The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress.

Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism in starving cells. Although altered autophagy has been observed in various heart diseases, including cardiac hypertrophy and heart failure, it remains unclear whether autophagy plays a beneficial or detrimental role in the heart. Here, we report that the cardiac-specific loss of autophagy causes cardiomyopathy in mice. In adult mice, temporally controlled cardiac-specific deficiency of Atg5 (autophagy-related 5), a protein required for autophagy, led to cardiac hypertrophy, left ventricular dilatation and contractile dysfunction, accompanied by increased levels of ubiquitination. Furthermore, Atg5-deficient hearts showed disorganized sarcomere structure and mitochondrial misalignment and aggregation. On the other hand, cardiac-specific deficiency of Atg5 early in cardiogenesis showed no such cardiac phenotypes under baseline conditions, but developed cardiac dysfunction and left ventricular dilatation one week after treatment with pressure overload. These results indicate that constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function, and that upregulation of autophagy in failing hearts is an adaptive response for protecting cells from hemodynamic stress.

Apoptosis signal-regulating kinase 1/p38 signaling pathway negatively regulates physiological hypertrophy.

BACKGROUND: Mechanical stress on the heart can lead to crucially different outcomes. Physiological stimuli such as exercise cause adaptive cardiac hypertrophy, characterized by a normal cardiac structure and normal or enhanced cardiac function. Pathological stimuli such as hypertension and aortic valvular stenosis cause maladaptive cardiac remodeling and ultimately heart failure. Apoptosis signal-regulating kinase 1 (ASK1) is known to be involved in pathological cardiac remodeling, but it has not been determined whether ASK1 pathways coordinate the signaling cascade leading to physiological type cardiac growth. METHODS AND RESULTS: To evaluate the role of ASK1 in the physiological form of cardiac growth, mice lacking ASK1 (ASK1-/-) were exercised by swimming for 4 weeks. ASK1-/- mice showed exaggerated growth of the heart accompanied by typical characteristics of physiological hypertrophy. Their swimming-induced activation of Akt, a key molecule in the signaling cascade of physiological hypertrophy, increased more than that seen in wild-type controls. The activation of p38, a downstream kinase of ASK1, was suppressed selectively in the swimming-exercised ASK1-/- mice. Furthermore, the inhibition of ASK1 or p38 activity enhanced insulin-like growth factor 1-induced protein synthesis in rat neonatal ventricular cardiomyocytes, and the treatment with a specific inhibitor of p38 resulted in enhancement of Akt activation and suppression of protein phosphatase 2A activation. The cardiac-specific p38alpha-deficient mice developed an exacerbated form of cardiac hypertrophy in response to swimming exercise. CONCLUSIONS: These results indicate that the ASK1/p38 signaling pathway negatively regulates physiological hypertrophy.

Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis.

The Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway regulates diverse cellular processes such as proliferation, differentiation, and apoptosis and is implicated as an important contributor to the pathogenesis of cardiac hypertrophy and heart failure. To examine the in vivo role of Raf-1 in the heart, we generated cardiac muscle-specific Raf-1-knockout (Raf CKO) mice with Cre-loxP-mediated recombination. The mice demonstrated left ventricular systolic dysfunction and heart dilatation without cardiac hypertrophy or lethality. The Raf CKO mice showed a significant increase in the number of apoptotic cardiomyocytes. The expression level and activation of MEK1/2 or ERK showed no difference, but the kinase activity of apoptosis signal-regulating kinase 1 (ASK1), JNK, or p38 increased significantly compared with that in controls. The ablation of ASK1 rescued heart dysfunction and dilatation as well as cardiac fibrosis. These results indicate that Raf-1 promotes cardiomyocyte survival through a MEK/ERK-independent mechanism.

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload.

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

Presenilin 2 regulates the systolic function of heart by modulating Ca2+ signaling.

Genetic studies of families with familial Alzheimer's disease have implicated presenilin 2 (PS2) in the pathogenesis of this disease. PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice to elucidate a role of PS2 in hearts. PS2KO mice developed normally with no evidence of cardiac hypertrophy and fibrosis. Invasive hemodynamic analysis revealed that cardiac contractility in PS2KO mice increased compared with that in their littermate controls. A study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice than those in their littermate controls. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins. Since it has been demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK-293 cells overexpressing these proteins or in mouse hearts. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z-lines. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low extracellular [Ca2+] but not at high levels of [Ca2+]. Taken together, our results suggest that PS2 plays an important role in cardiac excitation-contraction coupling by interacting with RyR2.

Inhibition of autophagy in the heart induces age-related cardiomyopathy.

Constitutive autophagy is important for control of the quality of proteins and organelles to maintain cell function. Damaged proteins and organelles accumulate in aged organs. We have previously reported that cardiac-specific Atg5 (autophagy-related gene 5)-deficient mice, in which the gene was floxed out early in embryogenesis, were born normally, and showed normal cardiac function and structure up to 10 weeks old. In the present study, to determine the longer-term consequences of Atg5-deficiency in the heart, we monitored cardiac-specific Atg5-deficient mice for further 12 months. First, we examined the age-associated changes of autophagy in the wild-type mouse heart. The level of autophagy, as indicated by decreased LC3-II (microtubule-associated protein 1 light chain 3-II) levels, in the hearts of 6-, 14- or 26-month-old mice was lower than that of 10-week-old mice. Next, we investigated the cardiac function and life-span in cardiac-specific Atg5-deficient mice. The Atg5-deficient mice began to die after the age of 6 months. Atg5-deficient mice exhibited a significant increase in left ventricular dimension and decrease in fractional shortening of the left ventricle at the age of 10 months, compared to control mice, while they showed similar chamber size and contractile function at the age of 3 months. Ultrastructural analysis revealed a disorganized sarcomere structure and collapsed mitochondria in 3- and 10-month-old Atg5-deficient mice, with decreased mitochondrial respiratory functions. These results suggest that continuous constitutive autophagy has a crucial role in maintaining cardiac structure and function.

Reduction in hemoglobin-oxygen affinity results in the improvement of exercise capacity in mice with chronic heart failure.

OBJECTIVES: This study examined whether a reduction in hemoglobin-oxygen affinity improves exercise capacity in mice with heart failure. BACKGROUND: Exercise intolerance is a major determinant of quality of life in patients with chronic heart failure. One of the major goals of the treatment for chronic heart failure is to improve quality of life. METHODS: Four weeks after left coronary ligation, we transplanted bone marrow cells isolated from the transgenic mice expressing a hemoglobin variant with low oxygen affinity, Presbyterian, into the lethally irradiated mice with heart failure or administered a synthetic allosteric modifier of hemoglobin. The mice were then exercised on a treadmill. RESULTS: Four weeks after the left coronary artery ligation, mice showed cardiac dysfunction and chamber dilation, which were characteristics of heart failure. The transplantation led to a reduction in hemoglobin-oxygen affinity and an increase in oxygen supply for skeletal muscle without changes in muscle properties. The transplanted mice showed improved running performance on a treadmill despite impaired cardiac contractility. Furthermore, administration of the synthetic allosteric modifier of hemoglobin showed a similar effect. CONCLUSIONS: Allosteric modification of hemoglobin represents a therapeutic option for improving exercise capacity in patients with chronic heart failure. One mechanism of improvement in exercise capacity is enhanced oxygen delivery in the skeletal muscle.

The antioxidant edaravone attenuates pressure overload-induced left ventricular hypertrophy.

The free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is used to treat patients with ischemic brain damage. We and others reported previously that in vitro and in vivo reactive oxygen species (ROS) act as second messengers to develop cardiac hypertrophy. In this study, we used an in vivo murine model of pressure overload-induced cardiac hypertrophy to examine the effects of edaravone on left ventricular hypertrophy. The animals were subjected to the transverse thoracic aorta constriction, and edaravone (10 mg/kg) was infused intraperitoneally twice daily. Seven days after the operation, we observed a significant increase in ROS production in hearts, which was eliminated by the treatment with edaravone. Pressure-overloaded hearts showed a significant increase in left ventricular weight/body weight ratio and the expression level of atrial natriuretic factor mRNA, which were attenuated by edaravone. It also reduced perivascular and intermuscular fibrosis and inhibited pressure overload-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream kinases of c-Jun N-terminal protein kinase and p38 mitogen-activated protein kinase. Edaravone attenuated the hypertrophic response even when the treatment was started after the onset of cardiac hypertrophic response. These findings indicate that edaravone significantly attenuates pressure overload-induced cardiac hypertrophy mediated through its antioxidative function and subsequent inhibition of ASK1 signaling pathway.

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death.

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.

CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy.

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.