Richard N. Kitsis

Akt Promotes Survival of Cardiomyocytes In Vitro and Protects Against Ischemia-Reperfusion Injury in Mouse Heart

BACKGROUND IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1-regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo. METHODS AND RESULTS IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1-mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1-induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury. CONCLUSIONS These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.

The MEK1–ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

Members of the mitogen‐activated protein kinase (MAPK) cascade such as extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac‐restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus‐infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus‐infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1–ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.

A mechanistic role for cardiac myocyte apoptosis in heart failure

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80-250 myocytes per 10(5) nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 10(5) nuclei, compared with 1.5 myocytes per 10(5) nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Cell Death in the Pathogenesis of Heart Disease: Mechanisms and Significance

Cell death was once viewed as unregulated. It is now clear that at least a portion of cell death is a regulated cell suicide process. This type of death can exhibit multiple morphologies. One of these, apoptosis, has long been recognized to be actively mediated, and many of its underlying mechanisms have been elucidated. Moreover, necrosis, the traditional example of unregulated cell death, is also regulated in some instances. Autophagy is usually a survival mechanism but can occur in association with cell death. Little is known, however, about how autophagic cells die. Apoptosis, necrosis, and autophagy occur in cardiac myocytes during myocardial infarction, ischemia/reperfusion, and heart failure. Pharmacological and genetic inhibition of apoptosis and necrosis lessens infarct size and improves cardiac function in these disorders. The roles of autophagy in ischemia/reperfusion and heart failure are unresolved. A better understanding of these processes and their interrelationships may allow for the development of novel therapies for the major heart syndromes.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction.

Death begets failure in the heart

Recently, low--but abnormal--rates of cardiomyocyte apoptosis have been observed in failing human hearts. Genetic and pharmacological studies suggest that this cell death is causally linked to heart failure in rodent models. Herein, we review these data and discuss potential therapeutic implications.

The Mitochondrial Apoptotic Pathway Is Activated by Serum and Glucose Deprivation in Cardiac Myocytes

Many cell types undergo apoptosis under conditions of ischemia. Little is known, however, about the molecular pathways that mediate this response. A cellular and biochemical approach to elucidate such signaling pathways was undertaken in primary cultures of cardiac myocytes, a cell type that is especially sensitive to ischemia-induced apoptosis. Deprivation of serum and glucose, components of ischemia in vivo, resulted in myocyte apoptosis, as determined by nuclear fragmentation, internucleosomal cleavage of DNA, and processing of caspase substrates. These manifestations of apoptosis were blocked by zVAD-fmk, a peptide caspase inhibitor, indicating that caspase activity is necessary for the progression of apoptosis in this model. In contrast to control cells, apoptotic myocytes exhibited cytoplasmic accumulation of cytochrome c, indicating release from the mitochondria. Furthermore, both caspase-9 and caspase-3 were processed to their active forms in serum-/glucose-deprived myocytes. Caspase processing, but not cytochrome c release, was inhibited by zVAD-fmk, placing the latter event upstream of caspase activation. This evidence demonstrates that components of ischemia activate the mitochondrial death pathway in cardiac myocytes.

Calcineurin-Mediated Hypertrophy Protects Cardiomyocytes From Apoptosis In Vitro and In Vivo An Apoptosis-Independent Model of Dilated Heart Failure

We have previously shown that the calcium-calmodulin-regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-x(L) gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion-induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gqalpha-transgenic mice or in cultured cardiomyocytes. Collectively, these data identify a calcineurin-dependent mouse model of dilated heart failure that is independent of apoptosis.

Inhibition of Cardiac Myocyte Apoptosis Improves Cardiac Function and Abolishes Mortality in the Peripartum Cardiomyopathy of Gαq Transgenic Mice

Background—Although the occurrence of cardiac myocyte apoptosis during heart failure has been documented, its importance in pathogenesis is unknown. Transgenic mice with cardiac-restricted overexpression of G&agr;q exhibit a lethal, peripartum cardiomyopathy accompanied by apoptosis. To test whether apoptosis is causally linked to heart failure, we assessed whether inhibiting this cell death would improve left ventricular function and survival in the G&agr;q peripartum cardiomyopathy model. Methods and Results—The potent polycaspase inhibitor IDN-1965 or vehicle was administered subcutaneously to G&agr;q mice by osmotic minipump beginning on day 12 of pregnancy and continuing through euthanasia at day 14 postpartum. As expected, IDN-1965 markedly suppressed cardiac caspase-3–like activity (86.5%; P <0.01), accompanied by reduction in the frequency of cardiac myocyte apoptosis from 1.9±0.3% to 0.2±0.1% (P <0.01). Animals receiving IDN-1965 exhibited significant improvements in left ventricular end-diastolic dimension (vehicle, 4.7±0.1 mm; IDN-1965, 4.2±0.1 mm; P <0.01), fractional shortening (vehicle, 30.7±1.2%; IDN-1965, 38.9±1.0%; P <0.01), positive (vehicle, 3972±412; IDN-1965, 5870±295; P <0.01) and negative (vehicle, 2365±213; IDN-1965, 3413±201; P <0.01) dP/dt, and complete suppression of mortality (vehicle, 6 of 20 died; IDN-1965, 0 of 14 died; P <0.05). Conclusions—Reduction in cardiac myocyte apoptosis by caspase inhibition improved left ventricular function and survival in pregnant G&agr;q mice. These data indicate that cardiac myocyte apoptosis plays a causal role in the pathogenesis of cardiomyopathy in this model. Caspase inhibition may provide a novel therapeutic target for heart failure.

Echocardiographic Assessment of Left Ventricular Mass and Systolic Function in Mice

The increasing use of transgenic mouse models for investigating the mechanisms of cardiac growth and function has made it important to develop noninvasive methods for assessing murine cardiac anatomy, size, and function. At present, murine cardiac mass can be determined only at necropsy. Left ventricular (LV) function can be assessed by use of various catheterization techniques, but these approaches are usually terminal procedures and provide no information about chamber anatomy and dimensions. Although transthoracic echocardiography has been used to study the LVs of rats and larger animals, the considerably smaller LV masses and somewhat faster heart rates of mice pose significant challenges to obtaining good-quality echocardiograms. In this study we tested the hypothesis that transthoracic echocardiography can image the murine LV as well as provide assessments of LV mass and function. Our results in a series of 33 mice, including normal, transgenic, and aortic-banded subgroups, demonstrate the capability of transthoracic two-dimensionally directed M-mode echocardiography in mice to (1) obtain good-quality images, (2) produce estimates of LV mass having good correlations with directly determined LV mass in normal mice, (3) detect LV hypertrophy noninvasively in different experimental models, and (4) identify impaired LV systolic function. Thus, echocardiography appears to be a promising approach for noninvasively assessing LV mass and function in mice.