Yiru Guo

Opposing cardioprotective actions and parallel hypertrophic effects of δPKC and ɛPKC

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.

Protein Kinase Cε Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria

Abstract— Although functional coupling between protein kinase C&egr; (PKC&egr;) 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 PKC&egr; 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 PKC&egr; 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 PKC&egr; 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 PKC&egr; complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKC&egr; can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKC&egr; resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKC&egr; in mice, which is cardioprotective, greatly increased interaction of PKC&egr; with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKC&egr; did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKC&egr; transgenesis. Collectively, these data demonstrate that PKC&egr; forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKC&egr;-induced cardioprotection.

Cells Expressing Early Cardiac Markers Reside in the Bone Marrow and Are Mobilized Into the Peripheral Blood After Myocardial Infarction

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 Protective Effect of Late Preconditioning Against Myocardial Stunning in Conscious Rabbits Is Mediated by Nitric Oxide Synthase: Evidence That Nitric Oxide Acts Both as a Trigger and as a Mediator of the Late Phase of Ischemic Preconditioning

Seventy-four conscious rabbits undergoing a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3) were assigned to nine groups. In group I (controls, n = 8), the recovery of systolic wall thickening (WTh) after the sixth reperfusion was markedly improved on days 2 and 3 compared with day 1, indicating late preconditioning (PC) against myocardial stunning; the total deficit of WTh after the sixth reperfusion was reduced by 56% on day 2 and 50% on day 3 compared with day 1 (P < .01). Administration on day 2 of the nonselective NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NA) (group II, n = 8) or of the selective inducible NOS inhibitors aminoguanidine (AG) (group IV, n = 8) and S-methylisothiourea sulfate (SMT) (group VI, n = 6) completely abrogated late PC against stunning on day 2. On day 3, the expected PC effect became manifest in all groups. Administration of L-NA, AG, or SMT on day 1 (groups III [n = 7], V [n = 6], and VII [n = 5], respectively) had no discernible effect on the deficit of WTh on day 1, indicating that these agents do not augment the severity of myocardial stunning in nonpreconditioned myocardium. In group VIII (n = 7), the abrogation of late PC by SMT on day 2 was completely reversed by the concomitant administration of L-arginine (595 mg/kg IV), indicating that it was not due to nonspecific NOS-unrelated actions. Administration of L-arginine alone on day 2 (group IX [n = 5]) had no effect on the deficit of WTh. Furthermore, administration of L-NA on day 1 (group III) prevented the appearance of the PC effect on day 2, whereas AG (group V) and SMT (group VI) did not, suggesting that the development of late PC on day 1 is triggered by the endothelial (type III) isoform of NOS. This study demonstrates that three structurally different NOS inhibitors (L-NA, AG, and SMT), given 24 hours after the PC ischemia, consistently abrogate late PC against myocardial stunning in conscious rabbits, indicating that this cardioprotective effect is mediated by the activity of NOS. The results obtained with AG and SMT specifically implicate the inducible (type II) isoform as the mediator of the protection on day 2. Previous studies have shown that NO triggers the development of late PC. The present results indicate that NO plays a dual role in late PC against stunning, acting initially as the trigger and subsequently as the mediator of the protection.

The nitric oxide hypothesis of late preconditioning

Abstract Ischemic preconditioning (PC) occurs in two phases: an early phase, which lasts 2–3 h, and a late phase, which begins 12–24 h later and lasts 3–4 days. The mechanism for the late phase of PC has been the subject of intensive investigation. We have recently proposed the “NO hypothesis of late PC”, which postulates that NO plays a prominent role both in initiating and in mediating this cardioprotective response. The purpose of this essay is to review the evidence supporting the NO hypothesis of late PC and to discuss its implications. We propose that, on day 1, a brief ischemic stress causes increased production of NO (probably via eNOS) and ·O2–, which then react to form ONOO–, ONOO–, in turn, activates the ɛ isoform of protein kinase C (PKC); either directly or via its reactive byproducts such as ·OH. Both NO and secondary species derived from ·O2– could also stimulate PKC ɛ independently. PKC ɛ activation triggers a complex signaling cascade that involves tyrosine kinases (among which Src and Lck appear to be involved) and probably other kinases, the transcription factor NF-κB, and most likely other as yet unknown components, resulting in increases transcription of the iNOS gene and increased iNOS activity on day 2, which is responsible for the protection during the second ischemic challenge. Tyrosine kinases also appear to be involved on day 2, possibly by modulating iNOS activity. According to this paradigm, NO plays two completely different roles in late PC: on day 1, it initiates the development of this response, whereas on day 2, it protects against myocardial ischemia. We propose that two different NOS isoforms are sequentially involved in late PC, with eNOS generating the NO that initiates the development of the PC response on day 1 and iNOS then generating the NO that protects against recurrent ischemia on day 2. The NO hypothesis of late PC puts forth a comprehensive paradigm that can explain both the initiation and the mediation of this complex phenomenon. Besides its pathophysiological implications, this hypothesis has potential clinical reverberations, since NO donors (i.e., nitrates) are widely used clinically and could be used to protect the ischemic myocardium in patients.

An essential role of the JAK-STAT pathway in ischemic preconditioning

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.

Stromal Cell–Derived Factor-1α Confers Protection Against Myocardial Ischemia/Reperfusion Injury Role of the Cardiac Stromal Cell–Derived Factor-1α–CXCR4 Axis

Background— Stromal cell–derived factor-1&agr; (SDF-1&agr;) 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-1&agr;–CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown. Methods and Results— In mice subjected to ischemic preconditioning, myocardial SDF-1&agr; mRNA was found to be increased 3 hours later (P<0.05). Myocardial SDF-1&agr; and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1&agr; 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-1&agr; resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1&agr; 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-1&agr; 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-1&agr; administration also was blocked by AMD3100. Conclusions— We conclude that SDF-1&agr; 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.

Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning

More than 10 years after its discovery, the function of cyclooxygenase-2 (COX-2) in the cardiovascular system remains largely an enigma. Many scholars have assumed that the allegedly detrimental effects of COX-2 in other systems (e.g. proinflammatory actions and tumorigenesis) signify a detrimental role of this protein in cardiovascular homeostasis as well. This view, however, is ill-founded. Recent studies have demonstrated that ischemic preconditioning (PC) upregulates the expression and activity of COX-2 in the heart, and that this increase in COX-2 activity mediates the protective effects of the late phase of PC against both myocardial stunning and myocardial infarction. An obligatory role of COX-2 has been observed in the setting of late PC induced not only by ischemia but also by delta-opioid agonists and physical exercise, supporting the view that the recruitment of this protein is a central mechanism whereby the heart protects itself from ischemia. The beneficial actions of COX-2 appear to be mediated by the synthesis of PGE(2) and/or PGI(2). Since inhibition of iNOS in preconditioned myocardium blocks COX-2 activity whereas inhibition of COX-2 does not affect iNOS activity, COX-2 appears to be downstream of iNOS in the protective pathway of late PC. The results of these studies challenge the widely accepted paradigm that views COX-2 activity as detrimental. The discovery that COX-2 plays an indispensable role in the anti-stunning and anti-infarct effects of late PC demonstrates that the recruitment of this protein is a fundamental mechanism whereby the heart adapts to stress, thereby revealing a novel, hitherto unappreciated cardioprotective function of COX-2. From a practical standpoint, the recognition that COX-2 is an obligatory co-mediator (together with iNOS) of the protection afforded by late PC has implications for the clinical use of COX-2 selective inhibitors as well as nonselective COX inhibitors. For example, the possibility that inhibition of COX-2 activity may augment myocardial cell death by obliterating the innate defensive response of the heart against ischemia/reperfusion injury needs to be considered and is the object of much current debate. Furthermore, the concept that the COX-2 byproducts, PGE(2) and/or PGI(2), play a necessary role in late PC provides a basis for novel therapeutic strategies designed to enhance the biosynthesis of these cytoprotective prostanoids in the ischemic myocardium. From a conceptual standpoint, the COX-2 hypothesis of late PC expands our understanding of the function of this enzyme in the cardiovascular system and impels a critical reassessment of current thinking regarding the biologic significance of COX-2.

Nitric Oxide Donors Induce Late Preconditioning Against Myocardial Stunning and Infarction in Conscious Rabbits via an Antioxidant-Sensitive Mechanism

The goal of this study was to test the hypothesis that the cardioprotective effects of the late phase of ischemic preconditioning (PC) can be mimicked by treatment with NO donors. In phase I (studies of myocardial stunning), conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In group I (controls, n=6), the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 54% on days 2 and 3 compared with day 1 (P<0.05), indicating a late PC effect against myocardial stunning. When rabbits were given the NO donors diethylenetriamine/NO (DETA/NO, 0.1 mg/kg i.v., 4 times [group II, n=5]) or S-nitroso-N-acetylpenicillamine (SNAP, 2.5 microg x kg(-1) x min(-1) i.v. for 75 minutes [group III, n=51) 24 hours before the first sequence of occlusion/reperfusion cycles, the deficit of WTh on day 1 was 60% (group II) and 54% (group III) less than that observed in controls (P<0.05 for both). In both groups II and III, there was no further improvement in the deficit of WTh on days 2 and 3 compared with day 1. The protective effect of DETA/NO was completely abrogated when this agent was given in conjunction with the ONOO- and .OH scavenger mercaptopropionyl glycine (MPG) (group IV, n=5). In phase II (studies of myocardial infarction), conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. When rabbits were preconditioned 24 hours earlier with six 4-minute occlusion/4-minute reperfusion cycles, infarct size was reduced by 43% (33.2+/-2.7% versus 58.3+/-4.1% of the region at risk in controls, P<0.05), indicating a late PC effect against myocardial infarction. When rabbits were pretreated with DETA/NO (group VII, n=8) or SNAP (group IX, n=7) 24 hours before the 30-minute occlusion, infarct size was reduced by a similar degree (29.3+/-3.6% and 32.0+/-3.3% of the region at risk, respectively; P<0.05 versus controls). The degree of protection could not be increased by doubling the dose of DETA/NO (group VIII, n=5). Coadministration of MPG completely abrogated the infarct-sparing action of DETA/NO (group X, n=7). Taken together, these results demonstrate that in conscious rabbits the administration of 2 structurally unrelated NO donors induces protection 24 hours later against both reversible (stunning) and irreversible (infarction) ischemia/reperfusion injury and that the magnitude of this protection is indistinguishable from that observed during the late phase of ischemic PC. The fact that the late phase of ischemic PC can be mimicked by NO donors provides direct evidence that NO in itself is sufficient to elicit this cardioprotective mechanism. The fact that NO donor-induced late PC was abrogated by MPG indicates that the mechanism whereby NO induces this phenomenon involves the generation of oxidant species, possibly ONOO- and/or .OH. Since a relatively brief treatment with hemodynamically inactive doses of NO donors can induce long-lasting protective effects, these agents could be useful for preconditioning the heart in patients.

Demonstration of an early and a late phase of ischemic preconditioning in mice

It is unknown whether ischemic preconditioning (PC; either early or late) occurs in the mouse. The goal of this study was to answer this question and to develop a reliable and physiologically relevant murine model of both early and late ischemic PC. A total of 201 mice were used. In nonpreconditioned open-chest animals subjected to 30 min of coronary occlusion followed by 24 h of reperfusion, infarct size (tetrazolium staining) averaged 52% of the region at risk. When the 30-min occlusion was performed 10 min after a PC protocol consisting of six cycles of 4-min occlusion and 4-min reperfusion, infarct size was reduced by 75%, indicating an early PC effect. When the 30-min occlusion was performed 24 h after the same PC protocol, infarct size was reduced by 48%, indicating a late PC effect. In mice in which the 30-min occlusion was followed by 4 h of reperfusion, infarct size was similar to that observed after 24 h of reperfusion, indicating that a 4-h reperfusion interval is sufficient to detect the final extent of cell death in this model. Fundamental physiological variables (body temperature, arterial oxygenation, acid-base balance, heart rate, and arterial pressure) were measured and found to be within normal limits. Taken together, these results demonstrate that, in the mouse, a robust infarct-sparing effect occurs during both the early and the late phases of ischemic PC, although the early phase is more powerful. This murine model is physiologically relevant, provides reliable measurements, and should be useful for elucidating the cellular mechanisms of ischemic PC in genetically engineered animals.It is unknown whether ischemic preconditioning (PC; either early or late) occurs in the mouse. The goal of this study was to answer this question and to develop a reliable and physiologically relevant murine model of both early and late ischemic PC. A total of 201 mice were used. In nonpreconditioned open-chest animals subjected to 30 min of coronary occlusion followed by 24 h of reperfusion, infarct size (tetrazolium staining) averaged 52% of the region at risk. When the 30-min occlusion was performed 10 min after a PC protocol consisting of six cycles of 4-min occlusion and 4-min reperfusion, infarct size was reduced by 75%, indicating an early PC effect. When the 30-min occlusion was performed 24 h after the same PC protocol, infarct size was reduced by 48%, indicating a late PC effect. In mice in which the 30-min occlusion was followed by 4 h of reperfusion, infarct size was similar to that observed after 24 h of reperfusion, indicating that a 4-h reperfusion interval is sufficient to detect the final extent of cell death in this model. Fundamental physiological variables (body temperature, arterial oxygenation, acid-base balance, heart rate, and arterial pressure) were measured and found to be within normal limits. Taken together, these results demonstrate that, in the mouse, a robust infarct-sparing effect occurs during both the early and the late phases of ischemic PC, although the early phase is more powerful. This murine model is physiologically relevant, provides reliable measurements, and should be useful for elucidating the cellular mechanisms of ischemic PC in genetically engineered animals.