Department of Pharmacology, Faculty of Medicine, Dalhousie University, 5850 College Street, P.O. Box 15000, Halifax B3H 4R2, Nova Scotia, Canada.
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, 5850 College Street, P.O. Box 15000, Halifax B3H 4R2, Nova Scotia, Canada.
J Mol Cell Cardiol. 2017 Oct;111:51-60. doi: 10.1016/j.yjmcc.2017.07.118. Epub 2017 Aug 1.
Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca currents were similar in myocytes from sham and OVX mice but Ca transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2μM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300μM IBMX), or an AC activator (10μM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50μM). Interestingly, selective inhibition of PDE4 increased Ca current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.
卵巢切除术 (OVX) 可促进心室肌细胞肌浆网 (SR) 钙超载。我们假设环磷酸腺苷 (cAMP)/蛋白激酶 A (PKA) 通路对此钙调节失常起作用。成年雌性 C57BL/6 小鼠行 OVX 或假手术 (约 1 个月时手术) 后分离心肌细胞。在电压钳制的心肌细胞中同时测量收缩、钙浓度 (fura-2) 和离子电流 (37°C，2Hz)。用酶免疫测定法测定细胞内 cAMP 水平；用 qPCR 检测磷酸二酯酶 (PDE) 和腺苷酸环化酶 (AC) 同工型的表达。OVX 组与假手术组的心肌细胞钙电流相似，但 OVX 组钙瞬变、兴奋-收缩 (EC) 偶联增益、SR 含量和收缩幅度均大于假手术组。为确定 cAMP/PKA 通路是否介导 OVX 诱导的 EC 偶联改变，用 PKA 抑制剂 H-89 (2μM) 孵育心肌细胞，该抑制剂消除了基础差异。虽然基础细胞内 cAMP 无差异，但在非选择性 PDE 抑制剂 (300μM IBMX) 或 AC 激活剂 (10μM forskolin) 存在时，OVX 组 cAMP 水平高于假手术组。这表明 OVX 增强了 AC 产生的 cAMP 及其被 PDE 分解。与此一致的是，与假手术组相比，OVX 组的 AC5 和 PDE4A mRNA 水平均升高。当 sham 和 OVX 细胞用含有相同浓度 8-溴-cAMP (50μM) 的 patched 管内液灌流时，钙稳态和收缩的差异被消除。有趣的是，选择性抑制 PDE4 仅增加 OVX 细胞的钙电流。综上所述，这些发现表明雌激素抑制 SR 钙释放，而 cAMP/PKA 通路至少部分调节该过程。当卵巢雌激素水平下降时，该 cAMP/PKA 通路的改变可能促进钙调节失常和心血管疾病。这些结果加深了我们对可能影响老年女性治疗干预反应的女性特有的心肌细胞机制的理解。
