Apoptosis Of Cardiac Muscle Cells Research Articles

Novel Antiarrhythmic and Cardioprotective Effects of Brilliant Blue G

In this study, we investigated the effects of the purinergic P2X7 receptor antagonist brilliant blue G (BBG) on cardiac arrhythmia and myocardial injury induced by intravenously (i.v.) administered epinephrine in anesthetized rats. We also examined the possible involvement of beta-adrenergic receptors or cholinergic mechanisms in the effects of BBG. Sprague-Dawley rats were treated with epinephrine (10 μg/kg, i.v.). Brilliant blue G (100 mg/kg) was intraperitoneally (i.p.) administered thirty minutes prior to i.v. epinephrine. The effects of pretreatment with propranolol (2 mg/kg, i.p.) or atropine (2 mg/kg, i.v.) given prior to BBG and epinephrine were examined. The control group received saline. Moreover, the effects of only BBG on electrocardiogram (ECG) parameters were investigated. Results showed that compared with the saline control, BBG caused significant bradycardia (from 405.8 ± 1.18 to 239.4 ± 6.69 beats/min), increased RR interval (from 0.149 ± 0.002 to 0.254± 0.007 sec) and PR interval (from 0.051 ± 0.0008 to 0.059 ± 0.0004 sec), increased R wave amplitude (from 0.238 ± 0.019 to 0.548 ± 0.009 mv), and shortened QTc interval (from 0.169 ± 0.006 to 0.141 ± 0.003 sec) over 15 minutes after of BBG administration. BBG did not cause cardiac arrhythmia. Meanwhile, epinephrine produced significant bradycardia (209.8 ± 28.78 vs. 405.8 ± 1.18 beats/min), increased PR interval, prolonged the QRS complex, shortened QTc interval, decreased R wave amplitude and induced ventricular tachycardia. Brilliant blue G given prior to epinephrine increased heart rate and completely suppressed the epinephrine-induced ventricular arrhythmia. The inhibitory effect of BBG on the arrhythmia caused by epinephrine was prevented by atropine. In contrast the epinephrine induced arrhythmia was completely suppressed with propranolol and BBG. The histopathological study showed that epinephrine caused necrosis and apoptosis of cardiac muscle cells, degeneration of cardiac muscle fibers, and interstitial haemorrhages. These changes were markedly prevented by BBG alone, propranolol/BBG and to a less extent by atropine/BBG pretreatment. The study provided the first evidence for a cardioprotective and anti-arrhythmogenic actions for BBG against epinephrine-induced arrhythmia and myocardial damage, and suggested that cholinergic mechanisms are involved in its anti-arrhythmogenic action.

Clinical value of BRE-AS1 in myocardial infarction and its role in myocardial infarction-induced cardiac muscle cell apoptosis

Objectives. The aim of this study was to investigate the expression of long non-coding RNA (lncRNA) brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in patients with acute myocardial infarction (AMI) and its effect on ischemia/reperfusion (I/R)-induced oxidative stress and apoptosis of cardiomyocytes. Methods. Serum BRE-AS1 levels in patients with AMI was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic and prognostic values of BRE-AS1 were evaluated. H9c2 cells were treated with hypoxia/reoxygenation to establish an in vitro myocardial infarction cell model. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined by commercial kits. Cell counting kit-8 (CCK-8) and flow cytometry were used to evaluate the cell viability and cell apoptosis. Results. The expression of BRE-AS1 in serum of patients with AMI is upregulated, which shows the clinical diagnostic value for AMI. In the I/R injury cell model, the knockout of BRE-AS1 can significantly alleviate the increase in TNF-α, IL-1β, and IL-6 levels, inhibit the production of LDH and MDA, increase the activities of SOD and GSH-Px, promote the cell viability and suppress cell apoptosis. Conclusions. Abnormally elevated BRE-AS1 has a high diagnostic value for AMI as well as a prognostic value for major adverse cardiovascular events (MACEs). The elevation of BRE-AS1 promoted oxidative stress injury and cell apoptosis in vitro.

Retracted: MicroRNA-489 Promotes the Apoptosis of Cardiac Muscle Cells in Myocardial Ischemia-Reperfusion Based on Smart Healthcare.

[This retracts the article DOI: 10.1155/2022/2538769.].

Single cell sequencing unveils endothelial alterations after cisplatin treatment

Abstract Introduction Cisplatin, one of the most potent anti-cancer chemotherapy drugs, is still broadly used in first-line chemotherapy regimens, which are subscribed to about 10 to 20% of total cancer patients. Nephrotoxicity is a particularly limiting factor for cancer patients to remain in the effective treatment due to the acute kidney injury. It has been reported that as early as 24 hours post treatment, testicular cancer patients receiving cisplatin developed endothelial dysfunction and vascular injury. A cross-sectional follow-up study published in 2008 suggested that testicular cancer survivors continued to experience endothelial dysfunction and vascular injury. A recent 30-year follow-up study on testicular cancer survivors suggested that testicular cancer survivors experienced worse diastolic function. Purpose This study is focused on identifying the specific genes altered in cardiac endothelial cells after the cisplatin treatment to unveil the molecular mechanisms of injury for potential new therapeutic development. Methods Cisplatin induced AKI mouse model was generated by i.p. injecting 25 mg/kg cisplatin to C57BL/6 mice. Saline injection was served as control. To evaluate blood vessel damage induced by cisplatin, mice were sacrificed 48 hours post injection. Hearts were collected and single cell suspensions were produced by using Multi Tissue Dissociation Kit 2. Freshly prepared single cell suspensions were used to created libraries by using 10X genomics kits, before sequencing. The CellRanger (10X genomics) was used for processing Single cell RNASeq outputs, before secondary Seurat and DE pathway analysis. Results The GO enrichment analysis suggested that, in endothelial cells, cisplatin treatment significantly altered cellular anatomical entity, intracellular anatomical structure, apical part of the cell, cell junction, and anchoring junction. Consequently, increased vascular permeability, signaling regulating monocyte differentiation, macrophage cytokine production, and cardiac muscle cell apoptosis were observed. At molecular level, cisplatin treatment significantly upregulated DNA damage (Ddit4, Acer2), hypoxia (Phlda3, Mt1, Slc3a2, Ier3, Klf9, Adipor2, UCP2), inflammatory responses (Timp4, Tns1, Gdf15, Neat1), cellular senescence (Cdkn1a), Cell cycle arrest (Trp53inpl), intrinsic and extrinsic apoptosis (Fas, Bax, Ei24, Tgm2), blood vessel remodeling (Pim-3), and angiogenesis (Timp3, Flt1). These results indicated that cisplatin treatment likely not only result in acute endothelial dysfunction, injury, and death, but also accelerated aging, which could contribute the cardiovascular complications in the cancer survivors. Conclusions Protecting endothelial cells from oxidative stress and inflammation caused by cisplatin treatment might prevent their irreversible injury and entering into premature cellular senescence, consequently, mitigating anti-cancer treatment induced cardiovascular complications in cancer survivors. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): NIH

MicroRNA-489 Promotes the Apoptosis of Cardiac Muscle Cells in Myocardial Ischemia-Reperfusion Based on Smart Healthcare.

With the development of information technology, the concept of smart healthcare has gradually come to the fore. Smart healthcare uses a new generation of information technologies, such as the Internet of Things (loT), big data, cloud computing, and artificial intelligence, to transform the traditional medical system in an all-around way, making healthcare more efficient, more convenient, and more personalized. miRNAs can regulate the proliferation, differentiation, and apoptosis of human cells. Relevant studies have also shown that miRNAs may play a key role in the occurrence and development of myocardial ischemia-reperfusion injury (MIRI). This study aims to explore the effects of miR-489 in MIRI. In this study, miR-489 expression in a myocardial ischemia-reperfusion animal model and H9C2 cells induced by H/R was detected by qRT-PCR. The release of lactate dehydrogenase (LDH) and the activity of creatine kinase (CK) was detected after miR-489 knockdown in H9C2 cells induced by H/R. The apoptosis of H9C2 cells and animal models were determined by ELISA. The relationship between miR-489 and SPIN1 was verified by a double fluorescence reporter enzyme assay. The expression of the PI3K/AKT pathway-related proteins was detected by Western blot. Experimental results showed that miR-489 was highly expressed in cardiac muscle cells of the animal model and in H9C2 cells induced by H/R of the myocardial infarction group, which was positively associated with the apoptosis of cardiac muscle cells with ischemia-reperfusion. miR-489 knockdown can reduce the apoptosis of cardiac muscle cells caused by ischemia-reperfusion. In downstream targeting studies, it was found that miR-489 promotes the apoptosis of cardiac muscle cells after ischemia-reperfusion by targeting the inhibition of the SPIN1-mediated PI3K/AKT pathway. In conclusion, high expression of miR-489 is associated with increased apoptosis of cardiac muscle cells after ischemia-reperfusion, which can promote the apoptosis after ischemia-reperfusion by targeting the inhibition of the SPIN1-mediated PI3K/AKT pathway. Therefore, miR-489 can be one of the potential therapeutic targets for reducing the apoptosis of cardiac muscle cells after ischemia-reperfusion.

Metabolic Changes Precede Radiation-Induced Cardiac Remodeling in Beagles: Using Noninvasive 18F-FDG (18F-Fludeoxyglucose) and 13N-Ammonia Positron Emission Tomography/Computed Tomography Scans.

BackgroundThis study was performed to characterize the metabolic, functional, and structural cardiac changes in a canine model of radiation‐induced heart disease by serial in vivo imaging and ex vivo analyses.Methods and ResultsThirty‐six dogs were randomly assigned to control or irradiated groups at 3 time points (months 3, 6, and 12 after radiation; each group comprised 6 dogs). The left anterior myocardium of dogs in irradiated groups was irradiated locally with a single dose of 20‐Gy X‐ray. The irradiated myocardial regions showed increased myocardial uptake of 18F‐FDG (18F‐fludeoxyglucose) in the irradiated beagles, but the increased uptake area decreased at months 6 and 12 compared with month 3 after radiation. Abnormality of myocardial perfusion and cardiac function were detected at month 6 after radiation. Compared with the control groups, the protein expression of GLUT4 (glucose transporter 4) was upregulated in the irradiated groups, correlating with significantly decreased CPT1 (carnitine acyltransferase 1) expression. Mitochondria degeneration, swelling, and count reduction in the irradiated groups were observed. The difference in CD68 of macrophage markers and the inflammatory cytokines (IL‐6 [interleukin 6], TNF‐α [tumor necrosis factor α]) between the irradiation and control groups was not significant. Furthermore, the progressive aggravation of apoptosis and fibrosis was displayed.ConclusionsElevated 18F‐FDG uptake occurred after irradiation and subsequently led to ventricular perfusion defects and dysfunction. The process was associated with myocardial metabolic substrate remodeling, cardiac muscle cell apoptosis, and myocardial fibrosis rather than inflammation.

Improvement of therapeutic effects of mesenchymal stem cells in myocardial infarction through genetic suppression of microRNA-142

Transplanted mesenchymal stem cells (MSCs) have been shown to contribute to myocardial repair after myocardial infarction (MI), primarily through production and secretion some growth factors and cytokines related to cell survival and regeneration. Further improvement of the therapeutic potential of MSCs appears to be an attractive strategy for MI treatment. CXC chemokine receptor (CXCR) 7 is the receptor for stromal cell-derived factor-1 (SDF-1), an important chemokine that is essential for tissue repair and angiogenesis. SDF-1/CXCR7 axis plays a critical role in the mobilization, recruitment and function of MSCs during tissue regeneration. Here, we depleted miR-142 that targets CXCR7 in MSCs cells through expression of antisense of miR-142, resulting in enhanced expression of CXCR7 in these miR-142-depleted MSCs (md-MSCs). In vitro, presence of md-MSCs reduced hypoxia-induced cardiac muscle cell apoptosis in a more pronounced manner than MSCs. In vivo, compared to transplantation of MSCs, transplantation of md-MSCs further enhanced cardiac re-vascularization and further improved cardiac functions after MI in mice. Together, our data suggest that depletion of miR-142 in MSCs may improve their therapeutic effects on MI.

Effect of stromal cell-derived factor-1 on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction

The aim of the study was to investigate the effect of stromal cell-derived factor-1 (SDF-1) on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction (AMI) and the mechanism of the Toll-like receptor (TLR)-4/nuclear factor-κB (NF-κB) signaling pathway. A total of 64 healthy male F344 rats were randomly divided into the sham operation, model, SDF-1 intervention and SDF-1 antibody groups, with 16 rats in each group. The method of Olivette was used to establish the AMI model by ligation of the left anterior descending artery. Day 1 after establishing the animal model, the rats in the SDF-1 intervention group were injected with 10 µl recombinant SDF-1 (400 ng/ml) in five regions including the myocardial infarction area and the four surrounding areas. The rats in the model group were injected with 10 µl normal saline including the myocardial infarction area and the four surrounding areas, and those in the SDF-1 antibody group were injected with 1 ml SDF-1 antibody (2 µg/ml). Four rats were sacrificed after 1, 3, 7 and 14 days after the intervention, and the analysis was carried out. TUNEL in situ labeled apoptotic cells were used for cell counting, and immunohistochemical staining was performed to measure vascular density. The animal echocardiographic measurement was for the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular fractional shortening (FS) and ejection fraction (EF) values. The results showed that the number of apoptotic cells in the SDF-1 treatment group was significantly lower than those in the other groups at each time-point. The vessel densities in the 3–14 days were significantly greater than those in other groups. At each time-point, the LVEDd and LVESd values were smaller compared with the model group, but greater than the sham operation group and decreased over time. FS and EF values were higher than those in the model group at each time-point, but less than those of the sham operation group and increased over time. The expression levels of TLR-4 and NF-κB at each time-point were significantly higher than those of the remaining groups (p<0.05). In conclusion, SDF-1 is capable of decreasing the apoptosis of cardiac muscle cells in AMI, promoting angiogenesis and improving cardiac function, which may be associated with the activation of the TLR-4/NF-κB signaling pathway.

Effects of cardiac biological activities on low-intensity physical training in doxorubicin-induced cardiotoxicity rat models

Objective: In the present study, we investigated the protective effects of low-intensity treadmill training in doxorubicin-induced cardiotoxicity rat models. Design: Randomized controlled trial. Methods: In this study, we randomly divided them into four groups. The normal group included non-cardiotoxicity normal control (n=10), the control group included non-treadmill training after doxorubicin-induced cardiotoxicity (n=10), the experimental group I included low-intensity treadmill training (3 m/min) after doxorubicin-induced cardiotoxicity (n=10), and the experimental group II included low-intensity treadmill training (8 m/min) after doxorubicin-induced cardiotoxicity (n=10). Rats in the treadmill training group underwent treadmill training, which began at 2 weeks after first intraperitoneal injection. We determined the body weight change for each rat on days 1 and 21. Biochemical markers (lactate dehydrogenase [LDH], creatine kinase [CK], glutathion, aspartate transaminase [AST], and alanine transaminase [ALT]) concentration in the serum change of rats from all four groups was examined at the end of the experiment. Results: The results showed that the experimental group I and II showed a significant increase in body weight as compared with that of the control group (p<0.05). We observed that the biochemical markers (LDH, CK, glutathion, AST, and ALT) were improved in the experimental group I than the experimental group II (p<0.05). There was no difference between the experimental groups. Conclusions: In conclusion, our data suggest that low-intensity treadmill training applied after doxorubicin treatment protects against cardiotoxicity following treatment, possibly by enhancing antioxidant defenses and inhibiting cardiac muscle cell apoptosis.

Thymoquinone enhances coronary flow and reduces infarct size in Langendorff‐perfused rat hearts through attenuation of inflammatory responce and apoptosis

The use of natural compounds to protect against a variety of cardiovascular diseases has been established. Thymoquinone (TQ), a constituent of the volatile oil derived from Nigella sativa seeds, possesses antioxidant, anti‐inflammatory, and vasodilating properties. TQ has demonstrated promising effects against ischemia‐reperfusion (IR) injury in different organs. However, the effect of TQ on myocardial IR injury (m‐IRI) has not been delineated. We show the efficacy and provide a mechanism of TQ effect on m‐IRI. Langendorff‐perfused rat hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. TQ was infused for 20 min either pre‐or post‐ischemia. Isolated hearts of TQ‐treated rats were also subjected to the same IR protocols. TQ significantly enhanced coronary flow and markedly reduced the infarct size. IR‐induced increase of TNF‐α, myocardial NF‐kB protein expression, and apoptosis of cardiac muscle cells were attenuated following TQ treatment. Moreover, TQ modulated the expression of myocardial Bcl2, and Bax proteins and also markedly reduced oxidative stress markers and LDH release. This study indicates that TQ induced inhibition of the pro‐inflammatory cytokines and oxidative stress modulated myocardial NF‐kB, Bcl2, and Bax expression, protecting the heart against IR injury. We provide mechanistic insight for understanding the molecular basis and efficacy of TQ on m‐IRI. (NIH # HL63744 to JLZ; Egyptian government)

Mitochondrial Akt-regulated mitochondrial apoptosis signaling in cardiac muscle cells

We recently reported translocation and activation of Akt in cardiac mitochondria. This study was to determine whether activation of Akt in mitochondria could inhibit apoptosis of cardiac muscle cells. Insulin stimulation induced translocation of phosphorylated Akt to the mitochondria in primary cardiomyocytes. A mitochondria-targeted constitutively active Akt was overexpressed via adenoviral vector and inhibited efflux of cytochrome c and apoptosis-inducing factor from mitochondria to cytosol and partially prevented loss of mitochondria cross-membrane electrochemical gradient. Activation of caspase 3 was suppressed in the cardiomyocytes transduced with mitochondria-targeted active Akt, whereas a mitochondria-targeted dominant negative Akt enhanced activation of caspase 3. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay showed that mitochondrial activation of Akt significantly reduced the number of apoptotic cells. When the endogenous Akt was abolished by LY294002, the antiapoptotic actions of mitochondrial Akt remained effective. These experiments suggested that mitochondrial Akt suppressed apoptosis signaling independent of cytosolic Akt in cardiac muscle cells.

Davallialactone protects against adriamycin-induced cardiotoxicity in vitro and in vivo

Adriamycin (ADR) is a potent anticancer drug. Its clinical applications are limited due to its cardiotoxicity. Oxidative stress is responsible for cardiomyopathy induced by ADR. Previous studies have demonstrated that davallialactone (DAVA), extracted from mushroom Inonotus xeranticus, has potential antiplatelet aggregation activity and free radical scavenging properties. In this study, we investigated whether DAVA has protective effects against ADR-induced free radical accumulation and apoptosis in cardiac muscle cells and compared the effects of DAVA with N-acetylcysteine, a potent antioxidant. We evaluated the effect of DAVA on ADR-induced cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and crystal violet staining, the reactive oxygen species (ROS) production by flow cytometry, and the expression of stress-related proteins like Cu/Zn superoxide dismutase (SOD), Mn-SOD, and the involvement of mitogen-activated protein kinase pathway by Western blot analysis. Apoptosis was assessed by nuclear condensation and the expression levels of pro-apoptotic proteins, such as caspase-3 and polyadenosine diphosphate-ribose polymerase (PARP). The cardio-protective effects of DAVA were also evaluated in an in vivo study in an animal model of ADR-induced acute cardiomyopathy. Our results showed that DAVA significantly increased the viability of doxorubicin-injured H9c2 cells and inhibited ADR-induced ROS production, apoptosis, and the expression of Cu/Zn SOD and Mn-SOD. DAVA also inhibited the expression of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), which was activated by ADR. In the in vivo animal model, treatment involving DAVA significantly reduced cardiomyocyte lesions. These results suggest that DAVA is a potentially protective agent for ADR-induced cardiotoxicity in cardiomyocytes and can be a potential candidate to protect against cardiotoxicity in ADR-treated cancer patients.

Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species

Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is known for its anti-oxidant properties. It has been identified as increased apoptosis in several cancer cell lines in combination with doxorubicin, but there are few studies about the effect of curcumin and doxorubicin on normal cardiac cells. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Preteatment with curcumin significantly increased DOX-induced apoptosis of cardiac muscle cells through down regulation of Bcl-2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1h pretreatment with curcumin. As well, curcumin increases ROS generation by DOX. In response to DOX, NF-κB was activated. However, curcumin was able to inhibit NF-κB activation. In conclusion, our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS.

Effects of acupuncture pretreatment on ischemic cardiac muscle cell apoptosis and gene expression in ischemia-reperfusion rats

Objective To investigate the protective effects of acupuncture pretreatment on ischemic myocardium, the protective mechanism of acupuncture pretreatment on ischemic myocardium was explored by observing the cardiac muscle cell apoptosis and the expression of HSP70 mRNA of ischemia-reperfusion injury rats treated with acupuncture pretreatment.

Plantainoside D protects adriamycin-induced apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS generation and NF-κB activation

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-κB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-κB. We also observed that the NF-κB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IκBα-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-κB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.

Effect of L-carnitine on cardiomyocyte apoptosis and cardiac function in patients undergoing heart valve replacement operation

The effects of L-carnitine, as an ingredient of cardioplegia solution, on cardiac function and cardiomyocyte apoptosis in patients undergoing heart valve replacement operation were investigated. Twenty-three cases undergoing heart valve replacement with cardiopulmonary bypass (CPB) were randomly allocated into two groups: L-carnitine group (n = 12, 12 g/L L-carnitine was put in the ST. Thomas cardioplegia) and control group (n = 11, identical to the L-carnitine group except that normal saline was administered instead of L-carnitine). Serum cardial troponin I (cTnI) levels, the left ventricular ejection fraction (LVEF), and cardiac index (CI) were measured perioperatively. A bit of myocardial tissue obtained from right atria was taken before CPB and by the end of intracardiac procedure to undergo electron microscopy examination and estimate apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). From the end of CPB to 3 days after operation, the serum levels of cTnI in the L-carnitine group was significantly lower than that in the control group (P < 0.05). Heart color ultrasonogram showed that the CI index and LVEF at 7th day postoperatively in the L-carnitine group were significantly higher than in the control group (P < 0.05). Compared to the control group, L-carnitine significantly alleviated the morphologic changes of cardiac muscle cells (electron microscopy examination) and decreased the amounts of apoptotic cardiac muscle cells (TUNEL). Furthermore, the dosage of vasoactive drugs used after operation was significantly less in the L-carnitine group (P < 0.01). It was concluded that L-carnitine cardioplegia solution could improve cardiac function in patients undergoing heart valve replacement operation and alleviate CPB-mediated apoptosis of cardiac muscle cells.

Metabolic and functional protection by selective inhibition of nitric oxide synthase 2 during ischemia-reperfusion in isolated perfused hearts.

Drugs that selectively block nitric oxide synthase (NOS) 2 enzyme activity by inhibiting dimerization of NOS2 monomers have recently been developed. To investigate whether selective inhibition of NOS2 is cardioprotective, rats were pretreated for 2 days with BBS2, an inhibitor of NOS2 dimerization, at 15 mg/kg SC. Isolated buffer-perfused hearts from treated (n=9) and control (n=7) hearts were subjected to 20 minutes of ischemia followed by 60 minutes of reperfusion. NOS2 protein was upregulated in all hearts at the end of ischemia and of reperfusion; NOS2 enzyme activity was 60% lower in hearts from the treated animals. In the treated hearts, the increase in end-diastolic pressure was significantly attenuated at the end of ischemia, and the return of developed pressure at reperfusion was greater (P<0.05). Creatine kinase release at reperfusion was lower in treated hearts than in controls (P=0.02). At the end of ischemia and of reperfusion, myocardial ATP levels were significantly higher in the treated hearts than in controls (P<0.05). In the treated hearts under ischemic conditions, lactate content was higher and the lactate/pyruvate ratio was lower than in controls (P<0.05); GAPDH activity was higher; and G-3-P and aldose reductase activity were lower. At reperfusion, in the treated hearts, there was less histological damage and less apoptosis of cardiac muscle cells. Pretreatment with BBS2, a selective inhibitor of NOS2, improves contractile performance, preserves myocardial ATP, and reduces damage and death of cardiac myocytes during ischemia and reperfusion of isolated buffer-perfused rat hearts.

Signal transduction of the protective effect of insulin like growth factor-1 on adriamycin-lnduced apoptosis in cardiac muscle cells

To determine whether Insulin-like growth factor (IGF-I) treatment represents a potential means of enhancing the survival of cardiac muscle cells from adriamycin (ADR)-induced cell death, the present study examined the ability of IGF-I to prevent cell death. The study was performed utilising the embryonic, rat, cardiac muscle cell line, H9C2. Incubating cardiac muscle cells in the presence of adriamycin increased cell death, as determined by MTT assay and annexin V-positive cell number. The addition of 100 ng/mL IGF-I, in the presence of adriamycin, decreased apoptosis. The effect of IGF-I on phosphorylation of PI, a substrate of phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase B (AKT), was also examined in H9C2 cardiac muscle cells. IGF-I increased the phosphorylation of ERK 1 and 2 and PKC zeta kinase. The use of inhibitors of PI 3-kinase (LY 294002), in the cell death assay, demonstrated partial abrogation of the protective effect of IGF-I. The MEK1 inhibitor-PD098059 and the PKC inhibitor-chelerythrine exhibited no effect on IGF-1-induced cell protection. In the regulatory subunit of PI3K-p85- dominant, negative plasmid-transfected cells, the IGF-1-induced protective effect was reversed. This data demonstrates that IGF-I protects cardiac muscle cells from ADR-induced cell death. Although IGF-I activates several signaling pathways that contribute to its protective effect in other cell types, only activation of PI 3-kinase contributes to this effect in H9C2 cardiac muscle cells.

Cyclic Adenosine Monophosphate Inhibits Nitric Oxide‐Induced Apoptosis of Cardiac Muscle Cells in a c‐Jun N‐Terminal Kinase‐Dependent Manner

Cyclic adenosine monophosphate (cAMP) modulates various agent‐induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)‐induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP‐induced apoptosis in H9c2 cells. In contrast, H‐89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP‐induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP‐activated p38 MAPK phosphorylation and did not affect SNP‐mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP‐induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP‐protective effect in SNP‐induced apoptosis. In addition, c‐AMP relating agents protected SNP‐induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP‐relating agent‐induced protective effect is not resricted in H9c2 cardiac muscle cells.

Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells

The development of doxorubicin cardiomyopathy involves apoptosis of cardiac muscle cells. This study was carried out to define the roles of two heat-shock proteins, Hsp10 and Hsp60, on doxorubicin-induced apoptosis in primary cardiomyocytes. Doxorubicin induces apoptosis of cardiomyocytes by activating mitochondria apoptosis signaling. Transducing cardiomyocytes with Hsp10 or Hsp60 with adenoviral vector suppressed the occurrence of apoptosis in the doxorubicin-treated cardiomyocytes. Overexpression of Hsp10 and Hsp60 increased the abundance of the anti-apoptotic Bcl-xl and Bcl-2, and reduced the protein content of the pro-apoptotic Bax. Hsp60 overexpression also significantly reduced doxorubicin induction of Bad, whereas overexpression of Hsp10 did not alter the expression of Bad in the doxorubicin-treated cells. Overexpression of Hsp10 and Hsp60, respectively, stabilized mitochondrial cross-membrane potential, inhibited Caspase 3, and suppressed PARP. These findings indicate that overexpression of Hsp10 and Hsp60 differentially modulated Bcl-2 family and in turn attenuate doxorubicin-induced cardiac muscle death. The effects of Hsp10 and Hsp60 on Bcl-2 family could not be explained by the abundance of Bcl-2 family mRNA levels. Hsp60 interacted with Bcl-xl and Bax in the cardiomyocytes in vivo. The effect of Hsp10 and Hsp60 on the abundance of Bcl-xl could not be blocked by cycloheximide. Moreover, Hsp10 and Hsp60 inhibited ubiquitination of Bcl-xl. These findings suggest that Hsp10 and Hsp60 modulated post-translational modification of Bcl-xl. Antisense Hsp60 reduced the abundance of endogenous Hsp60 in cardiomyocytes and amplified the cytotoxicity of doxorubicin. These data provide a novel link between Hsp10/Hsp60 and cardiac protection in doxorubicin cardiomyopathy.