Hui Ge

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.

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from beta-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressure-overload hypertrophy.

Type 5 adenylyl cyclase increases oxidative stress by transcriptional regulation of manganese superoxide dismutase via the SIRT1/FoxO3a pathway.

Background— For reasons that remain unclear, whether type 5 adenylyl cyclase (AC5), 1 of 2 major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol in AC5 transgenic (Tg) mice and the signaling mechanisms involved. Methods and Results— Chronic isoproterenol increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular ejection fraction fell 1.9-fold more than wild type, along with greater left ventricular dilation and increased fibrosis, apoptosis, and hypertrophy. Oxidative stress induced by chronic isoproterenol, detected by 8-OhDG was 15% greater, P=0.007, in AC5 Tg hearts, whereas protein expression of manganese superoxide dismutase (MnSOD) was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be attributable to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 knockout (KO) was lost in AC5 KO×MnSOD heterozyous KO mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. Conclusions— Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK, and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.

Genetic inhibition of calcineurin induces diastolic dysfunction in mice with chronic pressure overload

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase that induces myocardial growth in response to several physiological and pathological stimuli. Calcineurin inhibition, induced either via cyclosporine or genetically, can decrease myocardial hypertrophy secondary to pressure overload without affecting left ventricular (LV) systolic function. Since hypertrophy can also affect LV diastolic function, the goal of this study was to examine the effects of chronic pressure overload (2 wk aortic banding) in transgenic (Tg) mice overexpressing Zaki-4beta (TgZ), a specific endogenous inhibitor of calcineurin, on LV diastolic function. As expected, in the TgZ mice with calcineurin inhibitor overexpression, aortic banding reduced the degree of LV hypertrophy, as assessed by LV weight-to-body weight ratio (3.5 + or - 0.1) compared with that in non-Tg mice (4.6 + or - 0.2). LV systolic function remained compensated in both groups with pressure overload. However, the LV end-diastolic stress-to-LV end-diastolic dimension ratio, an index of diastolic stiffness and LV pressure half-time and isovolumic relaxation time, two indexes of isovolumic relaxation, increased significantly more in TgZ mice with aortic banding. Protein levels of phosphorylated phospholamban (PS16), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, phosphorylated ryanodine receptor, and the Na(+)/Ca(2+) exchanger were also reduced significantly (P < 0.05) in the banded TgZ mice. As expected, genetic calcineurin inhibition inhibited the development of LV hypertrophy with chronic pressure overload but also induced LV diastolic dysfunction, as reflected by both impaired isovolumic relaxation and increased myocardial stiffness. Thus genetic calcineurin inhibition reveals a new mechanism regulating LV diastolic function.

Cardiomyocyte overexpression of the α1a-adrenergic receptor in the rat phenocopies second but not first window preconditioning

We examined α(1A)-adrenergic receptor (AR) mediation of preconditioning in a novel α(1A)-AR cardiac transgenic (TG) rat model (α(1A)-TG). Compared with nontransgenic littermates (NTLs), in conscious α(1A)-TG rats, heart rate was reduced, contractility [left ventricle (LV) +dP/dt, ejection fraction, end-systolic elastance] was significantly enhanced, and triple product (LV systolic wall stress × LV +dP/dt × heart rate) was unchanged. However, infarct size (IS)/area at risk (AAR) in response to ischemia-reperfusion (30 min coronary occlusion/3 h reperfusion) was reduced to 35 ± 4.6% in α(1A)-TGs vs. 52 ± 2.2% in NTLs (P < 0.05). Second window preconditioning reduced IS/AAR in NTLs to 29 ± 2.7% but did not afford further protection in α(1A)-TGs. In contrast, with first window preconditioning, IS/AAR was reduced to similar levels in both α(1A)-TGs (12 ± 1.4%) and NTLs (10 ± 1.1%). In untreated α(1A)-TGs, cardioprotection was associated with enhanced myocardial phosphorylated (p)-mitogen/extracellular signal-regulated kinase (MEK), p-extracellular signal-regulated kinase (ERK), and inducible nitric oxide synthase (iNOS) at the protein level, along with a 1.3-fold increase in total nitric oxide synthase activity like in second window preconditioning. Affymetrix microarrays revealed that few genes (4.6% of 3,172 upregulated; 8.8% of 3,498 downregulated) showed directionally similar changes in α(1A)-TGs vs. NTLs subjected to second window preconditioning. Thus, second, but not first, window cardioprotection is evident in α(1A)-TGs in the absence of ischemic preconditioning and is mediated by iNOS activation associated with MEK/ERK phosphorylation. Transcriptionally, however, second window preconditioning is considerably more complex than α(1A)-TG preconditioning, with the alteration of thousands of additional genes affording no further protection than that already available in α(1A)-TG rats.

Type 5 Adenylyl Cyclase Increases Oxidative Stress by Transcriptional Regulation of Manganese Superoxide Dismutase via the SIRT1/FoxO3a PathwayClinical Perspective

Background— For reasons that remain unclear, whether type 5 adenylyl cyclase (AC5), 1 of 2 major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol in AC5 transgenic (Tg) mice and the signaling mechanisms involved. Methods and Results— Chronic isoproterenol increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular ejection fraction fell 1.9-fold more than wild type, along with greater left ventricular dilation and increased fibrosis, apoptosis, and hypertrophy. Oxidative stress induced by chronic isoproterenol, detected by 8-OhDG was 15% greater, P=0.007, in AC5 Tg hearts, whereas protein expression of manganese superoxide dismutase (MnSOD) was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be attributable to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 knockout (KO) was lost in AC5 KO×MnSOD heterozyous KO mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. Conclusions— Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK, and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.