Coronary artery disease leads to injury and loss of myocardial tissue by deprivation of blood flow (ischemia) and is a major underlying cause of heart failure. Prolonged ischemia causes necrosis and apoptosis of cardiac myocytes and vascular cells; however, the mechanisms of ischemia-mediated cell death are poorly understood. Ischemia is associated with both hypoxia and acidosis due to increased glycolysis and lactic acid production. We recently reported that hypoxia does not induce cardiac myocyte apoptosis in the absence of acidosis. We now report that hypoxia-acidosisassociated cell death is mediated by BNIP3, a member of the Bcl-2 family of apoptosis-regulating proteins. Chronic hypoxia induced the expression and accumulation of BNIP3 mRNA and protein in cardiac myocytes, but acidosis was required to activate the death pathway. Acidosis stabilized BNIP3 protein and increased the association with mitochondria. Cell death by hypoxia-acidosis was blocked by pretreatment with antisense BNIP3 oligonucleotides. The pathway included extensive DNA fragmentation and opening of the mitochondrial permeability transition pore, but no apparent caspase activation. Overexpression of wild-type BNIP3, but not a translocation-defective mutant, activated cardiac myocyte death only when the myocytes were acidic. This pathway may figure significantly in muscle loss during myocardial ischemia.heart ͉ apoptosis ͉ ischemia ͉ mitochondria ͉ antisense

No abstract

Expression of the human cardiac ~-actin gene (HCA) depends on the interactions of multiple transcriptional regulators with promoter elements. We report here that the tissue-specific expression of this promoter is determined by the simultaneous interaction of at least three specific protein-DNA complexes. The myogenic determinant gene MyoD1 activated the transcription of transfected HCA-CAT promoter constructs in nonmuscle cells, including CV-1 and HeLa cells. Gel mobility-shift and footprinting assays revealed that MyoD1 specifically interacted with a single consensus core sequence, CANNTG, at -50. Previously characterized sites interact with a protein identical with or related to the serum response factor (SRF) at -100 and Spl at -70. All three elements must be intact to support transcription in muscle cells: site-specific mutation within any one of these three elements eliminated transcriptional expression by the promoter. Furthermore, expression of the promoter in embryonic Drosophila melanogaster cells that lack MyoD1 andSpl is strictly dependent on all three sites remaining intact and on the presence of exogenously supplied Spl and MyoD1. These experiments suggest that the presence of three sequence-specific binding proteins, including MyoD1, and their intact target DNA sequences are minimal requirements for muscle-specific expression of the HCA gene.

Our group recently reported positive therapeutic benefit of human endometrium‐derived mesenchymal stem cells (EnMSCs) delivered to infarcted rat myocardium, an effect that correlated with enhanced secretion of protective cytokines and growth factors compared with parallel cultures of human bone marrow MSCs (BMMSCs). To define more precisely the molecular mechanisms of EnMSC therapy, in the present study, we assessed in parallel the paracrine and therapeutic properties of MSCs derived from endometrium, bone marrow, and adipose tissues in a rat model of myocardial infarction (MI). EnMSCs, BMMSCs, and adipose‐derived MSCs (AdMSCs) were characterized by fluorescence‐activated cell sorting (FACS). Paracrine and cytoprotective actions were assessed in vitro by coculture with neonatal cardiomyocytes and human umbilical vein endothelial cells. A rat MI model was used to compare cell therapy by intramyocardial injection of BMMSCs, AdMSCs, and EnMSCs. We found that EnMSCs conferred superior cardioprotection relative to BMMSCs or AdMSCs and supported enhanced microvessel density. Inhibitor studies indicated that the enhanced paracrine actions of EnMSCs were mediated by secreted exosomes. Analyses of exosomal microRNAs (miRs) by miR array and quantitative polymerase chain reaction revealed that miR‐21 expression was selectively enhanced in exosomes derived from EnMSCs. Selective antagonism of miR‐21 by anti‐miR treatment abolished the antiapoptotic and angiogenic effects of EnMSCs with parallel effects on phosphatase and tensin homolog (PTEN), a miR‐21 target and downstream Akt. The results of the present study confirm the superior cardioprotection by EnMSCs relative to BMMSCs or AdMSCs and implicates miR‐21 as a potential mediator of EnMSC therapy by enhancing cell survival through the PTEN/Akt pathway. The endometrium might be a preferential source of MSCs for cardiovascular cell therapy. Stem Cells Translational Medicine 2017;6:209–222

Hypoxia treatment enhances paracrine effect of mesenchymal stem cells (MSCs). The aim of this study was to investigate whether exosomes from hypoxia-treated MSCs (ExoH) are superior to those from normoxia-treated MSCs (ExoN) for myocardial repair. Mouse bone marrow-derived MSCs were cultured under hypoxia or normoxia for 24 h, and exosomes from conditioned media were intramyocardially injected into infarcted heart of C57BL/6 mouse. ExoH resulted in significantly higher survival, smaller scar size and better cardiac functions recovery. ExoH conferred increased vascular density, lower cardiomyocytes (CMs) apoptosis, reduced fibrosis and increased recruitment of cardiac progenitor cells in the infarcted heart relative to ExoN. MicroRNA analysis revealed significantly higher levels of microRNA-210 (miR-210) in ExoH compared with ExoN. Transfection of a miR-210 mimic into endothelial cells (ECs) and CMs conferred similar biological effects as ExoH. Hypoxia treatment of MSCs increased the expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. Blocking the activity of nSMase2 resulted in reduced miR-210 secretion and abrogated the beneficial effects of ExoH. In conclusion, hypoxic culture augments miR-210 and nSMase2 activities in MSCs and their secreted exosomes, and this is responsible at least in part for the enhanced cardioprotective actions of exosomes derived from hypoxia-treated cells.

Abstract-Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1␤, tumor necrosis factor-␣, and interferon-␥. These cytokines caused a time-dependent induction of cardiac myocyte apoptosis, but not necrosis, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase, reflecting caspase activation. Apoptosis was preceded by a Ͼ50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/g protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1␤; interferon-␥ and tumor necrosis factor-␣ had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species. (Circ Res. 1999;84:21-33.)Key Words: poly(ADP-ribose) polymerase Ⅲ protein kinase G Ⅲ nitric oxide Ⅲ Bcl-x(L) Ⅲ oxidative stress

Excess generation of reactive oxygen species (ROS) and cytosolic calcium accumulation play major roles in the initiation of programmed cell death during acute myocardial infarction. Cell death may include necrosis, apoptosis and autophagy, and combinations thereof. During ischemia, calcium handling between the sarcoplasmic reticulum and myofilament is disrupted and calcium is diverted to the mitochondria causing swelling. Reperfusion, while essential for survival, reactivates energy transduction and contractility and causes the release of ROS and additional ionic imbalance. During acute ischemia–reperfusion, the principal death pathways are programmed necrosis and apoptosis through the intrinsic pathway, initiated by the opening of the mitochondrial permeability transition pore and outer mitochondrial membrane permeabilization, respectively. Despite intense investigation, the mechanisms of action and modes of regulation of mitochondrial membrane permeabilization are incompletely understood. Extrinsic apoptosis, necroptosis and autophagy may also contribute to ischemia–reperfusion injury. In this review, the roles of dysregulated calcium and ROS and the contributions of Bcl-2 proteins, as well as mitochondrial morphology in promoting mitochondrial membrane permeability change and the ensuing cell death during myocardial infarction are discussed.

SUMMARY Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags)screened with cDNAs from aerobic and hypoxic cardiac myocytes identified>100 genes that were induced by >2-fold and ∼20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold(mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia–acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically,mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore,cardiac myocyte death during hypoxia–acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.