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We investigated whether microRNA-145 (miR-145) has a cardioprotective effect in a rabbit model of myocardial infarction (MI) and in H9c2 rat cardiomyoblasts. Rabbits underwent 30 min of coronary occlusion, followed by 2 days or 2 wk of reperfusion. Control microRNA (control group; 2.5 nmol/kg, n = 10) or miR-145 (miR-145 group, 2.5 nmol/kg, n = 10) encapsulated in liposomes was intravenously administered immediately after the start of reperfusion. H9c2 rat cardiomyoblasts were transfected with miR-145. The MI size was significantly smaller in the miR-145 group than in the control group at 2 days and 2 wk post-MI. miR-145 had improved the cardiac function and remodeling at 2 wk post-MI. These effects were reversed by chloroquine. Western blot analysis showed that miR-145 accelerated the transition of LC3B I to II and downregulated p62/SQSTM1 at 2 days or 2 wk after MI, but not at 4 wk, and activated Akt in the ischemic area at 2 days after MI. miR-145 inhibited the growth of H9c2 cells, accelerated the transition of LC3B I to II, and increased phosphorylated Akt in the H9c2 cells at 2 days after miR-145 transfection. Antagomir-145 significantly abolished the morphological change, the transition of LC3B I to II, and the increased phosphorylated Akt induced by miR-145 in H9c2 cells. We determined fibroblast growth factor receptor substrate 2 mRNA to be a target of miR-145, both in an in vivo model and in H9c2 cells. In conclusion, post-MI treatment with miR-145 protected the heart through the induction of cardiomyocyte autophagy by targeting fibroblast growth factor receptor substrate 2., (Copyright © 2015 the American Physiological Society.)

Although OPC-28326, 4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl) piperidine hydrochloride monohydrate, was developed as a selective peripheral vasodilator with α2-adrenergic antagonist properties, it also reportedly exhibits angiogenic activity in an ischemic leg model. The purpose of this study was to examine the effect of OPC-28326 on the architectural dynamics and function of the infarcted left ventricle during the chronic stage of myocardial infarction. Myocardial infarction was induced in male C3H/He mice, after which the mice were randomly assigned into two groups: a control group receiving a normal diet and an OPC group whose diet contained 0.05% OPC-28326. The survival rate among the mice (n = 18 in each group) 4 wk postinfarction was significantly greater in the OPC than control group (83 vs. 44%; P < 0.05), and left ventricular remodeling and dysfunction were significantly mitigated. Histologically, infarct wall thickness was significantly greater in the OPC group, due in part to an abundance of nonmyocyte components, including blood vessels and myofibroblasts. Five days postinfarction, Ki-67-positive proliferating cells were more abundant in the granulation tissue in the OPC group, and there were fewer apoptotic cells. These effects were accompanied by activation of myocardial Akt and endothelial nitric oxide synthase. Hypoxia within the infarct issue, assessed using pimonidazole staining, was markedly attenuated in the OPC group. In summary, OPC-28326 increased the nonmyocyte population in infarct tissue by increasing proliferation and reducing apoptosis, thereby altering the tissue dynamics such that wall stress was reduced, which might have contributed to a mitigation of postinfarction cardiac remodeling and dysfunction., (Copyright © 2015 the American Physiological Society.)

We investigated the effect of cardiac-targeting erythropoietin (EPO)-encapsulated liposomes with sialyl Lewis(X) (SLX) on myocardial infarct (MI) size, left ventricular (LV) remodeling and function, and its molecular mechanism for repairing infarcted myocardium. In rabbits, MI was induced by 30 min of coronary occlusion followed by reperfusion. EPO-encapsulated liposomes with SLX (L-EPO group), EPO-encapsulated liposomes without SLX (L-EPO without SLX group), liposomes with SLX without EPO (L group), or saline (saline group) were intravenously administered immediately after MI. MI sizes and numbers of microvessels were assessed 14 days after MI. Prosurvival proteins and signals were assessed by Western blot analysis 2 and 14 days after MI. Confocal microscopy and electron microscopy showed the specific accumulation of liposomes with SLX in the infarcted myocardium. MI and cardiac fibrosis areas were significantly smaller in the L-EPO group than in the other groups. LV function and remodeling were improved in the L-EPO group. The number of CD31-positive microvessels was significantly greater in the L-EPO group than in the other groups. Higher expressions of EPO receptors, phosphorylated (p)Akt, pERK, pStat3, VEGF, Bcl-2, and promatrix metalloproteinase-1 were observed in the infarct area in the L-EPO group than in the other groups. EPO-encapsulated liposomes with SLX selectively accumulated in the infarct area, reduced MI size, and improved LV remodeling and function through activation of prosurvival signals and by exerting antifibrotic and angiogenic effects. EPO-encapsulated liposomes with SLX may be a promising strategy for active targeting treatment of acute MI.

Ischemia is known to potently stimulate autophagy in the heart, which may contribute to cardiomyocyte survival. In vitro, transfection with small interfering RNAs targeting Atg5 or Lamp-2 (an autophagy-related gene necessary, respectively, for the initiation and digestion step of autophagy), which specifically inhibited autophagy, diminished survival among cultured cardiomyocytes subjected to anoxia and significantly reduced their ATP content, confirming an autophagy-mediated protective effect against anoxia. We next examined the dynamics of cardiomyocyte autophagy and the effects of manipulating autophagy during acute myocardial infarction in vivo. Myocardial infarction was induced by permanent ligation of the left coronary artery in green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) transgenic mice in which GFP-LC3 aggregates to be visible in the cytoplasm when autophagy is activated. Autophagy was rapidly (within 30 min after coronary ligation) activated in cardiomyocytes, and autophagic activity was particularly strong in salvaged cardiomyocytes bordering the infarcted area. Treatment with bafilomycin A1, an autophagy inhibitor, significantly increased infarct size (31% expansion) 24 h postinfarction. Interestingly, acute infarct size was significantly reduced (23% reduction) in starved mice showing prominent autophagy before infarction. Treatment with bafilomycin A1 reduced postinfarction myocardial ATP content, whereas starvation increased myocardial levels of amino acids and ATP, and the combined effects of bafilomycin A1 and starvation on acute infarct size offset one another. The present findings suggest that autophagy is an innate and potent process that protects cardiomyocytes from ischemic death during acute myocardial infarction.

Granulocyte colony-stimulating factor (G-CSF) has been reported to improve cardiac function after myocardial infarction. However, whether postinfarct acute effect of G-CSF is mediated through the same signaling pathways as those of ischemic postconditioning is still unclear. We examined the postinfarct acute effect of G-CSF on myocardial infarct size and its precise molecular mechanism. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Rabbits were intravenously injected 10 μg/kg of G-CSF (G-CSF group) or saline (control group) immediately after reperfusion. The wortmannin + G-CSF, PD-98059 + G-CSF, N(ω)-nitro-L-arginine methyl ester (l-NAME) + G-CSF, and 5-hydroxydecanoic acid sodium salt (5-HD) + G-CSF groups were respectively injected with wortmannin (0.6 mg/kg), PD-98059 (0.3 mg/kg), L-NAME (10 mg/kg), and 5-HD (5 mg/kg) 5 min before G-CSF administration. Myocardial infarct size was calculated as a percentage of the risk area of the left ventricle. Western blot analysis was performed to examine the signals such as protein kinase B (Akt), extracellular signal-regulated protein kinase (ERK), eNOS, p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK3β) in the ischemic myocardium after 48 h of reperfusion. The infarct size was significantly smaller in the G-CSF group (26.7 ± 2.7%) than in the control group (42.3 ± 4.6%). The infarct size-reducing effect of G-CSF was completely blocked by wortmannin (44.7 ± 4.8%), PD-98059 (38.3 ± 3.9%), L-NAME (42.1 ± 4.2%), and 5-HD (42.5 ± 1.7%). Wortmannin, PD-98059, L-NAME, or 5-HD alone did not affect the infarct size. Western blotting showed higher myocardial expression of phospho-Akt, phospho-ERK, phosho-eNOS, phosho-p70S6K, and phosho-GSK3β at 10 min and 48 h after reperfusion in the G-CSF group than in the control group. In conclusion, postreperfusion G-CSF administration reduces myocardial infarct size via activation of phosphatidylinositol 3-kinase-Akt and ERK prosurvival signaling pathways and their downstream targets eNOS, p70S6 kinase, GSK3β, and mitochondrial ATP-dependent K(+) channel.

Anemia may accelerate angiogenesis in ischemic organs through its ability to augment tissue hypoxia-induced generation of several known angiogenic factors and to increase erythropoietin levels, which are also potently angiogenic. We examined the effect of controlled phlebotomy (bloodletting) on blood flow in a mouse ischemic leg model. We ligated the right femoral artery of BALB/c mice. In the phlebotomy group, 200 microl of blood were drawn from the tail vein once a week. After 4 wk, blood flow in the ischemic leg was significantly better in the phlebotomy group (flow ratio of the ischemic to nonischemic leg, 0.87 + or - 0.04) than the control group (0.59 + or - 0.05, P < 0.05), and capillary density was significantly higher. Repeated phlebotomy increased serum erythropoietin levels as well as the expression of hypoxia-inducible transcription factor-1alpha and vascular endothelial growth factor and both the expression and activity of Akt and endothelial nitric oxide synthase (eNOS) in ischemic legs. Treatment with wortmannin or N(omega)-nitro-l-arginine methyl ester significantly attenuated the phlebotomy-induced improvement of blood flow. In addition, fluorescence-activated cell sorting analysis revealed an increase in circulating peripheral endothelial progenitor cells in the phlebotomy group, and treatment with AMD3100, a specific inhibitor of the chemokine receptor CXCR4, blocked the beneficial effect of phlebotomy. These findings suggest that repeated phlebotomy improves blood flow in ischemic legs through an angiogenic action that involves the Akt/eNOS pathway, endothelial progenitor cell mobilization, and their complicated cross talk. An adequately controlled phlebotomy might be one method by which to induce therapeutic angiogenesis.

The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-beta (TGF-beta) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorin's chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-beta1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-beta signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.

The insulin-sensitizing drug pioglitazone has been reported to be protective against myocardial infarction. However, its precise mechanism is unclear. Rabbits underwent 30 min of coronary occlusion followed by 48 h of reperfusion. Rabbits were assigned randomly to nine groups (n = 10 in each): the control group (fed a normal diet), pioglitazone group (fed diets containing 1 mg.kg(-1).day(-1) pioglitazone), pioglitazone + 5-hydroxydecanoic acid (HD) group [fed the pioglitazone diet + 5 mg/kg iv 5-HD, a mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker], pioglitazone + GW9662 group [fed the pioglitazone diet + 2 mg/kg iv GW9662, a peroxisome proliferator activated receptor (PPAR)-gamma antagonist], GW9662 group (fed a normal diet + iv GW9662), pioglitazone + wortmannin group [fed the pioglitazone diet + 0.6 mg/kg iv wortmannin, a phosphatidylinositol (PI)3-kinase inhibitor], wortmannin group (fed a normal diet + iv wortmannin), pioglitazone + nitro-l-arginine methyl ester (l-NAME) group [fed the pioglitazone diet + 10 mg/kg iv l-NAME, a nitric oxide synthase (NOS) inhibitor], and l-NAME group (fed a normal diet + iv l-NAME). All groups were fed the diets for 7 days. The risk area and nonrisk area of the left ventricle (LV) were separated by Evans blue dye, and the infarct area was determined by triphenyltetrazolium chloride staining. The infarct size was calculated as a percentage of the LV risk area. Western blotting was performed to assess levels of Akt and phospho-Akt and phospho-endothelial NOS (eNOS) in the myocardium following reperfusion. The infarct size was significantly smaller in the pioglitazone group (21 +/- 2%) than in the control group (43 +/- 3%). This effect was abolished by GW9662 (42 +/- 3%), wortmannin (40 +/- 3%), or l-NAME (42 +/- 7%) but not by 5-HD (24 +/- 5%). Western blotting showed higher levels of phospho-Akt and phospho-eNOS in the pioglitazone group. Pioglitazone reduces the myocardial infarct size via activation of PPAR-gamma, PI3-kinase, Akt, and eNOS pathways, but not via opening the mitochondrial K(ATP) channel. Pioglitazone may be a novel strategy for the treatment of diabetes mellitus with coronary artery disease.

We hypothesized that therapy, composed of antiapoptotic soluble Fas (sFas) gene transfer, combined with administration of the cardioprotective cytokine granulocyte colony-stimulating factor (G-CSF), would markedly mitigate cardiac remodeling and dysfunction following myocardial infarction (MI). On the 3rd day after MI induced by ligating the left coronary artery in mice, four different treatments were initiated: saline injection (Group C, n = 26); G-CSF administration (Group G, n = 27); adenoviral transfer of sFas gene (Group F, n = 26); and the latter two together (Group G+F, n = 26). Four weeks post-MI, Group G+F showed better survival than Group C (96 vs. 65%, P < 0.05) and the best cardiac function among the four groups. In Group G, the infarct scar was smaller and less fibrotic, whereas in Group F the scar was thicker, without a reduction in area, and contained abundant myofibroblasts and vascular cells; Group G+F showed both phenotypes. G-CSF exerted a beneficial effect on infarct tissue dynamics through antifibrotic and proliferative effects on granulation tissue; however, it also exerts an adverse proapoptotic effect that leads to thinning of the infarct scar. sFas appeared to offset the latter drawback. In vitro study using cultured myofibroblasts derived from the infarct tissue revealed that G-CSF increased proliferating activity of those cells accompanying activation of Akt and signal transducer and activator of transcription 3, while accelerating Fas-mediated apoptosis with increasing Bax-to-Bcl-2 ratio. The results suggest that combined use of G-CSF administration and sFas gene therapy is a potentially powerful tool against post-MI heart failure.

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

Hepatocyte growth factor (HGF) reportedly exerts beneficial effects on the heart following myocardial infarction and during nonischemic cardiomyopathy, but the precise mechanisms underlying the latter have not been well elucidated. We generated nonischemic cardiomyopathy in mice by injecting them with doxorubicin (15 mg/kg ip). Two weeks later, when cardiac dysfunction was apparent, an adenoviral vector encoding human HGF gene (Ad.CAG-HGF, 1x10(11) particles/mouse) was injected into the hindlimb muscles; LacZ gene served as the control. Left ventricular dilatation and dysfunction normally seen 4 wk after doxorubicin administration were significantly mitigated in HGF-treated mice, as were the associated cardiomyocyte atrophy/degeneration and myocardial fibrosis. Myocardial expression of GATA-4 and a sarcomeric protein, myosin heavy chain, was downregulated by doxorubicin, but the expression of both was restored by HGF treatment. The protective effect of HGF against doxorubicin-induced cardiomyocyte atrophy was confirmed in an in vitro experiment, which also showed that neither cardiomyocyte apoptosis nor proliferation plays significant roles in the present model. Upregulation of c-Met/HGF receptor was noted in HGF-treated hearts. Among the mediators downstream of c-Met, the activation of extracellular signal-regulated kinase (ERK) was reduced by doxorubicin, but the activity was restored by HGF. Levels of transforming growth factor-beta1 and cyclooxygenase-2 did not differ between the groups. Our findings suggest the HGF gene delivery exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium in cases of established cardiac dysfunction caused by doxorubicin. These beneficial effects appear to be related to HGF-induced ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins.

Although amlodipine, a long-acting L-type calcium channel blocker, reportedly prevents left ventricular remodeling and dysfunction after myocardial infarction, the mechanism responsible is not yet well understood. Myocardial infarction was induced in mice by ligating the left coronary artery. Treatment of mice with amlodipine (10 mg x kg(-1) x day(-1)), beginning on the third day postinfarction, significantly improved survival and attenuated left ventricular dilatation and dysfunction 4 wk postinfarction compared with treatment with saline or hydralazine. Although infarct sizes did not differ among the groups, the infarcted wall thickness was greater and the infarct segment length was smaller in the amlodipine-treated group, and cellular components, including vessels and myofibroblasts, were abundant within the infarcted area. Ten days postinfarction (the subacute stage), the proliferation of granulation tissue cells in the infarcted area was similar among the groups, but the incidence of apoptosis was significantly lower in the amlodipine-treated group, where Bad, a proapoptotic Bcl-2 family protein, was significantly phosphorylated (inactivated). Calcineurin, which dephosphorylates (activates) Bad, was upregulated in infarcted hearts, but its levels were significantly reduced by amlodipine treatment. In vitro, Fas stimulation augmented calcineurin activity and induced apoptosis among infarct tissue-derived myofibroblasts; both of those effects were strongly inhibited by amlodipine, two other calcium channel blockers (verapamil or nifedipine), and two calcineurin inhibitors (cyclosporin A or FK-506). Amlodipine inhibits Fas-mediated granulation tissue cell apoptosis in infarcted hearts, possibly by attenuating the activities of calcineurin and Bad. These findings may provide new insight into the mechanism by which calcium channel blockers attenuate postinfarction cardiac remodeling and dysfunction.

Blockade of angiotensin II type 1 receptor (AT1) signaling attenuates heart failure following myocardial infarction (MI), perhaps through reduction of fibrosis in the noninfarcted myocardium. However, its specific effect on the infarct tissue itself has not been fully clarified, which we examined in the present study. After MI induction in mice, treatment with the AT1 blocker olmesartan, beginning on the 3rd day post-MI, significantly improved survival (94%) 4 wk post-MI, compared with saline (53%) and hydralazine (73%). Olmesartan-treated mice also showed significant attenuation of left ventricular dilatation and dysfunction, as well as significantly greater infarct wall thickness, although the absolute size of the infarct scar was unchanged. In addition, significantly greater numbers of nonmyocytes (mainly vascular cells and myofibroblasts) were present within the infarct scar in olmesartan-treated hearts. Ten days post-MI, apoptosis among granulation tissue cells was significantly suppressed in the olmesartan-treated hearts, where expression of Fas, Bax, procaspase-3, and Daxx and activation of caspase-3, c-Jun NH(2)-terminal kinase, and c-Jun were all significantly attenuated. By contrast, expression of Fas ligand, Bcl-2, and Fas-associated death domain and activation of caspase-8 were unaffected, suggesting olmesartan exerts a negative regulatory effect on the alternate pathway downstream of Fas receptor. In vitro, olmesartan dose-dependently inhibited Fas-mediated apoptosis in granulation tissue-derived myofibroblasts. The present study proposes this antiapoptotic effect as another important mechanism for an AT1 blocker in improving post-MI ventricular remodeling, as well as its antifibrotic effect, and also suggests a significant link between renin-angiotensin and Fas/Fas ligand systems in postinfarction hearts.

Blockade of ANG II type 1A receptor (AT(1A)) is known to attenuate postinfarction [postmyocardial infarction (post-MI)] heart failure, accompanying reduction in fibrosis of the noninfarcted area. In the present study, we investigated the influence of AT(1A) blockade on the infarcted tissue itself. Consistent with earlier reports, AT(1A) knockout (AT(1A)KO) mice showed significantly attenuated left ventricular (LV) remodeling (dilatation) and dysfunction compared with wild-type (WT) mice. Morphometry revealed that the infarcted wall was thicker and had a smaller circumferential length in AT(1A)KO than WT hearts. In addition, significantly greater numbers of cells were present within infarcts in AT(1A)KO hearts 4 wk post-MI; most notably, there was an abundance of vessels and myofibroblasts. One week post-MI, the incidence of apoptosis among granulation tissue cells was fewer (3.3 +/- 0.4 vs. 4.4 +/- 0.5% in WT, P < 0.05), whereas vessel proliferation was higher in AT(1A)KO hearts, which likely explains the later abundance of cells within the scar tissue. Insulin-like growth factor receptor-I was upregulated and its downstream signal protein kinase B (Akt) was significantly activated in infarcted AT(1A)KO hearts compared with WT hearts. Inactivation of Akt with wortmannin partially but significantly prevented the benefits observed in AT(1A)KO. Collectively, in AT(1A)KO hearts, Akt-mediated granulation tissue cell proliferation and preservation resulting from antiapoptosis likely contributed to an abundant cell population that altered the infarct scar structure, thereby reducing wall stress and attenuating LV dilatation and dysfunction at the chronic stage. In conclusion, altered structural dynamics of infarct scar and increasing myocardial fibrosis may be responsible for the deleterious effects of AT(1A) signaling following MI.

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.