB activation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A and by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002. These results indicate that ANG II stimulates Akt/PKB activity via AT1 receptors in VSMC and that the activities of tyrosine kinase and PI3K are required for this activation.

Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AMActivation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601-605

Article

Jul 1999

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.

A Role for the Sarcolemmal Na+/H+ Exchanger in the Slow Force Response to Myocardial Stretch

Article

Nov 1999
CIRC RES

Jonathan C Kentish

Although the contractile performance of the myocardium is under continuous nervous and hormonal regulation, the myocardium possesses a number of intrinsic, load-dependent mechanisms by which it can adjust cardiac output to meet the needs of the circulation over periods ranging from seconds to years. In isolated hearts, an increase in ventricular end-diastolic volume (EDV), produced by increased venous return or decreased aortic outflow, leads immediately to a more powerful contraction via the Frank-Starling mechanism (“heterometric autoregulation”1 ), so that cardiac output increases over a few beats to match venous return. However, over the next few minutes, there is a further increase in myocardial performance, such that EDV returns toward its original value. This second autoregulatory mechanism, the “Anrep effect”2 or “homeometric autoregulation,”1 allows a given change in cardiac output to be achieved with a smaller change in EDV than if the Frank-Starling effect were the only compensatory mechanism. Finally, if the increase in EDV or wall stress is maintained, genes are switched on that eventually lead to myocardial cell hypertrophy. Much is known about the cellular and molecular basis of the Frank-Starling mechanism and of the initial stages of load-induced hypertrophy, but the processes responsible for the Anrep effect are poorly understood. In this issue of Circulation Research , Alvarez et al3 suggest a novel mechanism for the slow increase in myocardial contractility: a stretch-induced activation of the sarcolemmal Na+/H+ exchanger (NHE) by local autocrine/paracrine systems involving angiotensin II (Ang II) and endothelin-1 (ET-1). Our knowledge of the cellular mechanisms involved in the time-dependent increase in contractility after myocardial fiber stretch has come chiefly from studies in which fiber length is controlled (for reviews, see References 44 –6). Parmley and Chuck7 were the first to show that stretch of isolated papillary …

Akt Promotes Survival of Cardiomyocytes In Vitro and Protects Against Ischemia-Reperfusion Injury in Mouse Heart

Article

Mar 2000
CIRCULATION

IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1-regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo. IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1-mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1-induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury. These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.

Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists

Article

Jul 1999
AM J CARDIOL

Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy.

The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular Signal-Regulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways

Article

Apr 2000
CIRC RES

The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT(1)) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT(1) sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT(1) into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH(2)-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca(2+), whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca(2+) dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca(2+) and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT(1).

Dystrophin Associates With Caveolae of Rat Cardiac Myocytes: Relationship to Dystroglycan

Article

Oct 2000
CIRC RES

The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan. Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies. To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes. Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped. Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae. These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex.

Reverse Mode of the Na+-Ca2+ Exchange After Myocardial Stretch : Underlying Mechanism of the Slow Force Response

Article

Apr 2001
CIRC RES

This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na(+) concentration ([Na(+)](i)) were assessed in cat papillary muscles stretched from 92% to approximately 98% of L(max). The SFR was 120+/-0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na(+)](i). The SFR was markedly depressed by Na(+)-H(+) exchanger inhibition, AT(1) receptor blockade, nonselective endothelin-receptor blockade and selective ET(A)-receptor blockade, extracellular Na(+) removal, and inhibition of the reverse mode of the Na(+)-Ca(2+) exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na(+)](i). Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na(+)](i). The SFR and the increase in [Na(+)](i) after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na(+)](i). The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca(2+) entry through the reverse mode of Na(+)-Ca(2+) exchange.

Upregulation of ??3-Adrenoceptors and Altered Contractile Response to Inotropic Amines in Human Failing Myocardium

Article

Apr 2001
CIRCULATION

Contrary to beta(1)- and beta(2)-adrenoceptors, beta(3)-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of beta(3)-adrenoceptors in nonfailing and failing human hearts. We analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6+/-2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2+/-3%). beta(3)-Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to beta(1)-adrenoceptor mRNA, Western blot analysis of beta(3)-adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for Galpha(i-2) proteins that couple beta(3)-adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of beta(3)-preferential agonists was only mildly reduced. Opposite changes occur in beta(1)- and beta(3)-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.