Soluble adenylyl cyclase: A novel player in cardiac hypertrophy induced by isoprenaline or pressure overload

<div><p>Aims</p><p>In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems.</p><p>Methods and results</p><p>In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h β₁-adrenoceptor stimulation using isoprenaline (ISO) and a β₂-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy.</p><p>Conclusion</p><p>Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term β₁-adrenoceptor stimulation or to pressure overload.</p></div

CREB, B-Raf, Erk1/2 phosphorylation in adult rat cardiomyocytes.

<p>(A) Western blot analysis of phosphorylated CREB (pCREB,) from the lysates of untreated (-KH7) or KH7-treated (+KH7, 12.5 μmol/L) cardiomyocytes followed by optical band density analysis at different incubation times with ISO/ICI (n = 4). Data were normalized to the total proteins by gel staining as indicated in Methods; incubation times: 0, 15 min, 30 min, 1 h, 3 h, 6 h. Solid line indicates control, broken line—treatment with KH7. Western blot analysis of (B) phosphorylated B-Raf (pB-Raf; Ser445) and B-Raf (n = 3), of (C) phosphorylated Erk1/2 (pErk1, pErk2; TEY motif)) and Erk1/2 (n = 4) followed by the optical band density analysis (lower panel) are presented. Data were normalized to total protein and are expressed as means ± SEM. *(P<0.05), vs. ISO/ICI treated control cells. n refers to the number of cardiomyocyte preparations analyzed.</p

Effect of TAC on haemodynamic parameters in WT and sAC-KO mice.

<p>Analyses of (A) heart rate (HR), (B) systolic (SAP), (C) diastolic aortic pressure (DAP), (D) left ventricular systolic pressure (LVSP), (E) left ventricular end-diastolic pressure (LVEDP), rates of (F) contraction (+dp/dt) and (G) of relaxation (–dp/dt) were performed in anaesthetised WT or sAC KO mice 2 weeks after TAC or sham surgery. Data are expressed as means ± SEM, n = 9–12, ***(P< 0.001), *(P<0.05).</p

Hypertrophic parameters in sham and TAC operated WT and sAC KO mice.

<p>(A-C) Body weight, lung weight, heart weight and Tibia length (BW, LW, HW, TL, respectively) and their ratios (HW/BW;LW/BW and HW/TL) were analyzed 2 weeks after TAC or sham surgery in WT or sAC KO mice (n = 9–12). *(P< 0.05); **(P < 0.01). (D) Western blot analysis of ANP followed by optical band density analysis (n = 3 for sAC-WT and n = 5 for sAC-KO), ANP is normalized to tubulin. Equal experimental conditions as in A-C. Data are expressed as means ± SEM. **(P < 0.01) vs. TAC in wt mice. (E) Western blot analysis of pERK1/2 followed by optical band density analysis performed with cardiac tissue lysates from WT sham (n = 4), sAC WT TAC (n = 2), sAC KO sham (n = 4) and sAC KO TAC (n = 5) mice. Differences in total Erk1/2 band intensities are due to different total protein concentrations. Data are expressed as means ±SEM; **(P < 0.01) vs. TAC in WT mice.</p

sAC knockdown in ISO/ICI treated isolated adult cardiomyocytes.

<p>(A) Representative western blots followed by the optical band analysis of sAC in lysates of cardiomyocytes after the following treatments: ISO/ICI (24h), adenoviral transfections either with scrambled shRNA (scRNA) or with sAC-targeted shRNA (shRNA). Data were normalized to actin band intensity. n = 5,*** (P<0.0001). (B-D) Statistical analyses of the cross sectional cell area of 110 cells from three cardiomyocyte preparations, the RNA/DNA ratio (n = 3) and the protein/DNA ratio (n = 3) are shown. *(P<0.05) and **(P<0.01),vs. ISO/ICI-treated cells expressing scRNA. <i>All</i> data are expressed as means ± SEM. n refers to the number of cardiomyocyte preparations analyzed.</p

Effect of pharmacological sAC inhibition on ISO/ICI-induced hypertrophy of isolated cardiomyocytes.

<p>(A–D) Cardiomyocytes were cultured for 24 h under basal conditions or with the addition of ISO/ICI and/or KH7 (12.5 μmol/L). In (A) the cross sectional areas of 118 cells are given (n = 5). In (B) the RNA (ng/μL)/DNA (ng/μL) ratio (n = 3) and in (C) the protein (μg/μL)/DNA (ng/μL) ratio are presented (n = 3). (D) Western blot analysis followed by optical band density analysis of α-skeletal actin (n = 5). Data were normalized to GAPDH band density and to the control (no treatment, 100%). (A-D) Data are expressed as means ± SEM. *(P < 0.05) or ***(P < 0.001) vs. ISO/ICI-treated cells. (E) Isolated rat cardiomyocytes were incubated with ISO/ICI and KH7 (0.0, 6.0, 9.0, 12.5, 15.0 μmol/L) for 24 h. The protein (μg/μL)/ DNA (ng/μL) ratio was determined (n = 4 cardiomyocyte preparations). Data are expressed as means ± SEM. 6.0 and 9.0 μmol/L KH7 vs 0.0 μmol/L KH7: P<0.05; 12.5 and 15 μmol/L KH7 vs 0.0mKH7: P<0.01. n refers to the number of cardiomyocyte preparations.</p