Background-Peroxisome proliferator-activated receptors (PPARs) are transcription factors of the nuclear receptor superfamily. It has been reported that the thiazolidinediones, which are antidiabetic agents and high-affinity ligands for PPAR␥, regulate growth of vascular cells. In the present study, we examined the role of PPAR␥ in angiotensin II (Ang II)-induced hypertrophy of neonatal rat cardiac myocytes and in pressure overload-induced cardiac hypertrophy of mice. Methods and Results-Treatment of cultured cardiac myocytes with PPAR␥ ligands such as troglitazone, pioglitazone, and rosiglitazone inhibited Ang II-induced upregulation of skeletal ␣-actin and atrial natriuretic peptide genes and an increase in cell surface area. Treatment of mice with a PPAR␥ ligand, pioglitazone, inhibited pressure overload-induced increases in the heart weight-to-body weight ratio, wall thickness, and myocyte diameter in wild-type mice and an increase in the heart weight-to-body weight ratio in heterozygous PPAR␥-deficient mice. In contrast, pressure overload-induced increases in the heart weight-to-body weight ratio and wall thickness were more prominent in heterozygous PPAR␥-deficient mice than in wild-type mice. Conclusions-These results suggest that the PPAR␥-dependent pathway is critically involved in the inhibition of cardiac hypertrophy.

Activation of ␤-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked intracellular signal transduction pathways leading to activation of extracellular signalregulated kinases (ERKs) and cardiomyocyte hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that G s protein is involved in the activation. Inhibition of G i protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the G ␤␥ subunit binding domain of the ␤-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of G ␤␥ and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of dominant-negative mutants of Ras and Raf-1 kinase and of the ␤-adrenoreceptor mutant that lacks phosphorylation sites by PKA abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed by inhibitors for PKA, G i , tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy through two different G proteins, G s and G i . cAMP-dependent PKA activation through G s may phosphorylate the ␤-adrenoreceptor, leading to coupling of the receptor from G s to G i . Activation of G i activates ERKs through G ␤␥ , Src family tyrosine kinases, Ras, and Raf-1 kinase.

Background-Although anthracyclines, such as daunomycin (DM) and adriamycin, are potent chemotherapeutic agents, they have serious adverse effects, including cardiac toxicity. In the present study, we investigated the molecular mechanisms of DM-induced cardiomyocyte impairment. Methods and Results-When cultured cardiac myocytes of neonatal rats were exposed to 1 mol/L DM for 24 hours, many cells became positive for TUNEL staining, with morphological changes characteristic of apoptosis. Fragmentation of DNA into oligonucleosome-size fragments was recognized by agarose gel electrophoresis in DM-treated myocytes. DM activated 3 members of the mitogen-activated protein kinase (MAPK) family dose-dependently, such as extracellular signal-regulated protein kinases (ERKs), c-Jun NH 2 -terminal kinases, and p38 MAPK in cardiac myocytes. Oxyradical scavengers or Ca 2ϩ chelators inhibited DM-induced activation of ERKs and p38 MAPK. DM-induced activation of ERKs was also inhibited by overexpression of dominant negative mutants of Ras (D.N.Ras), and the p38 MAPK activation was attenuated by D.N.Rho. The number of DM-induced apoptotic cells was markedly increased when the ERK signaling pathway was selectively blocked by a specific MAPK/ERK kinase inhibitor, PD98059, whereas pretreatment with a specific inhibitor of p38 MAPK, SB203580, significantly reduced the amount of apoptosis. Conclusions-These results suggest that DM activates MAPKs through reactive oxygen species and Ca 2ϩ and that the MAPK family plays important roles in DM-induced apoptosis in cardiac myocytes. ERKs protect cardiomyocytes from apoptosis, whereas p38 MAPK is involved in the induction of cardiomyocyte apoptosis. (Circulation. 1999;100:2100-2107.)

Background-Cardiac hypertrophy is a fundamental adaptive response to hemodynamic overload; how mechanical load induces cardiac hypertrophy, however, remains elusive. It was recently reported that activation of a calcium-dependent phosphatase, calcineurin, induces cardiac hypertrophy. In the present study, we examined whether calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy. Methods and Results-Pressure overload produced by constriction of the abdominal aorta increased the activity of calcineurin in the rat heart and induced cardiac hypertrophy, including reprogramming of gene expression. Treatment of rats with a calcineurin inhibitor, FK506, inhibited the activation of calcineurin and prevented the pressure overload-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Load-induced expression of immediate-early-response genes and fetal genes was also suppressed by the FK506 treatment. Conclusions-The present results suggest that the calcineurin signaling pathway plays a pivotal role in load-induced cardiac hypertrophy and may pave the way for a novel pharmacological approach to prevent cardiac hypertrophy.

We have previously reported that stretching of cardiomyocytes activates the phosphorylation cascade of protein kinases, including Raf-1 kinase and mitogen-activated protein (MAP) kinases, followed by an increase in protein synthesis partly through enhanced secretion of angiotensin II and endothelin-1. Membrane proteins, such as ion channels and exchangers, have been postulated to first receive extracellular stimuli and evoke intracellular signals. The present study was performed to determine whether mechanosensitive ion channels and exchangers are involved in stretch-induced hypertrophic responses. Neonatal rat cardiomyocytes cultured on expandable silicone dishes were stretched after pretreatment with a specific inhibitor of stretch-sensitive cation channels (gadolinium and streptomycin), of ATP-sensitive K+ channels (glibenclamide), of hyperpolarization-activated inward channels (CsCl), or of the Na+-H+ exchanger (HOE 694). Pretreatment with gadolinium, streptomycin, glibenclamide, and CsCl did not show any inhibitory effects on MAP kinase activation by mechanical stretch. HOE 694, however, markedly attenuated stretch-induced activation of Raf-1 kinase and MAP kinases by approximately 50% and 60%, respectively, and attenuated stretch-induced increase in phenylalanine incorporation into proteins. In contrast, HOE 694 did not inhibit angiotensin II-and endothelin-1-induced Raf-1 kinase and MAP kinase activation. These results suggest that among many mechanosensitive ion channels and exchangers, the Na+-H+ exchanger plays a critical role in mechanical stress-induced cardiomyocyte hypertrophy.

Background-Extracellular signal-regulated kinases (ERKs) and calcineurin have been reported to play important roles in the development of cardiac hypertrophy. We examined here the relation between calcineurin and ERKs in cardiomyocytes. Methods and Results-Isoproterenol activated ERKs in cultured cardiomyocytes of neonatal rats, and the activation was abolished by chelation of extracellular Ca 2ϩ with EGTA, blockade of L-type Ca 2ϩ channels with nifedipine, or depletion of intracellular Ca 2ϩ stores with thapsigargin. Isoproterenol-induced activation of ERKs was also significantly suppressed by calcineurin inhibitors in cultured cardiomyocytes as well as in the hearts of mice. Isoproterenol failed to activate ERKs in either the cultured cardiomyocytes or the hearts of mice that overexpress the dominant negative mutant of calcineurin. Isoproterenol elevated intracellular Ca 2ϩ levels at both systolic and diastolic phases and dose-dependently activated calcineurin. Inhibition of calcineurin also attenuated isoproterenol-stimulated phosphorylation of Src, Shc, and Raf-1 kinase. The immunocytochemistry revealed that calcineurin was localized in the Z band, and isoproterenol induced translocation of calcineurin and ERKs into the nucleus. Conclusions-Calcineurin, which is activated by marked elevation of intracellular Ca 2ϩ levels by the Ca 2ϩ -induced Ca

These results suggest that calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy.

No abstract