Abstract-The endoplasmic reticulum (ER) stress response (ERSR) is activated when folding of nascent proteins in the ERlumen is impeded. Myocardial ischemia was recently shown to activate the ERSR; however, the role of this complex signaling system in the heart is not well understood. ER stress activates the transcription factor ATF6, which induces expression of proteins targeted to the ER, where they restore protein folding, thus fostering cytoprotection. We previously developed a transgenic mouse line that expresses a conditionally activated form of ATF6 in the heart. In this mouse line, ATF6 activation decreased ischemic damage in an ex vivo model of myocardial ischemia/reperfusion and induced numerous genes, including mesencephalic astrocyte-derived neurotrophic factor (MANF). In the present study, MANF expression was shown to be induced in cardiac myocytes and in other cell types in the hearts of mice subjected to in vivo myocardial infarction. Additionally, simulated ischemia induced MANF in an ATF6-dependent manner in neonatal rat ventricular myocyte cultures. In contrast to many other ER-resident ERSR proteins, MANF lacks a canonical ER-retention sequence, consistent with our finding that MANF was readily secreted from cultured cardiac myocytes. Knockdown of endogenous MANF with micro-RNA increased cell death upon simulated ischemia/ reperfusion, whereas addition of recombinant MANF to media protected cultured cardiac myocytes from simulated ischemia/reperfusion-mediated death. Thus, a possible function of the ERSR in the heart is the ischemia-mediated induction of secreted proteins, such as MANF, that can function in an autocrine/paracrine manner to modulate myocardial damage from ER stresses, including ischemia. Key Words: adenovirus Ⅲ cardiac myocytes Ⅲ endoplasmic reticulum stress Ⅲ hypoxia Ⅲ ischemia Ⅲ myocardial infarction Ⅲ unfolded protein response N umerous proteins that are critical to cellular function are synthesized on endoplasmic reticulum (ER) ribosomes and then folded and further posttranslationally modified in the ER lumen. Stresses that impede ER protein folding trigger the ER stress response (ERSR), 1-4 a signaling system that has not been studied extensively in the heart. ER stress activates the transcription factors X-box binding protein (XBP1) and activator of transcription-6 (ATF6), which induce numerous ERSR proteins designed to restore efficient ER protein folding, which contributes to resisting the stress. 5,6 Many ERSR genes are induced by ATF6 or XBP1, whereas others exhibit a requirement for only 1 of the 2 factors. 7 The folding of proteins in the ER lumen requires molecular oxygen, 8 suggesting that that hypoxia-mediated induction of ERSR proteins during myocardial ischemia may enhance ER protein folding and survival of cardiac myocytes and limit ischemic damage. Consistent with this hypothesis are recent studies showing that simulated ischemia (sI) activates the ERSR in cultured rat and mouse ventricular myocytes and that ER stress is activated in the surviving myocytes adj...

Key Words: Pim-1 Ⅲ mitochondria Ⅲ cardiomyocyte Ⅲ apoptosis C ardiovascular disease is the leading cause of death among men and women and affects approximately 33% of the US population. 1 A direct correlation between the decline in heart function and loss of cardiomyocytes via apoptosis involving the mitochondria occurs in cardiomyopathy, myocardial ischemia/reperfusion (I/R), and congestive heart failure. 2-9 Specifically, myocardial I/R injury generates calcium overload and oxidative stress, which initiate the intrinsic apoptotic pathway through activation of the mitochondrial permeability transition pore (mPTP). The ensuing chain of events result in dramatic changes to mitochondrial morphology associated with uncoupling of the electron transport chain, depolarization of the inner membrane, matrix swelling, unfolding of the cristae, and ultimately outer membrane rupture, with release of proapoptotic cytochrome c. 10 -15 Release of cytochrome c into the cytosol consequently activates apoptotic protease-activating factor, which mediates caspase cascade programmed cell death. 16 Thus, preservation of mitochondrial integrity is essential in designing molecular strategies to enhance cardiomyocyte cell survival by blunting injury attributed to cardiomyopathic insult.Cardioprotection mediated by survival kinase signal transduction acts through multiple mechanisms including preservation of mitochondrial integrity. 17 Numerous studies have documented antiapoptotic actions of the serine/threonine kinase AKT, which acts in part through protecting mitochon-

Jin JK, Whittaker R, Glassy MS, Barlow SB, Gottlieb RA, Glembotski CC. Localization of phosphorylated ␣B-crystallin to heart mitochondria during ischemia-reperfusion. Am J Physiol Heart Circ Physiol 294: H337-H344, 2008. First published November 9, 2007 doi:10.1152/ajpheart.00881.2007.-The cytosolic small heat shock protein ␣B-crystallin (␣BC) is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses such as ischemia-reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to phosphorylation of ␣BC on Ser 59 (P-␣BC-S59), which increases its protective ability. ␣BC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. We investigated the hypothesis that protection from I/R damage in the heart by P-␣BC-S59 can be mediated by localization to mitochondria. We found that P-␣BC-S59 localized to mitochondria isolated from untreated mouse hearts and that this localization increased more than threefold when the hearts were subjected to ex vivo I/R. Mitochondrial P-␣BC-S59 decreased when hearts were treated with the p38 inhibitor SB-202190. Moreover, SB-202190-treated hearts exhibited more tissue damage and less functional recovery upon reperfusion than controls. I/R activates mitochondrial permeability transition (MPT) pore opening, which increases cell damage. We found that mitochondria incubated with a recombinant mutant form of ␣BC that mimics P-␣BC-S59 exhibited decreased calcium-induced MPT pore opening. These results indicate that mitochondria may be among the key components in stressed cells with which P-␣BC-S59 interacts and that this localization may protect the myocardium, in part, by modulating MPT pore opening and, thus, reducing I/R injury. mitochondrial permeability transition; cardioprotection

alphaB-crystallin (alphaBC) is a small heat shock protein expressed at high levels in the myocardium where it protects from ischemia-reperfusion damage. Ischemia-reperfusion activates p38 MAP kinase, leading to the phosphorylation of alphaBC on serine 59 (P-alphaBC-S59), enhancing its ability to protect myocardial cells from damage. In the heart, ischemia-reperfusion also causes the translocation of alphaBC from the cytosol to other cellular locations, one of which was recently shown to be mitochondria. However, it is not known whether alphaBC translocates to mitochondria during ischemia-reperfusion, nor is it known whether alphaBC phosphorylation takes place before or after translocation. In the present study, analyses of mitochondrial fractions isolated from mouse hearts subjected to various times of ex vivo ischemia-reperfusion showed that alphaBC translocation to mitochondria was maximal after 20 min of ischemia and then declined steadily during reperfusion. Phosphorylation of mitochondrial alphaBC was maximal after 30 min of ischemia, suggesting that at least in part it occurred after alphaBC association with mitochondria. Consistent with this was the finding that translocation of activated p38 to mitochondria was maximal after only 10 min of ischemia. The overexpression of alphaBC-AAE, which mimics alphaBC phosphorylated on serine 59, has been shown to stabilize mitochondrial membrane potential and to inhibit apoptosis. In the present study, infection of neonatal rat cardiac myocytes with adenovirus-encoded alphaBC-AAE decreased peroxide-induced mitochondrial cytochrome c release. These results suggest that during ischemia alphaBC translocates to mitochondria, where it is phosphorylated and contributes to modulating mitochondrial damage upon reperfusion.

The objective of this study was to explore the relationship between severity of depression and cardiovascular disease (CVD) risk factors among selected Latino patients within a primary care setting. We conducted a cross-sectional analysis of 164 low-income Latino patients at San Ysidro Health Center (SYHC) who had been recruited into a mental health program between January 2007 and March 2008. Patients were between the ages of 18 and 83 years, 54 were males and 109 females. Patients were screened using the 9-item patient health questionnaire (PHQ-9), a standardized instrument used to measure depression severity. We used regression models to analyze the relationship between severity of depression and CVD risk factors. Seventy-eight percent of the patients had at least mild depression based on PHQ-9 score categories. Significant age-adjusted pairwise associations were found with CVD risk factors; body mass index (BMI), diabetes status, serum cholesterol level, and serum triglyceride level were all associated with depression severity score. Regression models indicated that diabetic patients on anti-depressive medication with higher BMI scores and triglyceride levels had significantly higher depression severity scores. Clinicians should be made aware that depressive symptoms may impede efforts to modify BMI, serum triglycerides, anti-depression medication, and diabetes among Latino patients. They should, therefore, screen and treat depression among Latinos at risk to CVD.