Cardiac Myocyte Apoptosis Research Articles

Secreted Frizzled-related Protein 5 Diminishes Cardiac Inflammation and Protects the Heart from Ischemia/Reperfusion Injury

Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 KO and WT mice were subjected to ischemia/reperfusion (I/R). Although Sfrp5-KO mice exhibited no detectable phenotype when compared with WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis, and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression when compared with WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.

Signaling Pathways in Cardiac Myocyte Apoptosis.

Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation.

Sorcin Expression in the Epiphyseal Growth Plates of Mice

Sorcin is a small calcium-binding protein that is widely expressed in many tissues. Sorcin regulates cardiac myocyte apoptosis by modulating mitochondrial Ca2+ cycling and regulates fibroblast cell cycling and apoptosis via Ca2+ signaling through the endoplasmic reticulum (ER). During endochondral ossification, some chondrocytes undergo apoptosis after their terminal differentiation; however, Sorcin’s expression in these cells has not been characterized. Here we examined Sorcin’s gene expression in the chondrocytes derived from mouse growth plate by reverse transcription PCR (RT-PCR), and its protein localization in the chondrocytes of femoral growth plate using immunohistochemistry. Sorcin protein was detected in the chondrocytes, and was particularly abundant in the cytoplasm and nuclei of proliferative zone chondrocytes and in the nuclei of hypertrophic chondrocytes. Apoptotic analysis of the growth plate revealed that many of the hypertrophic chondrocytes undergo the DNA fragmentation. We report for the first time the localization of Sorcin in the epiphyseal growth plate in which one of the apoptotic phenomenon was detected.

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade.

Prevent diabetic cardiomyopathy in diabetic rats by combined therapy of aFGF-loaded nanoparticles and ultrasound-targeted microbubble destruction technique

Acidic fibroblast growth factor (aFGF) has shown the great potential to prevent the structural and functional injuries caused by diabetic cardiomyopathy (DCM). The present study sought to investigate the preclinical performance and mechanism of the combination therapy of aFGF-nanoparticles (aFGF-NP) and ultrasound-targeted microbubble destruction (UTMD) technique for DCM prevention. From Mason staining and TUNEL staining, aFGF-NP+UTMD group showed significant differences from the diabetes group and other groups treated with aFGF or aFGF-NP. The cardiac collagen volume fraction (CVF) and cardiac myocyte apoptosis index in aFGF-NP+UTMD group reduced to 4.15% and 2.31% respectively, compared with those in the diabetes group (20.5% and 11.3% respectively). Myocardial microvascular density (MCD) in aFGF-NP+UTMD group was up to 35n/hpf, much higher than that in the diabetes group (14n/hpf). The diabetes group showed similar results (MCD, CVF and cardiac myocyte apoptosis index) to other aFGF treatment groups (free aFGF±UTMD or aFGF-NP). Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis and metabolic could be suppressed by the different extents of twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-NP±UTMD), with the strongest improvements observed in the aFGF-NP+UTMD group. Western blot and immunohistochemical analyses of heart tissue samples further revealed the high efficiency of heart-targeted delivery and effective cardioprotection with this combination approach. Overall, this study has generated supportive data that are critical for the translation of a promising DCM prevention strategy.

Cardioprotective effects of baicalein on heart failure via modulation of Ca2 + handling proteins in vivo and in vitro

AimsBaicalein is a widely used Chinese herbal medicine extracted from Labiatae plants Scutellaria baicalensis Georgi's dry root, which has multiple pharmacological activities. However, the precise mechanism of baicalein against myocardial remodeling remains poorly understood. The aim of our study was to investigate the underlying mechanism of baicalein treatment in rats model of heart failure (HF) and rat myocardial cells (H9C2). Main methodsHF model was established by abdominal aorta constriction in rats and incubation with 50μM isoproterenol for 48h in H9C2 cells. Various molecular biological experiments were performed to assess the effects of baicalein on cardiac function, myocardial remodeling, apoptosis and Ca2+ handling proteins. Key findingsIn the present study, first we found that baicalein alleviated HF in vivo. Additionally, treatment with baicalein inhibited the myocardial fibrosis, restrained the expression and activity of MMP2 and MMP9, and suppressed apoptosis in heart tissue. Moreover, baicalein could inhibit the cardiac myocyte hypertrophy and apoptosis induced by isoproterenol in vitro. Finally we found that baicalein could modulate the expressions and activities of Ca2+ handling proteins, including downregulation of phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and expression of Na(+)/Ca(2+)-exchangers (NCX1), upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and ryanodine receptor 2 (RYR2). Baicalein also restrained the decreased SERCA activity induced by aortic banding. SignificanceOur studies suggested that baicalein alleviated myocardial remodeling and improved cardiac function via modulation of Ca2+ handling proteins, which may be a potential phytochemical flavonoid for therapeutics of HF.

Extracellular Ubiquitin: Role in Myocyte Apoptosis and Myocardial Remodeling.

Ubiquitin (UB) is a highly conserved low molecular weight (8.5 kDa) protein. It consists of 76 amino acid residues and is found in all eukaryotic cells. The covalent linkage of UB to a variety of cellular proteins (ubiquitination) is one of the most common posttranslational modifications in eukaryotic cells. This modification generally regulates protein turnover and protects the cells from damaged or misfolded proteins. The polyubiquitination of proteins serves as a signal for degradation via the 26S proteasome pathway. UB is present in trace amounts in body fluids. Elevated levels of UB are described in the serum or plasma of patients under a variety of conditions. Extracellular UB is proposed to have pleiotropic roles including regulation of immune response, anti-inflammatory, and neuroprotective activities. CXCR4 is identified as receptor for extracellular UB in hematopoietic cells. Heart failure represents a major cause of morbidity and mortality in western society. Cardiac remodeling is a determinant of the clinical course of heart failure. The components involved in myocardial remodeling include-myocytes, fibroblasts, interstitium, and coronary vasculature. Increased sympathetic nerve activity in the form of norepinephrine is a common feature during heart failure. Acting via β-adrenergic receptor (β-AR), norepinephrine is shown to induce myocyte apoptosis and myocardial fibrosis. β-AR stimulation increases extracellular levels of UB in myocytes, and UB inhibits β-AR-stimulated increases in myocyte apoptosis and myocardial fibrosis. This review summarizes intracellular and extracellular functions of UB with particular emphasis on the role of extracellular UB in cardiac myocyte apoptosis and myocardial remodeling.

Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4

Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that GRK2, a critical regulator of myocardial β-AR signaling, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (Iso) caused increased oxidative stress and apoptosis. There was also increased Nox4 expression, but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. Adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with Iso stimulation. These increases in oxidative stress were abolished by pre-treatment with the non-specific Nox inhibitor, apocynin, or siRNA knockdown of Nox4. Adenoviral-mediated expression of a GRK2 inhibitor prevented ROS production and apoptosis in response to Iso stimulation. β-Arrestins are signaling proteins that function downstream of GRK2 in β-AR uncoupling. Adenoviral-mediated overexpression of β-arrestins increased ROS production and Nox4 expression. Chronic β-agonist stimulation in mice increased Nox4 expression and apoptosis compared to PBS or AngII treatment. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in cardiac myocytes and provides an additional novel mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition to prevent the development of HF.

Dental pulp-derived stem cell conditioned medium reduces cardiac injury following ischemia-reperfusion.

Stem cells from human exfoliated deciduous teeth (SHEDs) can regenerate various tissues. We investigated the impact of SHED-conditioned medium (SHED-CM) on myocardial injury in a mouse model of ischemia-reperfusion (I/R). Wild-type (WT) mice were subjected to myocardial ischemia followed by reperfusion. SHED-CM was intravenously injected at 5 min after reperfusion. Administration of SHED-CM reduced myocardial infarct size as well as decreased apoptosis and inflammatory cytokine levels, such as TNF-α, IL-6, and IL-β, in the myocardium following I/R. In cultured cardiac myocytes, SHED-CM significantly suppressed apoptosis under hypoxia/serum-deprivation and reduced LPS-induced expression of pro-inflammatory genes. Furthermore, anti-apoptotic action of SHED-CM was stronger than bone marrow-derived stem cell (BMSC)-CM or adipose-derived stem cell (ADSC)-CM in cardiac myocytes. SHED-CM contains a higher concentration of hepatocyte growth factor (HGF) than BMSC-CM and ADSC-CM, and neutralization of HGF attenuated the inhibitory actions of SHED-CM on apoptosis in cardiac myocytes. Finally, WT mice were intravenously treated with an HGF-depleted SHED-CM, followed by myocardial I/R. HGF depletion significantly attenuated the inhibitory actions of SHED-CM on myocardial infarct size and apoptosis after I/R. SHED-CM protects the heart from acute ischemic injury because it suppresses inflammation and apoptosis. SHED-CM could be a useful treatment option for acute myocardial infarction.

Carvedilol Suppresses Apoptosis and Ion Channel Remodelling of HL-1 Cardiac Myocytes Expressing E334K cMyBPC.

Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling. To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol. We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry. Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC. Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

Cardiac-protective effects and the possible mechanisms of alatamine during acute myocardial ischemia.

Alatamine is a constituent in the extract of a traditional herbal medicine Ramulus euonymi widely used for cardiac protection. However, its precise effects remain unclear. In the present study, we found that alatamine was able to reduce acute myocardial ischemia (AMI)-induced cardiac dysfunction in a rat model, as reflected by significantly restored electrocardiograms, M-mode echocardiograms, and left ventricular hemodynamics. Also, Nagar Olsen staining revealed that alatamine markedly reduced AMI-induced cardiac injury and cardiac myocyte apoptosis. TUNEL and caspase-3 activity assay showed that cardiac myocytes underwent significant apoptosis during AMI, and levels of LDH and CK-MB increased in the serum. However, such changes were significantly inhibited by pre-administration of alatamine. Furthermore, such anti-apoptotic effects of alatamine was also confirmed in a cardiac myocyte model of isoproterenol (ISO)-induced damage. Mechanistically, it was also found that alatamine improved the expression and activity of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), which were inhibited during AMI, promoting contractility and relaxation. Meanwhile, alatamine decreased Bax and increased Bcl-2 expressions both in vivo and in vitro, therefore inhibiting cardiac myocyte apoptosis and preventing cardiac dysfunction caused by AMI at the cellular level. The present study revealed the beneficial role of alatamine in cardiac protection and highlighted it as a potential therapeutic reagent for reduction of AMI-induced cardiac injury.

STATIN THERAPY INITIATED PRE- INSTEAD OF POST-CHOLESTEROL DIET IS MORE BENEFECIAL IN ATTENUATING HYPERCHOLESTEROLEMIA-INDUCED CARDIOVASCULAR CELL APOPTOSIS

Hypercholesterolemia is a common health problem that significantly increases risk of cardiovascular disease in part through the activation of apoptotic pathways. We evaluated the impact of dietary and statin therapy modification (measures known to favorably influence hypercholesterolemia outcome) on the incidence of cardiovascular apoptosis and modulators of the apoptotic response, the S100 proteins B, A1, and A6 and their receptor, the receptor for advanced glycation end- products (RAGE). Male New Zealand white rabbits (16 weeks old) were fed either a normal diet for 8 weeks, a high cholesterol diet (HCD) (containing 2% cholesterol) for 8 weeks, simvastatin (SIM) (3 mg/kg/day) for 8 weeks, HCD for 8 weeks and SIM for an additional 8 weeks, or SIM for 8 weeks and HCD for an additional 8 weeks. HCD compared to a normal diet increased plasma and cardiovascular (cardiac and aortic) tissue cholesterol, triglycerides and LDL levels that were attenuated to a greater extent in the SIM+HCD compared to the HCD+SIM. HCD induced cardiovascular cell (cardiac myocyte and aortic smooth muscle cell) apoptosis as defined by increases in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive nuclei (1.75-fold), caspase-3 activity (1.3-fold), BAX/BCL2 mRNA ratio (3-fold), compared to control. Cardiovascular cell apoptosis was progressively more attenuated in SIM+HCD compared to HCD+SIM. HCD induced the pro-apoptotic S100B 8-fold, RAGE 5-fold and decreased anti-apoptotic S100A6 by 65 per cent. SIM+HCD attenuated the expression of S100B and RAGE (3-4 fold) and increased S100A6 (3-fold) to a greater extent than HCD+SIM. S100A1 was not affected by HCD or SIM therapy. Thus, we conclude that statin therapy, initiated prior to rather than after the development of hypercholesterolemia is more beneficial in attenuating hypercholesterolemia-induced cardiovascular cell apoptosis by a mechanism that may involve the up-regulation of anti-apoptotic S100A6 and down-regulation of pro-apoptotic S100B.

Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis.

Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

Abstract 175: Pro-survival Function of Mef2 in Cardiomyocytes is Enhanced by β-blockers

β1-adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes via activation of cAMP/ protein kinase A (PKA) signaling. β-adrenergic receptor antagonists, or β-blockers, oppose the action of PKA signaling by blocking the β1-receptor and effectively inhibit apoptosis and heart failure. The Myocyte Enhancer Factor 2 (MEF2) proteins have been implicated as nuclear targets for signaling cascades involved in muscle-gene expression and have important roles in proliferation, apoptosis and survival in multiple cell types. We previously reported that PKA signaling represses MEF2 activity. Here, we assessed whether β-blockers can inhibit neonatal cardiac myocyte apoptosis by interfering with PKA dependent MEF2 repression. We show that siRNA mediated MEF2 loss of function induced cardiomyocyte apoptosis. β1AR activation by isoproterenol treatment represses MEF2 transcriptional activity and promotes apoptosis in neonatal cardiomyocytes and, importantly, this effect was reversed in cells expressing a PKA resistant form of MEF2D (S121/190A), as indicated by FACS analysis. We also report that a β-blocker, Atenolol, antagonizes isoproterenol induced apoptosis and also acutely enhanced MEF2 transcriptional activity. We observed that β-adrenergic stimulation modulated MEF2 cellular localization in neonatal cardiomyocytes and this was reversed by atenolol treatment. In addition, we also document that Kru[[Unable to Display Character: ̈]]ppel-like factor 6 (KLF6) is an important MEF2 target gene and loss of function analysis using siRNA-mediated knockdown of KLF6 expression resulted in cardiomyocyte apoptosis. Collectively, these observations, establish that MEF2 plays an important pro-survival role in cardiomyocytes which can be modulated by β-adrenergic signaling. These observations have important clinical implications and may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology.

20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes.

Cardiomyocyte apoptosis is involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both Angiotensin II (Ang II) and 20-hydroxyeicosatetraenoic acid (20-HETE) induce apoptosis in cardiomyocytes. Here, we examined the relationship between 20-HETE and Ang II in cardiomyocyte apoptosis. Apoptosis was examined using flow cytometry in primary cultured rat cardiomyocytes treated with control, Ang II, and Ang II plus HET0016 (a 20-HETE formation inhibitor). The results demonstrated that the treatment of cardiomyocytes with Ang II or 20-HETE significantly increased the percentage of apoptotic cells and that Ang II-induced apoptosis was markedly attenuated by HET0016 or losartan (an AT1 receptor antagonist). In apoptotic mechanism experiments, Ang II or 20-HETE treatment significantly reduced mitochondrial membrane potential, indicating that a mitochondria-dependent mechanism is involved. Ang II-induced alteration in mitochondrial membrane potential was significantly attenuated by HET0016. Treatment of cardiomyocytes with Ang II also increased superoxide production, and this effect of Ang II was attenuated by HET0016. Treatment of cardiomyocytes with Ang II significantly increased CYP4A1 expression and 20-HETE production, as measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes by a mitochondrial superoxide-dependent pathway.

The effects of phosphodiesterase-5 inhibitor sildenafil against post-resuscitation myocardial and intestinal microcirculatory dysfunction by attenuating apoptosis and regulating microRNAs expression: essential role of nitric oxide syntheses signaling.

BackgroundRecent experimental and clinical studies have indicated the cardioprotective role of sildenafil during ischemia/reperfusion (I/R) injury. Sildenafil has been shown to attenuate postresuscitation myocardial dysfunction in piget models of ventricular fibrillation. This study was designed to investigate if administration of sildenafil will attenuate post-resuscitation myocardial dysfunction by attenuating apoptosis and regulating miRNA expressions, furthermore, ameliorating the severity of post-microcirculatory dysfunction.MethodsTwenty-four male pigs (weighing 30 ± 2 kg) were randomly divided into groups, sildenafil pretreatment (n = 8), saline (n = 8) and sham operation (sham, n = 8). Sildenafil pretreatment consisted of 0.5 mg/kg sildenafil, administered once intraperitoneally 30 min prior to ventricular fibrillation (VF). Eight minutes of untreated VF was followed by defibrillation in anesthetized, closed-chest pigs. Hemodynamic status and blood samples were obtained at 0 min, 0.5, 1, 2, 4 and 6 h after return of spontaneous circulation (ROSC). Surviving pigs were euthanatized at 24 h after ROSC, and hearts were removed for analysis by electron microscopy, western blotting, quantitative real-time polymerase chain reaction (PCR), and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Intestinal microcirculatory blood flow was visualized by a sidestream dark-field imaging device at baseline and 0.5, 1, 2, 4, and 6 h after ROSC.ResultsCompared with the saline group, the sildenafil group had a higher 24-hour survival (7/8 versus 3/8 survivors, p < 0.05) and a better outcome in hemodynamic parameters. The protective effect of sildenafil also correlated with reduced cardiomyocyte apoptosis, as evidenced by reduced TUNEL-positive cells, increased anti-apoptotic Bcl-2/Bax ratio and inhibited caspase-3 activity in myocardium. Additionally, sildenafil treatment inhibited the increases in the microRNA-1 levels and alleviated the decreases in the microRNA-133a levels which negatively regulates pro-apoptotic genes. At 6 h after ROSC, post-resuscitation perfused vessel density and microcirculatory flow index were significantly lower in the saline group than in the sildenafil group.ConclusionsThe major findings of this study are as follows: (1) sildenafil improved post-resuscitation perfusion of the heart, and thus reduced cardiac myocyte apoptosis and improved cardiac function; (2) sildenafil treatment inhibited the increases in the microRNA-1 levels, but alleviated the decreases in the microRNA-133a levels.

Epigallocatechin-3-gallate and zinc provide anti-apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells.

It has previously been demonstrated that phospha-tidylinositol-3-kinase (PI3K)/Akt and cleaved caspase-3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin-3-gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription-1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3-h hypoxia, followed by 1-h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti-apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R-induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice.

Osteopontin‐Stimulated Apoptosis in Cardiac Myocytes Involves Reactive Oxygen Species and Mitochondrial Pathway

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we have shown that OPN stimulates cardiac myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. The objective of this study was to evaluate the production and source of reactive oxygen species (ROS), and their role in OPN‐stimulated myocyte apoptosis. Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) induced protein oxidation as analyzed by protein carbonylation assay. Direct monitoring of ARVMs using total ROS detection kit and DCFDA (2',7'‐dichlorofluorescin diacetate; a fluorogenic dye) staining showed increased number of ROS‐positive cells in response to OPN. OPN induced mitochondrial depolarization, and decreased the expression of mitochondrial antioxidant enzyme, manganese superoxide dismutase‐2 (SOD‐2). Transmission electron microscopy revealed that OPN induces mitochondrial abnormalities including swelling and reorganization of cristae. OPN‐mediated increase in the levels of cytosolic cytochrome c and apoptosis were significantly inhibited by N‐acetyl‐cysteine (antioxidant), apocynin (NADPH oxidase inhibitor), MnTBAP [Mn(III)tetrakis (4‐benzoic acid) porphyrin; SOD mimic] and glibenclamide (mitochondrial KATP channel blocker). These data suggest that OPN induces ROS production, and affects mitochondrial morphology and integrity. Mitochondria may serve as a source of ROS production through the opening of mKATP channels.Supported by Department of Veterans Affairs (BX002332) and NIH

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes.

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.

Preventive effect of yuzu and hesperidin on left ventricular remodeling and dysfunction in rat permanent left anterior descending coronary artery occlusion model.

Left ventricular (LV) remodeling, which includes ventricular dilatation and increased interstitial fibrosis after myocardial infarction (MI), is the critical process underlying the progression to heart failure. Therefore, a novel approach for preventing LV remodeling after MI is highly desirable. Yuzu is a citrus plant originating in East Asia, and has a number of cardioprotective properties such as hesperidin. However, no study has proved whether yuzu can prevent LV remodeling. The aim of this study was to determine the effects of yuzu on heart failure (HF) and its potential impact on the LV remodeling process after MI. Our in vivo study using the permanent left anterior descending coronary artery (LAD) occlusion model demonstrate that one week pre-treatment with yuzu or its major metabolite hesperidin before LAD occlusion significantly attenuated cardiac dysfunction, myocyte apoptosis and inflammation. Not only yuzu but also hesperidin inhibited caspase-3 activity, myeloperoxidase expression, α-smooth muscle actin expression, and matrix metalloproteinase-2 activity in a permanent LAD occlusion rat model. To our knowledge, our findings provide the first evidence that yuzu and hesperidin prevent MI-induced ventricular dysfunction and structural remodeling of myocardium.