Ahmar Ayub

Implantation of bone marrow stem cells reduces the infarction and fibrosis in ischemic mouse heart

Myocardial infarction may cause sudden cardiac death and heart failure. Adult cardiac myocytes do not replicate due to lack of a substantive pool of precursor, stem, or reserve cells in an adult heart. Ventricular myocytes following myocardial infarction are replaced by fibrous tissue and this leads to congestive heart failure in severe cases. Anversa et al. described that resident cardiac stem cells are present in the heart, and can repair the damaged mycardium by myocyte regeneration. Recent findings suggest the feasibility of cardiac repair using cell transplantation. However, it remains controversial which cell types are the best for cell transplantation in the ischemic heart. In this study, we demonstrate that cultured bone marrow stromal cells (MSCs) and Lin(-) bone marrow cells upon transplantation differentiate into myocytes and endothelial cells in the ischemic heart, eventually reducing both infarct size and fibrosis.

Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes

Background—Bone marrow stromal cells (BMSCs) have the potential to differentiate into various cells and can transdifferentiate into myocytes if an appropriate cellular environment is provided. However, the molecular signals that underlie this process are not fully understood. In this study, we show that BMSC differentiation is dependent on communication with cells in their microenvironment. Methods and Results—BMSCs were isolated from green fluorescent protein (GFP)–transgenic mice and cocultured with myocytes in a ratio of 1:40. Myocytes were obtained from neonatal rat ventricles. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcription–polymerase chain reaction. Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After 7-day coculture with myocytes, some BMSCs became α-actinin–positive and formed gap junctions with native myocytes. However, BMSCs separated from myocytes by a semipermeable membrane were still negative for α-actinin. Transdifferentiated myocytes from BMSCs were microdissected from cocultures by laser captured microdissection to determine the changes in gene expression. BMSCs cocultured with myocytes expressed mouse cardiac transcription factor GATA-4. Conclusions—When cocultured with myocytes, BMSCs can transdifferentiate into cells with a cardiac phenotype. Differentiated myocytes express cardiac transcription factors GATA-4 and myocyte enhancer factor-2. The transdifferentiation processes rely on intercellular communication of BMSCs with myocytes.

Downregulation of Protein Kinase C Inhibits Activation of Mitochondrial KATP Channels by Diazoxide

Background—The mitochondrial KATP (mitoKATP) channel has been shown to confer short- and long-term cardioprotection against prolonged ischemia via protein kinase C (PKC) signaling pathways. However, the exact association between PKC or its isoforms and mitoKATP channels has not yet been clarified. The present study tested the hypothesis that the activity and translocation of PKC to the mitochondria are important for cardiac protection elicited by mitoKATP channels. Methods and Results—PKC was downregulated by prolonged (24-hour) treatment with phorbol 12-myristate 13-acetate (4 &mgr;g/kg body weight) before subsequent experiments in rats. Langendorff-perfused rat hearts were subjected to 40 minutes of ischemia followed by 30 minutes of reperfusion. Effects of PKC downregulation on the activation of mitoKATP channels and other interventions on hemodynamic, biochemical, and pathological changes were assessed. Subcellular localization of PKC isoforms by Western blot analysis and immunocytochemistry demonstrated that PKC-&agr; and PKC-&dgr; were translocated to the sarcolemma and that PKC-&dgr; was translocated to the mitochondria after diazoxide treatment. In hearts treated with diazoxide (80 &mgr;mol/L), a significant improvement in cardiac function and an attenuation of cell injury were observed. In PKC-downregulated hearts, protection was abolished because mitoKATP channels could not be activated by diazoxide. Conclusions—These data suggest that PKC activation is required for the opening of mitoKATP channels during protection against ischemia and that this effect is linked to isoform-specific translocation of PKC-&dgr; to the mitochondria.

Role of Mitochondrial Membrane Potential in Cardiac Protection against Ischemia

Our previous study has indicated that diazoxide protected myocardium against ischemia-reperfusion injury. This study tests the hypothesis that the maintenance of mitochondrial membrane potential (ΔΨm) in myocytes is responsible for cell protection against ischemia. This was specifically tested in myocytes after activation of the mitoKATP channel. Myocyte damage by 3 hrs anoxia and 2 hrs reoxygenation (A-R) was evaluated by cell viability, membrane permeability and apoptosis. Mitochondrial function was indicated by the concentration of ATP. Mitochondrial morphology was observed by staining myocytes with Mito Tracker Orange CMTMRos and by electron microscopy. Immunostaining was used to determine the distribution of cytochrome c. ΔΨm was assayed by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and observed by con-focal microscopy. Results show that 1) An extensive damage was observed in cultured myocytes as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis and decreased ATP concentration after A-R. 2) Mitochondria in A-R myocytes were swollen and exhibited a collapsed ΔΨm. Cytochrome c was released from mitochondria into the cytosol. 3) Diazoxide (100μmol/L) significantly prevented myocyte and mitochondrial damage, cytochrome c loss, and stabilized ΔΨm. 4) This protection was blocked by 5-hydroxydecanoate (5-HD, 500μmol/L), a mitoKATP channel selective inhibitor but not by HMR-1098 (30μmol/L), a putative sarcolemmal KATP channel selective inhibitor. 5) Diazoxide reduced ΔΨm in normal cultured myocytes in a concentration- and time-independent pattern. It is concluded that activation of mitoKATP channel with diazoxide prevented disruption of ΔΨm resulting in protection against A-R induced injury.

Cardioprotection by Mitochondrial KATP Channel in Both Early and Late Preconditioning

Activation of the mitochondrial KATP channel (mitoKATP) has been shown to confer an early and late phase of cardioprotection against ischemic injury via protein kinase C (PKC) signaling pathways. However, the exact relationship between PKC or its isoforms and mitoKATP channels has not yet been clarified in both early and late preconditioning (PC). The hypothesis of this study is that the translocation of PKC to mitochondria is important for early cardiac protection elicited by mitoKATP channels. PKC was down-regulated by treatment of PMA (4μg/kg) for 24-hours prior to subsequent experiments. Langendorff-perfused rat hearts were subjected to 40-min of ischemia followed by 30-min reperfusion. In late PC, nitric oxide was hypothesized to be trigger for the opening of mitoKATP channels to enable the heart to protect itself against ischemic damage. This was tested in iNOS knockout mouse hearts. Effects of PKC down-regulation or blockade of PKC activity by chelerythrine on the activation of mitoKATP channel and other interventions on hemodynamic, biochemical and pathological changes were assessed. Subcellular distribution of PKC isoforms by immunocy-tochemistry demonstrates that immunostaining of PKC-δ was observed in the mitochondria after diazoxide (DE, 80μmol/L) pretreatment, PKC-e was translocated to intercalated disc and PKC-β1 was translocated to the nucleus. In DE treated hearts, a significant improvement in cardiac function and attenuation of cell injury was observed. In the PKC down-regulated hearts or hearts pretreated with chelerythrine, the protection was abolished because mitoKATP channels could not be activated by DE. These data suggest that PKC activation is required for the opening of mitoKATP channels in the protection against ischemia and this effect is linked with isoform specific translocation of δ to mitochondria. NO is an important trigger for the opening of mitoKATP channels after 24 hours of initial preconditioning stimulus. DE was found to be totally ineffective in iNOS knockout mice. DE activated NF-kB via PKC signaling pathway and that lead to NOS up-regulation for further activation of the mitoKATP after 24 hours upon ischemic stimulus.