Activation of cardiac adenylyl cyclase expression increases function of the failing ischemic heart in mice

Abstract

Objectives: This study sought to evaluate whether increased left ventricular (LV) adenylyl cyclase VI (AC(VI)) expression, at a time when severe congestive heart failure (CHF) was present, would increase function of the actively failing heart.

Background: Increased LV AC(VI) content markedly reduces mortality and increases LV function after acute myocardial infarction (MI) in mice. However, the effects of increased cardiac AC(VI) content in the setting of severe heart failure caused by ischemic cardiomyopathy are unknown.

Methods: Mice with cardiac-directed and regulated expression of AC(VI) underwent coronary artery ligation to induce severe CHF 5 weeks later. AC(VI) expression was then activated in 1 group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 5 weeks later, and LV samples were assessed for alterations in calcium and beta-adrenergic receptor signaling, apoptosis, and cardiac troponin I phosphorylation.

Results: The LV systolic and diastolic function was increased 5 weeks after activation of AC(VI) expression. Improved LV function was associated with normalization of cardiac troponin I phosphorylation and reduced apoptosis.

Conclusions: Activation of cardiac AC(VI) expression in mice with ischemic cardiomyopathy and severe CHF improves function of the failing heart.

Figure 1. Experimental Design

See the text for a full description. AC = adenylyl cyclase; MI = myocardial infarction.

Figure 2. LV AC_VI Content

The Western blot shows a marked increase in AC_VI protein in LV samples from mice 5 weeks after activation of AC_VI transgene expression (n = 8 for both groups). The graph summarizes data from Western blotting. Bars = mean values; error bars = 1 SE; number above bars = probability value (Student unpaired t test, 2-tailed). AC_VI = adenylyl cyclase VI; LV= left ventricular.

Figure 3. Contractile Function

The ESPVR was measured in intact mice 5 weeks after activation of AC_VI expression in animals with severe CHF. Increased cardiac AC_VI expression was associated with substantial increases in LV contractility as reflected in increased slope of the ESPVR. Cardiac loops, generated by altering loading conditions of the LV, are shown with the slope of the end-systolic pressure-volume point depicted. The graph below summarizes data from all animals. Numbers in bars denote animals studied. Bars = mean values; error bars = 1 SE; number above bars = probability value (Student unpaired t test, 2-tailed). CHF = congestive heart failure; ESPVR = end-systolic pressure-volume relationship; other abbreviations as in Figure 2.

Figure 4. LV cAMP Production and PKA Activity

(Left) Increased AC_VI expression was associated with increased cAMP generation in response to NKH477 (10 μM) stimulation of LV samples. Basal cAMP generation was not changed by AC_VI expression. (Right) Increased cardiac AC_VI expression was associated with increased PKA activity in LV samples. Numbers in bars = animals studied; bars = mean values; error bars = 1 SE; number above bars = probability value (Student unpaired t test, 2-tailed). cAMP = cyclic adenosine monophosphate; PKA = protein kinase A; other abbreviations as in Figure 2.

Figure 5. LV cTnI Phosphorylation

The Western blot shows a marked increase in cTnI phosphorylation in LV samples from mice 5 weeks after activation of AC_VI transgene expression. Summary of data from Western blotting is shown in the lower panel. Numbers in bars = animals studied; bars = mean values; error bars = 1 SE; number above bars = probability value (Student unpaired t test, 2-tailed). Abbreviations as in Figure 2.

Figure 6. Apoptosis

Rates of LV apoptosis (TUNEL staining) were increased in both border and remote zones compared with the normal rate in murine LV of 50 TUNEL-positive nuclei per 100,000 cells (1). Activation of AC_VI expression reduced the rate of apoptosis by about 50% in both regions. Numbers in bars = animals studied; bars = mean values; error bars = 1 SE; p value = probability value for gene effect (2-way analysis of variance). TUNEL = terminal 2′-deoxyuridine 5′-triphosphate nick-end labeling; other abbreviations as in Figure 2.