Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

肾缺血预处理对心脏缺血／再灌注损伤的保护作用

目的：探讨肾缺血预处理对家兔心脏缺血／再灌注（I／R）损伤的影响及意义。方法：32只大耳白家兔随机分为假手术（SO）、心脏I／R、经典缺血预处理（CIPC）及肾缺血预处理（RIPC）4组。观察各组心肌梗塞面积、左室舒缩功能、心脏超微结构及心律失常发生率的变化。结果：CIPC、RIPC组,心肌梗塞面积、再灌性心律失常发生率较I／R组明显降低,左室舒缩功能明显恢复（P＜0.01）,心脏超微结构损伤明显减轻。结论：RIPC可诱导出与CIPC类似的心脏保护效应。     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10-30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5'-[gamma-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.     

ZFP580对大鼠心肌缺血/再灌注后心室重塑的影响

目的：探究锌指转录因子（ZFP580）与心肌缺血/再灌注损伤后心室重塑的关系。方法：72只SD大鼠随机分为假手术（sham）组（n=8）和心肌缺血/再灌注（I/R）组（n=64）,其中I/R组分别在再灌注后的0.5 h、1 h、2 h、4h、1 d,7 d,14 d,28 d处死后取材,观察心肌组织中ZFP580的表达。培养大鼠H9C2心肌细胞,每组设3个复孔,分别在转化生长因子β1（TGF-β1）刺激0 h、8 h、16 h、24 h后观察心肌细胞肥大情况,并检测心肌细胞中β-MHC、心房利钠肽（ANP）以及ZFP580 mRNA的表达。利用慢病毒介导的基因转染获得高表达ZFP580的H9C2心肌细胞,转染72h后,检测心肌细胞中基质金属蛋白酶3（MMP-3）的表达。结果：成功建立心肌缺血/再灌注损伤模型,大鼠心肌I/R损伤后第14天,心肌组织大面积梗死,心肌细胞呈嗜酸性变。大鼠心肌组织中ZFP580及TGF-β1表达上调。TGF-β1（5 ng/ml）刺激H9C2心肌细胞后诱导心肌细胞肥大,心肌细胞肥大标志蛋白β-MHC、ANP表达上调,且心肌细胞中ZFP580mRNA表达上调（P < 0.05）。高表达ZFP580的H9c2心肌细胞中MMP-3表达下调（P < 0.05）。结论：锌指转录因子ZFP580可能参与了心肌缺血/再灌注后心室重塑的过程,其作用可能与参与TGF-β1诱导的心肌细胞肥大过程以及抑制心肌细胞产生MMP-3有关。     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Protective effects of circulating microvesicles derived from ischemic preconditioning on myocardial ischemia/reperfusion injury in rats by inhibiting endoplasmic reticulum stress

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<?1 µm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats respectively. Additionally, treatment with IPC-MVs significantly alleviated damage of myocardium, and restored cardiac function of I/R injury rats, as evidenced by increased heart rate, and decreased the elevation of ST-segment. The size of myocardial infarction, lactate dehydrogenase activity, and the number of apoptotic cardiomyocytes were also reduced significantly with IPC-MVs treatment, coincident with the above function amelioration. Moreover, IPC-MVs decreased the activity of caspase 3, and the expression of endoplasmic reticulum stress (ERS) markers, GRP78, CHOP and caspase 12 indicating the involvement of ERS-specific apoptosis in I/R injury, and cardioprotective effects of IPC-MVs. In summary, our study demonstrated a novel mechanism of IPC in which circulating IPC-MVs could protect hearts from I/R injury in rats through attenuation of ERS-induced apoptosis. These findings provide new insight into therapeutic potential of IPC-induced MVs in cardioprotection against I/R injury.     

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.     

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.     

Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress,serum deprivation or hypoxia mimetic CoCl2

Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5′ untranslated region (5′ UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5′ UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl₂, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5′ UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed.     

Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the alpha2-, alpha3-, beta1-, and beta2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the alpha1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase alpha2-, alpha3-, and beta2-isoforms induced by I/R, without affecting the alpha1- and beta1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase alpha2-, alpha3-, and beta1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+ exchanger in hearts after I/R.     

Regulation of metabolism by mitochondrial enzyme acetylation in cardiac ischemia-reperfusion injury

Ischemia reperfusion injury (I/R injury) contributes significantly to morbidity and mortality following myocardial infarction (MI). Although rapid reperfusion of the ischemic myocardium was established decades ago as a highly beneficial therapy for MI, significant cell death still occurs after the onset of reperfusion. Mitochondrial dysfunction is closely associated with I/R injury, resulting in the uncontrolled production of reactive oxygen species (ROS). Considerable efforts have gone into understanding the metabolic perturbations elicited by I/R injury. Recent work has identified the critical role of reversible protein acetylation in maintaining normal mitochondrial biologic function and energy metabolism both in the normal heart and during I/R injury. Several studies have shown that modification of class I HDAC and/or Sirtuin (Sirt) activity is cardioprotective in the setting of I/R injury. A better understanding of the role of these metabolic pathways in reperfusion injury and their regulation by reversible protein acetylation presents a promising way forward in improving the treatment of cardiac reperfusion injury. Here we briefly review some of what is known about how acetylation regulates mitochondrial metabolism and how it relates to I/R injury.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.     

Myocardial Ablation of G Protein–Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein–coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.