Background-Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied. Methods and Results-We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes. Conclusions-Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases. Key Words: apoptosis Ⅲ ischemia Ⅲ microRNA Ⅲ myocardial infarction Ⅲ reperfusion M ore than 1 million Americans suffer from myocardial infarction every year. 1 Both human autopsy data and evidence from rodent models of myocardial infarction indicate that most cell death happens by apoptosis during the initial 2 to 4 hours after coronary occlusion. 2,3 Clinical treatment of myocardial infarction by thrombolytic therapy and revascularization by percutaneous coronary intervention or coronary artery bypass graft surgery are effective. 1,3 However, given the health, economic, and personal burden caused by ischemic heart disease, research into additional treatment modalities is imperative. Furthermore, the molecular mechanisms that regulate gene expression during myocardial ischemia/reperfusion (I/R) are still not completely understood. Clinical Perspective on p 2366MicroRNAs (miRNAs) are a class of endogenous nonprotein-coding RNAs comprising Ϸ22 nucleotides. 4 -6 They regulate gene expression via RNA-induced silencing complexes, targeting them to mRNAs where they inhibit translation or direct destructive cleavage. 4 -6 Increasing evidence indicates the importa...

Background-Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against ␤-agonistinduced apoptosis. Methods and Results-To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region-to-risk region ratio was 8.1Ϯ1.1% in TG hearts (nϭ7), compared with 19.5Ϯ2.1% in WT hearts (nϭ11, PϽ0.001). Conclusions-Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.

Abstract-Doxorubicin (DOX) is a widely used antitumor drug, but its application is limited because of its cardiotoxic side effects. Heat shock protein (Hsp)20 has been recently shown to protect cardiomyocytes against apoptosis, induced by ischemia/reperfusion injury or by prolonged ␤-agonist stimulation. However, it is not clear whether Hsp20 would exert similar protective effects against DOX-induced cardiac injury. Actually, DOX treatment was associated with downregulation of Hsp20 in the heart. To elucidate the role of Hsp20 in DOX-triggered cardiac toxicity, Hsp20 was first overexpressed ex vivo by adenovirus-mediated gene delivery. Increased Hsp20 levels conferred higher resistance to DOX-induced cell death, compared to green fluorescent protein control. Furthermore, cardiac-specific overexpression of Hsp20 in vivo significantly ameliorated acute DOX-triggered cardiomyocyte apoptosis and animal mortality. Hsp20 transgenic mice also showed improved cardiac function and prolonged survival after chronic administration of DOX. The mechanisms underlying these beneficial effects were associated with preserved Akt phosphorylation/activity and attenuation of DOX-induced oxidative stress. Coimmunoprecipitation studies revealed an interaction between Hsp20 and phosphorylated Akt. Accordingly, BAD phosphorylation was preserved, and cleaved caspase-3 was decreased in DOX-treated Hsp20 transgenic hearts, consistent with the antiapoptotic effects of Hsp20. Parallel ex vivo experiments showed that either infection with a dominant-negative Akt adenovirus or preincubation of cardiomyocytes with the phosphatidylinositol 3-kinase inhibitors significantly attenuated the protective effects of Hsp20. Taken together, our findings indicate that overexpression of Hsp20 inhibits DOX-triggered cardiac injury, and these beneficial effects appear to be dependent on Akt activation. Thus, Hsp20 may constitute a new therapeutic target in ameliorating the cardiotoxic effects of DOX treatment in cancer patients. (Circ Res. 2008;103:1270-1279.)

Abstract-Ischemic heart disease, which remains the leading cause of morbidity and mortality in the Western world, is invariably characterized by impaired cardiac function and disturbed Ca 2ϩ homeostasis. Because enhanced inhibitor-1 (I-1) activity has been suggested to preserve Ca 2ϩ cycling, we sought to define whether increases in I-1 activity in the adult heart may ameliorate contractile dysfunction and cellular injury in the face of an ischemic insult. To this end, we generated an inducible transgenic mouse model that enabled temporally controlled expression of active I-1 (T35D). Active I-1 expression in the adult heart elicited significant enhancement of contractile function, associated with preferential phospholamban phosphorylation and enhanced sarcoplasmic reticulum Ca 2ϩ -transport. Further phosphoproteomic analysis revealed alterations in proteins associated with energy production and protein synthesis, possibly to support the increased metabolic demands of the hyperdynamic hearts. Importantly, on ischemia/reperfusion-induced injury, active I-1 expression augmented contractile function and recovery. Further examination revealed that the infarct region and apoptotic as well as necrotic injuries were significantly attenuated by enhanced I-1 activity. These cardioprotective effects were associated with suppression of the endoplasmic reticulum stress response. The present findings indicate that increased I-1 activity in the adult heart enhances Ca 2ϩ cycling and improves mechanical recovery, as well as cell survival after an ischemic insult, suggesting that active I-1 may represent a potential therapeutic strategy in myocardial infarction. Key Words: ischemia Ⅲ reperfusion Ⅲ protein phosphatase-1 inhibitor-1 Ⅲ phospholamban Ⅲ ER stress I schemic heart disease remains the leading cause of cardiovascular disease and mortality in the Western world. In the United States alone, the incidence of myocardial infarction has reached an alarming 8.1 million. 1 Even though restoration of coronary flow alleviates the detriment of the ischemic insult, it is invariably accompanied by reperfusion-induced contractile dysfunction and cellular damage. The causes for these deleterious effects are multifactorial, but disturbed Ca 2ϩ homeostasis has been proposed as a central contributor to postischemic injury. 2,3 The sarcoplasmic reticulum (SR) is a crucial regulator of Ca 2ϩ handling in the cardiomyocyte, and elucidation of its role in ischemia/reperfusion (I/R)-induced injury has been the focus of several investigations. Experimental evidence indicates that SR Ca 2ϩ cycling is depressed in the postischemic myocardium 4,5 ; yet the functional manifestations of this alteration remain controversial. On the one hand, it has been reported that intracellular Ca 2ϩ overload, during reperfusion, and the subsequent futile cycles of Ca 2ϩ release and reuptake by the SR are critical events that lead to contractile dysfunction, necrosis, and mitochondrial-dependent apoptosis. 6 -8 As such, depressed SR function may be an adaptive m...