TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8 h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia–reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.     

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3β, and ERK1/2. There was also maintenance of cardiac myocyte β-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

c-Jun N-terminal kinase (JNK-1) confers protection against brief but not extended ischemia during acute myocardial infarction

Brief periods of ischemia do not damage the heart and can actually protect against reperfusion injury caused by extended ischemia. It is not known what causes the transition from protection to irreversible damage as ischemia progresses. c-Jun N-terminal kinase-1 (JNK-1) is a stress-regulated kinase that is activated by reactive oxygen and thought to promote injury during severe acute myocardial infarction. Because some reports suggest that JNK-1 can also be protective, we hypothesized that the function of JNK-1 depends on the metabolic state of the heart at the time of reperfusion, a condition that changes progressively with duration of ischemia. Mice treated with JNK-1 inhibitors or transgenic mice wherein the JNK-1 gene was ablated were subjected to 5 or 20 min of ischemia followed by reperfusion. When JNK-1 was inactive, ischemia of only 5 min duration caused massive apoptosis, infarction, and negative remodeling that was equivalent to or greater than extended ischemia. Conversely, when ischemia was extended JNK-1 inactivation was protective. Mechanisms of the JNK-1 switch in function were investigated in vivo and in cultured cardiac myocytes. In vitro there was a comparable switch in the function of JNK-1 from protective when ATP levels were maintained during hypoxia to injurious when reoxygenation followed glucose and ATP depletion. Both apoptotic and necrotic death pathways were affected and responded reciprocally to JNK-1 inhibitors. JNK-1 differentially regulated Akt phosphorylation of the regulatory sites Ser-473 and Thr-450 and the catalytic Thr-308 site in vivo. The studies define a novel role for JNK-1 as a conditional survival kinase that protects the heart against brief but not protracted ischemia.     

Myocardial infarction and heart failure in the db/db diabetic mouse

Clinical studies have reported that the incidence and severity of myocardial infarction is significantly greater in diabetics compared with nondiabetics after correction for all other risk factors. The majority of studies investigating the pathophysiology of myocardial ischemia-reperfusion injury have focused on otherwise healthy animals. At present, there is a paucity of experimental investigations on the pathophysiology of heart failure in diabetic animals. We hypothesized that the severity of myocardial reperfusion injury and the development of congestive heart failure would be markedly enhanced in the db/db diabetic mouse. Accordingly, we studied the effects of varying durations of in vivo myocardial ischemia and reperfusion on the incidence of heart failure in db/db diabetic mice. Nondiabetic and db/db diabetic mice (10 wk of age) were subjected to 30, 45, or 60 min of left coronary artery occlusion and 28 days of reperfusion. Survival at 24 h of reperfusion was 100% in nondiabetic mice subjected to 30 min of myocardial ischemia and 88% in nondiabetic mice subjected to 45 min of myocardial ischemia. In contrast, survival was 53% in db/db diabetic mice subjected to 30 min of myocardial ischemia and 44% in db/db mice after 45 min of myocardial ischemia. Prolonged survival in nondiabetic mice was not significantly attenuated when compared during the 28-day follow-up period with all groups experiencing >90% survival. Prolonged survival was significantly decreased in db/db mice after both 30 and 45 min of myocardial ischemia compared with sham controls. Furthermore, we observed a significant degree or left ventricular dilatation, cardiac hypertrophy, and cardiac contractile dysfunction in db/db mice subjected to 45 min of myocardial ischemia and 28 days reperfusion. In nondiabetic mice subjected to 45 min of myocardial ischemia, we failed to observe any changes in left ventricular dimensions or fractional shortening. These studies provide a feasible experimental model system for the investigation of heart failure secondary to acute myocardial infarction in the db/db diabetic mouse.     

Effects of endotoxin on neutrophil-mediated ischemia/reperfusion injury in the rat heart in vivo

We have previously shown that a nonlethal dose of lipopolysaccharide (LPS) decreases L-selectin expression of neutrophils (PMNs), thereby preventing PMN-mediated reperfusion injury in the isolated heart. In the present study we determined whether or not that dose of LPS would protect hearts during in vivo ischemia and reperfusion by preventing PMN-induced reperfusion injury. Rats receiving saline vehicle showed marked myocardial injury (necrotic area/area at risk = 82%+/-2%) and significant depression in left ventricular function as assessed in the isolated isovolumic heart preparation at constant flow rates of 5, 10, 15, and 20 ml/min. The administration of LPS (100 microg/kg body wt) 7 hr prior to ischemia resulted in a reduction in myocardial damage (necrotic area/area at risk = 42%+/-3%) and preservation of function. Myocardial function was similar to that of sham ischemic saline- and LPS-treated rats. Moreover, PMN infiltration as determined by histology was quantitatively more severe in hearts of saline-treated rats than in hearts of LPS-treated rats. Isolated hearts from vehicle- and LPS-treated animals undergoing sham ischemia in vivo recovered to the same extent after in vitro ischemia/reperfusion, suggesting that LPS did not induce protection by altering intrinsic properties of the heart. Our results indicate that LPS-induced protection of the heart from in vivo PMN-mediated ischemia/reperfusion injury may be due to decreased L-selectin expression of PMNs in LPS-treated animals.     

Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion

Reperfusion after myocardial ischemia is associated with a rapid influx of calcium, leading to activation of various enzymes including calpain. Isolated perfused adult rabbit hearts subjected to global ischemia and reperfusion were studied. Calpain or a calpain-like activity was activated within 15 min after reperfusion, and preconditioning suppressed calpain activation. In contrast, caspase activation was not detected although cytochrome c was released after ischemia and reperfusion. The pro-apoptotic BH3-only Bcl-2 family member, Bid, was cleaved during ischemia/reperfusion in the adult rabbit heart. Recombinant Bid was cleaved by calpain to a fragment that was able to mediate cytochrome c release. The calpain cleavage site was mapped to a region within Bid that is extremely susceptible to proteolysis. These findings suggest that there is cross-talk between apoptotic and necrotic pathways in myocardial ischemia/reperfusion injury.     

Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 mug/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 mug/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 +/- 3.5% (vehicle) to 11.0 +/- 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 +/- 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.     

Metformin preferentially provides neuroprotection following cardiac ischemia/reperfusion in non-diabetic rats

Following acute myocardial infarction, re-establishment of coronary perfusion aggravates further injuries in the heart and remote organs including the brain as a consequence of ischemia/reperfusion (I/R) injury. Since pretreatment with metformin attenuated both cardiac and cerebral I/R injury via AMP-activated protein kinase (AMPK) pathways, we hypothesized that metformin given after ischemia mitigates both cardiac and brain pathologies following cardiac I/R. Male Wistar rats were subjected to either cardiac I/R (30 min-ischemia/120 min-reperfusion; n = 30) or sham operation (n = 5). Metformin 200 mg/kg was given intravenously to the cardiac I/R group (n = 10/group), either during ischemia (D-MET) or at the onset of reperfusion (R-MET). Left ventricular ejection fraction (LVEF) and arrhythmia scores were determined. The heart and brain tissues were collected to determine the extent of injury, mitochondrial function, and apoptosis. Additionally, microglial morphology, Alzheimer's proteins, and dendritic spine density were determined in the brain. Cardiac I/R led to not only reduced LVEF, cardiac mitochondrial dysfunction, and arrhythmias, but also brain mitochondrial dysfunction, apoptosis, Alzheimer's protein aggregation, microglial activation, and dendritic spine loss. A single dose of metformin did not alter p-AMPK/AMPK in both organs. In the heart, impaired LVEF, arrhythmias, infarct size expansion, mitochondrial dysfunction, and apoptosis were not alleviated. On the contrary, metformin attenuated brain mitochondrial dysfunction, apoptosis, and Alzheimer's protein levels. Microglial morphology and dendritic spine density were additionally preserved in D-MET group. In conclusion, metformin given during ischemia preferentially provides neuroprotection against brain mitochondrial dysfunction, apoptosis, microglial activation, and dendritic spine loss in an AMPK-independent manner following cardiac I/R injury.     

HDAC Inhibition Elicits Myocardial Protective Effect through Modulation of MKK3/Akt-1

We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3^−/−, Akt-1^−/− mice received intraperitoneal injections of trichostatin A (0.1mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection.     

Initiation of mitochondrial-mediated apoptosis during cardiac reperfusion

Reperfusion of myocardial tissue can result in programmed cell death. Nevertheless, relatively little information exists concerning pathways initiated in vivo that ultimately commit cardiac cells to apoptosis during ischemia/reperfusion. The goal of the present study was to determine whether mitochondrial-mediated mechanisms of apoptosis are initiated during in vivo cardiac ischemia/reperfusion. We provide evidence that the content of cytochrome c in the cytosol increases exclusively during reperfusion. Over the same time interval Bax, a pro-apoptotic protein implicated in release of cytochrome c from mitochondria, was found to disappear from cytosolic extracts. This was associated with the appearance of tightly associated Bax in the mitochondrial fraction. Cytochrome c from reperfused cytosolic extracts is present as a high molecular weight oligomer consistent with formation of the apoptosome. In addition, pro-caspase-9 was found to disappear exclusively during reperfusion. Therefore, the results of the current study indicate that the mitochondrial-mediated pathway of apoptosis is initiated as a result of in vivo cardiac ischemia/reperfusion.     

Protection against myocardial ischemia/reperfusion injury in TLR4-deficient mice is mediated through a phosphoinositide 3-kinase-dependent mechanism

TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4(-/-) mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4(-/-) cardioprotection following I/R injury. TLR4(-/-) and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4(-/-) vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4(-/-) mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4(-/-) mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4(-/-) mice, when compared with control TLR4(-/-) mice. These data indicate that protection against myocardial I/R injury in TLR4(-/-) mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4(-/-) myocardium may involve increased phosphorylation/inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3beta. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.     

CTRP9 protein protects against myocardial injury following ischemia-reperfusion through AMP-activated protein kinase (AMPK)-dependent mechanism

Ischemic heart disease is the major cause of death in Western countries. CTRP9 (C1q/TNF-related protein 9) is a fat-derived plasma protein that has salutary effects on glucose metabolism and vascular function. However, the functional role of CTRP9 in ischemic heart disease has not been clarified. Here, we examined the regulation of CTRP9 in response to acute cardiac injury and investigated whether CTRP9 modulates cardiac damage after ischemia and reperfusion. Myocardial ischemia-reperfusion injury resulted in reduced plasma CTRP9 levels and increased plasma free fatty acid levels, which were accompanied by a decrease in CTRP9 expression and an increase in NADPH oxidase component expression in fat tissue. Treatment of cultured adipocytes with palmitic acid or hydrogen peroxide reduced CTRP9 expression. Systemic administration of CTRP9 to wild-type mice, before the induction of ischemia or at the time of reperfusion, led to a reduction in myocardial infarct size following ischemia-reperfusion. Administration of CTRP9 also attenuated myocyte apoptosis in ischemic heart, which was accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Treatment of cardiac myocytes with CTRP9 protein reduced apoptosis in response to hypoxia/reoxygenation and stimulated AMPK phosphorylation. Blockade of AMPK activity reversed the suppressive actions of CTRP9 on cardiomyocyte apoptosis. Knockdown of adiponectin receptor 1 diminished CTRP9-induced increases in AMPK phosphorylation and survival of cardiac myocytes. Our data suggest that CTRP9 protects against acute cardiac injury following ischemia-reperfusion via an AMPK-dependent mechanism.     

Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways

12/15-Lipoxygenase (LOX) is a member of the LOX family that catalyzes the step from arachidonic acid to hydroxy-eicosatetraenoic acids (HETEs). Previous studies demonstrated that 12/15-LOX plays a critical role in the development of atherosclerosis, hypertension, heart failure, and other diseases; however, its role in myocardial ischemic injury was contraversal. Here, we investigated the inhibition of 12/15-LOX by baicalein on acute cardiac injury and dissected its molecular mechanism. In a mouse model of acute ischemia/reperfusion (I/R) injury, 12/15-LOX was significantly upregulated in the peri-infarct area surrounding the primary infarction. In cultured cardiac myocytes, baicalein suppressed apoptosis and caspase 3 activity in response to simulated ischemia/reperfusion (I/R). Moreover, administration of 12/15-LOX inhibitor, baicalein, significantly attenuated myocardial infarct size induced by I/R injury. Moreover, baicalein treatment significantly inhibited cardiomyocyte apoptosis, inflammatory responses and oxidative stress in the heart after I/R injury. The mechanisms underlying these effects were associated with the activation of ERK1/2 and AKT pathways and inhibition of activation of p38 MAPK, JNK1/2, and NF-kB/p65 pathways in the I/R-treated hearts and neonatal cardiomyoctes. Our data indicated that 12/15-LOX inhibitor baicalein can prevent myocardial I/R injury by modulation of multiple mechanisms, and suggest that baicalein could represent a novel therapeutic drug for acute myocardial infarction.     

黑木耳多糖对抗离体心脏缺血/再灌注损伤的研究

目的:探讨黑木耳多糖(AAP)对离体大鼠心脏缺血/再灌注(I/R)损伤的防护作用及其机制。方法:健康雄性SD大鼠灌胃黑木耳多糖(50,100,200mg/(kg.d))4周后,采用离体心脏Langendorff灌流方法,全心停灌30min,复灌120min建立I/R模型。测定左心室动力学指标和再灌注各时间点冠脉流出液中乳酸脱氢酶(LDH)含量;实验结束测定心肌组织甲月赞(formazan)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性的变化。结果:与单纯I/R组相比,AAP预处理明显提高心肌细胞的formazan含量,降低再灌注期间冠脉流出液中LDH含量,明显增强左室发展压、左心室内压最大上升速率和心率与发展压乘积的恢复,缓解冠脉流量的减少;高剂量AAP改善I/R心肌功能的作用要好于丹参预处理(4ml/(kg.d),gastricperfusion)组。中剂量AAP(100mg/(kg.d))预处理4周后明显抑制I/R心肌MDA的增加和SOD活性的减弱(P0.01),其效果要好于丹参阳性对照组。结论:在大鼠离体心脏灌流模型上,黑木耳多糖预处理具有抗心脏I/R损伤的作用,这种保护作用可能与其增加心肌SOD活性,减少脂质过氧化损伤有关。     

Diverse effects of L-arginine on cardiac function of rats subjected to myocardial ischemia and reperfusion in vivo

In vivo administration of L-arginine at different time points during the course of myocardialischemia and reperfusion(MI/R)has been shown to differentially regulate postischemic apoptosis.Cardiacfunction is one of the most important indexes used to judge the degree of myocardial injury.The presentstudy attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has adiverse influence on cardiac function of ischemic reperfused hearts and,if so,to investigate the mechanismsinvolved.Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion.Anintravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion(early treatment)or3 h and 4 h after reperfusion(late treatment).Early treatment with L-arginine markedly increased the leftventricular systolic pressure(LVSP)and dP/dt_(max),and decreased myocardial nitrotyrosine content.In strictcontrast,late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dt_(max)from 4 h to 5h after reperfusion,and increase in toxic peroxynitrite formation as measured by nitrotyrosine.These resultssuggest that the administration of L-arginine at different time points during the course of MI/R leads todiverse effects on cardiac dysfunction.Early supplementation decreased the nitrative stress and improvedleft ventricular function.However,late treatment with L-arginine increased the formation of peroxynitriteand aggravated cardiac functional injury.     

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and risk of arsenic-induced urothelial carcinoma in residents of southwestern Taiwan

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Apelin-13 protects the heart against ischemia-reperfusion injury through inhibition of ER-dependent apoptotic pathways in a time-dependent fashion

Endoplasmic reticulum (ER) stress is activated during and contributes to ischemia-reperfusion (I/R) injury. Attenuation of ER stress-induced apoptosis protects the heart against I/R injury. Using apelin, a ligand used to activate the apelin APJ receptor, which is known to be cardioprotective, this study was designed to investigate 1) the time course of changes in I/R injury after ER stress; 2) whether apelin infusion protects the heart against I/R injury via modulation of ER stress-dependent apoptosis signaling pathways; and 3) how phosphatidylinositol 3-kinase (PI3K)/Akt, endothelial nitric oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and ERK activation are involved in the protection offered by apelin treatment. The results showed that, using an in vivo rat I/R model induced by 30 min of ischemia followed by reperfusion, infarct size (IS) increased from 2 h of reperfusion (34.85 ± 2.14%) to 12 h of reperfusion (48.98 ± 3.35, P < 0.05), which was associated with an abrupt increase in ER stress-dependent apoptosis activation, as evidenced by increased CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and JNK activation (CHOP: 2.49-fold increase, caspase-12: 2.09-fold increase, and JNK: 3.38-fold increase, P < 0.05, respectively). Administration of apelin at 1 μg/kg not only completely abolished the activation of ER stress-induced apoptosis signaling pathways at 2 h of reperfusion but also significantly attenuated time-related changes at 24 h of reperfusion. Using pharmacological inhibition, we also demonstrated that PI3K/Akt, AMPK, and ERK activation were involved in the protection against I/R injury via inhibition of ER stress-dependent apoptosis activation. In contrast, although eNOS activation played a role in decreasing IS at 2 h of reperfusion, it failed to modify either IS or ER stress-induced apoptosis signaling pathways at 24 h after reperfusion.     

Cardioprotection requires flipping the 'posttranslational modification' switch

Minimizing damage during reperfusion of the heart following an ischemic event is an important part of the recovery process, as is preventing future recurrences; however, restoring blood perfusion to the heart following ischemia can lead to apoptosis, necrosis, and finally, diminished cardiac function. Exercise reduces risk of heart disease and has been shown to improve the recovery of the heart following ischemia and reperfusion. Brief intermittent ischemic events administered prior to or following a myocardial infarction have also been demonstrated to reduce the infarct size and improve cardiac function, thereby providing cardioprotection. Many signaling transduction pathways are known to regulate cardioprotection, including but not limited to calcium regulation, antioxidant scavenging, and kinase activation. Although posttranslational modifications (PTM) such as phosphorylation, O-GlcNAcylation, methylation, and acetylation are essential regulators of these pathways, their contributions are often overlooked in the literature. This review will examine how PTMS are important regulators of cardioprotection and demonstrate why they should be targeted when developing future therapies for the minimization of damage caused by cardiac ischemia and reperfusion.     

Ankyrin-B Protein in Heart Failure: IDENTIFICATION OF A NEW COMPONENT OF METAZOAN CARDIOPROTECTION

Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, β(II)-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly, implicate ankyrin-B as a molecular component for cardioprotection following ischemia.