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Signal transduction pathways implicated in the regulation of apoptosis in cardiac myocytes This fi gure summarizes some of the signaling mechanisms by which apoptosis may be regulated in the heart as described in the text
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Apoptosis is a complex and highly regulated form of cell death, and believed to contribute to the continuous decline of ventricular function in heart failure. Apoptotic cell death is observed in a variety of cardiovascular diseases, including myocardial infarction, ischemia-reperfusion injury, end-stage heart failure, arrhythmias, and adriamycin ca...
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... ̊ B. GUSTAFSSON 1 and ROBERTA A. GOTTLIEB Cardiovascular disease is the leading cause of death in North America and predicted to O become more prevalent as our population ages. Cardiovascular disease can be initiated by multiple factors, PR but in recent years it has become clear that a major contributing factor is the loss of myocardial uncontrolled cells. manner Cell R via death necrosis can or occur by a in highly a destructive, regulated cess process that termed is characterized FO apoptosis . by Necrosis swelling is an of irreversible the cell and pro- its organelles, and disruption of the cell membrane (1). The ensuing release of cytoplasmic contents into the extracellular space provokes inflammation and can cause damage to neighboring cells. In contrast, apoptosis is a distinct form of cell death in which the cell commits to a sui- cide program, which leads to the elimination of the cell without inducing an inflammatory response. Apoptosis is characterized by cell shrinkage, chromatin condensation, formation DNA of apoptotic fragmentation, LY bodies (2). membrane Until recently, blebbing, the loss and of myocytes was attributed to necrosis; however, it is now clear that N apoptosis may play an important role in the pathogenesis O of a variety of cardiovascular diseases. For instance, apoptosis has been detected in myocardial samples G obtained from patients with end-stage congestive heart failure (3), arrhythmogenic right ventricular dysplasia (4), and myocardial infarction (5, 6). In addition, apoptosis has been detected in cardiac myocytes under hypoxia/reoxygenation (7), mechanical stretch (8), and in animal models of cardiac ischemia/reperfusion injury (9, 10). Importantly, cardiac myocytes are termi- nally differentiated; once destroyed they are not replaced. Consequently, with fewer myocytes, the ability of the myocardium to sustain contractile function is reduced. Cardiac myocyte apoptosis may lead to the loss of contractile function during heart failure, and thus, a better understanding of the complex, highly regulated apoptotic process in the heart may lead to the identification of novel therapeutic targets for cardiovascular disease. In this pa- per, we review the current evidence for apoptosis in the heart and provide an overview of the literature involving the molecular mechanisms that regulate apoptosis in the heart. Apoptosis may be induced in response to a variety of stimuli; one of the best characterized pathways is the death receptor pathway. These death receptors are a class of cell membrane receptors belonging to the tumor necrosis factor (TNF) receptor gene superfamily. Upon binding by their cognate ligand, they initiate the cellular apoptosis machinery. The death receptors contain a distinct conserved cytoplasmic domain, the death domain, which is critical for proapoptotic function (11, 12). After ligand binding, death receptors, such as Fas and TNFR1, form a homotrimeric complex, which leads to the recruitment of adaptor proteins that interact through the death domain. For Fas and TNFR1, these adaptor proteins are FADD (Fas-associated death domain) and TRADD (TNFR-associated death domain), respectively (13, 14). Activation of these two pathways leads to the recruitment and subsequent activation of caspase-8 (15, 16). The activation of caspase-8 then activates downstream effector caspases, culminating in cell death by apoptosis (17, 18). In addition to the pro-apoptotic pathway, TNFR1 also activates the transcription factor NF- κ B (19), which may induce the expression of survival genes and counteract the apoptotic pathway (20 – 22). Recent studies suggest a potential role for Fas in heart disease. Soluble Fas ligand (FasL) was elevated in patients with advanced congestive heart failure (23, 24), and Fas mRNA was expressed in failing human myocardium (25). Moreover, increased expression of Fas was reported in association with increased cardiac myocytes apoptosis in models of hypoxia (7), ischemia/reperfusion (26), and overstretched myocardium (8) (Fig. 1). Many studies have reported that cardiac myocytes are generally resistant to Fas-mediated apoptosis, and only low levels of apoptosis are observed in response to FasL (27 – 30). Wollert et al. (28) reported that stimulation of cultured cardiac myocytes with recombinant soluble FasL (sFasL) did not promote cell death. Moreover, systemic injection of an antagonistic anti-Fas antibody-induced se- vere liver damage resulting from extensive hepatocyte apoptosis but did not promote cardiac myocyte apoptosis in the myocardium (28, 31). Also, Hayakawa et al. (27) observed that cardiac myocytes as well as noncar- diomyocytes of the heart were relatively resistant to apoptosis even when sFasL was directly injected into the heart; only at very high doses did FasL induce apoptosis. However, there is strong evidence that the Fas/FasL system participates in various types of stress-induced apoptosis in the heart, where stress may sensitize cardiac myocytes to Fas. For instance, cultured neonatal cardiac myocytes are normally resistant to hypoxia-induced apoptosis (32); however, in contrast to control normoxic cells, which were resistant to FasL-induced apoptosis, neonatal myocytes subjected to hypoxia were sensitive to FasL (30). Moreover, cardiac myocytes became susceptible to FasL- induced apoptosis in the presence of nonlethal doses of doxorubicin (29). In a model of adriamycin-induced cardiomyopathy, overexpression of Fas was detected in the heart and a neutralizing FasL antibody reduced cardiac myocyte apoptosis (33). Injection of an adenovirus encod- ing FasL into the myocardium of beating hearts led to FasL expression and increased cell death of cardiac myocytes (34). Furthermore, isolated hearts from mice lacking functional Fas displayed a marked reduction in cell death (35) and infarct size following ischemia and reperfusion (34). Jeremias et al. (35) detected signi fi cant increases in FasL in the coronary ef fl uents from postischemic hearts during reperfusion. This suggests that the Fas/FasL system may be important in modulating apoptosis in the heart following ischemia/reperfusion. Signaling through the TNF receptor may also play a role in the progression of heart disease. TNF- α was present in increased levels in congestive heart failure (36), myocardial infarction (37), and ischemia/reperfusion injury (38). Furthermore, there appears to be a direct relation- ship between disease severity and circulating levels of TNF- α (39). Pentoxifylline, a drug known to inhibit TNF- α production, reportedly improved symptoms and left- ventricular function in patients with idiopathic dilated cardiomyopathy, supporting the hypothesis that elevated TNF- α contributes to the pathogenesis of cardiac dysfunction (40). Cardiac myocytes also may express TNF- α at times of stress (41, 42). Cardiac myocytes express functional TNFR1 (43, 44), and can undergo apoptosis after stimulation with TNF- α in vitro (45). As such, part of the effect of TNF- α may be due to the induction of cell death. In contrast, several studies have presented evidence of a bene fi cial role for TNF- α in the heart. TNF- α pretreatment was protective in a rat model of ischemia/reperfusion injury, where hearts from TNF- pretreated animals had signi fi cantly lower lactate dehydrogenase (LDH) release after ischemia than did control hearts (46). Subsequent studies have demonstrated that TNF- α can protect adult cardiac myocytes against hypoxia or ischemic injury (47, 48). For instance, treatment of cultured adult cardiac myocytes with TNF- α prior to hypoxia followed by reoxygenation attenuated LDH release compared to untreated hypoxic cells (47). Mice lacking TNFR had an increased infarct size and showed an increased number of apoptotic myocytes after ischemia/reperfusion injury, implying a protective effect of antiapoptotic pathways generating from the TNFR also in vivo (48). Clearly, TNFR activates multiple signal transduction pathways in the heart; however, it is not clear whether the net effect of TNF- α will be pro- or antiapoptotic and needs further investigation. The importance of NF- κ B in mediating protection against TNF- α was demonstrated by Beg and Baltimore (20), who found that embryonic fi broblasts derived from mice de fi cient in the NF- κ B subunit, p65, were sensitive to TNF- α -induced apoptosis, whereas wild-type embryonic fi broblasts were resistant to TNF- α It is thought that NF- κ B activation suppresses TNF- α -mediated apoptosis by inducing expression of proteins that interfere directly with the TNF- α death signaling pathway. In fact, Wang et al. (49) reported that NF- κ B activation blocked the activation of caspase-8 in the TNFR signaling cascade through the transcriptional regulation of factors such as TRAF (TNFR-associated factor) and IAP (Inhibitor of Apoptosis protein). Thus, the apoptotic effect of TNF- α may depend on whether NF- κ B signaling is inactivated in addition to the recruitment and activation of ...
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Apoptosis, or programmed cell death, plays a pivotal role in the elimination of unwanted, damaged, or infected cells in multicellular organisms and also in diverse biological processes, including development, cell differentiation, and proliferation. Apoptosis is a highly regulated form of cell death, and dysregulation of apoptosis results in pathol...
Citations
... Excessive reactive oxidative stress, symbolized by oxygen species (ROS) production, is recognized as an important pathological process of the APAP-induced liver injury (AILI) (Burke 2017). In response to particular signals such as ROS, the outer membrane of mitochondria becomes sufficiently permeable to release apoptosis-inducing proteins and accelerate cell death (Gustafsson and Gottlieb 2003). On the other hand, the proton gradient collapses and ATP production is disrupted as a result of the mitochondrial permeability transition pore (mPTP) opening (Gustafsson and Gottlieb 2008;Chiong et al. 2011). ...
Aims
Purinergic signaling-mediated mitochondria dysfunction and innate immune-mediated inflammation act as triggers during acetaminophen (APAP)-induced liver injury (AILI). However, the underlying mechanisms by which purinoceptor regulates mitochondria function and inflammation response in the progression of AILI remains unclear.
Methods
First, the hepatic level of purinergic receptor P2X 1 (P2RX1) was identified in the DILI patients and APAP-induced WT mice. P2rx1 knockout (KO) mice ( P2rx1 −/− ) with 300 mg/kg APAP challenge were used for the analysis of the potential role of P2RX1 in the progression of AILI. Administration of DMX, the activator of stimulator of interferon genes (STING), was performed to investigate the effects of the STING-related pathway on APAP-treated P2rx1 −/− mice.
Results
The elevated hepatic P2RX1 levels were found in DILI patients and the AILI mice. P2rx1 depletion offered protection against the initial stages of AILI, mainly by inhibiting cell death and promoting inflammation resolution, which was associated with alleviating mitochondria dysfunction. Mechanistically, P2rx1 depletion could inhibit STING-TANK-binding kinase 1 (TBK1)-P65 signaling pathways in vivo. We then showed that DMX-mediated STING activation could greatly aggravate the liver injury of P2rx1 −/− mice treated with APAP.
Conclusion
Our data confirmed that P2RX1 was inducted during AILI, identified P2RX1 as a novel regulator in mitochondria dysfunction and STING pathways, and suggested a promising therapeutic approach for AILI involving the blockade of P2RX1.
Graphical abstract
1. It first demonstrated the protective effects of P2rx1 deficiency on acetaminophen-induced liver injury (AILI).
2. P2rx1 knockout alleviates mitochondria function and promotes inflammation resolution after APAP treatment.
3. It first reported the regulation of P2RX1 on the STING signaling pathway in the progress of AILI.
4. P2RX1 blockade is a promising therapeutic strategy for AILI.
... Unlike light microscopic preparations, electron microscopy can detect ultrastructural alterations associated with apoptosis, such as nuclear fragmentation (karyorrhexis) and modest cytoplasmic organelle abnormalities [92]. In cardiomyocytes, three primary apoptosis signaling routes have been identified: (a) endoplasmic reticulum system, (b) mitochondrial pathway, and (c) extrinsic death receptor pathway (e.g., TNF and FAS) [93,94]. ...
This study compared the cardioprotective action of mesenchymal stem cells (MSCs) and PUFAs in a rat model of gentamicin (GM)-induced cardiac degeneration. Male Wistar albino rats were randomized into four groups of eight rats each: group I (control group), group II (gentamicin-treated rats receiving gentamicin intraperitoneally (IP) at dose of 100 mg/kg/day for 10 consecutive days), group III (gentamicin and PUFA group receiving gentamicin IP at dose of 100 mg/kg/day for 10 consecutive days followed by PUFAs at a dose of 100 mg/kg/day for 4 weeks), and group IV (gentamicin and MSC group receiving gentamicin IP at dose of 100 mg/kg/day followed by a single dose of MSCs (1 × 106)/rat IP). Cardiac histopathology was evaluated via light and electron microscopy. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), caspase-3 (apoptosis), Bcl2, and Bax expression was performed. Moreover, cardiac malonaldehyde (MDA) content, catalase activity, and oxidative stress parameters were biochemically evaluated. Light and electron microscopy showed that both MSCs and PUFAs had ameliorative effects. Their actions were mediated by upregulating PCNA expression, downregulating caspase-3 expression, mitigating cardiac MDA content, catalase activity, and oxidative stress parameters. MSCs and PUFAs had ameliorative effects against gentamicin-induced cardiac degeneration, with MSCs showing higher efficacy compared to PUFAs.
... 18,19 Of note, cardiomyocyte apoptosis has been indicated to result in continuous increase of ventricular dysfunction in heart failure, including cardiomyopathy, myocardial infarction and ischaemia-reperfusion injury. 20 Therefore, we made a hypothesis in the present study that ln-cRNA SNHG1 may orchestrate the DNMT1/Bcl-2 axis to affect the sepsis-induced myocardial injury. ...
Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.
... e ventricular fibrillation threshold was measured by electrical stimulation, which showed that Wenxin Granule treatment increased the ventricular fibrillation threshold, reducing the occurrence of arrhythmias (Figure 3). e ERS response is a primary pathological mechanism associated with myocardial infarction [22][23][24], and Wenxin Granules may play a protective role by reducing the activation of the UPR under ERS conditions. e ERS-initiated UPR exerts biological effects through three pathways: IRE1, PERK, and ATF6. ...
Background. Arrhythmia after myocardial infarction is the leading cause of death in clinical heart disease. Increasing studies have shown that the response to endoplasmic reticulum (ER) stress (ERS) caused by myocardial infarction is related to prognosis and the development of arrhythmias. The unfolded protein response (UPR) could serve as an important regulatory signaling pathway following myocardial infarction. The traditional Chinese medicine Wenxin Granules improve arrhythmias following myocardial infarction, which may be related to ERS intervention and the activation of the UPR and apoptosis. We aimed to investigate the involvement of Wenxin Granules in the activation of the UPR and apoptosis following myocardial infarction. Left coronary artery ligation was established as a rat model of myocardial infarction. The rats were randomly divided into the model group, low-dose Wenxin Granule group, high-dose Wenxin Granule group, and metoprolol group. Rats with only wire insertion and no ligature were used as the sham group. Small animal ultrasound systems were used to detect changes in heart structure and function, and the electrical stimulation threshold for ventricular fibrillation was detected. The expression of glucose-regulated protein (GRP)78, activating transcription factor (ATF)6, X-box binding protein (XBP)1, protein kinase–like ER kinase (PERK), phosphorylated (p)-PERK, Bax, Bcl2, C/EBP homologous protein (CHOP), caspase 12, caspase 8, and caspase 3 were detected by western blot, and terminal deoxynucleotidyl transferase dUTP Nick end labeling (TUNEL) was used to determine the cardiomyocyte apoptosis index. Compared with the sham group, rats in the model group displayed immediate ST-segment elevation and pathological Q waves after 24 hours. After 2 weeks, the left ventricular (LV) anterior wall thickness (LVAW) became thinner, and the inner diameter (LVID) increased. The end-diastolic LVAW (LVAWd), end-systolic LVAW (LVAWs), ejection fraction (EF), and fractional shortening (FS) were significantly reduced (), whereas the LVIDd, LVIDs, diastolic LV volume (LV Vold), and systolic LV volume (LV Vols) significantly increased (). The ventricular fibrillation threshold decreased significantly (). ERS proteins GRP78, p-PERK, PERK, ATF6, and XBP1 and apoptotic proteins CHOP, Bax, caspase 12, caspase 8, and caspase 3 significantly increased (, ), whereas Bcl-2 expression and the Bcl-2/Bax ratio decreased (). Compared with the sham group, LVAWd, LVAWs, FS, and Bcl-2 protein expression were significantly increased in the low-dose Wenxin Granule group (, ), and p-PERK and ATF6 decreased (, ). Compared with the sham group, LVAWd, LVAWs, EF, FS, and the ventricular fibrillation threshold significantly increased in the high-dose Wenxin Granule and metoprolol groups (, ), whereas LVIDs, LV Vols, and ERS proteins were significantly decreased (, ). CHOP, Bax, caspase 12, caspase 8, and caspase 3 protein expression decreased in the Wenxin Granule group (, ), whereas Bcl-2 and the Bcl-2/Bax ratio increased (, ). LVIDd and Bax decreased in the metoprolol group (, ), and the Bcl-2/Bax ratio increased (). The cardiomyocyte apoptosis index values for the low- and high-dose Wenxin Granule and metoprolol groups were significantly reduced (). This study suggested that the UPR is an essential mechanism underlying pathological injury after myocardial infarction. Wenxin Granule treatment can improve ventricular remodeling and cardiac function and inhibit arrhythmia by preventing excessive ERS from activating the UPR and apoptosis.
1. Introduction
Cardiovascular disease is a major cause of morbidity and mortality worldwide, and an urgent need exists for the development of new drug therapies. Growing evidence suggests that traditional Chinese medicine (TCM) can play a preventive or therapeutic role for cardiovascular disease through complex mechanisms, such as inhibiting oxidative stress, mitigating damage due to inflammation, promoting angiogenesis and antiapoptosis activities, and regulating autophagy, the gut microflora, and metabolites [1–4]. TCM has the potential to prevent a variety of cardiovascular diseases, such as hypertension, myocardial infarction, cardiomyopathy, arrhythmias, and cardiac remodeling [5–8].
The endoplasmic reticulum (ER) is the primary site of protein synthesis and transport in eukaryotic cells. When cells are stimulated by internal and external factors, the ER functional balance can change, leading to the activation of the ER stress (ERS) mechanism, resulting in the upregulation of specific ERS genes to restore homeostasis. Under conditions of persistent ERS, cells will initiate apoptosis. The ERS can be divided into the unfolded protein response (UPR), which represents the best-studied ERS mechanism, the ER overload reaction, and the cholesterol regulatory cascade reaction. ERS can activate the UPR through three different signaling pathways, including the ER transmembrane protein kinase inositol-requiring enzyme 1 (IRE1), the double-stranded RNA-dependent protein kinase–like ER kinase (PERK), and activated transcription factor 6 (ATF6). UPR activation regulates protein synthesis, degradation, and secretion to mitigate ERS by altering cellular transcription and translation programs [9–11]. ERS often leads to protein misfolding and UPR activation, and the upregulated expression of UPR components is often used as an ERS indicator. Cells activate different signaling molecules depending on the duration and intensity of ERS, which ultimately determine whether the cell adapts or dies.
ERS has also been found to regulate the inflammatory response and the apoptosis signaling pathway through the UPR, maintaining the dynamic ER balance under normal conditions and adapting to balance induced by external stimuli [10]. Excessive or long-lasting ERS leads to impaired cell function and disease development. The contributions of both intracellular and extracellular stress factors following myocardial infarction lead to the accumulation of misfolded or unfolded proteins in the ER lumen. Early or mild ERS induces the UPR, which promotes correct protein folding to alleviate ERS and plays a protective role. However, long-term or severe ERS can induce apoptosis. ERS is an important pathogenic contributor after myocardial infarction, suggesting that interventions that target UPR-related pathways may be important for the prevention and treatment of myocardial infarction.
The bidirectional function of ERS and the synergistic inflammatory response play complex and critical roles in the occurrence and development of disease. The TCM Wenxin Granules are composed of five drugs: Codonopsis, Rhizoma Polygoni, Panax notoginseng, Amber, and Gansong. Wenxin Granules replenish qi, activates blood, nourishes yin, removes blood stasis, and restores the pulse, which can be used to reduce the occurrence of arrhythmia [12, 13]. Relevant pharmacological studies have shown that the clinical effects of Wenxin Granules are related to the inhibition of myocardial remodeling and the regulation of cardiac conduction systems [14, 15]. ERS-activated UPR signaling has been identified as the pathological basis damage due to myocardial infarction through effects on myocardial remodeling and ion channel activity [16–18], which are closely related to the occurrence of arrhythmias. However, little work has examined the contributions of the UPR to arrhythmia development. In this study, we used Sprague Dawley rats to construct a rat model of myocardial infarction by ligating the anterior descending branch of the left coronary artery to clarify the induction of ERS and cell damage by myocardial infarction and further investigate the contributions of Wenxin Granules to ERS and cell death induced by myocardial infarction.
2. Materials and Methods
2.1. Animals
Specific-pathogen-free male Sprague Dawley rats were used at 6 weeks old and weighing 180 ± 20 g. Rats were obtained from the Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and were raised in a controlled environment in the animal room of Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine. The animal experiment was approved by the Laboratory Animal Welfare and Ethics Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (approval number 17-12-01). Experimental animal license number is SCXK (Beijing) 2016–0006.
2.2. Medicines
Wenxin Keli (WXKL) Granules were produced by Shandong Buchang Pharmaceutical Co., Ltd. (SFDA approval number Z10950026). The main ingredients and quality control of WXKL were those described previously [19], and the product batch number was 2007005. The medicine consists of Codonopsis, Panax notoginseng, Amber, Gansong, and Polygonatum. Metoprolol tartrate tablets were produced by AstraZeneca Pharmaceutical Co., Ltd. (National Medicine Standard H32025391).
2.3. Main Reagents and Instruments
The following reagents and instruments were used: pentobarbital sodium (Beijing Chemical Reagent Company, batch number: C06818794), hematoxylin and eosin (HE) staining solution (Nanjing Jiancheng Technology Co., Ltd., product number: D006), protease phosphatase inhibitor mixture (Beyotime Company, product number: P1045), bicinchoninic acid (BCA) protein concentration determination kit (LABLEAD company, product number: B5000), 4 × sodium dodecyl sulfate (SDS) protein loading buffer (LABLEAD company, product number: G2526-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Proteintech company, product number: 60004-1-Ig), anti-rabbit secondary antibody, anti-mouse secondary antibody (LABLEAD company, product number: s0101-1, s0100-1), PERK and p-PERK antibodies (CST company, product numbers: 3192s and 3179s, resp.), ATF6 and XBP1 antibodies (Abcam company, product numbers: ab203119 and ab37152, resp.), GRP78, CHOP, Bcl-2, Bax, caspase 3, and caspase 8 antibodies (Proteintech company, product numbers: 11587-1AP, 15204-1-AP, 26591-1-Ap, 60267-1-Ig, 19677-1-AP, and 13423-1-AP, resp.), caspase 12 antibody (Abcam, product number: ab62484), DeadEnd™ terminal deoxynucleotidyl transferase dUTP Nick end labeling (TUNEL) fluorescence detection reagent kit (Promega, product number: G3250), small animal ventilator (Shanghai Alcott Biotechnology Co., Ltd., model number: ALC-V8S), multichannel automatic analysis electrocardiograph (Beijing Futian Electronic Medical Instrument Co., Ltd., model number: FX-7202), biological function experiment signal acquisition system (Chengdu Taimeng Software Co., Ltd., model number: BL-420S), electronic balance (Shanghai Precision Scientific Instrument Co., Ltd., model number: JA1003 N), microplate reader (Thermo Fisher, USA, model number: Varioskan LUX), inverted optical microscope (Olympus, Japan, model number: Olympus BX60), small animal ultrasound system (Visual Sonics, model number: Vevo 2100), electrophoresis tank, and electrorotor tank (Beijing Longfang Technology Co., Ltd., product number: LF-mini3, LF-ZY01).
2.4. Model Preparation
A previously published paper [20] was referenced for the establishment of a rat model of myocardial infarction. First, the rats were anesthetized with 1% sodium pentobarbital (0.5 ml/100 g) injected intraperitoneally, fixed in a supine position, and intubated through the larynx and trachea. The third and fourth intercostals of the anterior area were opened laterally. The left anterior descending coronary artery was ligated using a 5/0 surgical suture approximately 2 mm below the pulmonary artery cone and the left atrial appendage. The left ventricular anterior wall (LVAW) became white, and the ST-segment of the electrocardiogram (ECG) became elevated. The chest was closed using sutures, layer by layer. A pathological Q wave appeared in the ECG 24 hours after the operation, indicating that the ligation operation was successful. The sham operation group was operated in parallel without ligation. To prevent infection, 40 U penicillin was administered by injection for 3 consecutive days after the operation.
2.5. Group Administration
The rats were randomly divided into the sham operation group, the model group, the low-dose Wenxin Granule group, the high-dose Wenxin Granule group, and the metoprolol group. The sham group received the sham operation, and all other groups were subjected to ligation. The Wenxin Granule doses were established according to the “Pharmacological Experiment Methodology” by Xu Shuyun [21], and the human clinical daily dose was converted into an equivalent dose for rats (approximately 6 times the human clinical dose) for the low-dose group, which was doubled for the high-dose group (approximately 12 times the human clinical dose). The metoprolol group received an equivalent dose (approximately 6 times the human clinical dose). Intragastric drug administration was started 24 hours after the operation and was performed once per day for 2 consecutive weeks. The model group and sham operation group were administered an equal volume of deionized water each day.
2.6. Small Animal Ultrasound Detection
Two weeks after the operation, 1% sodium pentobarbital was injected intraperitoneally to anesthetize the animals. After skin preparation, the animals were fixed in a dorsal position to perform ECG. The MS201 probe was placed on the left chest of the rat to obtain a satisfactory two-dimensional image of the long-axis of the left ventricle (LV). The probe was rotated 90° clockwise to obtain the short-axis view at the level of the papillary muscle. The short-axis LVAW end-diastolic thickness (LVAWd), LVAW end-systolic thickness (LVAWs), left ventricular inner diameter end-diastolic thickness (LVIDd), left ventricular end-systolic thickness (LVIDs), posterior wall end-diastolic thickness (LVPWd), posterior wall end-systolic thickness (LVPWs), left ventricular ejection fraction (EF), fractional shortening (FS), diastolic left ventricular volume (LV Vold), and systolic left ventricular volume (LV Vols) were measured 5 times for each animal, and the average was used as a representative value.
2.7. The Ventricular Electrical Stimulation Fibrillation Threshold
First, a cut was made between ribs 4 and 5 of the rat chest, and two pairs of tweezers were used to bluntly separate the muscles and ribs. After fully exposing the heart, the apex of the heart was connected to the positive pole of the stimulating electrode, and the negative pole was connected to the bottom of the heart, approximately 3 mm away from the positive pole. BL-420 software was used in stimulation mode, using the following parameters: (a) mode: coarse voltage; (b) delay: 5 ms; (c) frequency: 30 Hz; (d) method: string stimulation; (e) wave width: 5 ms; (f) intensity: 1 V; and (g) string length: 10 stimulation waves. Program control information was set according to the following parameters: (a) type: automatic amplitude direction increase; (b) increment: 1 V; (c) main period: 10 seconds; and (d): number of stops: 20. According to the ECG results, the voltage of ventricular fibrillation first appeared as a ventricular threshold.
2.8. Western Blot
The radioimmunoprecipitation assay (RIPA) protein lysis method was used to extract the total protein from the tissue, and the BCA method was used to determine the protein concentration. All protein samples were adjusted to the same concentration according to the measurement results and stored at −20°C. Western blot assays were performed within 2 weeks of protein lysis. SDS-polyacrylamide gel electrophoresis (PAGE) was used to separate the proteins, followed by a wet transfer to a nitrocellulose membrane, which was blocked in 5% skim milk at room temperature with shaking for 1 h. Membranes were incubated with preconfigured primary antibodies overnight at 4°C. The next day, after washing the membrane with Tris-buffered saline containing Tween 20 (TBST), the corresponding secondary antibody was added and incubated for 1 h at room temperature. After washing with TBST, the protein band was visualized using enhanced chemiluminescence (ECL). ImageJ was used to analyze the bands using GAPDH as the internal reference.
2.9. TUNEL Method to Determine the Cell Apoptosis Index
TUNEL staining was performed according to the kit instructions. Sections were fully deparaffinized, hydrated, and fixed in 4% paraformaldehyde for 15 min. Sections were washed with phosphate-buffered saline (PBS) and incubated with 20 μg/ml proteinase K at room temperature for 10 min. The balance solution was equilibrated at room temperature for 10 min, and the reaction solution was prepared on ice, followed by incubation at 37°C for 60 min. The stop solution was added for 15 min, and sections were washed with PBS solution to remove unincorporated fluorescein-12-dUTP. A drop of anti-fluorescence attenuation sealing liquid containing 4′,6-diamidino-2-phenylindole (DAPI) was added to the myocardial tissue, which was sealed with cover glass. Image acquisition was performed immediately under a fluorescence microscope (A1, ZEISS, Germany), using a standard fluorescein filter to observe green fluorescence at 520 nm, which visualized apoptotic nuclei; intact blue nuclei were visualized at 460 nm. ImageJ software was used to count the numbers of apoptotic cell nuclei and normal cell nuclei to calculate the apoptosis index.
2.10. Statistical Analysis
SPSS 23.0 software was used for all statistical analyses. Data conforming to the normal distribution are expressed as the mean ± standard deviation and assessed by one-way analysis of variance (ANOVA). Comparisons between groups were performed using the least significant difference (LSD) method. The Kruskal–Wallis rank-sum test was used for comparisons between groups that did not conform to the normal distribution. was considered significant.
3. Results
3.1. ECG Results of Rats in the Sham Operation and Model Groups
Compared with the sham operation group, the rats in the model group presented with ST-segment elevation immediately after the operation, and a pathological Q wave appeared 24 hours after the operation (Figure 1(a)).
(a)
... hücre içi iyon konsantrasyonunda oluşan değişimler ile hücre şeklinin bozulduğu ve hücre canlılığının azaldığı belirlenmiştir(41). Sonuç olarak, 2.45 GHz Wireless frekansında EMA'nın kalp dokusu üzerindeki etkisi sıcaklık artışı ve oksidatif stres aracılığı ile meydana gelmektedir. ...
Gelişen teknoloji ile birlikte günlük hayatın bir çok alanında elektromanyetik alanlar ( EMA) karşımıza çıkmaktadır. İnsanlar, yerkürenin statik manyetik alanına ek olarak, yapay EMA kaynaklarından farklı seviyelerde EMA’ya maruz kalırlar. Bir EMA dalga formu olan radyofrekans (RF) dalga aralığında bulunan 2.45 GHz elektromanyetik radyasyonun (EMR) endüstriyel, askeri ve bilimsel alanlarda kullanımı yaygındır. Bu çalışmada, EMA’nın kalp dokusu üzerine etkisi ve C vitamininin (C vit) olası koruyucu rolünü değerlendirmek için 18 dişi sıçan kontrol, EMA ve EMA + C vit olarak gruplara ayrıldı. Sıçanların hareketini kısıtlayan bir düzenek yardımıyla EMA ve EMA + C vit gruplarına 30 gün süreyle günde 60 dakika 2.24 mW/kg gücündeki 2.45 GHz EMA maruziyeti sağlandı. Tedavi grubuna ayrıca 30 gün boyunca C vit uygulandı. Deney sonunda kalp dokularında yapılan analizlerde, EMA grubunun TAS değerlerinde kontrol grubuna göre sayısal bir azalma gözlenirken, tedavi grubundaki TAS değerlerinde hem kontrol hem de EMA grubuna göre istatistiksel olarak anlamlı bir artış gözlendi. EMA grubunda TOS ve OSI kontrol grubuna göre istatistiksel olarak anlamlı yüksek iken, EMA+ C vit grubunda TOS değerindeki azalma istatistiksel olarak anlamlı bulunmadı. Histopatolojik olarak, EMA grubundaki belirgin hiperemi dışında major bir anomali gözlenmedi. EMA grubunda immunhistokimyasal olarak tesbit edilen kaspas-3 ve kaspas-8 ekspresyonları, tedavi grubunda izlenmedi. Gelişen teknoloji ile birlikte, EMA maruziyeti her geçen gün artmaktadır. Teknoloji kullanımının önlenmesinin neredeyse imkansız olacağı düşünüldüğünde, EMA’ya bağlı gelişebilecek hasarın en aza indirilmesinde C vitamini alternatif bir yöntem olarak görülebilir.
... So autophagy scavenges protein aggregates that have been accumulated during ischemia [36]. The third mechanism is that autophagy in cardiomyocytes plays an important role in reducing ROS, as well as removal of damaged organelles such as mitochondria that may release proapoptotic factors such as cytochrome c [18]. ...
Despite remarkable advances in our knowledge about the function of autophagy in myocardial ischemia/reperfusion (I/R) injury, the debate continues over whether autophagy is protective or deleterious in cardiac I/R. Due to the complexity of autophagy signaling, autophagy can play a dual role in the pathological processes of myocardial I/R injury. Thus, more researches are needed to shed light on the complex roles of autophagy in cardioprotection for the future clinical development. Such researches can lead to the finding of new therapeutic strategies for improving cardiac I/R outcomes in patients. Several preclinical studies have targeted autophagy flux as a beneficial strategy against myocardial I/R injury. In this review, we aimed to discuss the complex contribution of autophagy in myocardial I/R injury, as well as the therapeutic agents that have been shown to be useful in reducing myocardial I/R injury by targeting autophagy. For this reason, we provided an updated summary of the data from in vivo, ex vivo, and in vitro experimental studies about the therapeutic agents that exert positive effects against myocardial I/R injury by modulating autophagy flux. By addressing these valuable studies, we try to provide a motivation for the promising hypothesis of “autophagy modulation as a therapeutic strategy against cardiac I/R” in the future clinical studies.
... After an animal is slaughtered, protein fragmentation is initiated, which is generally regulated by the caspase-3 enzyme [40]. In a normal cell, activation of apoptosis occurs to remove harmful compounds and pathogens associated with hypoxia and ischemia [41]. The effect of the caspase-3 enzyme on meat tenderization varies and is likely species-dependent. ...
Objective:
The effects of a crude protease extracted from Cordyceps militaris (CM) mushrooms on the postmortem tenderization mechanism and quality improvement in spent hen breast were investigated.
Methods:
Different percentages of the crude protease extracted from CM mushrooms were introduced to spent hen breast via spray marination, and its effects on tenderness-related indexes and proteolytic enzymes were compared to papain.
Results:
The results indicated that there was a possible improvement by the protease extracted from CM mushroom through the upregulation of endogenous proteolytic enzymes involved in the calpain system, cathepsin-B, and caspase-3 coupled with its nucleotide-specific impact. However, the effect of the protease extracted from CM mushroom was likely dose-dependent, with significant improvements at a minimum level of 4%. Marination with the protease extracted from CM mushroom at this level led to increased protein solubility and an increased myofibrillar fragmentation index. The sarcoplasmic protein and collagen contents seemed to be less affected by the protease extracted from CM mushroom, indicating that substrate hydrolysis was limited to myofibrillar protein. Furthermore the protease extracted from CM mushroom intensified meat product taste due to increasing the inosinic acid content, a highly effective salt that provides umami taste.
Conclusion:
The synergistic results of the proteolytic activity and nucleotide-specific effects following treatments suggest that the exogenous protease derived from CM mushroom has the potential for improving the texture of spent hen breast.
... NF-κBderived pro-oxidants and the antioxidant Nrf2 are coreleased during inflammation; the latter becomes an essential response in an attempt to reduce the excess ROS, mitochondrial damage, and the NLRP3 inflammasome activation (Garstkiewicz et al. 2017;Liu et al. 2017;Vilhardt et al. 2017). It was reported by us that the lack of the NLRP3 inflammasome in nlrp3 −/− mice converts sepsis to a moderate inflammatory disease with a reduction in the myocardial impairment during sepsis (Martin et al. 2000;Gustafsson and Gottlieb 2003;Kensler et al. 2007;Abdullah et al. 2012). Thus, this model may yield further information regarding the Nrf2/NLRP3 inflammasome connection in sepsis. ...
... In addition to NF-κB and NLRP3 inflammasome, excessive ROS can modify the mitochondrial membrane potential and induce the release of cytochrome c into the cytosol, eventually leading to apoptosis of cardiomyocytes (Kaltschmidt et al. 2000;Gustafsson and Gottlieb 2003). Here, we found a significant reduction in mitochondrial function, including reduced RCR and ATP production, with increased proton leak in nlrp3 +/+ mice, but not in mutant mice. ...
Melatonin improved the outcome of septic cardiomyopathy by inhibiting NLRP3 priming induced by reactive oxygen species. To get insights into these events, we studied the melatonin/Nrf2 antioxidant pathways during sepsis in the heart of NLRP3-deficient mice. Sepsis was induced by cecal ligation and puncture and melatonin was given at a dose of 30 mg/kg. Nuclear turnover of Nrf2 and p-Ser40 Nrf2 and expression of ho-1 were enhanced in nlrp3+/+ and nlrp3-/- mice during sepsis. Sepsis caused higher mitochondria impairment, apoptotic and autophagic events in nlrp3+/+ mice than in nlrp3-/- animals. These findings were accompanied by greater levels of Parkin and PINK-1, and lower Mfn2/Drp-1 ratio in nlrp3+/+ than in nlrp3-/- mice during sepsis, supporting less mitophagy in the latter. Ultrastructural analysis of myocardial tissue further confirmed these observations. The activation of NLRP3 inflammasome accounted for most of the deleterious effects of sepsis, whereas the Nrf2-dependent antioxidative response activation in response to sepsis was unable to neutralize these events. In turn, melatonin further enhanced the Nrf2 response in both mice strains and reduced the NLRP3 inflammasome activation in nlrp3+/+ mice, restoring myocardial homeostasis. The data support that the anti-inflammatory efficacy of melatonin against sepsis depends, at least in part, on Nrf2 activation.
... Apoptotic proteins p53 and caspase 9 were found to be increased in EA and OA vs. Y mice in both WT and NLP3 −/− mice ( Figure 9C,D and Figure 10C,D). Oxidative stress that occurs during aging has been shown to induce apoptosis, mitochondria dysfunction in cardiomyocytes, and ultimately heart failure [99][100][101][102]. The Bax/Bcl2 ratio confirmed the increase in apoptosis with aging in WT mice. ...
Aging is a major risk for cardiovascular diseases (CVD). Age-related disorders include oxidative stress, mitochondria dysfunction, and exacerbation of the NF-κB/NLRP3 innate immune response pathways. Some of the molecular mechanisms underlying these processes, however, remain unclear. This study tested the hypothesis that NLRP3 inflammasome plays a role in cardiac aging and melatonin is able to counteract its effects. With the aim of investigating the impact of NLRP3 inflammasome and the actions and target of melatonin in aged myocardium, we analyzed the expression of proteins implied in mitochondria dynamics, autophagy, apoptosis, Nrf2-dependent antioxidant response and mitochondria ultrastructure in heart of wild-type and NLRP3-knockout mice of 3, 12, and 24 months-old, with and without melatonin treatment. Our results showed that the absence of NLRP3 prevented age-related mitochondrial dynamic alterations in cardiac muscle with minimal effects in cardiac autophagy during aging. The deficiency of the inflammasome affected Bax/Bcl2 ratio, but not p53 or caspase 9. The Nrf2-antioxidant pathway was also unaffected by the absence of NLRP3. Furthermore, NLRP3-deficiency prevented the drop in autophagy and mice showed less mitochondrial damage than wild-type animals. Interestingly, melatonin treatment recovered mitochondrial dynamics altered by aging and had few effects on cardiac autophagy. Melatonin supplementation also had an anti-apoptotic action in addition to restoring Nrf2-antioxidant capacity and improving mitochondria ultrastructure altered by aging.
... It is reported that mitochondrial pro-apoptosis protein Cytochrome c (Cyt-c) is released into the cytoplasm in response to apoptotic stimuli, which is associated with formation of apoptosomes induced by pro-caspase-9, triggering the activation of caspase-3 and apoptosis. 36 However, the release of Cyt-c can be reversed by anti-apoptosis protein Bcl-2, and its sequester Bax or other pro-apoptosis factors are integrated into the mitochondrial outer membrane. 37,38 There are also trustable indicators in oxidative stress, such as MDA contents and SOD activity, which are usually used to evaluate lipid peroxidation and inner antioxidant ability, respectively. ...
Objective
Coronary microembolization (CME) results in progressive contractile dysfunction associated with cardiomyocyte apoptosis. Alprostadil injection improves microcirculation, which is effective in treating various cardiovascular disorders. However, the therapeutic effects of alprostadil in CME-induced myocardia injury remain unknown. Therefore, we evaluated the effects of alprostadil injection on cardiac protection in a rat model of CME and explored the underlying mechanisms.
Methods
A rat model of CME was established by injecting polyethylene microspheres into the left ventricle. After injection of microspheres, rats in the alprostadil group received alprostadil via tail vein within 2 minutes. Cardiac function, histological alterations in myocardium, serum c-troponin I (cTnI) levels, myocardium adenosine triphosphate (ATP) concentrations, the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content in myocardium, and myocardial apoptosis-related proteins were detected 12 hours after CME modeling.
Results
Compared with the Sham group, ATP concentrations, SOD activity in the myocardium, and cardiac function were significantly decreased in a rat model of CME. In addition, serum cTnI levels, MDA content, expression levels of pro-apoptotic proteins, and the number of TUNEL-positive nuclei were remarkably higher in CME group than those in the Sham group. However, alprostadil treatment notably reduced serum cTnI levels and expression levels of pro-apoptotic proteins, while noticeably improved cardiac function, and accelerated SOD activity in the myocardium following CME. Additionally, it was unveiled that the protective effects of alprostadil injection inhibit CME-induced myocardial apoptosis in the myocardium potentially through regulation of the GSK-3β/Nrf2/HO-1 signaling pathway.
Conclusion
Alprostadil injection seems to significantly suppress oxidative stress, alleviate myocardial apoptosis in the myocardium, and improve cardiac systolic and diastolic functions following CME by regulating the GSK-3β/Nrf2/HO-1 signaling pathway.
