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 β1 or β3 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.

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 β-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.

BackgroundAtaxia‐telangiectasia results from mutations in ataxia telangiectasia mutated kinase (ATM) gene. We recently reported that ATM deficiency attenuates left ventricular (LV) dysfunction and dilatation 7 days after myocardial infarction (MI) with increased apoptosis and fibrosis. Here we investigated the role of ATM in the induction of inflammatory response, and activation of survival signaling molecules in the heart acute post‐MI.Methods and ResultsLV structure, function, inflammatory response, and biochemical parameters were measured in wild‐type (WT) and ATM heterozygous knockout (hKO) mice 1 and 3 days post‐MI. ATM deficiency had no effect on infarct size. MI‐induced decline in heart function, as measured by changes in percent fractional shortening, ejection fraction and LV end systolic and diastolic volumes, was lower in hKO‐MI versus WT‐MI (n=10 to 12). The number of neutrophils and macrophages was significantly lower in the infarct LV region of hKO versus WT 1 day post‐MI. Fibrosis and expression of α‐smooth muscle actin (myofibroblast marker) were higher in hKO‐MI, while active TGF‐β1 levels were higher in the WT‐MI 3 days post‐MI. Myocyte cross‐sectional area was higher in hKO‐sham with no difference between the two MI groups. MMP‐9 protein levels were similarly increased in the infarct LV region of both MI groups. Apoptosis was significantly higher in the infarct LV region of hKO at both time points. Akt activation was lower, while Bax expression was higher in hKO‐MI infarct.ConclusionATM deficiency results in decreased dilative remodeling and delays inflammatory response acute post‐MI. However, it associates with increased fibrosis and apoptosis.

β-Adrenergic receptor (β-AR) stimulation increases extracellular ubiquitin (UB) levels, and extracellular UB inhibits β-AR-stimulated apoptosis in adult cardiac myocytes. This study investigates the role of exogenous UB in chronic β-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-3β 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 β-AR-stimulated myocardial remodeling with effects on LV function, fibrosis, and myocyte apoptosis.

Objective Extracellular ubiquitin (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. Methods Cardiac microvascular endothelial cells (CMECs) and aortic tissue were isolated from rats to measure changes in angiogenic protein levels and to assess angiogenic responses to extracellular Ub. Results In CMECs, extracellular Ub increased protein levels of vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-2 (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. Conclusion 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.

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-3β (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.

β-Adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-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 β-adrenergic receptors increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-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.

The data presented here suggest that UB interacts with CXCR4, and UB/CXCR4 interaction affects intracellular signaling, and modulates fibroblast phenotype and function.