Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, δPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of ɛPKC, δPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, δPKC and ɛPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of ɛPKC caused cardioprotection whereas activation of δPKC increased damage induced by ischemia in vitro and in vivo . In contrast, δPKC and ɛPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Although functional coupling between protein kinase Cε (PKCε) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCε remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCε and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCε with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCε complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCε can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCε resulted in a significant inhibition of Ca ²⁺ -induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCε in mice, which is cardioprotective, greatly increased interaction of PKCε with the pore components and inhibited Ca ²⁺ -induced pore opening. In contrast, cardiac expression of kinase-inactive PKCε did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCε transgenesis. Collectively, these data demonstrate that PKCε forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCε-induced cardioprotection.

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo . A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant ( P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent l -citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that ( i ) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and ( ii ) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo .

Background— Stromal cell–derived factor-1α (SDF-1α) binding to its cognate receptor, CXCR4, regulates a variety of cellular functions such as stem cell homing, trafficking, and differentiation. However, the role of the SDF-1α–CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown. Methods and Results— In mice subjected to ischemic preconditioning, myocardial SDF-1α mRNA was found to be increased 3 hours later ( P <0.05). Myocardial SDF-1α and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1α production increased significantly after 1 or 4 hours of hypoxia and 18 hours of reoxygenation in cultured myocytes ( P <0.05) but did not change in fibroblast cultures. In isolated myocytes, CXCR4 activation by SDF-1α resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1α were resistant to hypoxia/reoxygenation damage, exhibiting less lactate dehydrogenase release, trypan blue uptake, and apoptotic cell death (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay) ( P <0.05). This protective effect was blocked by the CXCR4 selective antagonist AMD3100. In vivo, administration of SDF-1α before 30 minutes of coronary occlusion followed by 4 hours of reperfusion decreased infarct size ( P <0.05). The decrease in infarct size with SDF-1α administration also was blocked by AMD3100. Conclusions— We conclude that SDF-1α and its receptor, CXCR4, constitute a paracrine or autocrine axis in cardiac myocytes that is activated in response to preconditioning and hypoxic stimuli, recruiting the antiapoptotic kinases ERK and AKT and promoting an antiapoptotic program that confers protection against ischemia/reperfusion damage.

The concept that bone marrow (BM)–derived cells participate in cardiac regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. In this study, we report that the postnatal BM contains a mobile pool of cells that express early cardiac lineage markers (Nkx2.5/Csx, GATA-4, and MEF2C). These cells are present in significant amounts in BM harvested from young mice but their abundance decreases with age; in addition, the responsiveness of these cells to gradients of motomorphogens SDF-1, HGF, and LIF changes with age. FACS analysis, combined with analysis of early cardiac markers at the mRNA and protein levels, revealed that cells expressing these markers reside in the nonadherent, nonhematopoietic CXCR4 ⁺ /Sca-1 ⁺ /lin ⁻ /CD45 ⁻ mononuclear cell (MNC) fraction in mice and in the CXCR4 ⁺ /CD34 ⁺ /AC133 ⁺ /CD45 ⁻ BMMNC fraction in humans. These cells are mobilized into the peripheral blood after myocardial infarction and chemoattracted to the infarcted myocardium in an SDF-1-CXCR4–, HGF-c-Met–, and LIF-LIF-R–dependent manner. To our knowledge, this is the first demonstration that the postnatal BM harbors a nonhematopoietic population of cells that express markers for cardiac differentiation. We propose that these potential cardiac progenitors may account for the myocardial regenerative effects of BM. The present findings provide a novel paradigm that could reconcile current controversies and a rationale for investigating the use of BM-derived cardiac progenitors for myocardial regeneration.

The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)–signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 ± 53%) and JAK2 (+238 ± 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 ± 61%) and pTyr-STAT3 (+253 ± 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 γ-IFN activation site DNA-binding activity (+606 ± 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress.