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Regional differences in Ca²⁺-independent, depolarization-activated K⁺ currents in isolated adult mouse left ventricular myocytes. Outward K⁺ currents were recorded as described in Fig. 1 during 4.5-s depolarizing voltage steps to potentials between −40 and +60 mV from a holding potential of −70 mV; the records displayed are from three different cells: one isolated from the apex (A) and the other two from the septum (B and C). As is evident, peak outward current amplitudes in A are substantially larger than those in B and C. In addition, the decay phases of the outward K⁺ currents in C are slower than those in A or B and the rapidly inactivating transient K⁺ current, Ito,f that is so prominent in A is not evident in the records in C (see text). Scale bars, 2 nA and 500 ms.
Source publication
In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three...
Citations
... Potassium currents were sampled at 5 kHz. Individual I K components were isolated kinetically as described before (Xu et al. 1999). Details of the isolation of I K components is provided in the supplementary methods. ...
Hypercholesterolemia is one of the most important risk factors for cardiovascular diseases. However, it is mostly associated with vascular dysfunction and atherosclerotic lesions, while evidence of direct effects of hypercholesterolemia on cardiomyocytes and heart function is still incomplete and controversial. In this study, we assessed the direct effects of hypercholesterolemia on heart function and the electro-contractile properties of isolated cardiomyocytes. After 5 weeks, male Swiss mice fed with AIN-93 diet added with 1.25% cholesterol (CHO), developed an increase in total serum cholesterol levels and cardiomyocytes cholesterol content. These changes led to altered electrocardiographic records, with a shortening of the QT interval. Isolated cardiomyocytes displayed a shortening of the action potential duration with increased rate of depolarization, which was explained by increased IK, reduced ICa.L and altered INa voltage-dependent inactivation. Also, reduced diastolic [Ca²⁺]i was found with preserved adrenergic response and cellular contraction function. However, contraction of isolated hearts is impaired in isolated CHO hearts, before and after ischemia/reperfusion, although CHO heart was less susceptible to arrhythmic contractions. Overall, our results demonstrate that early hypercholesterolemia-driven increase in cellular cholesterol content is associated with direct modulation of the heart and cardiomyocytes’ excitability, Ca²⁺ handling, and contraction.
... Any further responses from the reviewers can be found at the end of the article temperature-dependent action potential (Vornanen and Hassinen, 2016). Significant differences in the intracellular electrophysiology of mouse cardiomyocytes, caused by contrasting expression and activation of the delayed rectifier and transient outward K + currents as well as the voltage-gated sodium and calcium channels, are broadly illustrated in their comparatively rapid heart rate of between 500-700 bpm (Xu et al., 1999;Niwa and Nerbonne, 2010;Blechschmidt et al., 2008). Consequently, the modelling of cardiac diseases such as arrhythmia, cardiomyopathy and heart failure, which often manifest through concomitant electrical and structural remodelling, is often hampered in mice due to distinct species-dependent differences. ...
Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.
... Zebrafish offer distinct advantages including optical transparency and rapid development but are limited in their capacity as a cardiac model by their two-chambered heart and temperaturedependent action potential (Vornanen and Hassinen, 2016). Significant differences in the intracellular electrophysiology of mouse cardiomyocytes, caused by contrasting expression and activation of the delayed rectifier and transient outward K + currents as well as the voltage-gated sodium and calcium channels, are broadly illustrated in their comparatively rapid heart rate of between 500-700 bpm (Xu et al., 1999;Niwa and Nerbonne, 2010;Blechschmidt et al., 2008). Consequently, the modelling of cardiac diseases such as arrhythmia, cardiomyopathy and heart failure, which often manifest through concomitant electrical and structural remodelling, is often hampered in mice due to distinct species-dependent differences. ...
Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.
... Because of the relative ease at which their genetics can be altered, mice have been useful models for studying cardiac electric signaling (Milani-Nejad & Janssen, 2014;Nerbonne, 2004). In ventricular and atrial mouse cardiomyocytes, there are three major components of I Ksum : rapidly inactivating transient outward K þ currents (I Kto,f and/or I Kto,s ), which are conducted through K v 4.2 and K v 1.4 respectively, slowly inactivating delayed rectifier K þ currents (I Kslow1 and I Kslow2 ), which are conducted through K v 1.5/K v 2.1, and the non-inactivating steady-state K þ current (I Kss ) (Xu et al., 1999). I Kto,s (K v 1.4) is primarily found in septal cardiomyocytes (Bondarenko et al., 2004). ...
... In all myocytes isolated from the right ventricle (RV) or the apex of the left ventricle (LV), for example, there is a rapid component of current decay, consistent with the presence of a transient outward K + current (I to ), as described in cardiac myocytes in several species (Campbell et al., 1995). In adult mouse ventricular myocytes, however, this current is referred to as the "fast" transient outward current, I to,fast (I to,f ) to distinguish it from another transient outward current (see later) with slower kinetics (Xu et al., 1999a;Brunet et al., 2004). The decay phases of the Kv currents in adult mouse RV and LV myocytes ( Figure 9.2A) are well described by the sum of two exponentials with mean decay time constants (τ decay ) of ~70 ms and ~1200 ms, and a non-inactivating, "steady-state" component, I ss (Xu et al., 1999a;Brunet et al., 2004); neither time constant displays any appreciable voltage dependence (Figure 9.2B). ...
... In adult mouse ventricular myocytes, however, this current is referred to as the "fast" transient outward current, I to,fast (I to,f ) to distinguish it from another transient outward current (see later) with slower kinetics (Xu et al., 1999a;Brunet et al., 2004). The decay phases of the Kv currents in adult mouse RV and LV myocytes ( Figure 9.2A) are well described by the sum of two exponentials with mean decay time constants (τ decay ) of ~70 ms and ~1200 ms, and a non-inactivating, "steady-state" component, I ss (Xu et al., 1999a;Brunet et al., 2004); neither time constant displays any appreciable voltage dependence (Figure 9.2B). The rapidly decaying (τ decay ≈ 70 ms) component is I to,f and the slowly inactivating component (τ decay ≈ 1200 ms) is referred to as I K,slow (London et al., 1998;Xu et al., 1999a;Brunet et al., 2004). ...
... The decay phases of the Kv currents in adult mouse RV and LV myocytes ( Figure 9.2A) are well described by the sum of two exponentials with mean decay time constants (τ decay ) of ~70 ms and ~1200 ms, and a non-inactivating, "steady-state" component, I ss (Xu et al., 1999a;Brunet et al., 2004); neither time constant displays any appreciable voltage dependence (Figure 9.2B). The rapidly decaying (τ decay ≈ 70 ms) component is I to,f and the slowly inactivating component (τ decay ≈ 1200 ms) is referred to as I K,slow (London et al., 1998;Xu et al., 1999a;Brunet et al., 2004). The peak amplitudes and the waveforms of the Kv currents evoked in cells isolated from the interventricular septum (IVS) are quite different (Figure 9.2A) from those recorded Patch-Clamp Recordings of Native Membrane Currents from RV and LV cells (Xu et al., 1999a;Brunet et al., 2004). ...
... Myocytes were isolated from the LV of adult (10-20-wk-old) male and female WT, Fgf13 floxed (female Fgf13 fl/fl and male Fgf13 fl/y ), cFgf13KO (female cFgf13 −/− and male cFgf13 −/y ), and Fgf12KO mice by enzymatic and mechanical dissociation using previously described methods (Xu et al. 1999;Brunet et al., 2004). In addition, LV myocytes were isolated from cFgf13KO mice (4-6 wk) after retro-orbital (eGFP-+ FGF12B-expressing) AAV9 injections. ...
Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I–IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11–iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.
... reduction in I K,slow1 , which is a major repolarizing current that is active throughout the AP duration in mouse myocardium 50 . On the other hand, cardiomyocytes from the Tmem65 KD hearts displayed increases in I to,f which contributes primarily to early repolarization in the mouse heart 51,52 . The slow component of I to (i.e. ...
... The slow component of I to (i.e. I to,s ) was also increased following 3 weeks of Tmem65 silencing, but this is unlikely to influence APD because the slow recovery from inactivation of I to,s limits current magnitude in the rapidly beating mouse heart 51,52 . Regardless, the APD prolongation, while very modest, is consistent generally with previous studies showing APD prolongation in cardiomyopathy and heart disease. ...
The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3–7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel β subunit (β1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na⁺ currents but did show reduced Ca²⁺ and K⁺ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.
... Thus, the group of cells utilized in this study is as homogeneous as possible being from the same region of the heart. Previous published studies have shown that cardiomyocytes isolated from the ventricular septum show nearly 50 % reduction in I to currents when compared to cardiomyocytes isolated from the ventricular apex region [27,31]. Our data exclusively show recordings compatible with I to currents isolated from the apex area, validating our specific isolation from apex ventricular cardiomyocytes. ...
... A change in voltage-dependence as seen here for I K , with a shift of >20 mV in the conductance curve, attest the significance of the data presented here. First, ventricular cardiomyocytes from different parts of the heart ventricle may have their K + conductance perhaps similarly changed in amplitude, but changes in the currents' voltage dependence has not been reported [18,[31][32][33]. Second, we hypothesize that beyond a change in membrane expression of these proteins as indirectly induced by the absence of Gpc1, a different amount of heparan sulfate GAG may be in place since Gpc1 is one of the glypicans that has the GAG anchoring moieties the closest to the membrane, holding GAG negative charges close to the membrane, therefore close to the channels' extracellular surface. ...
Background
Glypican 1 (Gpc1) is a heparan sulfate proteoglycan attached to the cell membrane via a glycosylphosphatidylinositol anchor, where it holds glycosaminoglycans nearby. We have recently shown that Gpc1 knockout (Gpc1−/−) mice feature decreased systemic blood pressure. To date, none has been reported regarding the role of Gpc1 on the electrical properties of the heart and specifically, in regard to a functional interaction between Gpc1 and voltage-gated K⁺ channels.
Methods
We used echocardiography and in vivo (electrocardiographic recordings) and in vitro (patch clamping) electrophysiology to study mechanical and electric properties of mice harts. We used RT-PCR to probe K⁺ channels' gene transcription in heart tissue.
Results
Gpc1−/− hearts featured increased cardiac stroke volume and preserved ejection fraction. Gpc1−/− electrocardiograms showed longer QT intervals, abnormalities in the ST segment, and delayed T waves, corroborated by longer action potentials in isolated ventricular cardiomyocytes. In voltage-clamp, these cells showed decreased Ito and IK voltage-activated K⁺ current densities. Moreover, IK showed activation at less negative voltages, but a higher level of inactivation at a given membrane potential. Kcnh2 and Kcnq1 voltage-gated K⁺ channels subunits' transcripts were remarkably more abundant in heart tissues from Gpc1−/− mice, suggesting that Gpc1 may interfere in the steps between transcription and translation in these cases.
Conclusion
Our data reveals an unprecedented connection between Gpc1 and voltage-gated K⁺ channels expressed in the heart and this knowledge contributes to the understanding of the role of this HSPG in cardiac function which may play a role in the development of cardiovascular disease.
... The cultured H9C2 cells were grown to 80-90% in a Petri dish and were exposed to EMFs for 24 h, 48 h, and 72 h. Cardiac myocytes were isolated from male 10-week-old C57BL6 mice using the method described by Xu et al. [23]. Briefly, hearts were perfused by the Langendorff method with HEPES-buffered Earle's balanced salt solution (GIBCO-BRL) supplemented with 6 mM glucose, amino acids and vitamins (buffer A), and then with buffer A containing 0.8 mg/mL collagenase B (Boehringer-Mannheim, Mannheim, Germany) and 10 µM CaCl 2 . ...
Exposure to electromagnetic fields (EMFs) is a sensitive research topic. Despite extensive research, to date there is no evidence to conclude that exposure to EMFs influences the cardiovascular system. In the present study, we examined whether 915 MHz EMF exposure affects myocardial antioxidative and apoptotic status in vitro and in vivo. No statistically significant difference in the apoptotic cell profile and antioxidant capacity was observed between controls and short-term EMF-exposed mouse cardiomyocytes and H9C2 cardiomyoblasts. Compared with sham-exposed controls, mice subjected to a 915 MHz EMF for 48 h and 72 h had no significant effect on structural tissue integrity and myocardial expression of apoptosis and antioxidant genes. Therefore, these results indicate that short-term exposure to EMF in cardiac cells and tissues did not translate into a significant effect on the myocardial antioxidant defense system and apoptotic cell death.
... 4h, 5c) that led to an increase in I Ks (Supplementary Fig. 6c). In contrast, in cells with short APs (<100 ms), such as Ex293 (Supplementary Fig. 2b, c), serum-free cultured NRVMs (Fig. 6e), and adult mouse CMs (Fig. 8m), h2SheP current yielded APD prolongation likely due to low I Ks expression in these cells [75][76][77] . Moreover, in h2SheP-expressing mouse CMs, no increase in APA, APD 20 , or APD 50 (Fig. 8j-l) along with APD 90 prolongation, suggested that the relatively large transient outward K + current (I to ) 78-80 additionally opposed h2SheP current to prevent an increase in repolarizing K + currents. ...
Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders. In this in vitro, in silico, and in vivo study Nguyen and colleagues show that specific and stable viral gene delivery of engineered prokaryotic voltage-gated sodium channels (BacNav) to cardiomyocytes can directly augment cardiac tissue excitability and conduction.
