ATM deficiency differentially affects expression of proteins related to fatty acid oxidation and oxidative stress in a sex-specific manner in response to Western-type diet prior to and following myocardial infarction.

AIMS: Published work has shown that ataxia-telangiectasia mutated kinase (ATM) deficiency is associated with cardioprotective effects in Western-type diet (WD)-fed female mice. This study assessed the expression of proteins related to fatty acid oxidation (FAO) and oxidative stress in WD-fed male and female mouse hearts, and investigated if sex-specific cardioprotective effects in WD-fed female ATM-deficient mice are maintained following myocardial infarction (MI). MAIN METHODS: Wild-type (WT) and ATM-deficient (hKO) mice (both sexes) were placed on WD for 14 weeks. Myocardial tissue from a subset of mice was used for western blot analyses, while another subset of WD-fed mice underwent MI. Heart function was analyzed by echocardiography prior to and 1 day post-MI. KEY FINDINGS: CPT1B (mitochondrial FAO enzyme) expression was lower in male hKO-WD, while it was higher in female hKO-WD vs WT-WD. WD-mediated decrease in ACOX1 (peroxisomal FAO enzyme) expression was only observed in male WT-WD. PMP70 (transports fatty acyl-CoA across peroxisomal membrane) expression was lower in male hKO-WD vs WT-WD. Catalase (antioxidant enzyme) expression was higher, while Nox4 (pro-oxidant enzyme) expression was lower in female hKO-WD vs WT-WD. Heart function was better in female hKO-WD vs WT-WD. However, post-MI heart function was not significantly different among all MI groups. Post-MI, CPT1B and catalase expression was higher in male hKO-WD-MI vs WT-WD-MI, while Nox4 expression was higher in female hKO-WD-MI vs WT-WD-MI. SIGNIFICANCE: Increased mitochondrial FAO and decreased oxidative stress contribute towards ATM deficiency-mediated cardioprotective effects in WD-fed female mice which are abolished post-MI with increased Nox4 expression.

Vagus nerve stimulation alleviates cardiac dysfunction and inflammatory markers during heart failure in rats.

Vagus nerve stimulation (VNS) is under clinical investigation as a therapy for heart failure with reduced ejection fraction (HFrEF). This study aimed to investigate its therapeutic effects on three main components of heart failure: cardiac function, cardiac remodeling and central neuroinflammation using a pressure overload (PO) rat model. Male Sprague-Dawley rats were divided into four groups: PO, PO + VNS, PO + VNS sham, and controls. All rats, except controls, underwent a PO surgery to constrict the thoracic aorta (~50 %) to induce HFrEF. Open loop VNS therapy was continuously administered to PO + VNS rats at 20 Hz, 1.0 mA for 60 days. Evaluation of cardiac function and structure via echocardiograms showed decreases in stroke volume and relative ejection fraction and increases in the internal diameter of the left ventricle during systole and diastole in PO rats (p < 0.05). However, these PO-induced adverse changes were alleviated with VNS therapy. Additionally, PO rats exhibited significant increases in myocyte cross sectional areas indicating hypertrophy, along with significant increases in myocardial fibrosis and apoptosis, all of which were reversed by VNS therapy (p < 0.05). Furthermore, VNS mitigated microglial activation in two central autonomic nuclei: the paraventricular nucleus of the hypothalamus and locus coeruleus. These findings demonstrate that when VNS therapy is initiated at an early stage of HFrEF progression (<10 % reduction in relative ejection fraction), the supplementation of vagal activity is effective in restoring multi organ homeostasis in a PO model.

Post-ischemic cardioprotective potential of exogenous ubiquitin in myocardial remodeling late after ischemia/reperfusion injury.

AIMS: Pretreatment with ubiquitin (UB) associates with preservation of heart function 3 days post-ischemia/reperfusion (I/R) injury. This study investigated the cardioprotective potential of exogenous UB late after myocardial I/R injury. To enhance the clinical relevance, UB treatment was started at the time of reperfusion and continued for 28 days post-I/R. MAIN METHODS: Mice underwent ligation of the left anterior descending coronary artery for 45 min. At the time of reperfusion, mice were treated with UB or saline which was continued until 28 days post-I/R. Heart function was measured at 3, 7, 14 and 28 days post-I/R using echocardiography. Biochemical parameters of the heart and serum cytokines/chemokines levels were measured 28 days post-I/R. KEY FINDINGS: I/R decreased heart function and induced LV dilation at all time points post-I/R. However, I/R + UB exhibited improved heart function throughout the observation period, while LV dilation was lower in I/R + UB group at 3, 14 and 28 days post-I/R. I/R-mediated increase in myocardial fibrosis, hypertrophy and apoptosis were significantly lower in I/R + UB vs. I/R. Collagen-1α1 and MMP-2 expression was lower, while MMP-9 and TIMP-2 expression was higher in I/R + UB vs. I/R. MYH-7B (hypertrophy marker) expression was lower in I/R + UB vs. I/R. GSK3ß activation was lower (vs. Sham), while activation of ERK1/2 (vs. I/R) and AKT (vs. Sham) was higher in I/R + UB. Serum levels of IL-6, G-CSF and IL-2 were lower in I/R + UB vs. I/R. SIGNIFICANCE: Post-ischemic UB treatment improves heart function, and associates with decreased myocardial fibrosis, apoptosis, hypertrophy and serum cytokine/chemokine levels.

Deficiency of ataxia-telangiectasia mutated kinase attenuates Western-type diet-induced cardiac dysfunction in female mice.

Chronic consumption of Western-type diet (WD) induces cardiac structural and functional abnormalities. Previously, we have shown that WD consumption in male ATM (ataxia-telangiectasia mutated kinase) deficient mice associates with accelerated body weight (BW) gain, cardiac systolic dysfunction with increased preload, and exacerbation of hypertrophy, apoptosis, and inflammation. This study investigated the role of ATM deficiency in WD-induced changes in functional and biochemical parameters of the heart in female mice. Six-week-old wild-type (WT) and ATM heterozygous knockout (hKO) female mice were placed on WD or NC (normal chow) for 14 weeks. BW gain, fat accumulation, and cardiac functional and biochemical parameters were measured 14 weeks post-WD. WD-induced subcutaneous and total fat contents normalized to body weight were higher in WT-WD versus hKO-WD. Heart function measured using echocardiography revealed decreased percent fractional shortening and ejection fraction, and increased LV end systolic diameter and volume in WT-WD versus WT-NC. These functional parameters remained unchanged in hKO-WD versus hKO-NC. Myocardial fibrosis, myocyte hypertrophy, and apoptosis were higher in WT-WD versus WT-NC. However, apoptosis was significantly lower and hypertrophy was significantly higher in hKO-WD versus WT-WD. MMP-9 and Bax expression, and Akt activation were higher in WT-WD versus WT-NC. PARP-1 (full-length) expression and mTOR activation were lower in WT-WD versus hKO-WD. Thus, ATM deficiency in female mice attenuates fat weight gain, preserves heart function, and associates with decreased cardiac cell apoptosis in response to WD.

Deficiency of ataxia-telangiectasia mutated kinase modulates functional and biochemical parameters of the heart in response to Western-type diet.

Ataxia-telangiectasia mutated (ATM) kinase deficiency exacerbates heart dysfunction late after myocardial infarction. Here, we hypothesized that ATM deficiency modulates Western-type diet (WD)-induced cardiac remodeling with an emphasis on functional and biochemical parameters of the heart. Weight gain was assessed in male wild-type (WT) and ATM heterozygous knockout (hKO) mice on weekly basis, whereas cardiac functional and biochemical parameters were measured 14 wk post-WD. hKO-WD mice exhibited rapid body weight gain at weeks 5, 6, 7, 8, and 10 versus WT-WD. WD decreased percent fractional shortening and ejection fraction, and increased end-systolic volumes and diameters to a similar extent in both genotypes. However, WD decreased stroke volume, cardiac output, peak velocity of early ventricular filling, and aortic ejection time and increased isovolumetric relaxation time (IVRT) and Tei index versus WT-NC (normal chow). Conversely, IVRT, isovolumetric contraction time, and Tei index were lower in hKO-WD versus hKO-NC and WT-WD. Myocyte apoptosis and hypertrophy were higher in hKO-WD versus WT-WD. WD increased fibrosis and expression of collagen-1α1, matrix metalloproteinase (MMP)-2, and MMP-9 in WT. WD enhanced AMPK activation, while decreasing mTOR activation in hKO. Akt and IKK-α/ß activation, and Bax, PARP-1, and Glut-4 expression were higher in WT-WD versus WT-NC, whereas NF-κB activation and Glut-4 expression were lower in hKO-WD versus hKO-NC. Circulating concentrations of IL-12(p70), eotaxin, IFN-γ, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were higher in hKO-WD versus WT-WD. Thus, ATM deficiency accelerates weight gain, induces systolic dysfunction with increased preload, and associates with increased apoptosis, hypertrophy, and inflammation in response to WD.NEW & NOTEWORTHY Ataxia-telangiectasia mutated (ATM) kinase deficiency in humans associates with enhanced susceptibility to ischemic heart disease. Here, we provide evidence that ATM deficiency accelerates body weight gain and associates with increased cardiac preload, hypertrophy, and apoptosis in mice fed with Western-type diet (WD). Further investigations of the role of ATM deficiency in WD-induced alterations in function and biochemical parameters of the heart may provide clinically applicable information on treatment and/or nutritional counseling for patients with ATM deficiency.

Cardioprotective Potential of Exogenous Ubiquitin.

Ischemic heart disease (IHD) accounts for the majority of heart disease-related deaths worldwide. Ubiquitin (UB), found in all eukaryotic cells, is a highly conserved low molecular weight (~8.5 kDa) protein. A well-known intracellular function of UB is to regulate protein turnover via the UB-proteasome system. UB is a normal constituent of plasma, and elevated levels of UB are observed in the serum of patients under a variety of pathological conditions. Recent studies provide evidence for cardioprotective potential of exogenous UB in the remodeling process of the heart in IHD, including effects on cardiac myocyte apoptosis, inflammatory response, and reorganization of the vasculature and extracellular matrix. This review summarizes functions of UB with an emphasis on the role of exogenous UB in myocardial remodeling in IHD.

Exogenous ubiquitin attenuates hypoxia/reoxygenation-induced cardiac myocyte apoptosis via the involvement of CXCR4 and modulation of mitochondrial homeostasis.

Exogenous ubiquitin (UB) plays a protective role in ß-adrenergic receptor-stimulated and ischemia/reperfusion (I/R)-induced myocardial remodeling. Here, we report that UB treatment inhibits hypoxia/reoxygenation (H/R)-induced apoptosis in adult rat ventricular myocytes (ARVMs). The activation of Akt was elevated, whereas the activation of glycogen synthase kinase-3ß was reduced in UB-treated cells post-H/R. The level of oxidative stress was lower, whereas the number of ARVMs with polarized mitochondria was significantly greater in the UB-treated samples. ARVMs express CXCR4 with majority of CXCR4 localized in the membrane fraction. CXCR4 antagonism using AMD3100, and siRNA-mediated knockdown of CXCR4 negated the protective effects of UB. Two mutated UB proteins (unable to bind CXCR4) had no effect on H/R-induced apoptosis, activation of Akt and GSK-3ß, or oxidative stress. UB treatment enhanced mitochondrial biogenesis, and inhibition of mitochondrial fission using mdivi1 inhibited H/R-induced apoptosis. Ex vivo, UB treatment significantly decreased infarct size and improved functional recovery of the heart following global I/R. Activation of caspase-9, a key player of the mitochondrial death pathway, was significantly lower in UB-treated hearts post-I/R. UB, most likely acting via CXCR4, plays a protective role in H/R-induced myocyte apoptosis and myocardial I/R injury via modulation of mitochondrial homeostasis and the mitochondrial death pathway of apoptosis.

Exogenous ubiquitin reduces inflammatory response and preserves myocardial function 3 days post-ischemia-reperfusion injury.

ß-Adrenergic receptor (ß-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases ß-AR-stimulated myocyte apoptosis and myocardial fibrosis. Here, we hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after ischemia-reperfusion (I/R) injury. C57BL/6 mice infused with vehicle or UB (1 µg·g-1·h-1) were subjected to myocardial I/R injury. Functional and biochemical parameters of the heart were examined 3 days post-I/R. Heart weight-to-body weight ratios were similarly increased in I/R and UB + I/R groups. The area at risk and infarct size were significantly lower in UB + I/R versus I/R groups. Measurement of heart function using echocardiography revealed that I/R decreases percent fractional shortening and percent ejection fraction. However, the decrease in fractional shortening and ejection fraction was significantly lower in the UB + I/R group. The UB + I/R group displayed a significant decrease in inflammatory infiltrates, neutrophils, and macrophages versus the I/R group. Neutrophil activity was significantly lower in the UB + I/R group. Analysis of the concentration of a panel of 23 cytokines/chemokines in the serum using a Bio-Plex assay revealed a significantly lower concentration of IL-12 subunit p40 in the UB + I/R versus I/R group. The concentration of monocyte chemotactic protein-1 was lower, whereas the concentration of macrophage inflammatory protein-1α was significantly higher, in the UB+I/R group versus the sham group. Expression of matrix metalloproteinase (MMP)-2 and activity of MMP-9 were higher in the UB + I/R group versus the I/R group. Levels of ubiquitinated proteins and tissue inhibitor of metalloproteinase 2 expression were increased to a similar extent in both I/R groups. Thus, exogenous UB plays a protective role in myocardial remodeling post-I/R with effects on cardiac function, area at risk/infarct size, the inflammatory response, levels of serum cytokines/chemokines, and MMP expression and activity. NEW & NOTEWORTHY Stimulation of ß-adrenergic receptors increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases ß-adrenergic receptor-stimulated myocyte apoptosis and myocardial fibrosis. Here, we provide evidence that exogenous UB decreases the inflammatory response and preserves heart function 3 days after myocardial ischemia-reperfusion injury. Further identification of the molecular events involved in the anti-inflammatory role of exogenous UB may provide therapeutic targets for patients with ischemic heart disease.

Extracellular ubiquitin modulates cardiac fibroblast phenotype and function via its interaction with CXCR4.

AIMS: ß-adrenergic receptor (ß-AR) stimulation increases extracellular levels of ubiquitin (UB), and exogenous UB plays an important role in ß-AR-stimulated myocardial remodeling with effects on heart function, fibrosis and myocyte apoptosis. Cardiac fibroblasts are vital for maintaining the normal function of the heart, and in the structural remodeling of the heart in response to injury. Here we hypothesized that extracellular UB modulates cardiac fibroblast phenotype and function via its interaction with CXC chemokine receptor type 4 (CXCR4). MAIN METHODS: Serum starved adult cardiac fibroblasts were used to identify CXCR4 as a receptor for UB. Fluorescent microscopy, co-immunoprecipitation, western blot, proliferation, migration and collagen contraction assays were performed to investigate the role of UB/CXCR4 axis on cell signaling, and modulation of fibroblast phenotype and function. KEY FINDINGS: Using fluorescent microscopy and co-immunoprecipitation assay, we provide evidence that extracellular UB interacts with CXCR4. CXCR4 antagonist, AMD3100, inhibited interaction of UB with CXCR4. UB activated ERK1/2, not Akt. It enhanced VEGF-A expression, while decreasing ß3 integrins expression. Two mutated UB proteins (V70A and F4A; unable to interact with CXCR4) failed to affect the expression of VEGF-A and ß3 integrins. UB treatment inhibited migration of cells into the wound and FBS-stimulated cell proliferation. UB enhanced expression of α-smooth muscle actin (marker of myofibroblast differentiation) and contraction of fibroblast-populated collagen gel pads. Most of the effects of UB were negated by AMD3100. SIGNIFICANCE: The data presented here suggest that UB interacts with CXCR4, and UB/CXCR4 interaction affects intracellular signaling, and modulates fibroblast phenotype and function.

NF2 signaling pathway plays a pro-apoptotic role in ß-adrenergic receptor stimulated cardiac myocyte apoptosis.

METHODS AND RESULTS: Treatment of adult rat ventricular myocytes (ARVMs) with ß-AR agonist (isoproterenol) for 15 min increased phosphorylation (serine-518) and sumoylation of NF2. Co-immunoprecipitation assay confirmed ß-AR-stimulated sumoylation of NF2. ß-AR stimulation enhanced nuclear translocation of phosphorylated and sumoylated NF2. Specific inhibition of ß1-AR and protein kinase A (PKA) decreased ß-AR-stimulated increase in NF2 post-translational modifications, while inhibition of ß2-AR had no effect. Activation of adenylyl cyclase using forskolin (FSK) mimicked the effects of ß-AR stimulation. ß-AR stimulation and expression of wild-type (WT)-NF2 using adenoviruses increased phosphorylation of mammalian sterile like kinase-1/2 (MST1/2) and yes activated protein (YAP), downstream targets of NF2. Knockdown of NF2 using siRNA in H9C2 cardiomyocytes decreased ß-AR-stimulated increase in NF2 and YAP phosphorylation. siRNA-mediated knockdown of NF2 decreased ß-AR-stimulated increase in apoptosis, while expression of WT-NF2 induced apoptosis in ARVMs. Expression of WT-NF2 stimulated the mitochondrial death pathway as evidenced by activation of c-Jun N-terminal Kinases (JNKs), and increase in cytosolic cytochrome c levels and Bax expression. CONCLUSION: ß-AR stimulation affects post-translational modifications of NF2 via the involvement ß1-AR/PKA/cAMP pathway, and NF2 plays a pro-apoptotic role in ß-AR-stimulated myocyte apoptosis via the phosphorylation (inactivation) of YAP and involvement of mitochondrial death pathway.

Ataxia telangiectasia mutated kinase deficiency impairs the autophagic response early during myocardial infarction.

Ataxia telangiectasia mutated kinase (ATM) is activated in response to DNA damage. We have previously shown that ATM plays a critical role in myocyte apoptosis and cardiac remodeling after myocardial infarction (MI). Here, we tested the hypothesis that ATM deficiency results in autophagic impairment in the heart early during MI. MI was induced in wild-type (WT) and ATM heterozygous knockout (hKO) mice by ligation of the left anterior descending artery. Structural and biochemical parameters of the heart were measured 4 h after left anterior descending artery ligation. M-mode echocardiography revealed that MI worsens heart function, as evidenced by reduced percent ejection fraction and fractional shortening in both groups. However, MI-induced increase in left ventricular end-diastolic and end-systolic diameters and volumes were significantly lower in hKO hearts. ATM deficiency resulted in autophagic impairment during MI, as evidenced by decreased microtubule-associated protein light chain 3-II increased p62, decreased cathepsin D protein levels, and increased aggresome accumulation. ERK1/2 activation was only observed in WT-MI hearts. Activation of Akt and AMP-activated protein kinase (AMPK) was lower, whereas activation of glycogen synthase kinase (GSK)-3ß and mammalian target of rapamycin (mTOR) was higher in hKO-MI hearts. Inhibition of ATM using KU-55933 resulted in autophagic impairment in cardiac fibroblasts, as evidenced by decreased light chain 3-II protein levels and formation of acidic vesicular organelles. This impairment was associated with decreased activation of Akt and AMPK but enhanced activation of GSK-3ß and mTOR in KU-55933-treated fibroblasts. Thus, ATM deficiency results in autophagic impairment in the heart during MI and cardiac fibroblasts. This autophagic impairment may occur via the activation of GSK-3ß and mTOR and inactivation of Akt and AMPK. NEW & NOTEWORTHY Ataxia telangiectasia mutated kinase (ATM) plays a critical role in myocyte apoptosis and cardiac remodeling after myocardial infarction (MI). Here, we provide evidence that ATM deficiency results in autophagic impairment during MI. Further investigation of the role of ATM in autophagy post-MI may provide novel therapeutic targets for patients with ataxia telangiectasia suffering from heart disease.

Ataxia telangiectasia-mutated kinase deficiency exacerbates left ventricular dysfunction and remodeling late after myocardial infarction.

Ataxia telangiectasia-mutated kinase (ATM), a cell cycle checkpoint protein, is activated in response to DNA damage and oxidative stress. We have previously shown that ATM deficiency is associated with increased apoptosis and fibrosis and attenuation of cardiac dysfunction early (1-7 days) following myocardial infarction (MI). Here, we tested the hypothesis that enhanced fibrosis and apoptosis, as observed early post-MI during ATM deficiency, exacerbate cardiac dysfunction and remodeling in ATM-deficient mice late post-MI. MIs were induced in wild-type (WT) and ATM heterozygous knockout (hKO) mice by ligation of the left anterior descending artery. Left ventricular (LV) structural and functional parameters were assessed by echocardiography 14 and 28 days post-MI, whereas biochemical parameters were measured 28 days post-MI. hKO-MI mice exhibited exacerbated LV dysfunction as observed by increased LV end-systolic volume and decreased percent fractional shortening and ejection fraction. Infarct size and thickness were not different between the two genotypes. Myocyte cross-sectional area was greater in hKO-MI group. The hKO-MI group exhibited increased fibrosis in the noninfarct and higher expression of α-smooth muscle actin (myofibroblast marker) in the infarct region. Apoptosis and activation of GSK-3ß (proapoptotic kinase) were significantly lower in the infarct region of hKO-MI group. Matrix metalloproteinase 2 (MMP-2) expression was not different between the two genotypes. However, MMP-9 expression was significantly lower in the noninfarct region of hKO-MI group. Thus ATM deficiency exacerbates cardiac remodeling late post-MI with effects on cardiac function, fibrosis, apoptosis, and myocyte hypertrophy.

Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK.

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

Osteopontin: At the cross-roads of myocyte survival and myocardial function.

Heart failure represents a major cause of morbidity and mortality in Western society. Cardiac myocyte loss due to apoptosis plays a significant role in the progression of heart failure. The extracellular matrix (ECM) maintains the structural integrity of the heart and allows the transmission of electrical and mechanical signals during cardiac contraction and relaxation. Matricellular proteins, a class of non-structural ECM proteins, play a significant role in ECM homeostasis and intracellular signaling via their interactions with cell surface receptors, structural proteins, and/or soluble extracellular factors such as growth factors and cytokines. Osteopontin (OPN), also called cytokine Eta-1, is a member of the matricellular protein family. The normal heart expresses low levels of OPN. However, OPN expression increases markedly under a variety of pathophysiological conditions of the heart. Many human and transgenic mouse studies provide evidence that increased OPN expression, specifically in myocytes, is associated with increased myocyte apoptosis and myocardial dysfunction. This review summarizes OPN expression in the heart, and its role in myocyte apoptosis and myocardial function.

Osteopontin stimulates apoptosis in adult cardiac myocytes via the involvement of CD44 receptors, mitochondrial death pathway, and endoplasmic reticulum stress.

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with ß1 or ß3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Extracellular ubiquitin increases expression of angiogenic molecules and stimulates angiogenesis in cardiac microvascular endothelial cells.

Extracellular Ub is an immune modulator that plays a role in suppression of inflammation, organ injury, myocyte apoptosis, and fibrosis. The purpose of this study was to investigate the effects of extracellular Ub on the process of cardiac angiogenesis. CMECs and aortic tissue were isolated from rats to measure changes in angiogenic protein levels and to assess angiogenic responses to extracellular Ub. In CMECs, extracellular Ub increased protein levels of VEGF-A and MMP-2, known angiogenesis regulators. CMECs demonstrated enhanced rearrangement of fibrillar actin and migration in response to Ub treatment. Ub-treated CMECs demonstrated an increase in tube network formation which was inhibited by the CXCR4 receptor antagonist, AMD3100. Methylated Ub, unable to form polyubiquitin chains, enhanced tube network formation. Aortic ring sprouting assays demonstrated that Ub increases microvessel sprouting in the Matrigel. The results of our study suggest a novel role for extracellular Ub in cardiac angiogenesis, providing evidence that extracellular Ub, at least in part acting via the CXCR4 receptor, has the potential to facilitate the process of angiogenesis in myocardial endothelial cells.

Deficiency of ataxia telangiectasia mutated kinase modulates cardiac remodeling following myocardial infarction: involvement in fibrosis and apoptosis.

UNLABELLED: Ataxia telangiectasia mutated kinase (ATM) is a cell cycle checkpoint protein activated in response to DNA damage. We recently reported that ATM plays a protective role in myocardial remodeling following ß-adrenergic receptor stimulation. Here we investigated the role of ATM in cardiac remodeling using myocardial infarction (MI) as a model. METHODS AND RESULTS: Left ventricular (LV) structure, function, apoptosis, fibrosis, and protein levels of apoptosis- and fibrosis-related proteins were examined in wild-type (WT) and ATM heterozygous knockout (hKO) mice 7 days post-MI. Infarct sizes were similar in both MI groups. However, infarct thickness was higher in hKO-MI group. Two dimensional M-mode echocardiography revealed decreased percent fractional shortening (%FS) and ejection fraction (EF) in both MI groups when compared to their respective sham groups. However, the decrease in %FS and EF was significantly greater in WT-MI vs hKO-MI. LV end systolic and diastolic diameters were greater in WT-MI vs hKO-MI. Fibrosis, apoptosis, and α-smooth muscle actin staining was significantly higher in hKO-MI vs WT-MI. MMP-2 protein levels and activity were increased to a similar extent in the infarct regions of both groups. MMP-9 protein levels were increased in the non-infarct region of WT-MI vs WT-sham. MMP-9 protein levels and activity were significantly lower in the infarct region of WT vs hKO. TIMP-2 protein levels similarly increased in both MI groups, whereas TIMP-4 protein levels were significantly lower in the infarct region of hKO group. Phosphorylation of p53 protein was higher, while protein levels of manganese superoxide dismutase were significantly lower in the infarct region of hKO vs WT. In vitro, inhibition of ATM using KU-55933 increased oxidative stress and apoptosis in cardiac myocytes.

Exogenous ubiquitin modulates chronic ß-adrenergic receptor-stimulated myocardial remodeling: role in Akt activity and matrix metalloproteinase expression.

ß-Adrenergic receptor (ß-AR) stimulation increases extracellular ubiquitin (UB) levels, and extracellular UB inhibits ß-AR-stimulated apoptosis in adult cardiac myocytes. This study investigates the role of exogenous UB in chronic ß-AR-stimulated myocardial remodeling. l-Isoproterenol (ISO; 400 µg·kg(-1)·h(-1)) was infused in mice in the presence or absence of UB (1 µg·g(-1)·h(-1)). Left ventricular (LV) structural and functional remodeling was studied 7 days after infusion. UB infusion enhanced serum UB levels. In most parts, UB alone had no effect on morphometric or functional parameters. Heart weight-to-body weight ratios were increased to a similar extent in the ISO and UB + ISO groups. Echocardiographic analyses showed increased percent fractional shortening, ejection fraction, and LV circumferential stress and fiber-shortening velocity in the ISO group. These parameters were significantly lower in UB + ISO vs. ISO. Isovolumic contraction and relaxation times and ejection time were significantly lower in ISO vs. UB + ISO. The increase in the number of TUNEL-positive myocytes and fibrosis was significantly higher in ISO vs. UB + ISO. Activation of Akt was higher, whereas activation of GSK-3ß and JNKs was lower in UB + ISO vs ISO. Expression of MMP-2, MMP-9, and TIMP-2 was higher in UB + ISO vs ISO. In isolated cardiac fibroblasts, UB enhanced expression of MMP-2 and TIMP-2 in the presence of ISO. Neutralizing UB antibodies negated the effects of UB on MMP-2 expression, whereas recombinant UB enhanced MMP-2 expression. UB activated Akt, and inhibition of Akt inhibited UB + ISO-mediated increases in MMP-2 expression. Thus, exogenous UB plays an important role in ß-AR-stimulated myocardial remodeling with effects on LV function, fibrosis, and myocyte apoptosis.

Β-adrenergic receptor stimulation induces endoplasmic reticulum stress in adult cardiac myocytes: role in apoptosis.

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that ß-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs). ß-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. ß-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ~50% below basal levels at 3 h after ß-AR stimulation. This decline in eIF-2α phosphorylation was prevented by ß1-AR, not by ß2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited ß-AR-stimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited ß-AR-stimulated and thapsigargin-induced apoptosis. In vivo, ß-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased ß-AR-stimulated apoptosis in the heart. Thus, ß-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role.

Role of osteopontin in heart failure associated with aging.

Cardiovascular disease is one of the leading causes of death in the elderly. Much of the morbidity and mortality in the elderly is attributable to acute ischemic events leading to myocardial infarction (MI) and death of cardiac myocytes. Evidence has been provided that aging associated with adverse remodeling post MI as demonstrated by less effective myocardial repair, greater infarct expansion, and septal hypertrophy. Expression of osteopontin (OPN) increases in the heart post MI. Transgenic mice studies suggest that increased expression of OPN plays a protective role in post-MI LV remodeling by modulating collagen deposition and fibrosis. OPN, a multifunctional protein, has the potential to influence the molecular and cellular changes associated with infarct healing. The post-MI infarct healing process involves temporarily overlapping phases that include the following--(1) inflammation with migration and adhesion of neutrophils and macrophages, phagocytosis and inflammatory gene expression; (2) tissue repair with fibroblast adhesion and proliferation, myofibroblast differentiation, extracellular matrix deposition and scar formation; and (3) structural and functional remodeling of infarcted and non-infarcted myocardium through cardiac myocyte apoptosis, hypertrophy and myocardial angiogenesis. This review is focused on the expression of OPN in the heart post MI and its role in various phases of infarct healing.