Miho Mizukami

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.

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.

Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle‐derived cells expressed cardiac‐specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac‐enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle‐derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte‐like action potentials were recorded from beating skeletal muscle‐derived cells. Treatment of nifedipine or culture in Ca2+‐free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac‐specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell‐to‐cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors prevent the development of cardiac hypertrophy and heart failure in rats

OBJECTIVES The aim of the present study was to determine whether 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have preventive effects on the development of cardiac hypertrophy and heart failure. BACKGROUND Statins have been reported to have various pleiotropic effects, such as inhibition of inflammation and cell proliferation. METHODS Dahl rats were divided into three groups: LS, the rats fed the low-salt diet (0.3% NaCl); HS, the rats fed the high-salt diet (8% NaCl) from the age of 6 weeks; and CERI, the rats fed the high-salt diet with cerivastatin 1 mg/kg/d by gavage from the age of 6 weeks. RESULTS In HS rats, cardiac function was markedly impaired and all rats showed the signs of heart failure within 17 weeks of age. In CERI rats, cardiac function was better than that of HS and no rats were dead up to 17 weeks of age. The development of cardiac hypertrophy and fibrosis was attenuated, and the number of apoptotic cells and expression of proinflammatory cytokine interleukin (IL)-1beta gene were less as compared with HS rats. Pretreatment of cerivastatin suppressed the adriamycin-induced apoptosis of cultured cardiomyocytes of neonatal rats. CONCLUSIONS These results suggest that statins have a protective effect on cardiac myocytes and may be useful to prevent the development of hypertensive heart failure.

Heat Shock Transcription Factor 1 Protects Cardiomyocytes From Ischemia/Reperfusion Injury

Background—Because cardiomyocyte death causes heart failure, it is important to find the molecules that protect cardiomyocytes from death. The death trap is a useful method to identify cell-protective genes. Methods and Results—In this study, we isolated the heat shock transcription factor 1 (HSF1) as a protective molecule by the death trap method. Cell death induced by hydrogen peroxide was prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42°C for 60 minutes activated HSF1, which played a critical role in survival of cardiomyocytes from oxidative stress. In the heart of transgenic mice overexpressing a constitutively active form of HSF1, ischemia followed by reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct size was smaller, and cardiomyocyte death was less than wild-type mice. Protein kinase B/Akt was more strongly activated, whereas Jun N-terminal kinase and caspase 3 were less activated in transgenic hearts than wild-type ones. Conclusions—These results suggest that HSF1 protects cardiomyocytes from death at least in part through activation of Akt and inactivation of Jun N-terminal kinase and caspase 3.

Functional Analyses of Three Csx/Nkx-2.5 Mutations That Cause Human Congenital Heart Disease

A homeodomain-containing transcription factorCsx/Nkx-2.5 is an important regulator of cardiogenesis in mammals. Three different mutants, Gln170ter (designated A) and Thr178Met (designated B) in the helix 2 of the homeodomain and Gln198ter mutation (designated C) just after homeodomain, have been reported to cause atrial septal defect with atrial ventricular block. We here examined the functions of these three mutants of Csx/Nkx-2.5. The atrial natriuretic peptide (ANP) promoter was activated by wild typeCsx/Nkx-2.5 (WT, ∼8-fold), B(∼2-fold), and C (∼6-fold) but not by A. When A, B, or C was cotransfected into COS-7 cells with the same amount of WT,WT-induced activation of the ANP promoter was attenuated by A and B (A >B), whereas C further enhanced the activation. Immunocytochemical analysis using anti-Myc tag antibody indicated that transfected Myc-tagged WT, B, and Cwere localized in the nucleus of both COS-7 cells and cardiomyocytes of neonatal rats, whereas A was distributed diffusely in the cytoplasm and nucleus in COS-7 cells. Electrophoretic mobility shift assay showed that Csx/Nkx-2.5-binding sequences were bound strongly by WT and C, weakly by B, but not by A. Immunoprecipitation and GST pull-down assay revealed that WT and all mutants interacted with GATA-4. The synergistic activation of the ANPpromoter by WT and GATA-4 was further enhanced by C but was inhibited by A and B. In the cultured cardiomyocytes, overexpression of C but notWT, A, or B, induced apoptosis. These results suggest that although the three mutants induce the same cardiac phenotype, transactivation ability and DNA binding ability are different among the three mutants and that apoptosis may be a cause forC-induced cardiac defect.

Calcineurin Inhibitor Attenuates the Development and Induces the Regression of Cardiac Hypertrophy in Rats With Salt-Sensitive Hypertension

BackgroundIt remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. Methods and ResultsIn the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg twice daily) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg twice daily but not with 0.01 mg/kg twice daily of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. ConclusionsThese results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

Sodium calcium exchanger plays a key role in alteration of cardiac function in response to pressure overload

The Na+‐Ca2+ exchanger (NCX) on the plasma membrane is thought to be the main calcium extrusion system from the cytosol to the extracellular space in many mammalian excitable cells, including cardiac myocytes. However, the pathophysiological role of NCX in the heart is still unclear because of the lack of known specific inhibitors of NCX. To determine the role of NCX in cardiac contraction and the development of cardiac hypertrophy, we imposed pressure overload on the heart of heterozygous NCX knockout (KO) mice by constricting transverse aorta, and examined cardiac function and morphology 3 wk after operation. Although there was no difference in cardiac function between sham‐operated KO mice and sham‐operated wild‐type (WT) mice, KO mice showed higher left ventricular pressure and better systolic function than WT mice in response to pressure overload. Northern blot analysis revealed that mRNA levels of sarcoplasmic reticulum Ca2+‐ATPase were reduced by pressure overload in left ventricles of WT but not of KO mice. However, hypertrophic changes with interstitial fibrosis were more prominent in KO mice than WT mice. These results suggest that reduction of NCX results in supernormalized cardiac function and causes marked cardiac hypertrophy in response to pressure overload.—Takimoto, E., Yao, A., Toko, H., Takano, H., Shimoyama, M., Sonoda, M., Wakimoto, K., Takahashi, T., Akazawa, H., Mizukami, M., Nagai, T., Nagai, R., Komuro, I. Sodium calcium exchanger plays a key role in alteration of cardiac function in response to pressure overload. FASEB J. 16, 373–378 (2002)

Ca2+—Dependent Signaling Pathways Through Calcineurin and Ca2+ Calmodulin—Dependent Protein Kinase in Development of Cardiac Hypertrophy

Cardiac hypertrophy is induced by a variety of cardiovascular diseases such as hypertension, valvular diseases, myocardial infarction, cardiomyopathy, and endocrine disorders. Although cardiac hypertrophy may be initially a beneficial response that normalizes wall stress and maintains normal cardiac function, prolonged hypertrophy becomes a leading cause of heart failure and sudden death. A number of studies have elucidated molecules responsible to the development of cardiac hypertrophy, including protein kinase C (PKC), protein kinase A (PKA), Raf-1 kinase, mitogen-activated protein (MAP) kinase family, and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) family, Ras, and Rho family. It has been reported that Ca2+ regulates a number of cellular processes including cardiac hypertrophy. Since most hypertrophic signaling pathways are associated with an increase in intracellular Ca2+, Ca2+-dependent signaling pathways may be critical targets for therapies designed to prevent the progression of cardiac hypertrophy. Recently, a Ca2+/calmodulin-dependent protein kinase, and a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, have attracted much attention as critical molecules that induce cardiac hypertrophy. In this review, we summarize the Ca2+-dependent signaling pathways through Ca2+/calmodulin-dependent protein kinase and calcineurin in cardiac hypertrophy.

Gene expression profile revealed different effects of angiotensin II receptor blockade and angiotensin-converting enzyme inhibitor on heart failure

Summary: Although recent clinical studies have indicated that angiotensin II receptor blocker is as effective in treating heart failure as an angiotensin‐converting enzyme inhibitor, it is unknown whether their effects are different. Dahl salt‐sensitive rats were treated with an angiotensin‐converting enzyme inhibitor benazepril, and an angiotensin II receptor blocker candesartan from 11 weeks old. We examined cardiac geometry and function by echocardiography, and histology and gene expression by high‐density oligonucleotide arrays using Affymetrix U34 (Affymetrix, Santa Clara, CA, U.S.A.). Dahl salt‐sensitive rats fed a high salt diet showed a marked increase in blood pressure and developed concentric hypertrophy at 11 weeks, followed by left ventricle dilation and congestive heart failure by 20 weeks after birth. Although both medications had only a mild antihypertensive effect, they strongly suppressed the development of cardiac hypertrophy, fibrosis and heart failure to the same extent. Gene expression pattern examined by Affymetrix GeneChip (Affymetrix) is quite different between the two drug groups, indicating that angiotensin II receptor blocker and angiotensin‐converting enzyme inhibitor prevent heart failure by different mechanisms.