Stephen F. Vatner

Type 5 Adenylyl Cyclase Disruption Increases Longevity and Protects Against Stress

Mammalian models of longevity are related primarily to caloric restriction and alterations in metabolism. We examined mice in which type 5 adenylyl cyclase (AC5) is knocked out (AC5 KO) and which are resistant to cardiac stress and have increased median lifespan of approximately 30%. AC5 KO mice are protected from reduced bone density and susceptibility to fractures of aging. Old AC5 KO mice are also protected from aging-induced cardiomyopathy, e.g., hypertrophy, apoptosis, fibrosis, and reduced cardiac function. Using a proteomic-based approach, we demonstrate a significant activation of the Raf/MEK/ERK signaling pathway and upregulation of cell protective molecules, including superoxide dismutase. Fibroblasts isolated from AC5 KO mice exhibited ERK-dependent resistance to oxidative stress. These results suggest that AC is a fundamentally important mechanism regulating lifespan and stress resistance.

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure

A small molecule improves cardiac function by accelerating the transition of myosin into a force-producing state. Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5′-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Activation of the Cardiac Proteasome During Pressure Overload Promotes Ventricular Hypertrophy

Background— The adaptation of cardiac mass to hemodynamic overload requires an adaptation of protein turnover, ie, the balance between protein synthesis and degradation. We tested 2 hypotheses: (1) chronic left ventricular hypertrophy (LVH) activates the proteasome system of protein degradation, especially in the myocardium submitted to the highest wall stress, ie, the subendocardium, and (2) the proteasome system is required for the development of LVH. Methods and Results— Gene and protein expression of proteasome subunits and proteasome activity were measured separately from left ventricular subendocardium and subepicardium, right ventricle, and peripheral tissues in a canine model of severe, chronic (2 years) LVH induced by aortic banding and then were compared with controls. Both gene and protein expressions of proteasome subunits were increased in LVH versus control (P<0.05), which was accompanied by a significant (P<0.05) increase in proteasome activity. Posttranslational modification of the proteasome was also detected by 2-dimensional gel electrophoresis. These changes were found specifically in left ventricular subendocardium but not in left ventricular subepicardium, right ventricle, or noncardiac tissues from the same animals. In a mouse model of chronic pressure overload, a 50% increase in heart mass and a 2-fold increase in proteasome activity (both P<0.05 versus sham) were induced. In that model, the proteasome inhibitor epoxomicin completely prevented LVH while blocking proteasome activation. Conclusions— The increase in proteasome expression and activity found during chronic pressure overload in myocardium submitted to higher stress is also required for the establishment of LVH.

Aging Increases Aortic MMP-2 Activity and Angiotensin II in Nonhuman Primates

Abstract— To seek evidence that the nonhuman primate arterial wall, as it ages in the absence of atherosclerosis, exhibits alterations in pathways that are involved in the pathogenesis of experimental atherosclerosis, we assessed aortic matrix metalloproteinase‐2 (MMP‐2) and its regulators, ie, membrane type‐1 of matrix metalloproteinase (MT1‐MMP) and tissue inhibitor of matrix metalloproteinase‐2 (TIMP‐2), and the expression of angiotensin II (Ang II), angiotensin‐converting enzyme (ACE), and chymase in young (6.4±0.7 years) and old (20.0±1.9 years) male monkeys. With advancing age, (1) the intimal thickness increased 3‐fold and contained numerous vascular smooth muscle cells and matrix, but no inflammatory cells; (2) the intimal MMP‐2 antibody–staining fraction increased by 80% (P <0.01); (3) in situ zymography showed that MMP‐2 activity, mainly confined to the intima, increased 3‐fold (P <0.01); (4) the MT1‐MMP antibody–staining fraction increased by 150% (P <0.001), but the TIMP‐2 antibody–staining fraction did not significantly change; (5) steady levels of the mRNA‐staining fraction (via in situ hybridization) for MMP‐2 increased 7‐fold, for MT1‐MMP increased 9‐fold, and for TIMP‐2 increased 2‐fold (all P <0.001); and (6) intimal Ang II and ACE immunofluorescence were increased 5‐fold and 5.6‐fold, respectively, and colocalized with MMP‐2. Thus, age‐associated arterial remodeling and the development and progression of experimental atherosclerosis in young animals share common mechanisms, ie, MMP‐2 activation and increased Ang II signaling. This might explain, in part, the dramatically exaggerated prevalence and severity of vascular diseases with aging.

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.

Program of Cell Survival Underlying Human and Experimental Hibernating Myocardium

Hibernating myocardium refers to chronically dysfunctional myocardium in patients with coronary artery disease in which cardiac viability is maintained and whose function improves after coronary revascularization. It is our hypothesis that long-term adaptive genomic mechanisms subtend the survival capacity of this ischemic myocardium. Therefore, the goal of this study was to determine whether chronic repetitive ischemia elicits a gene program of survival protecting hibernating myocardium against cell death. Accordingly, we measured the expression of survival genes in hibernating myocardium, both in patients surgically treated for hibernation and in a chronic swine model of repetitive ischemia reproducing the features of hibernation. Human hibernating myocardium was characterized by an upregulation of genes and corresponding proteins involved in anti-apoptosis (IAP), growth (VEGF, H11 kinase), and cytoprotection (HSP70, HIF-1&agr;, GLUT1). In the swine model, the same genes and proteins were upregulated after repetitive ischemia, which was accompanied by a concomitant decrease in myocyte apoptosis. These changes characterize viable tissue, because they were not found in irreversibly injured myocardium. Our report demonstrates a novel mechanism by which the activation of an endogenous gene program of cell survival underlies the sustained viability of the hibernating heart. Potentially, promoting such a program offers a novel opportunity to salvage postmitotic tissues in conditions of ischemia.

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein’s anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

Caloric Restriction Reduces Growth of Mammary Tumors and Metastases

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells. CR also decreased proliferation and angiogenesis and increased apoptosis in tumors. CR reduced levels of insulin, leptin, insulin-like growth factor 1, insulin-like growth factor binding protein 3 and increased adiponectin in tumors. We also demonstrated that tumors from CR mice possessed lower levels of transforming growth factor-β, lower intratumor deposition of collagen IV and reduced invasiveness due to a decrease in tumor secretion of active matrix metalloproteinase 9. Our results suggest that CR-induced metabolic and signaling changes affect the stroma and the tumor cells resulting in a microenvironment that prevents proliferation of breast tumors and their metastases.

AUTOPHAGY: A NOVEL PROTECTIVE MECHANISM IN CHRONIC ISCHEMIA

During the search for cardioprotective mechanisms in a porcine model of chronic myocardial ischemia and hibernating myocardium, we discovered evidence for autophagy, which could be involved in the protection against apoptosis. Autophagy is a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which become sequestered in a double-membrane-bound vesicle, termed autophagosome, and subsequently degrade upon fusion with lysosomes. The dauer phase in C. elegans shares similarities with the induction of autophagy in chronically ischemic (hibernating) myocardium. In this sense, autophagy is an essential mechanism for survival which is activated by environmental stresses and confers stress resistance to the organism. Our study provided insight into understanding of the protective mechanism of autophagy in chronic ischemia.

Endocytosis Machinery Is Required for β1-Adrenergic Receptor-Induced Hypertrophy in Neonatal Rat Cardiac Myocytes

AIMS Cardiac hypertrophy by activation of the beta-adrenergic receptor (beta AR) is mediated more efficiently by the beta1-AR than by the beta2-AR. We investigated the signalling mechanism by which the beta1-AR mediates cardiac hypertrophy. METHODS AND RESULTS Experiments were performed in cultured neonatal rat cardiomyocytes. Hypertrophy was determined by the protein/DNA content and atrial natriuretic factor transcription. Phosphorylation of Akt and Src was assessed by immunoblotting. Isoproterenol (ISO, 10 microM), a non-selective beta-AR agonist, caused selective downregulation of the beta1-AR (control beta1 vs. beta2: 35 vs. 65%, Bmax 78 +/- 4 fmol/mg; 4 h, 10 vs. 90%, 61 +/- 5 fmol/mg). Concanavalin A (Con A, 0.5 microg/mL), an inhibitor of endocytosis, prevented downregulation of beta1-ARs by ISO treatment (4 h, 35 vs. 65%, 73 +/- 8 fmol/mg), suggesting that beta1-ARs selectively undergo endocytosis. Interference with beta1-AR endocytosis by Con A, carboxyl terminal peptide of beta-AR kinase-1, dominant negative (DN) beta-arrestin-1, or DN dynamin inhibited beta-adrenergic hypertrophy, suggesting that the endocytosis machinery plays a key role in mediating beta-adrenergic hypertrophy. Activation of Akt by the beta1-AR was blocked by inhibition of the endocytosis machinery, suggesting that endocytosis mediates activation of Akt. Akt plays a critical role in beta-adrenergic hypertrophy, since DN Akt blocked ISO-induced hypertrophy. beta-Adrenergic activation of Akt is mediated by Src, which associates with the endocytosis machinery and is necessary and sufficient to mediate beta-adrenergic hypertrophy. CONCLUSION Activation of the endocytosis machinery is required for activation of Akt, which, in turn, critically mediates beta1-AR-induced cardiac hypertrophy.