Dr. Stephen F. Vatner presented the results of his mouse studies on the role of adenylyl cyclase type 5 (AC5) on cardiac stress and longevity. Dr. Vatner explained that sympathetic agonists were developed for treating heart failure because it is known that acutely stimulating the sympathetic nervous system in patients with heart failure is beneficial. Catecholamines, which are depleted in heart failure, augment myocardial contractility, which is depressed in heart failure. Thus, beta adrenergic receptor agonists were developed for treating heart failure. However, evidence from animal and clinical studies indicates that chronic sympathetic stimulation is deleterious.

In one study, the effects of dobutamine and nitroprusside therapy in patients with heart failure were compared. Mortality was much higher in the dobutamine-treated patients than in those treated with nitroprusside. Studies using transgenic mice overexpressing beta 2 (b2) adrenergic receptors show that when young, these mice have enhanced cardiac function, with higher than normal left ventricular ejection fraction (LVEF). When the mice get old, they develop cardiomyopathy and heart failure. Mice with overexpressed b1 or b2 receptors have much earlier mortality (10 months) than wild-type (WT) mice (24-26 months). Mice with overexpressed Gsa (heterotrimeric G protein subunit that activates adenylate cyclase) also have higher mortality. Treatment with the beta blocker propranolol prevented the Gsa mice from dying. Similarly, a clinical study in patients with heart failure showed that patients treated with carbetolol had almost 100% survival in the first year of treatment.

Although there have been many studies on b adrenergic receptor signaling, resistance to stress, and the development of heart failure, there has been little research on adenylyl cyclase. The two major isoforms of adenylyl cyclase in the heart are AC5 and AC6. Dr. Vatner and his colleagues developed transgenic mice that overexpress AC5 in the heart. The mice were examined at baseline and in response to chronic pressure overload induced by transverse aortic banding. At baseline, the young AC5-overexpressing mice had enhanced cardiac function. After one week of pressure overload, the WT mice had no change in function, while the AC5-overexpressing mice showed decompensation, with more hypertrophy, decreased LVEF, pulmonary congestion, and cellular degeneration and necrosis. Mice with cardiac overexpression of AC2, normally not well expressed in the heart, also developed heart failure when banded.

Dr. Vatner’s group next assessed the effects of pressure overload on AC5-knockout (KO) mice. Aortic banding produced a similar pressure gradient in the AC5 KO and WT mice. Three weeks after aortic banding, the WT mice had decreased LVEF but the AC5 KO mice showed no decrease in LVEF. Cardiac apoptosis, known to increase with aortic banding, increased to a much lesser degree in the AC5 KO mice than in the WT mice. These experiments showed that the stress induced by aortic banding at 3 weeks impaired cardiac function in WT mice but AC5 KO mice were protected. These results led to a new hypothesis: AC5 does not participate in the desensitization, or downregulation, of b-receptor signaling that occurs with cardiac stress and enhanced sympathetic activity, but actually may have the opposite effect.

To determine the extent to which chronic catecholamine stimulation induces desensitization of AC5, isoproterenol was administered continuously over 2 weeks in the AC5 KO mice. After 2 weeks, the isoproterenol pumps were removed and the effects of acute isoproterenol challenges were examined. With each acute challenge, there was less of an increase in LVEF in the AC5 KO mice than in the WT mice, showing that they desensitized more fully to the chronic catecholamine stimulation. Histologic analysis showed that the AC5 KO mice were protected from apoptosis, which normally increases dramatically with chronic isoproterenol administration.

In response to chronic isoproterenol administration, AC6 decreased in WT mice and to a lesser extent in AC5 KO mice. In contrast, the WT mice had a paradoxical upregulation of AC5. Dr. Vatner hypothesized that this upregulation impedes the desensitization in WT mice.

These experiments show that chronic isoproterenol induces more effective desensitization in AC5 KO mice. One mechanism for the difference is the paradoxical upregulation of AC5 in WT mice. The absence of this effect in AC5 KO mice results in more effective desensitization and protects the heart from chronic stress induced by either catecholamines or pressure overload.

Dr. Vatner examined the effects of stress on longevity in AC5 KO mice. Comparison of survival of WT and AC5 KO mice showed that about 50% of the WT mice died at 25-26 months of age, the expected lifespan. The remaining WT mice died by 33 months. The AC5 KO mice lived about one-third longer, with a 50% survival of 33 months. Like humans, WT mice develop a cardiomyopathy of aging, with increased apoptosis, hypertrophy, and LV to body weight ratio, and decreased LVEF. However, the AC5 KO mice are protected against the development of cardiomyopathy.

According to Dr. Vatner, AC5 knockout also has extra-cardiac effects, including preservation of bone integrity. X-rays of older WT mouse legs showed osteoporosis, while bone integrity was preserved in the legs of older AC5 KO mice. Older AC5 KO mice also had much better bone strength than older WT mice. Additionally, both young and old AC5 KO mice had better exercise endurance than the corresponding age WT mice. Examination of food intake and weight showed that the AC5 KO mice ate more food but weighed less than WT mice.

Using proteomic techniques, heart, brain, and kidney tissue from young and old AC5 KO and WT mice were analyzed. Alterations in MAP kinase signaling and resistance to oxidative stress were found in the AC5 KO mice. Further analysis found upregulation of phospho-MEK and phospho-ERK in the old AC5 KO mice. The effects of hydrogen peroxide and ultraviolet light (which induce free radicals), on cell viability in neonatal myocytes were studied next. The AC5 KO mouse myocytes had much better viability than the WT myocytes in response to these stresses. Additionally, fibroblasts from the AC5 KO mice were protected from the free radical formation, showing less apoptosis than in WT mouse fibroblasts. AC5 KO mice had less cyclic AMP and protein kinase A (PKA) leading to upregulation of the antioxidative stress mechanisms RAF, MEK, and ERK, which results in less apoptosis and increased cell survival.

Dr. Vatner crossed AC5 KO mice with mice that overexpress b2 receptors. After about 12 months, the LVEF was dramatically higher in the crossed mice than in the mice with b2 overexpression, showing that AC5 knockout rescued the crossed mice from developing cardiomyopathy. Exercise endurance was depressed in the b2 overexpressing mice but not in the crossed mice with AC5 knockout. Apoptosis is a cardinal feature of overexpression of b2 receptors, and as expected, was high in the b2 overexpressing mice but practically nonexistent in the crossed mice. At 15 months, mortality was high in the b2 overexpressing mice compared with no mortality in the crossed mice.

Dr. Vatner concluded that mice with AC5 disruption exhibit both stress resistance and prolonged longevity. Knockout of type 5 AC can rescue the cardiomyopathy induced by chronic b adrenergic receptor stimulation. Thus, the AC5 KO model provides a new concept linking AC5 to stress resistance and longevity, and adds further support to the concept that inhibition of b adrenergic signaling is beneficial, not only for the pathogenesis of heart failure but also for extending lifespan. Inhibition of AC5 may be a novel therapy for heart failure since it can rescue the cardiomyopathy induced by chronic b adrenergic receptor stimulation.