Myocardial Caspase-3 Activity Research Articles

Overexpression of miR-431 inhibits cardiomyocyte apoptosis following myocardial infarction via targeting HIPK3.

Acute myocardial infarction (AMI) is a common acute cardiovascular crisis. Although the diagnosis and treatment of AMI are constantly improving, the mortality of AMI is still very high, and its pathogenesis is still unclear. This article focuses on the role of microRNA-431 (miR-431) in regulating myocardial apoptosis after myocardial infarction (MI) and its potential molecular mechanism. We constructed cell models and animal models of MI. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to detect miR-431 expression in myocardium after MI. Western blot, cell counting kit-8 (CCK-8) assay, flow cytometry and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining were performed to detect myocardial apoptosis; pathological sections of myocardium, serum lactate dehydrogenase (LDH) levels and Caspase-3 activity in myocardium were employed to evaluate myocardial injury of MI rats; echocardiography was utilized to assess cardiac function of rats. We revealed that miR-431 expression was decreased in H2O2-treated H9c2 cells and myocardium of MI rats. The expression of Cleaved Caspase-3 (C-Caspase-3) in H9c2 cells treated with H2O2 was significantly increased, the cell viability was dramatically decreased, the apoptosis rate and the percentage of TUNEL positive cells were notably increased, but up-regulation of miR-431 could reverse these effects. At the same time, compared with the sham group, serum LDH levels were observably increased, myocardial Caspase-3 activity was also increased, and cardiac function was greatly reduced, while overexpression of miR-431 could reduce myocardial injury and improve cardiac function of MI rats. Through the Luciferase reporter gene experiment, we found that miR-431 could directly target HIPK3. In summary, overexpression of miR-431 can inhibit apoptosis after myocardial infarction via targeting HIPK3, thereby reducing myocardial injury and improving cardiac function in MI rats.

MicroRNA-98 negatively regulates myocardial infarction-induced apoptosis by down-regulating Fas and caspase-3

Acute myocardial infarction (MI) is the leading cause of sudden death worldwide. MicroRNAs (miRs) is a novel class of regulators of cardiovascular diseases such as MI. This study aimed to explore the role of miR-98 in MI and its underlying mechanisms. We found that miR-98 was downregulated both in infarcted and ischemic myocardium of MI mice as well as H2O2-treated neonatal rat ventricular myocytes (NRVCs). miR-98 overexpression remarkably increased cell viability and inhibited apoptosis of H2O2-treated NRVCs. Meanwhile, overexpression of miR-98 reversed H2O2-induced Bcl-2 downregulation and Bax elevation and significantly reduced JC-1 monomeric cells. Meanwhile, miR-98 overexpression attenuated the upregulation of Fas and caspase-3 in H2O2-treated cardiomyocytes at the mRNA and protein levels. Dual-luciferase reporter assay showed that miR-98 directly targeted to Fas 3′-UTR. Furthermore, MI mice injected with miR-98-agomir had a significant reduction of apoptotic cells, the serum LDH levels, myocardial caspase-3 activity, Fas and caspase-3 expression in heart tissues. Administration of miR-98-agomir also showed decreased infarct size and improved cardiac function. Collectively, miR-98 is downregulated in the MI heart and NRVCs in response to H2O2 stress, and miR-98 overexpression protects cardiomyocytes against apoptosis. Anti-apoptotic effects of miR-98 are associated with regulation of Fas/Caspase-3 apoptotic signal pathway.

Doxorubicin mediated cardiotoxicity in rats: Protective role of felodipine on cardiac indices

Anthracyclines find vital uses in the treatment of solid tumors and other kind of malignancies. A typical side effect observed with few agents of this class is dose-dependent cardiotoxicity. Doxorubicin is one such agent which backs the generation of free radicals through metabolism of its quinone structure. This effect combined with induction of apoptotic and necrotic pathways leads to the development of irreversible cardiotoxicity. Reports showing the cardioprotective effects of felodipine have been published in the past. We chose to evaluate protective effect of felodipine in acute cardiotoxicity in rats induced by single dose of doxorubicin. Felodipine was assessed against doxorubicin-induced cardiotoxicity and we found that felodipine not only improves cardiac marker enzymes (P<0.001 for LDH; P<0.01 for CK-MB) but also prevents damage to myocardial tissue (20.61% necrosed area in doxorubicin intoxication; 11.52% necrosed area in felodipine treated group). Activation of apoptotic pathways is decelerated which is indicated by a significant reduction in myocardial caspase-3 activity (P<0.05) following felodipine pretreatment. Felodipine pretreatment was able to maintain normal cardiac morphology and histoarchitecture. Gravimetric analysis revealed beneficial effects following felodipine pretreatment. Abnormalities seen in the ECG after doxorubicin treatment were normalized to a significant extent (ST interval normalization was significant at P<0.01) in felodipine treated rats. In itself, felodipine was not found to have any detrimental effects on the myocardium or hemodynamic parameters of rats. Findings of the study suggest that pretreatment with felodipine prevents doxorubicin induced cardiotoxicity.

The role of the Hsp90/Akt pathway in myocardial calpain-induced caspase-3 activation and apoptosis during sepsis

BackgroundRecent studies have demonstrated that myocardial calpain triggers caspase-3 activation and myocardial apoptosis in models of sepsis, whereas the inhibition of calpain activity down-regulates myocardial caspase-3 activation and apoptosis. However, the mechanism underlying this pathological process is unclear. Therefore, in this study, our aim was to explore whether the Hsp90/Akt signaling pathway plays a role in the induction of myocardial calpain activity, caspase-3 activation and apoptosis in the septic mice.MethodsAdult male C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Next, myocardial caspase-3 activity and the levels of Hsp90/p-Akt (phospho-Akt) proteins were detected, and apoptotic cells were assessed by performing the TUNEL assay.ResultsIn the septic mice, there was an increase in myocardial calpain and caspase-3 activity in addition to an increase in the number of apoptotic cells; however, there was a time-dependent decrease in myocardial Hsp90/p-Akt protein levels. The administration of calpain inhibitors (calpain inhibitor-Ш or PD150606) prevented the LPS-induced degradation of myocardial Hsp90/p-Akt protein and its expression in cardiomyocytes in addition to inhibiting myocardial caspase-3 activation and apoptosis. The inhibition of Hsp90 by pretreatment with 17-AAG induced p-Akt degradation, and the inhibition of Akt activity by pretreatment with wortmannin resulted in caspase-3 activation in wildtype C57 murine heart tissues.ConclusionsMyocardial calpain induces myocardial caspase-3 activation and apoptosis in septic mice via the activation of the Hsp90/Akt pathway.

Glycyrrhizin protects rat heart against ischemia-reperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway

Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity, and protect spinal cord, liver and brain against I/R-induced injury in experimental animals. The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms. Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion. The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion. Serum HMGB1, TNF-α and IL-6 levels, and hemodynamic parameters were measured at the onset and different time points of reperfusion. At the end of the experiment, the heart was excised, and the infarct size and histological changes were examined. The levels of Bcl2, Bax and cytochrome c, as well as phospho-ERK1/2, phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis, and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit. Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size, but did not change the hemodynamic parameters at different time points during reperfusion. GL significantly decreased the levels of serum HMGB1, TNF-α and IL-6. GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue, resulting in inhibition of myocardial apoptosis. Moreover, expression of phospho-JNK, but not ERK1/2 and P38 was decreased by GL in the heart tissue. All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg). Intravenous administration of SP600125, a selective phospho-JNK inhibitor (0.5 mg/kg), attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis. These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly inhibiting extracellular HMGB1 cytokine activity and blocking the phospho-JNK/Bax pathway.

CALPAIN ACTIVATION CONTRIBUTES TO ISCHAEMIA/REPERFUSION-INDUCED MYOCARDIAL APOPTOSIS

ObjectivesCalpains belong to a family of calcium-dependent intracellular cysteine proteases. Previous studies demonstrated that calpain activation contributes substantially to ischaemia/reperfusion (I/R) injury. However, it remains unclear whether administration of calpain...

Abstract P061: The Possible Apoptosis Pathways Involved in Mechanical Trauma-Induced Secondary Heart Injury

Background: Trauma induces not only primary heart injury but also secondary heart injury. Our previous study has demonstrated that mechanical trauma could cause cardiomyocyte apoptosis which contributes to posttraumatic secondary cardiac dysfunction. The aim of the present study was to elaborate the potential mechanism involved in secondary post-traumatic cardiac dysfunction. Methods: Male adult rats were subjected to Noble-Collip drum with a total of 200 revolutions at a rate of 40 rpm. The activities of cardiomyocyte Caspase 3, 8, 9, 12 were detected respectively by Fluorometric assays. The cardiac function in vitro was determined using a Langendorff isolated heart perfusion system. Results: No direct mechanical traumatic injury was observed in the heart immediately after trauma. (1)Compared with the sham group, myocardial Caspase-3 activity in the traumatized rats significantly increased 6h after trauma (51±4 vs. 16±2, P &lt;0.01). 12h after trauma myocardial Caspase-3 activity reached the peak (69±4 vs. Sham, P &lt;0.01), which still remained at high level (46±3 vs. Sham, P &lt;0.01) 24h after trauma. (2) Administration of Z-VAD-FMK, a broad-spectrum caspase inhibitor, could reverse post-traumatic cardiac dysfunction evidenced by increased +dP/dt max [(4111±189) mmHg/sec, vs. sham (3414±208) mmHg/sec, P &lt;0.01], and decreased -dP/dt max [(-4997±351) mmHg/sec, vs. sham group (-3301±458) mmHg/sec, P &lt;0.01] 24h after trauma. (3) Compared with the sham group, myocardial Caspase-12 activity significantly increased 3h after trauma (66±8 vs. 27±10, P &lt;0.01). 6h after trauma Caspase-12 activity reached the peak (89±16, P &lt;0.01 vs. Sham) , but 12h after trauma it decreased ( P &gt;0.05 vs. Sham). Both myocardial Caspase-8 activity and Caspase-9 activity markedly increased 24h after trauma (2312±648 vs. 1449±296, P &lt;0.01 vs. Sham; 875±460 vs. 470±222, P &lt;0.01 vs. Sham, respectively). Conclusion: Caspase-dependent apoptotic pathway played an important role in post-traumatic secondary cardiac dysfunction. Moreover, in the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).

Endogenous κ-Opioid Peptide Mediates the Cardioprotection Induced by Ischemic Postconditioning

The aim of this study was to investigate the underlying mechanism that dynorphin, an endogenous kappa opioid receptor (κ-OR) agonist, triggers antiapoptotic effect of postconditioning (Postcon). In addition to vehicle treatment, Sprague Dawley rats (n = 6) underwent a 30-minute left anterior descending occlusion followed by 2 hours of reperfusion with or without a Postcon stimulus. The selective κ-OR antagonist nor-binaltorphimine (Nor-BNI) was administered intravenously 5 minutes before reperfusion. Infarct size was determined by using 2,3,5-triphenyltetrazolium chloride staining. Blood plasma concentrations of creatine kinase (CK) and lactate dehydrogenase (LDH) and myocardial caspase-3 activity were analyzed spectrophotometrically. Myocardial apoptosis was analyzed by the detection of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling. Immunoreactive dynorphin in blood serum and myocardium was measured by means of an antigen-competitive enzyme-linked immunosorbent assay. Infarction size, caspase-3 activity, apoptotic index, and CK and LDH levels were significantly higher in the ischemic/reperfusion group than in the vehicle group (P < 0.01). Postcon significantly reduced infarction size, caspase-3 activity, apoptotic index, CK and LDH levels (P < 0.01 vs. ischemic/reperfusion). Dynorphin content significantly increased after Postcon (P < 0.01). All the effects described above were abolished by Nor-BNI, with the exception of dynorphin content. We found that cardiac protection and antiapoptotic effect of Postcon is mediated by the activation of κ-OR. Effect of Postcon is mediated, at least partially, by enhanced dynorphin expression.

Erythropoietin is equally effective as fresh-blood transfusion at reducing infarct size in anemic rats

We recently demonstrated that transfusion of anemic animals up to 100 g/L hemoglobin with fresh blood protects the heart from ischemic injuries following myocardial infarction. Erythropoietin has cardioprotective effects independent of its erythropoietic activity. The objective of this study was to compare the cardioprotective effects of erythropoietin treatment to fresh-blood transfusion in anemic rats after acute myocardial infarction. Randomized animal study. University laboratory. Male Sprague-Dawley rats weighing 200-300 g. Myocardial infarction was induced by coronary artery ligation in 76 rats, 55 of which were anemic (80-90 g/L) and 21 of which had normal hemoglobin levels. Animals were randomized to erythropoietin (2000 units/kg), fresh-blood transfusion to 100 g/L hemoglobin, or saline-treatment groups immediately following myocardial infarction. At 24 hrs after myocardial infarction, cardiac function and infarct size were determined. Myocardial apoptosis was determined by caspase-3 activity and terminal deoxynucleotidyl transferase d-UTP nick end labeling (TUNEL) assay. Infarct size was significantly decreased in anemic rats treated with erythropoietin or blood transfusion compared to those in the saline-treatment group. Cardiac function, as measured by maximal positive and minimal negative first derivatives of left ventricular pressure, was better preserved in the normal hemoglobin groups and the erythropoietin- or transfusion-treated anemic animals compared to saline-treated anemic animals. Myocardial caspase-3 activity and TUNEL-positive nuclei were significantly increased in anemic rats but were decreased by erythropoietin treatment or red blood cell transfusion. Erythropoietin treatment is equally effective as fresh-blood transfusion in anemic rats after acute myocardial infarction at reducing infarct size, myocardial apoptosis, and improving cardiac function.

Molecular MRI Detects Low Levels of Cardiomyocyte Apoptosis in a Transgenic Model of Chronic Heart Failure

The ability to image cardiomyocyte (CM) apoptosis in heart failure could facilitate more accurate diagnostics and optimize targeted therapeutics. We thus aimed to develop a platform to image CM apoptosis quantitatively and specifically in heart failure in vivo. The myocardium in heart failure, however, is characterized by very low levels of CM apoptosis and normal vascular permeability, factors thought to preclude the use of molecular MRI. Female mice with overexpression of Gaq were studied. Two weeks postpartum, these mice develop a cardiomyopathy characterized by low levels of CM apoptosis and minimal myocardial necrosis or inflammation. The mice were injected with the annexin-labeled nanoparticle (AnxCLIO-Cy5.5) or a control probe (CLIO-Cy5.5) and imaged in vivo at 9.4 T. Uptake of AnxCLIO-Cy5.5 occurred in isolated clusters, frequently in the subendocardium. Myocardial T2* was significantly lower (7.6+/-1.5 versus 16.8+/-2.7 ms, P<0.05) in the mice injected with AnxCLIO-Cy5.5 versus CLIO-Cy5.5, consistent with the uptake of AnxCLIO-Cy5.5 by apoptotic CMs. A strong correlation (r(2)=0.86, P<0.05) was seen between in vivo T2* (AnxCLIO-Cy5.5 uptake) and myocardial caspase-3 activity. The ability of molecular MRI to image sparsely expressed targets in the myocardium is demonstrated in this study. Moreover, a novel platform for high-resolution and specific imaging of CM apoptosis in heart failure is established. In addition to providing novel insights into the pathogenesis of CM apoptosis, the developed platform could facilitate the development of novel antiapoptotic therapies in heart failure.

Propofol limits rat myocardial ischemia and reperfusion injury with an associated reduction in apoptotic cell death in vivo

Propofol, a rapidly acting, short duration, intravenous hypnotic anesthetic induction agent, is often used in clinical situations where myocardial ischemia/ reperfusion (I/R) injury is a threat. The aim of the present study was to evaluate the protective effect of propofol on myocardial I/R injury in rat due to apoptosis. Myocardial I/R injury were induced by occluding the left anterior descending (LAD) coronary artery for 25 min followed by either 2 h or 6 h reperfusion. Apoptosis was evaluated by Western blot analysis (Bcl-2, Bax expression), DNA strand breaks, TUNEL analysis and measuring myocardial caspase-3 activity. Propofol significantly reduced infarct size and improved I/R-induced myocardial contractile dysfunction by improving left ventricular diastolic pressure and positive and negative maximal values of the first derivative (+ d p/d t) of left ventricular pressure. Propofol increased Bcl-2/Bax expression ratio and decreased caspase-3 activity in I/R rat hearts, which resulted in reduction of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. In an in vitro study, propofol increased H9c2 cell viability against oxidative stress induced by glucose oxidase (GOX) in a dose-dependent manner. These data suggest propofol limits I/R injury with an associated reduction in apoptotic cell death in vivo.

Anti-Apoptotic Effect of Magnolol in Myocardial Ischemia and Reperfusion Injury Requires Extracellular Signal-Regulated Kinase1/2 Pathways in Rat In Vivo

Magnolol, an active component extracted from Magnolia officinalis, has been reported to have protective effect on ischemia and reperfusion (I/R)-induced injury in experimental animals. The aim of the present investigation was to further evaluate the mechanism(s) by which magnolol reduces I/R-induced myocardial injury in rats in vivo. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 24 h (for infarct size and cardiac function analysis). In some experiments, reperfusion was limited to 1 h or 6 h for analysis of biochemical and molecular events. Magnolol and DMSO solution (vehicle) were injected intra-peritoneally 1 h prior to I/R insult. The infarct size was measured by TTC technique and heart function was monitored by Millar Catheter. Apoptosis related events such as p-ERK, p-Bad, Bcl-xl and cytochrome c expression were evaluated by Western blot analysis and myocardial caspase-3 activity was also measured. Magnolol (10 mg/kg) reduced infarct size by 50% (P < 0.01 versus vehicle), and also improved I/R-induced myocardial dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in magnolol-treated rats. Magnolol increased the expression of phosphor ERK and Bad which resulted in inhibition of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. Application of PD 98059, a selective MEK1/2 inhibitor, strongly antagonized the effect of magnolol. Taken together, we concluded that magnolol inhibits apoptosis through enhancing the activation of ERK1/2 and modulation of the Bcl-xl proteins which brings about reduction of infarct size and improvement of cardiac function in I/R-induced injury.

Abstract 162: c-fos Protects the Myocardium from Ischemic Injury and Improves Cardiac Function

Background: c-fos is an immediate early response gene. c-Fos proteins form heterodimers with Jun family proteins, and the resulting AP-1 complexes regulate transcription by binding to the AP-1 sequence found in many cellular genes. c-fos is activated in cardiomyocytes following myocardial infarction. However, the role of c-fos in regulating cardiomyocyte survival and cardiac function post myocardial infarction (MI) is not known. In the present study, we hypothesized that c-fos protects the myocardium from ischemic injury and improves cardiac function. Methods and Results: The generation of mice with cardiomyocyte specific c-fos −/ − was achieved by crossing the floxed c-fos mice with mice over-expressing Cre recombinase under the control of α-myosin heavy chain. Wild-type (WT) littermates were used as controls. MI was induced by coronary artery ligation. Infarct size, myocardial apoptosis and cardiac function were determined at 2 days post-MI. While area at risk was similar between the 2 groups, infarct size was significantly increased in c-fos −/ − compared to WT mice (58 ± 4% vs. 44 ± 3%, P&lt; 0.05). Myocardial caspase-3 activity and cytosolic DNA fragments in the peri-infarct region were significantly increased while Bcl-2/Bax protein ratio was significantly decreased in c-fos −/− mice ( P&lt; 0.05). LV pressure volume relationship was assessed in vivo using a Millar pressure conductance catheter. LV end-systolic elastance ( E es ) and +d P /dt max were significantly decreased in c-fos −/− compared to WT mice (1.7 ± 0.4 vs. 5.1 ± 1.0 mmHg/μL; 4776 ± 567 vs. 7006 ± 319 mmHg/s, P&lt; 0.01). Conclusions: Deficiency in c-fos increases infarct size and myocardial apoptosis leading to impaired cardiac function post-MI. Our results suggest that c-fos protects the myocardium from ischemic injury and improves cardiac function.

Tumor necrosis factor-α-induced caspase activation mediates endotoxin-related cardiac dysfunction*

Sepsis-induced cardiac dysfunction is a serious clinical syndrome characterized by hypotension, decreased systemic vascular resistance, and elevated cardiac index. Although cytokines such as tumor necrosis factor (TNF)-alpha have been shown to play a significant role early in this response, the downstream effects of TNF-alpha signaling on cardiac function, specifically its relationship to apoptosis, have not been fully elucidated. Previous studies from our laboratory have identified endotoxin-induced apoptosis in cardiac cells in vitro. To further determine the role of lipopolysaccharide-induced apoptosis in vivo, mice were injected intraperitoneally with lipopolysaccharide (4 mg/kg), and cardiac apoptosis was detected and inhibited using a broad-spectrum caspase inhibitor. University research laboratory. Adult male wild-type (B6:129PF1/J) and TNF receptor 1/receptor 2 (TNFR-1/2) knockout mice (B6;129S-Tnfrsf1aTnfrsf1b). We sought to determine the dependence of cardiac apoptosis on TNF-alpha signaling and determine the physiologic role of caspase activation on lipopolysaccharide-induced cardiac dysfunction. Cardiac apoptosis was determined at baseline and at 2, 4, 8, and 24 hrs by detection of capase-3 and -8 activity, cytoplasmic levels of Bax/Bcl-2, cleaved caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL) staining of histologic sections in wild-type and TNFR-1/2 knockout mice. To determine the role of caspase activation in lipopolysaccharide-induced cardiac dysfunction, a broad-spectrum caspase inhibitor Z-Val-Ala-Asp (ome)-FMK (sad) was given, and cardiac function was determined in isolated beating hearts (Langendorff preparation). Our experiments determined that caspase-3-dependent apoptosis was active in cardiac tissue by 2 hrs and that this activation was completely mediated by TNFR-1/2. The Bax/Bcl-2 ratios supported the finding and time course of apoptosis, whereas TUNEL staining of cardiac tissue sections identified sporadic apoptotic ventricular cells. The administration of zVAD significantly inhibited myocardial caspase-3 activity and preserved cardiac physiologic function (Langendorff preparation). Endotoxin induces a TNF-alpha-dependent apoptotic cascade in the myocardium, which contributes to the development of cardiac dysfunction.

Critical timing of -arginine treatment in post-ischemic myocardial apoptosis?role of NOS isoforms

Background: The role of nitric oxide (NO) in apoptotic cell death has been extensively studied in recent years. However, reported results are inconsistent and often controversial, and the mechanisms underlying its diverse effects in apoptosis regulation remain unidentified. The present study attempted to determine whether in vivo administration of l-arginine, the substrate for NOS, at different time points during the course of myocardial ischemia and reperfusion may differentially regulate post-ischemic myocardial apoptosis, and if so, to investigate the mechanisms involved. Methods and results: Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. l-Arginine was administered as a bolus at either 10 min before (early treatment) or 3 h after reperfusion (late treatment). There was no difference in myocardial eNOS expression between any groups studied. Myocardial iNOS expression was detected at 3 h after reperfusion but not at 1 h after reperfusion. Administration of l-arginine 10 min before reperfusion markedly decreased TUNEL-positive staining cardiomyocytes, reduced myocardial caspase-3 activity, inhibited iNOS expression, and reduced myocardial nitrotyrosine content. In strict contrast, administration of l-arginine 3 h after reperfusion, a time point when iNOS was expressed, resulted in a significant increase in myocardial NOx content, myocardial injury, and toxic peroxynitrite formation as measured by nitrotyrosine. Conclusion: Our results demonstrated for the first time that l-arginine administered at different time points during ischemia/reperfusion exerted different effects on post-ischemic myocardial injury, and suggests that stimulation of eNOS reduces nitrative stress and decreases apoptosis whereas stimulation of iNOS increases nitrative stress and enhances myocardial reperfusion injury.

Simvastatin preserves cardiac function in genetically determined cardiomyopathy.

Endothelial dysfunction characterizes heart failure (HF). Simvastatin (Sim) increases endothelial nitric oxide (NO) independent of lipid-lowering. We evaluated the effect of Sim on cardiac function, apoptosis, and NO availability in HF. Five-month-old cardiomyopathic (CM) hamsters were divided into 2 groups: Sim (20 mg/kg, 6 weeks, n = 6) and Untreated (n = 6). Age-matched normal hamsters served as controls (n = 6). Serial echocardiograms were performed to measure LV function. Myocardial apoptosis, eNOS, and capillary density were measured at 6 weeks. Cardiomyopathic hamsters had lower LV shortening fraction (SF) compared with controls (17 +/- 3% vs 59 +/- 2%), higher LV end-diastolic volume (30 +/- 3 vs 6 +/- 2 mL/m2), and lower LV mass/volume ratio (0.5 +/- 0.04 vs 0.72 +/- 0.02 mg/ml, P < 0.001). During follow-up, SF decreased (9 +/- 2%) and LV volume increased (38 +/- 1 mL/m2) in untreated hamsters (P < 0.05 from baseline) but did not change significantly in the Sim group (P < 0.05 vs untreated). Myocardial caspase-3 activity was higher and apoptotic nuclear density was lower in Sim compared with untreated CM hamsters (0.072 +/- 0.02% vs 0.107 +/- 0.03%, P < 0.01). Myocardial capillary density was highest in the Sim group (P < 0.05). eNOS expression was not different between groups. Sim retards the progression of HF in CM hamsters. This may be related to an increase in coronary microvasculature, increase in NO availability, and decreased apoptosis.

Development of heart failure and congenital septal defects in mice lacking endothelial nitric oxide synthase.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in the regulation of cell growth, apoptosis, and tissue perfusion. Recent studies showed that mice deficient in eNOS developed abnormal aortic bicuspid valves. The aim of the present study was to additionally investigate the role of eNOS in heart development. We examined postnatal mortality, cardiac function, and septum defects in eNOS(-/-), eNOS(+/-), and wild-type mice. Postnatal mortality was significantly increased in eNOS(-/-) (85.1%) and eNOS(+/-) (38.3%) compared with wild-type mice (13.3%, P<0.001). Postmortem examination found severe pulmonary congestion with focal alveolar edema in mice deficient in eNOS. Heart shortening determined by ultrasound crystals was significantly decreased in eNOS(-/-) compared with wild-type mice (P<0.05). Congenital atrial and ventricular septal defects were found in neonatal hearts. The incidence of atrial or ventricular septal defects was significantly increased in eNOS(-/-) (75%) and eNOS(+/-) (32.4%) neonates compared with those of the wild-type mice (4.9%). At embryonic days 12.5 and 15.5, cardiomyocyte apoptosis and myocardial caspase-3 activity were increased in the myocardium of eNOS(-/-) compared with wild-type embryos (P<0.01), and increases in apoptosis persisted to neonatal stage in eNOS(-/-) mice. Deficiency in eNOS results in heart failure and congenital septal defects during cardiac development, which is associated with increases in cardiomyocyte apoptosis. Our data demonstrate that eNOS plays an important role in normal heart development.

Nitric oxide regulates the apoptotic pathway in explanted failing human hearts

Cardiomyocyte apoptosis causes ventricular remodeling in heart failure. Caspases are intracellular enzymes that mediate apoptosis. We investigated nitric oxide (NO) mediated regulation of caspase activity in failing human myocardium to determine if loss of NO production in heart failure contributes to apoptosis. Methods: Ventricular myocardium was obtained from 10 explanted failing human hearts at heart transplantation. 5 patients (pts) were on chronic angiotensin-converting enzyme inhibitors (ACEI) prior to transplantation. Caspase-3 activity was induced using recombinant caspases 8 and 9 in cytosolic extracts of myocardium and measured colorimetrically. Effect of following on caspase activity was measured-exogenous NO donor, S-nitroso-N-acetyl penicillamine (SNAP, 10-5M, 10-4M), endogenous NO inhibitor, nitro-L-arginine methyl ester (L-NAME, 10-4M), and ACEI (enalapril, 10-4M). Results: (i) Caspase 8 and 9 induced caspase-3 activity in myocardial cell lysates (5.44+/-0.6 pmol/10 mcg/min). Exogenous NO donor, SNAP, caused a direct, dose-dependent decrease in induced caspase 3 activity downstream of caspases 8 and 9 (0.52+/-0.5 pmol/10mcg/min at highest dose, p<0.01) (ii) Caspase-3 activity was lower in the 5 pts on chronic ACEI therapy and increased significantly with addition of L-NAME, indicating that ACEIs may decrease caspase activity through increased NO production. This was confirmed by the ability of exogenously added enalapril to reduce myocardial caspase-3 activity (2.48+/-0.4 pmol/10mcg/min). (iii) Caspase activity was significantly higher in nonischemic (9.86+/-1) versus ischemic hearts (5.14+/-0.6) (p<0.05) including caspase-9 induced caspase-3 activity suggesting upregulation of the mitochondrial pathway of apoptosis. Conclusion: NO regulates apoptosis by inhibiting downstream caspase-3 activity in explanted failing human hearts and can potentially rescue a cell from apoptosis even after activation of the caspase cascade. This may have implications for use of NO agonists to inhibit apoptotis in heart failure.