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The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-i (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2-1 μmol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 μmol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-l mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-l. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a transwell system. The culture medium of STS-treated myocytes recruited monocytes in a MCP- I-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 mm of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 ± 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP- I antibody showed that 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-l protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-l, which have a critical role in the active recruitment of macrophages. [ABSTRACT FROM AUTHOR]

Objectives: The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. Background: Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. Methods: Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. Results: Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a significantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidification which stimulates apoptosis. However, acidification-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. Conclusions: We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidification. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis. [Copyright &y& Elsevier]

Examines the role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis. Importance of intracellular energy levels in determining the form of cell death in cardiac myocytes; Definition of apoptosis; Loss of Bax translocation in apoptotic and neurotic cells.

Abstract: Nicorandil has been shown to inhibit myocyte apoptosis by opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels and nitrate-like effect against oxidative stress. However, the detailed mechanism of nicorandil-mediated cardioprotection under hypoxic conditions remains to be largely unknown. The present study examined whether nicorandil can inhibit apoptosis via regulation of Bcl-2 family proteins in hypoxic myocytes. Neonatal rat cardiac myocytes were exposed to hypoxia for 7 hours. Hypoxia-induced myocyte apoptosis (13.9±0.9%) under glucose-rich conditions. Myocyte apoptosis was accompanied by loss of mitochondrial membrane potential (Δψm), cytochrome c release from mitochondria into cytosol, and activation of caspase-3. Hypoxia also significantly increased Bax and decreased Bcl-2 mRNA and protein expression, thereby increasing Bax/Bcl-2 ratio. Nicorandil 100 μmol/l significantly decreased the percentage of apoptotic myocytes (7.2±0.5%) by inhibiting loss of Δψm and translocation of cytochrome c. These effects of nicorandil were partially but significantly inhibited by cotreatment of either 500 μmol/l 5-hydroxydecanoate, a selective mitoKATP channel antagonist, or 10 μmol/l 1H-[1,2,4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Moreover, nicorandil significantly inhibited the hypoxia-induced changes in Bax and Bcl-2 expression, and concomitant increased Bax and decreased Bcl-2 immunoreactivity in mitochondria. These effects of nicorandil in Bax and Bcl-2 expression were significantly blunted by cotreatment of ODQ and 5-HD, respectively. Cotreatment of KT5823, an inhibitor of protein kinase G, significantly blocked the effect of nicorandil on Bax expression and 8-bromo-cyclic guanosine 3′,5′ monophosphate (8-bromo-cGMP), a cGMP analog, mimicked the effect of nicorandil on Bax expression. The present study demonstrates that nicorandil regulates Bcl-2 family proteins via opening of mitoKATP channels and nitric oxide-cGMP signaling and inhibits hypoxia-induced mitochondrial death pathway. [Copyright &y& Elsevier]

: ObjectivesWe examined whether amlodipine, a calcium channel antagonist with potent antioxidant activity, inhibits doxorubicin-induced apoptosis in cultured neonatal rat cardiac myocytes.: BackgroundRecent studies have shown that doxorubicin induces apoptosis as well as necrosis in myocytes through generation of reactive oxygen species.: MethodsThe effects of amlodipine and several other antioxidants on doxorubicin-induced oxidative stress and mitochondria-mediated apoptosis were examined.: ResultsTreatment of myocytes with doxorubicin (10−6 mol/l) for 14 h increased the number of cells with elevated peroxides, as histochemically estimated by 2′,7′-dichlorofluorescin (DCF) diacetate, and the percentage of apoptotic myocytes, as estimated by Hoechst 33258 nuclear staining, compared with control myocytes (25.0 ± 1.6% vs. 5.2 ± 1.2%). Moreover, doxorubicin-induced myocyte apoptosis was also confirmed by annexin V–fluorescein isothiocyanate binding assay. Doxorubicin induced a reduction in myocyte adenosine 5′-triphosphate content, a loss of mitochondrial membrane potential, cytochrome c release from the mitochondria into the cytosol, and caspase-3 activation to 1.9-fold of control. Amlodipine significantly attenuated increased DCF fluorescence, inhibited the mitochondria-mediated apoptotic responses described earlier, and decreased apoptosis in the doxorubicin-treated myocytes in a dose-dependent fashion. Amlodipine at 10−6 mol/l significantly decreased apoptosis to 15.4 ± 0.7%, and this antiapoptotic action was more effective than that seen with other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. In contrast, the calcium channel antagonist nifedipine (10−6 mol/l) did not inhibit apoptosis. Catalase, glutathione, and N-acetylcysteine, but not mannitol or superoxide dismutase, significantly decreased DCF fluorescence and attenuated myocyte apoptosis induced by doxorubicin to 18.7 ± 1.2%, 19.1 ± 1.7%, and 18.7 ± 0.6%, respectively.: ConclusionsAmlodipine significantly inhibits doxorubicin-induced myocyte apoptosis by suppressing the mitochondrial apoptotic pathway. This effect is attributed to the antioxidant properties of amlodipine, affecting mainly hydrogen peroxide. [Copyright &y& Elsevier]