Peter M. Kang

The conserved phosphoinositide 3-kinase pathway determines heart size in mice

Phosphoinositide 3‐kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, we have created and characterized transgenic mice expressing constitutively active or dominant‐negative mutants of PI3K in the heart. Cardiac‐ specific expression of constitutively active PI3K resulted in mice with larger hearts, while dominant‐negative PI3K resulted in mice with smaller hearts. The increase or decrease in heart size was associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, were not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals.

Apoptosis and Heart Failure: A Critical Review of the Literature

This MiniReview is part of a thematic series on Apoptosis in the Cardiovascular System , which includes the following articles: Apoptosis and Heart Failure: A Critical Review of the Literature Vascular Cell Apoptosis in Remodeling, Restenosis, and Plaque Rupture Apoptosis During Cardiovascular Development Myocyte Apoptosis in Ischemic Heart Disease Endothelial Cell Apoptosis in Angiogenesis and Vessel Regression Richard Kitsis, Editor When the concept of apoptosis was introduced in the 1970s,1 it attracted only limited attention. However, less than two decades ago, Horvitz and colleagues2 3 4 identified its essential genetic components in the roundworm, Caenorhabditis elegans , and apoptosis emerged as a significant research front. The explosion of knowledge that took place is represented by the accumulation of >25 000 studies in the last 5 years alone. It is now clear that apoptosis is an important aspect of normal organ development and cellular regulation and that it plays a role in a wide variety of physiological and pathological conditions. However, there is still much debate and controversy concerning the role of apoptosis in heart failure. To address the issues of its presence in, significance for, and overall contribution to heart failure, we will review the currently available literature and then discuss its implications for future research and treatment strategies in heart failure. The etiology of heart failure involves multiple agents and conditions,5 but the progressive loss of cardiac myocytes is one of the most important pathogenic components. During the past few years, there has been accumulating evidence in both human and animal models suggesting that apoptosis may be an important mode of cell death during heart failure (Table 1⇓). Therefore, the possibility of limiting cardiac myocyte loss by inhibiting apoptosis has potentially important implications in the treatment of heart failure. View this table: Table 1. Heart Failure Models Associated With Increased …

Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice

ABSTRACT One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.

Phosphoinositide 3-kinase(p110α) plays a critical role for the induction of physiological, but not pathological, cardiac hypertrophy

An unresolved question in cardiac biology is whether distinct signaling pathways are responsible for the development of pathological and physiological cardiac hypertrophy in the adult. Physiological hypertrophy is characterized by a normal organization of cardiac structure and normal or enhanced cardiac function, whereas pathological hypertrophy is associated with an altered pattern of cardiac gene expression, fibrosis, cardiac dysfunction, and increased morbidity and mortality. The elucidation of signaling cascades that play distinct roles in these two forms of hypertrophy will be critical for the development of more effective strategies to treat heart failure. We examined the role of the p110α isoform of phosphoinositide 3-kinase (PI3K) for the induction of pathological hypertrophy (pressure overload-induced) and physiological hypertrophy (exercise-induced) by using transgenic mice expressing a dominant negative (dn) PI3K(p110α) mutant specifically in the heart. dnPI3K transgenic mice displayed significant hypertrophy in response to pressure overload but not exercise training. dnPI3K transgenic mice also showed significant dilation and cardiac dysfunction in response to pressure overload. Thus, PI3K(p110α) appears to play a critical role for the induction of physiological cardiac growth but not pathological growth. PI3K(p110α) also appears essential for maintaining contractile function in response to pathological stimuli.

Direct Activation of Mitochondrial Apoptosis Machinery by c-Jun N-terminal Kinase in Adult Cardiac Myocytes

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochromec release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.

Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH)2D2), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings. Compared with DSS rats fed a high-salt (HS) diet (6% NaCl for 6 weeks), HS+PC was associated with lower heart and lung weights, reduced LV mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Blood pressures did not significantly differ between the HS groups. Plasma brain natriuretic peptide levels, and cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin were significantly reduced in the HS+PC animals. Microarray analyses revealed 45 specific HS genes modified by PC. In a retrospective pilot study of hemodialysis patients, PC-treated subjects demonstrated improved diastolic function and a reduction in LV septal and posterior wall thickness by echocardiography compared with untreated patients. In summary, PC attenuates the development of LV alterations in DSS rats, and these effects should be examined in human clinical trials.

MicroRNA-1 Negatively Regulates Expression of the Hypertrophy-Associated Calmodulin and Mef2a Genes

ABSTRACT Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated posttranscriptionally in heart failure. The cardiomyocyte-restricted microRNA miR-1 inhibited the translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3′ untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through calcineurin to NFAT. miR-1 also negatively regulated the expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with the downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium signaling components calmodulin, Mef2a, and Gata4.

Morphological and Molecular Characterization of Adult Cardiomyocyte Apoptosis During Hypoxia and Reoxygenation

Apoptosis has been implicated in ischemic heart disease, but its mechanism in cardiomyocytes has not been elucidated. In this study, we investigate the effects of hypoxia and reoxygenation in adult cardiomyocytes and the molecular mechanism involved in cardiomyocyte apoptosis. Morphologically, reoxygenation induced rounding up of the cells, appearance of membrane blebs that were filled with marginated mitochondria, and ultrastructural findings characteristic of apoptosis. Reoxygenation (18 hours of reoxygenation after 6 hours of hypoxia) and prolonged hypoxia (24 hours of hypoxia) resulted in a 59% and 51% decrease in cellular viability, respectively. During reoxygenation, cell death occurred predominantly via apoptosis associated with appearance of cytosolic cytochrome c and activation of caspase-3 and -9. However, nonapoptotic cell death predominated during prolonged hypoxia. Both caspase inhibition and Bcl-2 overexpression during reoxygenation significantly improved cellular viability through inhibition of apoptosis but had minimal effect on hypoxia-induced cell death. Bcl-2 overexpression blocked reoxygenation-induced cytochrome c release and activation of caspase -3 and -9, but caspase inhibition alone did not block cytochrome c release. These results suggest that apoptosis predominates in cardiomyocytes after reoxygenation through a mitochondrion-dependent apoptotic pathway, and Bcl-2 prevents reoxygenation-induced apoptosis by inhibiting cytochrome c release from the mitochondria and prevents activation of caspase-3 and -9.

Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed up-regulation of genes associated with apoptosis and fibrosis, including members of the TGF-β pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.

Angiotensin II receptor antagonists: A new approach to blockade of the renin-angiotensin system

A-II exerts its activity on various target tissues by binding to its receptors. The discovery of local RASs and A-II receptors within various tissues has generated interest in the clinical usefulness of RAS inhibition by directly blocking the action of A-II at the receptor level. Different A-II receptor subtypes have been identified and subsequently termed AT1 and AT2. AT1-receptor subtypes are the predominant receptor subtypes existing in most organs and, by coupling to a transmembrane G protein, seem to be the main subtypes participating in the vasoactive responses of A-II. Saralasin, a peptide with specific A-II receptor-antagonistic activity, had limited practical long-term usefulness as a result of its short half-life, significant agonistic properties, and lack of oral bioavailability. The discovery of simple benzyl-substituted imidazoles, which possess weak but highly selective A-II receptor antagonistic properties, led to the development of losartan (DuP 753). Losartan is a potent, orally active, specific, competitive nonpeptide A-II receptor antagonist that appears to be an effective antihypertensive agent both in animal studies and in preliminary clinical trials. The therapeutic usefulness of losartan, however, is not limited to its antihypertensive effects. The potential benefits of A-II receptor antagonists include roles in postmyocardial infarction therapy, slowing A-II-induced cardiac hypertrophy, 154, 155 slowing the progression of heart failure, preventing postangioplasty restenosis, and in slowing the progression of renal disease. Furthermore, losartan, a selective A-II type 1 (AT1) receptor antagonist, has also been a valuable pharmacologic probe for studying the mechanism of A-II stimulation of its receptors. A-II receptor antagonism appears to be as effective as ACE inhibition in the treatment of hypertension and other pathologic processes that involve the RAS and may offer an alternative to those patients who cannot tolerate ACE inhibitors because of their side effects.