Abstract-In this study, we hypothesized that bone marrow stem cells (BMSCs) protect ischemic myocardium through paracrine effects that can be further augmented with preconditioning. In in vitro experiments, cell survival factors such as Akt and eNOS were significantly increased in BMSCs following anoxia. In the second series of experiments following coronary ligation in mice, left ventricles were randomly injected with the following: DMEM (G-1), BMSCs (G-2), and preconditioned BMSCs (G-3). Four days after myocardial infarction, BMSCs were observed within injured myocardium in G-2 and G-3. Apoptotic cardiomyocytes within periinfarct area were significantly reduced in G-3. Four weeks after myocardial infarction, smaller left ventricular (LV) dimension and increased LV ejection fraction were observed in G-3. Infarct area was significantly reduced in G-3. However, GFP ϩ cardiomyocytes were observed in low numbers within periinfarct area in G-2 and G-3. In conclusion, BMSCs secreted cell survival factors under ischemia, and they prevented apoptosis in cardiomyocytes adjacent to the infarcted area. Preconditioning of BMSCs enhanced their survival and ability to attenuate LV remodeling, which was attributable, in part, to paracrine effects. (Circ Res. 2006;98:1414-1421.)Key Words: stem cells Ⅲ paracrine effect Ⅲ preconditioning Ⅲ ischemia Ⅲ remodeling M yocardial infarction (MI) leads to cardiomyocyte loss and scar formation in the infarcted area. The large transmural infarction is associated with ventricular remodeling after MI. 1 Ventricular remodeling is characterized by changes in left ventricular (LV) geometry, mass, volume, and function, which include hypertrophy and cellular apoptosis of cardiomyocytes, in response to myocardial injury or alteration in load. 2 Ventricular remodeling is also a major factor in the progression of heart failure, and the prognosis for survival is poor.One approach proposed to reverse myocardial remodeling is regeneration of new cardiomyocytes. Recent reports have shown that bone marrow stem cells (BMSCs) have multilineage differentiation potential and potentially cross the lineage restriction to form various nonhematopoietic tissues including heart. 3 Most studies on BMSC therapy in experimental animal heart models 4 -6 and patients with acute MI 7-9 have shown an improvement in cardiac function, signifying the safety and feasibility of this approach. However, the mechanism of BMSC therapy is still controversial. BMSCs repair the ischemic myocardium primarily by angioblast-mediated vasculogenesis, 10,11 prevention of apoptosis of native cardiomyocytes, or direct regeneration of the lost cardiomyocytes.We, therefore, hypothesized that BMSCs protected ischemic myocardium through paracrine effects that could be further augmented with preconditioning. Materials and MethodsAn expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org. Coculture of BMSCs and CardiomyocytesIsolation and purification of BMSCs from C57B6 mice ...

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. Cardiac protection by mitoKATP channels is dependent on Akt translocation from cytosol to mitochondria during late preconditioning. Am J Physiol Heart Circ Physiol 290: H2402-H2408, 2006; doi:10.1152/ajpheart.00737.2005.-This investigation elucidates the Akt/mitochondrial ATP-sensitive K ϩ (mitoKATP) channel signaling pathway in late pharmacological preconditioning, using the mitoKATP channel openers BMS-191095 (BMS) and diazoxide (DE). BMS (1 mg/kg ip) and DE (7 mg/kg ip) alone or BMS plus wortmannin (WTN, 15 g/kg ip), an inhibitor of phosphatidylinositol 3-kinase, and BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoKATP channels, were administered to male mice. Twenty-four hours later, hearts were isolated and subjected to 40 min of ischemia and 120 min of reperfusion via Langendorff's apparatus. Both BMS and DE reduced left ventricular end-diastolic pressure and increased left ventricular developed pressure as well as reduced LDH release. Coadministration of BMS and WTN abolished the beneficial effects of BMS on cardiac function. Moreover, BMS and DE accelerated Akt phosphorylation in cardiac tissue as determined by Western blot analysis and also significantly reduced apoptosis compared with ischemic control. WTN significantly suppressed BMS-induced Akt phosphorylation, whereas 5-HD had no effect on Akt phosphorylation in cytosol, and the effect of BMS on apoptosis was abolished. It is concluded that the cardioprotective effect by mitoKATP channels is attributed to the translocation of phosphorylated Akt from cytosol to mitochondria. BMS-191095; diazoxide; apoptosis; mitochondrial ATP-sensitive K channels APOPTOSIS AND NECROSIS are generally responsible for myocyte death during ischemia and reperfusion. Necrosis is a rapidly occurring event of cell death that triggers a significant inflammatory response. Typically, cellular changes during necrosis include severe cellular and organelle swelling, denaturation and coagulation of cytoplasmic proteins, and breakdown of cell organelles. Also, there is a depletion of ATP that may be due to a lack of oxygen, a loss of calcium homeostasis, and defects in membrane permeability, which eventually leads to cell death (6). Apoptosis is a genetically controlled programmed cell death and is also responsible for cell injury in the ischemic myocardium. Simultaneous occurrence of both apoptosis and necrosis in myocardium determines the lethality of myocardial injury after ischemia and reperfusion (2,14,19). The cardioprotective effects of protein kinase B (Akt) have been mainly attributed to the reduction of myocardial apoptosis and have a pivotal role in vascular homeostasis and angiogenesis (21). The antiapoptotic activity of Akt is mediated through the activation of the phosphatidylinositol 3-kinase (PI3K) system (10). There is a strong support to the hypothesis that mitochondrial ATPsensitive K ϩ (mitoK ATP ) channel openers like BMS-191095 (BMS) and diazoxide (DE) activate PI3K/Akt pathway during late preconditioning. Bijur and Jope (3) have pro...

Wang, Yigang, Nauman Ahmad, Mitsuhiro Kudo, and Muhammad Ashraf. Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning. Am J Physiol Heart Circ Physiol 287: H1125-H1131, 2004. First published May 13, 2004; 10.1152/ajpheart.00183.2004.-The opening of mitochondrial ATP-sensitive K ϩ (mitoKATP) channels has a significant role in delayed ischemic preconditioning, and nitric oxide (NO) is a wellknown trigger for its activation. However, the source of NO remains unknown. Phosphorylation of endothelial NO synthase (eNOS) increases NO production and reduces apoptosis through the Akt signaling pathway. To elucidate the Akt signaling pathway involved in the opening and antiapoptotic effect of mitoK ATP channel during delayed pharmacological preconditioning, the mitoK ATP channel opener diazoxide (DE, 7 g/kg ip) alone or DE plus N -nitro-L-arginine methyl ester (L-NAME, 30 g/kg iv), an inhibitor of NOS, or wortmannin (WTN, 15 g/kg iv), an inhibitor of phosphatidylinositol 3Ј-kinase (PI3 kinase), was administered to wild-type (WT) or eNOS Ϫ/Ϫ mice during DE treatment. Twenty-four hours later, hearts were isolated and subjected to 40 min ischemia and 30 min reperfusion (I/R). The effect of DE and other interventions on hemodynamic, terminal dUTP nick-end labeling staining and biochemical changes during I/R was assessed in mouse hearts. Treatment with DE resulted in a 2.2-fold increase in phosphorylation of Akt and a significant increase in eNOS and inducible NOS (iNOS) proteins. Akt is upstream of NOS and the mitoK ATP channel as simultaneous pretreatment of WTN with DE abolished phosphorylation of Akt, which was not affected by L-NAME and 5-hydroxydecanoate. In hearts treated with DE, cardiac function was significantly improved after I/R, and apoptosis was also significantly decreased. WTN abolished the antiapoptotic effect of DE. Similarly, S-methylisothiourea, a specific iNOS inhibitor, when given to eNOS Ϫ/Ϫ mice that were pretreated with DE completely abolished the beneficial effects of DE on reduction of apoptotic death. DE was partially effective in eNOS Ϫ/Ϫ mice against the ischemic injury. It is concluded that DE activates Akt through the PI3 kinase signaling pathway and iNOS and eNOS is downstream of Akt. diazoxide; apoptosis NITRIC OXIDE (NO) is formed from L-arginine and oxygen by NO synthase (NOS), which is present in the coronary endothelium, endocardial endothelium, and cardiac myocytes (5). There are three known isoforms of NOS (13). Among them endothelial (eNOS), and neuronal cells (nNOS) are Ca 2ϩ dependent and remain inactive until intracellular calcium levels increase, resulting in enhanced calcium-calmodulin binding and subsequent activation (19), whereas the Ca 2ϩ -independent isoenzyme [inducible NOS (iNOS)] can be induced by cytokines (27) and other stimuli. Although NO is involved in late preconditioning (1, 24), it is still not clear which isoform is responsible for NO production, which serves as a trigger or mediator of preconditioning. Because the mit...

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