Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.

Abstract-Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of ␤1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of ␤1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the ␤1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte ␤1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of ␤1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old ␤1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt max and LV dP/dt min ) as compared with control groups (nϭ8 to 10 of each, PϽ0.01). Hemodynamic loading imposed by 7 days of transverse aortic constriction showed that the ␤1 integrin knockout mice were intolerant of this stress as they had 53% survival versus 88% in controls (nϭ15 each). Key Words: extracellular matrix Ⅲ homologous recombination Ⅲ Cre recombinase Ⅲ heart Ⅲ positron emission tomography I ntegrins are a large family of heterodimeric cell surface receptors composed of ␣ and ␤ subunits. They function in cell-extracellular matrix (ECM) adhesion and cell-cell adhesion, and signal bidirectionally across the cell membrane. 1,2 Further, they serve as mechanotransducers, converting mechanical signals to biochemical ones. 3 This combination of properties allows integrins to play important roles in cell growth, differentiation, migration, and survival 4 and also makes them attractive candidates for essential roles in the developing and postnatal heart.Our previous work has shown that ␤1 integrins are linked to the hypertrophic response of cultured ventricular myocytes and also that dominant-negative disruption of integrin function in transgenic mice resulted in cardiac fibrosis and abnormal cardiac function. 5-7 Ablation of ␤1 integrin expression in all murine tissues resulted in gastrulation defects and death by E5.5 of the 21-day gestation period. 8,9 Chimeric mice as well as embryoid bodies constructed from ␤1 integrin-null cells showed delayed development and differentiation of ␤1-deficient cells along the cardiac lineage, as well as abnormal sarcomerogenesis of these cardiac-like cells. 10 Although a few ␤1 integrin-null cells were detected in the myocardium of chimeric mice, cellular debris was always detected along with the null cells. These null cells were completely lost from the myocardium of the chimeric mouse heart by 6 months of age.To more specifically evaluate the role of ␤1 integrin in the myocardium, we used a Cre-loxP gene targeting approach. Cre recombinase expression driven by the myosin light chain-2 ventricular (MLC-2v) promoter caused ␤1 integrin gene excision exclusively in ventricular cardiac myocytes. 11 Our results in these ...

Many patients with Huntington's disease (HD) exhibit disturbances in their daily cycle of sleep and wake as part of their symptoms. These patients have difficulty sleeping at night and staying awake during the day, which has a profound impact on the quality of life of the patients and their care-givers. In the present study, we examined diurnal and circadian rhythms of four models of HD including the BACHD, CAG 140 knock-in and R6/2 CAG 140 and R6/2 CAG 250 lines of mice. The BACHD and both R6/2 lines showed profound circadian phenotypes as measured by wheel-running activity. Focusing on the BACHD line for further analysis, the amplitude of the rhythms in the BACHD mice declined progressively with age. In addition, the circadian regulation of heart rate and body temperature in freely behaving BACHD mice were also disrupted. Furthermore, the distribution of sleep as well as the autonomic regulation of heart rate was disrupted in this HD model. To better understand the mechanistic underpinnings of the circadian disruption, we used electrophysiological tools to record from neurons within the central clock in the suprachiasmatic nucleus (SCN). The BACHD mice exhibit reduced rhythms in spontaneous electrical activity in SCN neurons. Interestingly, the expression of the clock gene PERIOD2 was not altered in the SCN of the BACHD line. Together, this data is consistent with the hypothesis that the HD mutations interfere with the expression of robust circadian rhythms in behavior and physiology. The data raise the possibility that the electrical activity within the central clock itself may be altered in this disease.

Abstract-c-Myc, a protooncogene, mediates both proliferative and cellular growth in many cell types. Although not expressed in the adult heart under normal physiological conditions, Myc expression is rapidly upregulated in response to hypertrophic stimuli. Although Myc is capable of sustaining hyperplastic growth in fetal myocytes, the effects of its re-expression in adult postmitotic myocardium and its role in mediating cardiac hypertrophy are unknown.

Null mutation of dystrophin causes the lethal pathology of Duchenne muscular dystrophy (DMD) in which there is progressive pathology of skeletal and cardiac muscles. A large proportion of DMD patient deaths are attributable to cardiac dysfunction associated with ventricular fibrosis, arrhythmias and conduction abnormalities, although the relationships between the dystrophin mutation and the cardiac defects are unknown. Here, we tested whether cardiac pathology in dystrophin-deficient mdx mice can be corrected by the elevated production of nitric oxide (NO) by the myocardium. Dystrophin-deficient mdx mice were produced in which there was myocardial expression of a neuronal nitric oxide synthase (nNOS) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) attained by radiotelemetry of freely ambulatory mice showed that mdx mice displayed cardiac abnormalities that are characteristic of DMD patients, including deep Q-waves, diminished S:R ratios, polyphasic R-waves and frequent premature ventricular contractions. All of these ECG abnormalities in mdx mice were improved or corrected by nNOS transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by nNOS transgene expression. These findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.

c‐Myc (Myc) is highly expressed in developing embryos where it regulates body size by controlling proliferation but not cell size. However, Myc is also induced in many postmitotic tissues, including adult myocardium, in response to stress where the predominant form of growth is an increase in cell size (hypertrophy) and not number. The function of Myc induction in this setting is unproven. Therefore, to explore Myc's role in hypertrophic growth, we created mice where Myc can be inducibly inactivated, specifically in adult myocardium. Myc‐deficient hearts demonstrated attenuated stress‐induced hypertrophic growth, secondary to a reduction in cell growth of individual myocytes. To explore the dependence of Myc‐induced cell growth on CycD2, we created bigenic mice where Myc can be selectively activated in CycD2‐null adult myocardium. Myc‐dependent hypertrophic growth and cell cycle reentry is blocked in CycD2‐deficient hearts. However, in contrast to Myc‐induced DNA synthesis, hypertrophic growth is independent of CycD2‐induced Cdk2 activity. These data suggest that Myc is required for a normal hypertrophic response and that its growth‐promoting effects are also mediated through a CycD2‐dependent pathway.

The pocket protein family of tumor suppressors, and Rb specifically, have been implicated as controlling terminal differentiation in many tissues, including the heart. To establish the biological functions of Rb in the heart and overcome the early lethality caused by germ line deletion of Rb, we used a Cre/loxP system to create conditional, heart-specific Rb-deficient mice. Mice that are deficient in Rb exclusively in cardiac myocytes (CRb L/L ) are born with the expected Mendelian distribution, and the adult mice displayed no change in heart size, myocyte cell cycle distribution, myocyte apoptosis, or mechanical function. Since both Rb and p130 are expressed in the adult myocardium, we created double-knockout mice (CRb L/L p130 ؊/؊ ) to determine it these proteins have a shared role in regulating cardiac myocyte cell cycle progression. Adult CRb L/L p130 ؊/؊ mice demonstrated a threefold increase in the heart weight-to-body weight ratio and showed increased numbers of bromodeoxyuridine-and phosphorylated histone H3-positive nuclei, consistent with persistent myocyte cycling. Likewise, the combined deletion of Rb plus p130 up-regulated myocardial expression of Myc, E2F-1, and G 1 cyclin-dependent kinase activities, synergistically. Thus, Rb and p130 have overlapping functional roles in vivo to suppress cell cycle activators, including Myc, and maintain quiescence in postnatal cardiac muscle.

Abstract-The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca 2ϩ through voltage-dependent Ca 2ϩ channels. Ca 2ϩ influx induces a release of Ca 2ϩ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca 2ϩ homeostasis, Ca 2ϩ entry is balanced by efflux mediated by the sarcolemmal Na ϩ -Ca 2ϩ exchanger. In the absence of Na ϩ -Ca 2ϩ exchange, it would be expected that cardiac myocytes would overload with Ca 2ϩ . Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na ϩ -Ca 2ϩ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na ϩ -Ca 2ϩ exchanger as measured by both immunoblots and microarray analysis. Ca 2ϩ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca 2ϩ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca 2ϩ influx. Rather than upregulate other Ca 2ϩ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na ϩ -Ca 2ϩ exchanger by limiting Ca 2ϩ influx. With our current understanding of excitation-contraction coupling, it is almost inconceivable that myocardium could survive in the absence of Na ϩ -Ca 2ϩ exchange activity. Without a vigorous Ca 2ϩ efflux mechanism, cardiac myocytes should Ca 2ϩ overload, leading to a nonfunctional myocardium. Four laboratories, including ours, have reported that global knockout of the Na ϩ -Ca 2ϩ exchanger, NCX1, is embryonic lethal. 3-6 The result is not surprising and the lethality has been attributed to a cardiac phenotype. 3,5 One study, however, indicated that the lethality had an extracardiac origin. 7 Unexpectedly, heart tubes from NCX knockout embryos, dissected at day 9.5, had almost normal Ca 2ϩ transients in response to electrical stimulation. 5 homeostasis. The heart tubes were operating under low stress conditions (1 Hz stimulation at 26°C), however, and did not tolerate interventions (eg, increased stimulation rate) that increased the need for Ca 2ϩ efflux. Additionally, day 9.5 NCX knockout embryos are within 1 day of death and other adaptations of the myocardium have occurred. 8 Thus, embryonic heart tubes are a difficult preparation and not ideal for analysis of NCX ablation.We have now generated, using Cre/loxP technology, mice with a cardiac-specific knockout of NCX1. The exchanger is completely ablated in at least 80% of the myocytes. Strikingly, these mice survive to adulthood with diminished, yet surprisingly good, cardiac function. We...