Activated expression of cardiac adenylyl cyclase 6 reduces dilation and dysfunction of the pressure-overloaded heart

Abstract

Background and objective: Cardiac-directed adenylyl cyclase 6 (AC6) expression attenuates left ventricular (LV) hypertrophy and dysfunction in cardiomyopathy, but its effects in the pressure-overloaded heart are unknown.

Methods: Mice with cardiac-directed and regulated expression of AC6 underwent transaortic constriction (TAC) to induce LV pressure overload. Ten days prior to TAC, and for the duration of the 4 week study, cardiac myocyte AC6 expression was activated in one group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 4 weeks after TAC, and LV samples assessed for alterations in Ca2+ signaling.

Results: LV contractility, as reflected in the end-systolic pressure-volume relationship (Emax), was increased (p=0.01) by activation of AC6 expression. In addition, diastolic function was improved (p<0.05) and LV dilation was reduced (p<0.05). LV samples from AC-On mice showed reduced protein expression of sodium/calcium exchanger (NCX1) (p<0.05), protein phosphatase 1 (PP1) (p<0.01), and increased phosphorylation of phospholamban (PLN) at Ser16 (p<0.05). Finally, sarcoplasmic reticulum (SR) Ca2+ content was increased in cardiac myocytes isolated from AC-On mice (p<0.05).

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.

Fig. 1

(A, B) AC6 protein expression in LV homogenates in the presence or absence of doxycycline (DOX). Removing of doxycycline 10 days before TAC (-DOX) was associated with a 10-fold increase in AC6 protein expression 38 days later. (C) LV, lung and liver weight at necropsy normalized to tibial length (TL) (4 weeks after TAC). LV weight was increased 4 weeks after TAC in both groups. Lung weight was increased 4 weeks after TAC in AC-Off mice only. Liver weight was unaffected. No group difference in LV, lung or liver weight was observed. Error bars represent SE. *p < 0.05; two-way ANOVA with Bonferroni post hoc test compared to AC-Off sham.

Fig. 2

(A, B) Representative pressure–volume curves for an animal in each group (4 weeks after TAC). E_max was obtained from the slope of the end-systolic pressure–volume points on successive loops during inferior vena cava occlusion. (C) AC-On animals showed increased E_max values, indicative of increased LV contractility. Error bars represent SE.

Fig. 3

Isoproterenol stimulated caffeine-induced Ca²⁺ release in isolated cardiac myocytes 4 weeks after TAC. (A) Representative [Ca²⁺]_cyt traces in isoproterenol-stimulated myocytes, averaged from 5 to 8 individual cells. (B) Caffeine-induced rise in [Ca²⁺]_cyt, indicating Ca²⁺ release from SR. (C) The time required for a 50% reduction of the peak caffeine-induced intracellular Ca²⁺ level (T₅₀) was longer in AC-On when stimulated with isoproterenol. Error bars denote SE. Between-group comparisons were made using two-way ANOVA with Bonferroni post hoc test.

Fig. 4

LV protein expression 4 weeks after TAC. (A) Phospholamban (PLN) protein expression and phosphorylation at Ser16 or Thr17. Activation of AC6 expression increased p-PLN (Ser16), but not p-PLN (Thr17) or total PLN expression. (B) Sodium/calcium exchanger (NCX1) and type 1 protein phosphatase (PP1) expression. LV expression of NCX1 and PP1 were reduced by the activation of AC6 expression. In all graphs, bars represent mean values and error bars denote SE. Numbers above bars represent p-values from Student's t-test (2-tailed).