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.

Heart failure is a leading cause of morbidity and mortality in industrialized countries. Although infection with microorganisms is not involved in the development of heart failure in most cases, inflammation has been implicated in the pathogenesis of heart failure1. However, the mechanisms responsible for initiating and integrating inflammatory responses within the heart remain poorly defined. Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA2,3,4. Mitochondria damaged by external hemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes5. Here, we show that mitochondrial DNA that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated inflammatory responses in cardiomyocytes and is capable of inducing myocarditis, and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions, but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload. Early in the pathogenesis, DNase II-deficient hearts exhibited infiltration of inflammatory cells and increased mRNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of the inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Furthermore, Tlr9-ablation improved pressure overload-induced cardiac dysfunction and inflammation even in mice with wild-type Dnase2a alleles. These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.

Various stimuli, such as telomere dysfunction and oxidative stress, can induce irreversible cell growth arrest, which is termed 'cellular senescence'. This response is controlled by tumor suppressor proteins such as p53 and pRb. There is also evidence that senescent cells promote changes related to aging or age-related diseases. Here we show that p53 expression in adipose tissue is crucially involved in the development of insulin resistance, which underlies age-related cardiovascular and metabolic disorders. We found that excessive calorie intake led to the accumulation of oxidative stress in the adipose tissue of mice with type 2 diabetes-like disease and promoted senescence-like changes, such as increased activity of senescence-associated beta-galactosidase, increased expression of p53 and increased production of proinflammatory cytokines. Inhibition of p53 activity in adipose tissue markedly ameliorated these senescence-like changes, decreased the expression of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes-like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. Adipose tissue from individuals with diabetes also showed senescence-like features. Our results show a previously unappreciated role of adipose tissue p53 expression in the regulation of insulin resistance and suggest that cellular aging signals in adipose tissue could be a new target for the treatment of diabetes (pages 996-967).

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.

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.

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction. Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation. Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activated the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development.

A growing body of evidence has suggested that oxidative stress causes cardiac injuries during ischemia/reperfusion. Extracellular signal-regulated kinases (ERKs) have been reported to play pivotal roles in many aspects of cell functions and to be activated by oxidative stress in some types of cells. In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of ERKs in cultured cardiomyocytes of neonatal rats, and determined their role in oxidative stress-induced cardiomyocyte injuries. ERKs were transiently and concentration-dependently activated by hydrogen peroxide (H

Although Wingless (Wg)/Wnt signaling has been implicated in heart development of multiple organisms, conflicting results have been reported regarding the role of Wnt/␤-catenin pathway in cardiac myogenesis: Wg/armadillo signaling promotes heart development in Drosophila, whereas activation of Wnt/␤-catenin signaling inhibits heart formation in avians and amphibians. Using an in vitro system of mouse ES cell differentiation into cardiomyocytes, we show here that Wnt/␤-catenin signaling exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis. Activation of the Wnt/␤-catenin pathway in the early phase during embryoid body (EB) formation enhances ES cell differentiation into cardiomyocytes while suppressing the differentiation into hematopoietic and vascular cell lineages. In contrast, activation of Wnt/␤-catenin signaling in the late phase after EB formation inhibits cardiomyocyte differentiation and enhances the expression of hematopoietic/vascular marker genes through suppression of bone morphogenetic protein signaling. Thus, Wnt/␤-catenin signaling exhibits biphasic and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis, depending on the stage of development.cardiogenesis