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

AimsIn 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.Methods and resultsIn 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.ConclusionSoluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term β1-adrenoceptor stimulation or to pressure overload.研究目的：与膜结合型腺苷酸环化酶（membrane bound adenylyl cyclases）相比，可溶性腺苷酸环化酶（soluble adenylyl cyclase, sAC）可被碳酸氢根（bicarbonate）及包括钙离子（calcium）在内的二价离子（divalent ions）激活。该酶广泛分布于心肌（cardiac muscle）等多种组织的细胞质（cytosol）、细胞核（nuclei）与线粒体（mitochondria）中，但目前其在心脏病理（cardiac pathology）过程中的作用仍有待阐明。本研究通过两种不同的模型系统（model systems），探究sAC是否参与肥厚性生长（hypertrophic growth）的调控。 方法与结果：本研究在分离的成年大鼠心肌细胞（isolated adult rat cardiomyocytes）中，采用异丙肾上腺素（isoprenaline, ISO）联合β2肾上腺素能受体拮抗剂（β2-adrenoceptor antagonist）ICI118,551，通过24小时β1肾上腺素能受体（β1-adrenoceptor）刺激诱导细胞肥厚（hypertrophy）。为监测肥厚程度，我们分析了细胞体积（cell size）、RNA/DNA比值、蛋白/DNA比值以及α-骨骼肌肌动蛋白（α-skeletal actin）的表达水平。通过特异性抑制剂（specific inhibitor）KH7处理或基因敲降（knockdown）的方式，可抑制sAC的活性。无论是药物抑制（pharmacological inhibition）还是基因敲降，均可减弱ISO/ICI处理的大鼠心肌细胞的肥厚性生长，并降低α-骨骼肌肌动蛋白的表达水平。为探究其潜在细胞机制，我们通过蛋白质免疫印迹（western blot）检测了磷酸化cAMP反应元件结合蛋白（CREB）、B-Raf及Erk1/2的表达水平。结果显示，B-Raf参与了sAC介导的促肥厚作用（pro-hypertrophic action），而Erk1/2与CREB并未参与该过程。在野生型小鼠及sAC基因敲除小鼠中，我们通过主动脉弓缩窄（transverse aortic constriction, TAC）构建压力超负荷（pressure overload）模型，并检测了血流动力学（Hemodynamics）指标、心脏重量（heart weight）及心房利钠肽（atrial natriuretic peptide）的表达水平。实验结果一致表明，sAC基因敲除小鼠无法通过主动脉弓缩窄诱导心肌肥厚。机制分析进一步显示，Erk1/2可能在TAC诱导的心肌肥厚中发挥潜在作用。 结论：可溶性腺苷酸环化酶（soluble adenylyl cyclase, sAC）或可成为长期β1肾上腺素能受体刺激或压力超负荷诱导的心肌肥厚反应（cardiac hypertrophic response）中的关键新型调控靶点。