Biochemical and Biophysical Research Communications
Activated expression of cardiac adenylyl cyclase 6 reduces dilation and dysfunction of the pressure-overloaded heart
Research highlights
► Activation of cardiac adenylyl cyclase expression attenuates the deleterious effects on heart function associated with pressure overload. ► Both systolic and diastolic heart function were increased by activation of cardiac adenylyl cyclase expression. ► The predominant mechanism for these favorable effects is improved Ca2+ handling, a consequence of increased phospholamban phosphorylation, reduced expression of NCX1 and protein phosphatase-1, and increased SR Ca2+ content.
Introduction
Adenylyl cyclase (AC) is the key effector molecule that regulates cardiac function through generation of cAMP in cardiac myocytes. However, strategies to increase intracellular cAMP to enhance myocardial contractility have generally failed. For example, cardiac-directed expression of β-adrenergic receptors (βAR), the stimulatory GTP-binding protein (Gαs), and protein kinase A (PKA) – similar to inotropic agents – have resulted in left ventricular (LV) chamber dilation, cardiac fibrosis and heart failure [1], [2], [3]. Paradoxically, increased expression of AC type 6 (AC6), a predominant AC in cardiac myocytes, has pronounced favorable effects in genetically-induced cardiomyopathy, including increased LV function, increased survival, and prevention of deleterious LV remodeling [4], [5], [6]. These experimental data and others have provided the rationale for initiation of a clinical trial of AC6 gene transfer in patients with heart failure [ClinicalTrials.gov NCT00787059].
Heretofore, there have been very few studies of the effects of increased AC6 in the pressure-overloaded LV. There are several reasons to study the effects of AC6 expression in pressure-overload. First, increased expression of AC6 attenuates LV hypertrophy and improves LV function in genetically-induced cardiomyopathy [5], [7]. However, the pressure-overloaded LV may be a superior strategy to explore the effects of AC6 expression, vis-à-vis mechanistic insight, because of the weak correlation between clinical and genetic models of heart failure in mice. We achieved pressure overload using transverse aortic constriction [8]. Second, the ability to activate transgene expression when desired provides a means to study transgene effects on the pressure-overloaded heart at selected points in time, an advantage over constitutively expressed transgenes. In the present study, we used a transgenic line that provides cardiac-specific expression of AC6 under tet-regulation [9]. This enabled rapid activation of cardiac transgene AC6 when LV pressure overload was initiated.
Previous reports of the beneficial effects of increased levels of cardiac AC6 in murine cardiomyopathy led us to the hypothesis that activation of AC6 expression would be associated with beneficial effects on the pressure-overloaded LV. An additional goal was to determine mechanisms for improved LV function, if detected. We targeted Ca2+ handling, since disturbances of Ca2+ homeostasis have been shown to be important in the transition from LV hypertrophy to LV failure [10].
Section snippets
Animals
Animal use and care were in accordance with Institutional and NIH guidelines. Transgenic mice with cardiac-directed and regulated (tet-off) expression of AC6 (congenic C57BL6 background) were generated and previously described in detail [9]. In this line, cardiac transgene AC6 suppression is complete until doxycycline is removed from the water supply whereupon AC6 expression reaches a maximal level 10 days later [9].
A total of 125 mice (91 male, 34 female, 14.8 ± 0.6 weeks old) were used in the
Animal model, LV hypertrophy and survival
Activation of AC6 expression was associated with a 10-fold increase in LV AC6 protein content (Fig. 1; p < 0.0001), confirming the efficiency of tet-regulated transgene expression. TAC was associated with a similar degree of LV hypertrophy in both groups compared to sham operated animals. LV, lung and liver weight normalized to tibial length did not differ between groups (Fig. 1). The mortality 4 weeks after TAC was 37% (17/46) in AC-Off and 28% (13/47) in AC-On mice (p = 0.40).
Echocardiography
No group difference
Discussion
We found that AC6 expression reduces LV dilation and dysfunction in pressure-overload. Activation of AC6 expression was associated with improvements in both LV systolic and diastolic function. Cardiac myocytes from AC-On mice showed increased caffeine-induced SR Ca2+ release associated with increased PLN phosphorylation and reduced expression of NCX1 and PP1 – alterations that would be anticipated to increase LV function.
Increased caffeine-induced SR Ca2+ release was observed in cardiac
Conclusions
Activation of cardiac AC6 expression improves function of the pressure-overloaded and failing heart. The predominant mechanism for this favorable adaptation is improved Ca2+ handling, a consequence of increased PLN phosphorylation, reduced NCX1, reduced PP1 expression, and increased SR Ca2+ content.
Acknowledgments
This work was supported by NIH Grants 5P01HL066941, HL081741, and HL088426 (H.K.H.), Merit Review Awards from the Department of Veteran’s Affairs (H.K.H.), American Heart Association Western States Affiliate Grant-in-Aid 0765064Y (M.H.G.) and Postdoctoral Fellowship 09POST2250887 (Y.S.). We thank Diane Huang and Matthew Spellman for technical assistance and Dr. Glenn I. Fishman for providing the tTA mice.
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