Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3β (GSK-3β), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase–dependent protein kinase that phosphorylates GSK-3β on ser 9. Using adenovirus-mediated gene transfer of GSK-3β containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3β is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3β regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3β as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

In the failing heart, there is a clear prohypertrophic activity profile, likely occurring in response to increased systolic wall stress and neurohormonal mediators. However, with the activation of these hypertrophic pathways, potent proapoptotic and antiapoptotic signals may also be generated. Therapies directed at altering the balance of activity of these signaling pathways could potentially alter the progression of heart failure.

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.

Abstract-Female gender and estrogen-replacement therapy in postmenopausal women are associated with improved heart failure survival, and physiological replacement of 17␤-estradiol (E2) reduces infarct size and cardiomyocyte apoptosis in animal models of myocardial infarction (MI). Here, we characterize the molecular mechanisms of E2 effects on cardiomyocyte survival in vivo and in vitro. Ovariectomized female mice were treated with placebo or physiological E2 replacement, followed by coronary artery ligation (placebo-MI or E2-MI) or sham operation (sham) and hearts were harvested 6, 24, and 72 hours later. After MI, E2 replacement significantly increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis assessed by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining and caspase 3 activation. In vitro, E2 at 1 or 10 nmol/L caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis as measured by TUNEL staining, caspase 3 activation, and DNA laddering in cultured neonatal rat cardiomyocytes. The E2-mediated reduction in apoptosis was reversed by an estrogen receptor (ER) antagonist, ICI 182,780, and by phospho-inositide-3 kinase inhibitors, LY294002 and Wortmannin. Overexpression of a dominant negative-Akt construct also blocked E2-mediated reduction in cardiomyocyte apoptosis. These data show that E2 reduces cardiomyocyte apoptosis in vivo and in vitro by ER-and phospho-inositide-3 kinase-Aktdependent pathways and support the relevance of these pathways in the observed estrogen-mediated reduction in myocardial injury. Key Words: estrogen Ⅲ estrogen receptors Ⅲ myocardial infarction Ⅲ cardiomyocyte Ⅲ apoptosis Ⅲ Akt Ⅲ PI3 kinase H eart failure is a growing public health problem, 1 with several studies demonstrating that women with heart failure have a better prognosis than men. 2,3,4,5,6,7 Whether endogenous sex hormones contribute to these differences in prognosis remains unknown. However, observational studies have demonstrated that postmenopausal women taking estrogen after a myocardial infarction (MI) have a lower incidence of heart failure. 8,9 Furthermore, retrospective analyses of multicenter heart failure trials have shown that postmenopausal women taking estrogen have a better prognosis than women not on estrogen, 10,11 supporting that estrogen may improve heart failure prognosis.Several studies have demonstrated increased cardiomyocyte apoptosis in failing hearts, 12,13 and further evidence suggests that apoptosis contributes to heart failure progression. 14,15 Moreover, autopsy studies have shown that female gender is associated with less cardiomyocyte apoptosis in normal and failing hearts compared with males. 16,17,18,19 These observed gender differences in cardiomyocyte survival provide a plausible explanation for the beneficial effect of female gender on heart failure progression.We recently showed that physiological estrogen replacement in ovariectomized female mice reduces infarct size both early and late after left c...

␤-Catenin is a transcriptional activator that regulates embryonic development as part of the Wnt pathway and also plays a role in tumorigenesis. The mechanisms leading to Wnt-induced stabilization of ␤-catenin, which results in its translocation to the nucleus and activation of transcription, have been an area of intense interest. However, it is not clear whether stimuli other than Wnts can lead to important stabilization of ␤-catenin and, if so, what factors mediate that stabilization and what biologic processes might be regulated. Herein we report that ␤-catenin is stabilized in cardiomyocytes after these cells have been exposed to hypertrophic stimuli in culture or in vivo. The mechanism by which ␤-catenin is stabilized is distinctly different from that used by Wnt signaling. Although, as with Wnt signaling, inhibition of glycogen synthase kinase-3 remains central to hypertrophic stimulus-induced stabilization of ␤-catenin, the mechanism by which this occurs involves the recruitment of activated PKB to the ␤-catenin-degradation complex. PKB stabilizes the complex and phosphorylates glycogen synthase kinase-3 within the complex, inhibiting its activity directed at ␤-catenin. Finally, we demonstrate via adenoviral gene transfer that ␤-catenin is both sufficient to induce growth in cardiomyocytes in culture and in vivo and necessary for hypertrophic stimulus-induced growth. Thus, in these terminally differentiated cells, ␤-catenin is stabilized by hypertrophic stimuli acting via heterotrimeric G protein-coupled receptors. The stabilization occurs via a unique Wnt-independent mechanism and results in cellular growth.

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H 2 O 2 ) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H 2 O 2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H 2 O 2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H 2 O 2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/ reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H 2 O 2 . MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H 2 O 2 to a similar extent as with ischemia or ischemia/ reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SRMAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-␣-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.Ischemic and hypertensive myocardial disease is currently a major cause of mortality and morbidity. In these conditions, the heart is exposed to numerous cell stresses including increased production of reactive oxygen species (ROS), 1 ionic imbalances, osmotic stress, mechanical stress, and metabolic deprivation (reviewed in Refs. 1-4). In numerous cell lines, primary cultures, and tissues, cellular stresses activate "stress-regulated" mitogen-activated protein kinases (SR-MAPKs) (reviewed in Refs. 5 and 6). There are two relatively well characterized SR-MAPK families, the stress-activated protein kinases (SAPKs) and the p38-MAPKs. SAPKs are alternatively known as the c-Jun N-terminal kinases (JNKs), although, strictly speaking, the JNK terminology applies to the human enzymes, whereas the SAPK terminology applies to the rat enzymes.Substrates for SAPKs/JNKs include the transcription factors c-Jun (7, 8), ATF-2 (9 -12), and Elk-1 (13, 14). Phosphorylation of these transcription factors in their trans-activation domains leads to an increase in their ability to trans-activate transcription. p38-MAPK (15, 16) (alternatively known as cytokinesuppressive antiinflammatory drug-binding protein (17), reactivating kinase (18), Mxi2 (19), or stress-activated protein kinase-2 (20)) is a mammalian homolog of the yeast osmosensing protein kinase HOG-1. Like SAPKs/JNKs, p38-MAPKs also phosphorylate transcription factors (ATF2 (11), CHOP/ GADD153 (21), and MEF2C (22)),...

Glycogen synthase kinase (GSK) 3␤ is a negative regulator of stress-induced cardiomyocyte hypertrophy. It is not clear, however, if GSK-3␤ plays any role in regulating normal cardiac growth and cardiac function. Herein we report that a transgenic mouse expressing wild type GSK-3␤ in the heart has a dramatic impairment of normal post-natal cardiomyocyte growth as well as markedly abnormal cardiac contractile function. The most striking phenotype, however, is grossly impaired diastolic relaxation, which leads to increased filling pressures of the left ventricle and massive atrial enlargement. This is due to profoundly abnormal calcium handling, leading to an inability to normalize cytosolic [Ca 2؉ ] in diastole. The alterations in calcium handling are due at least in part to direct down-regulation of the sarcoplasmic reticulum calcium ATPase (SERCA2a) by GSK-3␤, acting at the level of the SERCA2 promoter. These studies identify GSK-3␤ as a regulator of normal growth of the heart and are the first of which we are aware, to demonstrate regulation of expression of SERCA2a, a critical determinant of diastolic function, by a cytosolic signaling pathway, the activity of which is dynamically modulated. De-regulation of GSK-3␤ leads to severe systolic and diastolic dysfunction and progressive heart failure. Because down-regulation of SERCA2a plays a central role in the diastolic and systolic dysfunction of patients with heart failure, these findings have potential implications for the therapy of this disorder.

It is well known that iodinated radiographic contrast media may cause kidney dysfunction, particularly in patients with preexisting renal impairment associated with diabetes. This dysfunction, when severe, will cause acute renal failure (ARF). We may define contrast-induced Acute Kidney Injury (AKI) as ARF occurring within 24–72 hrs after the intravascular injection of iodinated radiographic contrast media that cannot be attributed to other causes. The mechanisms underlying contrast media nephrotoxicity have not been fully elucidated and may be due to several factors, including renal ischaemia, particularly in the renal medulla, the formation of reactive oxygen species (ROS), reduction of nitric oxide (NO) production, and tubular epithelial and vascular endothelial injury. However, contrast-induced AKI can be prevented, but in order to do so, we need to know the risk factors. We have reviewed the risk factors for contrast-induced AKI and measures for its prevention, providing a long list of references enabling readers to deeply evaluate them both.