Haruhiro Toko

p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.

Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway.

The signaling pathway of insulin/insulin‐like growth factor‐1/phosphatidylinositol‐3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF‐16, a forkhead transcription factor. Although reduction‐of‐function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence‐like arrest of cell growth via a p53/p21‐dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt‐induced senescence may be involved in vascular pathophysiology.

Complement C1q Activates Canonical Wnt Signaling and Promotes Aging-Related Phenotypes

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.

Ras Induces Vascular Smooth Muscle Cell Senescence and Inflammation in Human Atherosclerosis

Background—Vascular cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related vascular disorders. Ras, an important signaling molecule involved in atherogenic stimuli, is known to promote aging in yeast and cellular senescence in primary human fibroblasts. The aim of this study was to investigate the role of Ras-induced vascular smooth muscle cell (VSMC) senescence in atherogenesis. Methods and Results—We introduced an activated ras allele (H-ras V12) into human VSMCs using retroviral infection. Introduction of H-ras V12 induced a growth arrest with phenotypic characteristics of cellular senescence, such as enlarged cell shapes and increases in expression of cyclin-dependent kinase inhibitors and senescence-associated &bgr;-galactosidase (SA-&bgr;-gal) activity. Activation of Ras drastically increased expression of proinflammatory cytokines, in part through extracellular signal-regulated kinase activation. To determine whether Ras activation induces cellular senescence in vivo, we transduced the adenoviral vector encoding H-ras V12 into rat carotid arteries injured by a balloon catheter. Introduction of Ras into the arteries enhanced vascular inflammation and senescence compared with mock-infected injured arteries. Moreover, SA-&bgr;-gal–positive VSMCs were detected in the intima of advanced human atherosclerotic lesions and exhibited increased levels of extracellular signal-regulated kinase activity and proinflammatory cytokine expression. Conclusions—Our results suggest that atherogenic stimuli mediated by Ras induce VSMC senescence and vascular inflammation, thereby contributing to atherogenesis. This novel mechanism of atherogenesis may provide insights into a new antisenescence treatment for atherosclerosis.

Calcineurin Plays a Critical Role in the Development of Pressure Overload–Induced Cardiac Hypertrophy

Background—Although activation of the Ca2+-dependent phosphatase calcineurin has been reported to induce cardiomyocyte hypertrophy, whether calcineurin is involved in pressure overload–induced cardiac hypertrophy remains controversial. Methods and Results—We examined in the present study the role of calcineurin in pressure overload–induced cardiac hypertrophy using transgenic mice that overexpress the dominant negative mutant of calcineurin specifically in the heart. There were no significant differences in body weight, blood pressure, heart rate, heart weight, and the cardiac calcineurin activity between the transgenic mice and their littermate wild-type mice at basal state. The activity of calcineurin was markedly increased by pressure overload produced by constriction of the abdominal aorta in the heart of wild-type mice but less increased in the heart of the transgenic mice. Pressure overload induced increases in heart weight, wall thickness of the left ventricle, and diameter of cardiomyocytes; reprogramming of expressions of immediate early response genes and fetal-type genes; activation of extracellular signal–regulated protein kinases; and fibrosis. All these hypertrophic responses were more prominent in the wild-type mice than in the transgenic mice. Conclusions—These results suggest that calcineurin plays a critical role in the development of pressure overload–induced cardiac hypertrophy.

Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents

Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overload-induced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.

Leukemia Inhibitory Factor Enhances Survival of Cardiomyocytes and Induces Regeneration of Myocardium After Myocardial Infarction

Background—Myocardial infarction (MI) is a leading cause of cardiac morbidity and mortality in many countries; however, the treatment of MI is still limited. Methods and Results—We demonstrate a novel gene therapy for MI using leukemia inhibitory factor (LIF) cDNA. We injected LIF plasmid DNA into the thigh muscle of mice immediately after inducing MI. Intramuscular injection of LIF cDNA resulted in a marked increase in circulating LIF protein concentrations. Two weeks later, left ventricular remodeling, such as infarct extent and myocardial fibrosis, was markedly attenuated in the LIF cDNA–injected mice compared with vehicle-injected mice. More myocardium was preserved and cardiac function was better in the LIF-treated mice than in the vehicle-injected mice. Injection of LIF cDNA not only prevented the death of cardiomyocytes in the ischemic area but also induced neovascularization in the myocardium. Furthermore, LIF cDNA injection increased the number of cardiomyocytes in cell cycle and enhanced mobilization of bone marrow cells to the heart and their differentiation into cardiomyocytes. Conclusions—The intramuscular injection of LIF cDNA may induce regeneration of myocardium and provide a novel treatment for MI.

Ca2+/Calmodulin-Dependent Kinase IIδ Causes Heart Failure by Accumulation of p53 in Dilated Cardiomyopathy

Background— Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown. Methods and Results— We established the mouse model of DCM by expressing a mutated cardiac &agr;-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase II&dgr; (CaMKII&dgr;). The inhibition of CaMKII&dgr; prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function. Conclusion— CaMKII&dgr; plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.