Pressure-overload Heart Failure Research Articles

Binding Affinity of Calmodulin to Cardiac Ryanodine Receptor, as the Novel Therapeutic Target in the Pressure-overloaded Heart Failure

Binding Affinity of Calmodulin to Cardiac Ryanodine Receptor, as the Novel Therapeutic Target in the Pressure-overloaded Heart Failure

Abstract 60: The Role of Mononuclear Phagocytes in Pressure Overload Heart Failure

We previously reported that chronic ischemic HF induced by coronary ligation results in an exacerbated pro-inflammatory cell profile in the systemic circulation, spleen, and failing heart. In addition, this pro-inflammatory cell profile was strongly influenced by the cardiosplenic axis; however, the role of this axis in other etiologies of HF is unknown. Immune cells infiltrate the heart early (1-4 weeks) after pressure-overload injury, but it is unknown whether these cells persist and are of pathological import during chronic pressure-overload HF. We hypothesized that phenotypic changes in mononuclear phagocytes are necessary for the transition from compensatory hypertrophy to chronic HF during sustained pressure-overload, and that inhibition of myocardial immune cell infiltration would ameliorate this progression. Pressure-overload was induced by transverse aortic constriction (TAC) in wild-type (WT) C57BL/6 mice, and in macrophage Fas-induced apoptosis (MAFIA) Tg mice, which allows ablation of c-fms expressing cells upon administration of the AP20187 dimerizer. Compared to sham mice, TAC mice developed progressive cardiac dysfunction beginning at 2 w with significantly (p<0.05) reduced LVEF (59 ± 10 vs 67 ± 3 %), increased left atrial size (2.29 ± 0.1 vs 2.04 ± 0.1 mm), and increased E/E’ Doppler ratio (31.8 ± 6 vs 24.0 ± 3). Circulating CD11b+Lineage- monocytes were elevated in TAC mice at 2 w (1.7 ± 0.2 vs 1.0 ± 0.1 %, p<0.05); however, these levels normalized by 8 w. Moreover, at 8 w, no significant differences in the levels of splenic and cardiac mononuclear phagocytes were seen between TAC and sham mice. Systemic ablation of mononuclear phagocytes beginning 2 w after TAC in MAFIA AP20187-treated mice did not rescue cardiac function or delay HF progression compared to TAC MAFIA vehicle-treated mice over the course of 16 w (LVEF 43 ± 18 vs 45 ± 18 %). We conclude that in contrast to chronic ischemic HF, inflammatory cell-mediated cardiac injury does not have primacy in chronic non-ischemic HF induced by pressure-overload. These findings highlight the importance of the index injury in regard to immune cell activation in HF, and suggest that therapeutic immunomodulatory approaches may not be equivalent across the various etiologies of this disease.

S2-1 Hydrogen sulfide in heart failure

Background Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro model systems and to promote vessel growth in the setting of chronic ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donors, SG-1002 and diallyl trisulfide (DATS), in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis. Studies were also performed to determine the mechanisms by which H2S protects the heart during heart failure. Methods and results Transverse aortic constriction was performed in mice (C57BL/6J; 8–10 weeks of age). Mice received either vehicle or diallyl trisulfide (200 μg/kg) or SG-1002 (20 mg/kg) starting 24 h after transverse aortic constriction and were followed for up to 12 weeks using echocardiography. H2S therapy with H2S donors significantly improved left ventricular remodeling and preserved left ventricular function in the setting of transverse aortic constriction. H2S therapy increased the expression of the proangiogenic factor, vascular endothelial cell growth factor, and decreased the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial NO synthase and the bioavailability of NO. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts. In additional studies we determined that H2S therapy resulted in the activation of endothelial nitric oxide synthase (eNOS) via phosphorylation of serine1177 and increased nitric oxide (NO) bioavailability. Interestingly, we determined that eNOS was uncoupled in CSE KO mice with reduced levels of H2S and NO coupled with an exacerbated injury response to acute myocardial infarction. Conclusions These results suggest that H2S therapy attenuates left ventricular remodeling and dysfunction in the setting of heart failure by creating a proangiogenic environment for the growth of new vessels. A major mechanism related to these cytoprotective actions is the activation of eNOS and increased nitric oxide bioavailability.

Oxidative post-translational modifications develop LONP1 dysfunction in pressure overload heart failure.

Mitochondrial compromise is a fundamental contributor to heart failure. Recent studies have revealed that several surveillance systems maintain mitochondrial integrity. The present study evaluated the role of mitochondrial AAA+ protease in a mouse model of pressure overload heart failure. The fluorescein isothiocyanate casein assay and immunoblotting for endogenous mitochondrial proteins revealed a marked reduction in ATP-dependent proteolytic activity in failing heart mitochondria. The level of reduced cysteine was decreased, and tyrosine nitration and protein carbonylation were promoted in Lon protease homolog (LONP1), the most abundant mitochondrial AAA+ protease, in heart failure. Comprehensive analysis revealed that electron transport chain protein levels were increased even with a reduction in the expression of their corresponding mRNAs in heart failure, which indicated decreased protein turnover and resulted in the accumulation of oxidative damage in the electron transport chain. The induction of mitochondria-targeted human catalase ameliorated proteolytic activity and protein homeostasis in the electron transport chain, leading to improvements in mitochondrial energetics and cardiac contractility even during the late stage of pressure overload. Moreover, the infusion of mitoTEMPO, a mitochondria-targeted superoxide dismutase mimetic, recovered oxidative modifications of LONP1 and improved mitochondrial respiration capacity and cardiac function. The in vivo small interfering RNA repression of LONP1 partially canceled the protective effects of mitochondria-targeted human catalase induction and mitoTEMPO infusion. Oxidative post-translational modifications attenuate mitochondrial AAA+ protease activity, which is involved in impaired electron transport chain protein homeostasis, mitochondrial respiration deficiency, and left ventricular contractile dysfunction. Oxidatively inactivated proteases may be an endogenous target for mitoTEMPO treatment in pressure overload heart failure.

Novel Protective Role of Endogenous Cardiac Myocyte P2X4 Receptors in Heart Failure

Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation-induced postinfarct or transverse aorta constriction-induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N(5)-(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection.

Activating Transcription Factor 3 Protects against Pressure-Overload Heart Failure via the Autophagy Molecule Beclin-1 Pathway

Activating transcription factor 3 (ATF3), a cAMP response element-binding protein/ATF family transcription factors member, has been implicated in the cardiovascular and inflammatory system and is rapidly induced by ischemic-reperfusion injuries. We performed transverse aortic banding (TAB) experiments using ATF3 gene-deleted mice (ATF3(-/-)) and wild-type (WT) mice to determine what effect it might have on heart failure induced by pressure overloading. Compared with the WT mice, ATF3(-/-) mice were found by echocardiography to have decreased left ventricular contractility with loss of normal cardiac hypertrophic remodeling. The ATF3(-/-) mice had greater numbers of terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling-positive cells and higher levels of activated caspase-3, as well as more apoptosis. Restoration of ATF3 expression in the heart of ATF3(-/-) mice by adenovirus-induced ATF3 treatment significantly improved cardiac contractility after TAB. The results from molecular and biochemical analyses, including chromatin immune-precipitation and in vitro /in vivo promoter assays, showed that ATF3 bound to the ATF/cAMP response element of the Beclin-1 promoter and that ATF3 reduced autophagy via suppression of the Beclin-1-dependent pathway. Furthermore, infusion of tert-butylhydroquinone (tBHQ), a selective ATF3 inducer, increased the expression of ATF3 via the nuclear factor erythroid 2-related transcriptional factor, inhibited TAB-induced cardiac dilatation, and increased left ventricular contractility, thereby rescuing heart failure. Our study identified a new epigenetic regulation mediated by the stress-inducible gene ATF3 on TAB-induced cardiac dysfunction. These findings suggest that the ATF3 activator tBHQ may have therapeutic potential for the treatment of pressure-overload heart failure induced by chronic hypertension or other pressure overload mechanisms.

The calcium release-activated calcium channel Orai1 represents a crucial component in hypertrophic compensation and the development of dilated cardiomyopathy

As exceptionally calcium selective store-operated channels, Orai channels play a prominent role in cellular calcium signaling. While most studied in the immune system, we are beginning to recognize that Orai1 provides unique calcium signaling pathways in numerous tissue contexts. To assess the involvement of Orai1 in cardiac hypertrophy we used transverse aortic constriction to model pressure overload cardiac hypertrophy and heart failure in Orai1 deficient mice. We demonstrate that Orai1 deficient mice have significantly decreased survival in this pressure overload model. Transthoracic echocardiography reveals that Orai1 deficient mice develop rapid dilated cardiomyopathy, with greater loss of function, and histological and molecular data indicate that this pathology is associated with significant apoptosis, but not major differences in cellular hypertrophy, fibrosis, and some major hypertrophic makers. Orai1 represents a crucial calcium entry mechanism in the compensation of the heart to pressure overload over-load, and the development of dilated cardiomyopathy.

Dissociation of Calmodulin from RyR2 Plays a Key Role in Diastolic Ca2+ Leak from Sarcoplasmic Reticulum in Pressure-overload Heart Failure

Calmodulin(CaM) plays a key role in modulating channel gating in ryanodine receptor(RyR2). Here, we investigated the pathogenic role of CaM in diastolic Ca2+ leak from sarcoplasmic reticulum in pressure-overload Heart Failure induced by transverse aortic constriction(TAC).

Abstract 100: Mechanisms That Mediate the Cardioprotective Activities of Resveratrol in Pressure Overload-Induced Heart Failure

We previously demonstrated that the potent antioxidant, resveratrol, significantly attenuates the adverse cardiac remodeling and dysfunction in pressure overload heart failure induced by transverse aortic constriction (TAC) in C57/BL6 mice. The purpose of this study was to identify the mechanisms that mediate the cardioprotective activities of resveratrol in this setting. Male C57BL6 mice (26-28 g) were subjected to either sham or TAC surgery. One group of TAC mice was given daily resveratrol treatment (oral gavage, 100 mg/kg/body weight for 28 days starting on day 2 after surgery. At the end of the protocol (28 days), the hearts were removed for analysis of markers of oxidative stress, inflammation, and apoptosis. Immunohistological staining for left ventricular (LV) 4-hydroxy-2-nonenal, a marker of lipid peroxidation was significantly elevated in the TAC mice (18.4±3.0 IOD/μm 2 ) compared to sham mice (3.4±1.0). Resveratrol treatment significantly attenuated this increase (14.4±2.0). In contrast, LV content of the antioxidant glutathione was significantly decreased in the TAC mice (5.3±0.2 nM/mg protein) compared to the sham mice (8.5±0.2). Resveratrol treatment significantly increased glutathione (7.6±0.3) to near sham levels. Likewise, LV activity of sodium oxide dismutase 2 was markedly reduced in TAC mice (7.3±0.9 U/mg protein) compared to sham mice (10.7±1.3) and was significantly increased by resveratrol treatment (7.6±0.3). LV macrophage and mast cell infiltration was markedly increased in the TAC mice (14.8±0.1 and 12.6±0.0 %) compared to sham mice (1.7±0.1 and 2.6±0.1). This increase was significantly attenuated by resveratrol administration (10.4±0.1 and 8.0±0.1). Apoptotic cell death, as determined by the TUNEL assay, was also increased in the TAC mice (0.13±0.01 % cells) compared to sham mice (0.01±0.01). This increase was significantly attenuated in the resveratrol treated group (0.06±0.01). A similar pattern was observed when activated LV caspase-3, a second marker of apoptosis, was measured. In conclusion, these data indicate that the cardioprotective effects of resveratrol in pressure overload-induced heart failure are mediated through the inhibition of oxidative stress, inflammation, and apoptotic cell death.

Hypoxia-Inducible Factor 1 and Cardiovascular Disease

Cardiac function is required for blood circulation and systemic oxygen delivery. However, the heart has intrinsic oxygen demands that must be met to maintain effective contractility. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that functions as a master regulator of oxygen homeostasis in all metazoan species. HIF-1 controls oxygen delivery, by regulating angiogenesis and vascular remodeling, and oxygen utilization, by regulating glucose metabolism and redox homeostasis. Analysis of animal models suggests that by activation of these homeostatic mechanisms, HIF-1 plays a critical protective role in the pathophysiology of ischemic heart disease and pressure-overload heart failure.

Oxygen Sensing, Hypoxia-Inducible Factors, and Disease Pathophysiology

Hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis, which is disrupted in disorders affecting the circulatory system and in cancer. The role of HIFs in these diseases has been elucidated by clinical studies and by analyses of mouse models. HIFs play a protective role in the pathophysiology of myocardial ischemia due to coronary artery disease, limb ischemia due to peripheral arterial disease, pressure-overload heart failure, wound healing, and chronic rejection of organ transplants. In contrast, HIFs contribute to the pathogenesis of pulmonary arterial hypertension, systemic hypertension associated with sleep apnea, ocular neovascularization, hereditary erythrocytosis, and cancer.

GW24-e0049 Electrical remodelling contributes to abnormal impulse induction and propagation in heart failure

ObjectivesThe increased abnormal activity in heart failure is one of the major mechanisms of arrhythmogenesis which contributes directly to the sudden cardiac death. The current study is to explore the...

Abstract 299: Simultaneous Cardiac And Adrenal Small Molecule GPCR-Gβ? Inhibition Halts The Progression Of Pressure Overload Heart Failure

Heart failure (HF) is a progressive disease with rapidly increasing rates of morbidity and mortality. Elevated sympathetic nervous system activity, a salient feature of HF progression, leads to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) due in part to Gβγ-mediated signaling. In the current study, we assessed the hypothesis that the small molecule Gβγ inhibitor “gallein” is salutary in treating pre-existing HF in a clinically relevant model (pressure-overload HF model of mouse transverse aortic constriction (TAC)) by simultaneously normalizing adrenergic receptor signaling in the heart and the adrenal gland. Four weeks post-TAC, mice received daily i.p. injections of vehicle or gallein for eight weeks (n=6-8 per group). Serial echocardiography was performed through out the study. At the end of the experiment, hemodynamic studies were performed, mice were sacrificed, blood, heart, and adrenal glands were harvested for further analysis. Gallein treatment improved survival and cardiac function and reduced cardiac hypertrophy, remodeling, and fetal genes expression in TAC mice. On the molecular level, gallein recovered membrane β-AR density and attenuated GRK2-PI3Kγ membrane recruitment, and Akt-GSK-3β signaling in TAC hearts. A salutary adrenal effect of gallein was obtained in cultured mice adrenal glands and human pheochromocytoma tissue (n=3), where direct gallein treatment restored α2-AR feedback inhibitory function and concurrently reduced catecholamine production. Moreover, gallein treatment attenuated adrenal hypertrophy in TAC mice and downregulated tyrosine hydroxylase and chromogranin A protein expression in adrenal glands from TAC mice and cultured pheochromocytoma tissue as well. In summary, our data suggest gallein as a systemic pharmacologic therapy with substantial therapeutic benefit in HF by simultaneously normalizing pathologic Gβγ-GRK2 signaling and recovering AR signaling in both the heart and the adrenal gland. In the heart, gallein mediated attenuation of cardiac remodeling probably involves inhibiting GRK2-PI3K-Akt signaling. Our data also suggest a role for small molecule Gβγ inhibition in other diseases of elevated catecholamine release, such as pheochromocytoma.

Alpha-calcitonin gene-related peptide is protective against pressure overload-induced heart failure

The sensory neuropeptide, α-calcitonin gene-related peptide (α-CGRP) is protective against hypertension-induced heart damage and cardiac ischemia/reperfusion injury. To determine whether this neuropeptide is also cardioprotective in heart failure, this study examined whether the absence of α-CGRP exacerbated the adverse cardiac remodeling, dysfunction and mortality in pressure overload heart failure induced by transverse aortic constriction (TAC). Male α-CGRP knockout (KO) and wild type (WT) mice had TAC or sham surgery at day 0 and were studied on days 3, 14, 21, and 28. The survival rate of TAC α-CGRP KO mice was lower than the TAC WT mice over the duration of the protocol. Left ventricular α-CGRP content in TAC WT mice was higher at days 3, 14, and 21 than sham WT mice. Echocardiography demonstrated greater adverse cardiac remodeling and dysfunction in the TAC α-CGRP KO compared to the TAC WT mice. The lung/body weight ratios and left ventricular masses were higher in TAC α-CGRP KO compared to the TAC WT mice. While there was increased cardiac fibrosis in the TAC WT mice compared to shams, the TAC α-CGRP KO mice had markedly increased fibrosis above that of the TAC WT mice. TAC WT mice had greater cardiac inflammation, cell death, and adaptive angiogenesis compared to sham mice. Importantly, the TAC α-CGRP KO mice had greater inflammation, cell death, and attenuation of angiogenesis compared to TAC WT hearts. Thus, α-CGRP plays a significant protective role in TAC-induced heart failure which may be mediated by decreased inflammation, cell death, and fibrosis.

Hydrogen Sulfide Attenuates Cardiac Dysfunction After Heart Failure Via Induction of Angiogenesis

Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro models and to promote vessel growth in the setting of hindlimb ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donor, diallyl trisulfide, in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis. Transverse aortic constriction was performed in mice (C57BL/6J; 8-10 weeks of age). Mice received either vehicle or diallyl trisulfide (200 µg/kg) starting 24 hours after transverse aortic constriction and were followed up for 12 weeks using echocardiography. H2S therapy with diallyl trisulfide improved left ventricular remodeling and preserved left ventricular function in the setting of transverse aortic constriction. H2S therapy increased the expression of the proangiogenic factor, vascular endothelial cell growth factor, and decreased the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial NO synthase and the bioavailability of NO. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts. These results suggest that H2S therapy attenuates left ventricular remodeling and dysfunction in the setting of heart failure by creating a proangiogenic environment for the growth of new vessels.

Potential Role of BNIP3 in Cardiac Remodeling, Myocardial Stiffness, and Endoplasmic Reticulum

Background— We have shown that BNIP3 expression is significantly increased in heart failure (HF). In this study, we tested the effects of BNIP3 manipulation in HF. Methods and Results— In a rat model of pressure overload HF, BNIP3 knockdown significantly decreased left ventricular (LV) volumes with significant improvement in LV diastolic and systolic function. There were significant decreases in myocardial apoptosis and LV interstitial fibrosis. Ultrastructurally, BNIP3 knockdown attenuated mitochondrial fragmentation and restored mitochondrial morphology and integrity. On the molecular level, there were significant decreases in endoplasmic reticulum (ER) stress and mitochondrial apoptotic markers. One of the mechanisms by which BNIP3 mediates mitochondrial dysfunction is via the oligomerization of the voltage-dependent anion channels causing a shift of calcium from the ER to mitochondrial compartments, leading to the decrease in ER calcium content, mitochondrial damage, apoptosis, and LV interstitial fibrosis, and hence contributes to both systolic and diastolic myocardial dysfunction, respectively. In systolic HF, the downregulation of SERCA2a (sarcoplasmic-endoplasmic reticulum calcium ATPase), along with an increased BNIP3 expression, further worsen myocardial diastolic and systolic function and contribute to the major remodeling seen in systolic HF as compared with diastolic HF with normal SERCA2a expression. Conclusions— The increase in BNIP3 expression contributes mainly to myocardial diastolic dysfunction through mitochondrial apoptosis, LV interstitial fibrosis, and to some extent to myocardial systolic dysfunction attributable to the shift of calcium from the ER to the mitochondria and to the decrease in ER calcium content. However, SERCA2a downregulation remains a prerequisite for the major LV remodeling seen in systolic HF.

Protective Effect of Qiliqiangxin Capsule on Energy Metabolism and Myocardial Mitochondria in Pressure Overload Heart Failure Rats

Qiliqiangxin capsule (QL) was developed under the guidance of TCM theory of collateral disease and had been shown to be effective and safe for the treatment of heart failure. The present study explored the role of and mechanism by which the herbal compounds QL act on energy metabolism, in vivo, in pressure overload heart failure. SD rats received ascending aorta constriction (TAC) to establish a model of myocardial hypertrophy. The animals were treated orally for a period of six weeks. QL significantly inhibited cardiac hypertrophy due to ascending aortic constriction and improved hemodynamics. This effect was linked to the expression levels of the signaling factors in connection with upregulated energy and the regulation of glucose and lipid substrate metabolism and with a decrease in metabolic intermediate products and the protection of mitochondrial function. It is concluded that QL may regulate the glycolipid substrate metabolism by activating AMPK/PGC-1α axis and reduce the accumulation of free fatty acids and lactic acid, to protect cardiac myocytes and mitochondrial function.

Cellular hypertrophy occurs before interstitial fibrosis in pressure-overload heart failure

Methods 11 mice underwent transverse aortic constriction (TAC). All mice were imaged at baseline, and a group of mice were examined early (2 weeks), intermediate (4 weeks) and late (7 weeks) post-TAC with histologic correlation. Mice were imaged at 9.8 Tesla with standard cine function sequences and T1 quantification via a Look-Locker cine experiment, pre and post gadolinium. The intra-cellular lifetime of water (τic), a-cell size dependent parameter, and the extracellular volume fraction (ECV) were determined by fitting R1 in tissue, against R1 in the blood pool, with a 2-site model for transcytolemmal water exchange. Minor (Dmin) and major (Dmaj) cell-diameters were determined by image analysis of FITC-wheat germ agglutinin stained sections.

Abstract 9711: Cardioprotection by Endogenous Cardiac Myocyte P2x4; Receptors in Pressure Overload and Ischemic Heart Failure

Despite progress in its therapy, heart failure is a leading cause of mortality and morbidity. In defining a biological role of the ligand-gated ion channel P2X4 receptor, we uncovered a protective role of this channel in heart failure. P2X4 gene was flanked by loxP sites bwteen exons 2 and 4. The second loxP site together with the Frt-PGKneo-Frt cassette was inserted in intron 1 in a direction opposite to P2X4 transcription. After targeting the ES cells, PGKneo was removed by breeding chimeric males with ROSA26-Flpe females to generate P2X4 flox/+ F1 pups. To create cardiac myocyte-specific knockout of P2X4 receptors, homozygous P2X4 floxed/floxed mice were bred with Tg mice (Myh6-cre/Esr1*, Jax stock no: 005650) containing a tamoxifen responsive Cre-estrogen receptor chimeric recombinase driven by the α-myosin heavy chain promoter. Knockout of P2X4 receptors (KO) was confirmed by lack of detectable P2X4 with immunoblotting and by lack of contractile response to the P2X agonist 2-methylthioATP. KO animals showed normal basal cardiac function but depressed contractile performance in both left coronary artery ligation and pressure overload models of heart failure by echocardiography and isolated working heart parameters. By contrast, cardiac myocyte-specific overexpression of the P2X4 receptor protected against pressure overload-induced and post-infarct heart failure. P2X4 receptors co-immunoprecipitated with nitric oxide synthase 3 (NOS3 or eNOS). Administration of eNOS inhibitor L-NIO blocked the salutary effect of cardiac P2X4 receptor overexpression in post-infarct heart failure as did eNOS knockout. The present study uncovered a protective role of endogenous cardiac myocyte P2X4 receptors in heart failure and demonstrated an important role of eNOS in mediating this protection.

Abstract 332: Phosphoprotein Abundance Changes in Hypertensive Cardiac Remodeling

There is over-whelming evidence that protein phosphorylations regulate cardiac function and remodeling. A wide variety of protein kinases, e.g., phosphoinositide 3-kinase (PI3K), Akt, GSK-3, TGFβ, and PKA, MAPKs, PKC, Erks, and Jaks, as well as phosphatases, e.g., phosphatase I (PP1) and calcineurin, control cardiomyocyte growth and contractility. In the present work, we used global phosphoprotein profiling to identify phosphorylated proteins associated with pressure overload (PO) cardiac hypertrophy and heart failure. Phosphoproteins from hypertrophic and systolic failing hearts from male hypertensive Dahl salt-sensitive rats; trans-aortic banded (TAC); Spontaneously hypertensive heart failure (SHHF) rats were analyzed. Profiling was performed by 2-dimensional difference in gel electrophoresis (2D-DIGE) on phosphoenriched proteins. A total of 25 common phosphoproteins with differences in abundance in 1) the 3 hypertrophic and /or 2) the 2 systolic failure heart models were identified (CI > 99%) by Matrix assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and Mascot analysis. Among these were 1) myofilament proteins, including alpha-tropomyosin and myosin regulatory light chain 2; cap Z interacting protein (cap ZIP), and tubulin β5; 2) mitochondrial proteins, including pyruvate dehydrogenase α, branch chain ketoacid dehydrogenase E1, and mitochondrial creatine kinase 3) phosphatases, including protein phosphatase 2A and protein phosphatase 1 regulatory subunit and 4) other proteins including proteosome subunits α type 3 and β type 7, , and eukaryotic translation initiation factor 1A (eIF1A). These include previously described and novel phosphoproteins in cardiac hypertrophy and systolic failure. The database of phosphoproteins identified may provide new insights and therapeutic targets for heart failure.