Mikhail A. Kolpakov

Increasing Cardiac Contractility After Myocardial Infarction Exacerbates Cardiac Injury and Pump Dysfunction

Rationale: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. Objectives: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca2+-handling defects could avert post-MI cardiac pump dysfunction. Methods and Results: MI was produced in mice with inducible, cardiac-specific expression of the &bgr;2a subunit of the L-type Ca2+ channel. Myocyte and cardiac function were compared in control and &bgr;2a animals before and after MI. &bgr;2a myocytes had increased Ca2+ current; sarcoplasmic reticulum Ca2+ load, contraction and Ca2+ transients (versus controls), and &bgr;2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in &bgr;2a versus control hearts after MI. &bgr;2a animals also had poorer survival after MI. Myocytes isolated from &bgr;2a hearts after MI did not develop depressed Ca2+ handling, and Ca2+ current, contractions, and Ca2+ transients were still above control levels (before MI). Conclusions: Maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

Sympathetic Activation Causes Focal Adhesion Signaling Alteration in Early Compensated Volume Overload Attributable to Isolated Mitral Regurgitation in the Dog

We reported that left ventricular (LV) dilatation after 4 weeks of isolated mitral regurgitation (MR) in the dogs is marked by extracellular matrix loss and an increase in adrenergic drive. Given that extracellular matrix proteins and their receptor integrins influence β-adrenergic receptor (β-AR) responses in vitro, we tested whether β1-AR activation modulates focal adhesion (FA) signaling and LV remodeling in these same dogs with isolated MR. Normal dogs were compared with dogs with MR of a 4-week duration and with MR dogs treated with β1-AR blockade (β1-RB) (extended-release metoprolol succinate, 100 mg QD) that was started 24 hours after MR induction. In MR LVs, a decrease in collagen accumulation compared with normal dogs was associated with a decrease in FA kinase tyrosine phosphorylation, along with FA kinase interaction with adapter and cytoskeletal proteins, p130Cas and paxillin, respectively, as determined by immunoprecipitation assays. There was increased phosphorylation of stress related molecules p38 mitogen-activated protein kinase (MAPK) and Hsp27 and survival signaling kinases extracellular signal-regulated kinase 1/2 and AKT, with no evidence of cardiomyocyte apoptosis. β1-RB attenuated FA signaling loss and prevented p38 MAPK, Hsp27, and AKT phosphorylation induced by MR and significantly increased LV epicardial collagen content. However, β1-RB did not improve LV endocardial collagen loss or LV dilatation induced by MR. Isolated myocytes from normal and MR dog hearts treated with β1- or β2-AR agonists demonstrated no difference in FA kinase, p38 MAPK, Hsp27, or AKT phosphorylation. These results showed that chronic stimulation of β1-AR during early compensated MR impairs FA signaling that may affect myocyte/fibroblast–extracellular matrix scaffolding necessary for LV remodeling.

Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130Cas and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

c-Cbl Ubiquitin Ligase Regulates Focal Adhesion Protein Turnover and Myofibril Degeneration Induced by Neutrophil Protease Cathepsin G

Background: The neutrophil protease cathepsin G induces myocyte anoikis. Results: Cathepsin G promotes c-Cbl activation and interaction with focal adhesion proteins that leads to focal adhesion and myofibril protein degradation and myocyte anoikis. Conclusion: c-Cbl is a key ligase required during cathepsin G-induced focal adhesion and myofibrillar protein degradation. Significance: This is a novel mechanism to regulate focal adhesion and myofibril stability and turnover. The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.

Intracoronary Cytoprotective Gene Therapy: A Study of VEGF-B167 in a Pre-Clinical Animal Model of Dilated Cardiomyopathy

BACKGROUND Vascular endothelial growth factor (VEGF)-B activates cytoprotective/antiapoptotic and minimally angiogenic mechanisms via VEGF receptors. Therefore, VEGF-B might be an ideal candidate for the treatment of dilated cardiomyopathy, which displays modest microvascular rarefaction and increased rate of apoptosis. OBJECTIVES This study evaluated VEGF-B gene therapy in a canine model of tachypacing-induced dilated cardiomyopathy. METHODS Chronically instrumented dogs underwent cardiac tachypacing for 28 days. Adeno-associated virus serotype 9 viral vectors carrying VEGF-B167 genes were infused intracoronarily at the beginning of the pacing protocol or during compensated heart failure. Moreover, we tested a novel VEGF-B167 transgene controlled by the atrial natriuretic factor promoter. RESULTS Compared with control subjects, VEGF-B167 markedly preserved diastolic and contractile function and attenuated ventricular chamber remodeling, halting the progression from compensated to decompensated heart failure. Atrial natriuretic factor-VEGF-B167 expression was low in normally functioning hearts and stimulated by cardiac pacing; it thus functioned as an ideal therapeutic transgene, active only under pathological conditions. CONCLUSIONS Our results, obtained with a standard technique of interventional cardiology in a clinically relevant animal model, support VEGF-B167 gene transfer as an affordable and effective new therapy for nonischemic heart failure.

c-Cbl Inhibition Improves Cardiac Function and Survival in Response to Myocardial Ischemia

Background— The proto-oncogene Casitas b-lineage lymphoma (c-Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and nonreceptor tyrosine kinases, resulting in their ubiquitination and downregulation. However, the function of c-Cbl in the control of cardiac function is currently unknown. In this study, we examined the role of c-Cbl in myocyte death and cardiac function after myocardial ischemia. Methods and Results— We show increased c-Cbl expression in human ischemic and dilated cardiomyopathy hearts and in response to pathological stress stimuli in mice. c-Cbl–deficient mice demonstrated a more robust functional recovery after myocardial ischemia/reperfusion injury and significantly reduced myocyte apoptosis and improved cardiac function. Ubiquitination and downregulation of key survival c-Cbl targets, epidermal growth factor receptors and focal adhesion kinase, were significantly reduced in c-Cbl knockout mice. Inhibition of c-Cbl expression or its ubiquitin ligase activity in cardiac myocytes offered protection against H2O2 stress. Interestingly, c-Cbl deletion reduced the risk of death and increased cardiac functional recovery after chronic myocardial ischemia. This beneficial effect of c-Cbl deletion was associated with enhanced neoangiogenesis and increased expression of vascular endothelial growth factor-a and vascular endothelial growth factor receptor type 2 in the infarcted region. Conclusions— c-Cbl activation promotes myocyte apoptosis, inhibits angiogenesis, and causes adverse cardiac remodeling after myocardial infarction. These findings point to c-Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling after myocardial ischemia.

Beta1-adrenergic receptors promote focal adhesion signaling downregulation and myocyte apoptosis in acute volume overload

Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (β(1)-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk(2) and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. β(1)-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with β(1)-AR agonists or adenoviral expression of β(1)-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by β(1)-ARs. Collectively, these data show that β(1)-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.

Pleiotropic effects of neutrophils on myocyte apoptosis and left ventricular remodeling during early volume overload

Most of the available evidence on the role of neutrophils on pathological cardiac remodeling has been pertained after acute myocardial infarction. However, whether neutrophils directly contribute to the pathogenesis of cardiac remodeling after events other than acute myocardial infarction remains unknown. Here we show that acute eccentric hypertrophy induced by aorto-caval fistula (ACF) in the rats induced an increase in the inflammatory response characterized by activation of the STAT pathway and increased infiltration of neutrophils in the myocardium. This early inflammation was associated with a decrease in interstitial collagen accumulation and an increase in myocyte apoptosis. Neutrophil infiltration blockade attenuated MMP activation, ECM degradation, and myocyte apoptosis induced by ACF at 24 hours and attenuated the development of eccentric hypertrophy induced by ACF at 2 and 3 weeks, suggesting a causal relationship between neutrophils and the ACF-induced cardiac remodeling. In contrast, sustained neutrophil depletion over 4 weeks resulted in adverse cardiac remodeling with further increase in cardiac dilatation and macrophage infiltration, but with no change in myocyte apoptosis level. These data support a functional role for neutrophils in MMP activation, ECM degradation, and myocyte apoptosis during eccentric cardiac hypertrophy and underscore the adverse effects of chronic anti-neutrophil therapy on cardiac remodeling induced by early volume overload.

Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury.

Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.