Bedioune Ibrahim, Gandon-Renard Marine, Dessillons Matthieu, Barthou Aurélien, Varin Audrey, Mika Delphine, Bichali Saïd, Cellier Joffrey, Lechène Patrick, Karam Sarah, Dia Maya, Gomez Susana, Pereira de Vasconcelos Walma, Mercier-Nomé Françoise, Mateo Philippe, Dubourg Audrey, Stratakis Constantine A, Mercadier Jean-Jacques, Benitah Jean-Pierre, Algalarrondo Vincent, Leroy Jérôme, Fischmeister Rodolphe, Gomez Ana-Maria, Vandecasteele Grégoire
Université Paris-Saclay, Inserm, Signaling and Cardiovascular Pathophysiology, UMR-S1180 (I.B., M.G.-R., M.D., A.B., A.V., D.M., S.B., J.C., P.L., S.K., M.D., S.G., W.P.d.V., P.M., A.D., J.-J.M., J.-P.B., V.A., J.L., R.F., A.-M.G., G.V.), Orsay, France.
Université Paris-Saclay, Inserm, UMS IPSIT (F.M.-N.), Orsay, France.
Circulation. 2024 Dec 17;150(25):2031-2045. doi: 10.1161/CIRCULATIONAHA.124.068858. Epub 2024 Oct 2.
The heart expresses 2 main subtypes of cAMP-dependent protein kinase (PKA; type I and II) that differ in their regulatory subunits, RIα and RIIα. Embryonic lethality of RIα knockout mice limits the current understanding of type I PKA function in the myocardium. The objective of this study was to test the role of RIα in adult heart contractility and pathological remodeling.
We measured PKA subunit expression in human heart and developed a conditional mouse model with cardiomyocyte-specific knockout of RIα (RIα-icKO). Myocardial structure and function were evaluated by echocardiography, histology, and ECG and in Langendorff-perfused hearts. PKA activity and cAMP levels were determined by immunoassay, and phosphorylation of PKA targets was assessed by Western blot. L-type Ca current (), sarcomere shortening, Ca transients, Ca sparks and waves, and subcellular cAMP were recorded in isolated ventricular myocytes (VMs).
RIα protein was decreased by 50% in failing human heart with ischemic cardiomyopathy and by 75% in the ventricles and in VMs from RIα-icKO mice but not in atria or sinoatrial node. Basal PKA activity was increased ≈3-fold in RIα-icKO VMs. In young RIα-icKO mice, left ventricular ejection fraction was increased and the negative inotropic effect of propranolol was prevented, whereas heart rate and the negative chronotropic effect of propranolol were not modified. Phosphorylation of phospholamban, ryanodine receptor, troponin I, and cardiac myosin-binding protein C at PKA sites was increased in propranolol-treated RIα-icKO mice. Hearts from RIα-icKO mice were hypercontractile, associated with increased and [Ca] transients and sarcomere shortening in VMs. These effects were suppressed by the PKA inhibitor, H89. Global cAMP content was decreased in RIα-icKO hearts, whereas local cAMP at the phospholamban/sarcoplasmic reticulum Ca ATPase complex was unchanged in RIα-icKO VMs. RIα-icKO VMs had an increased frequency of Ca sparks and proarrhythmic Ca waves, and RIα-icKO mice had an increased susceptibility to ventricular tachycardia. On aging, RIα-icKO mice showed progressive contractile dysfunction, cardiac hypertrophy, and fibrosis, culminating in congestive heart failure with reduced ejection fraction that caused 50% mortality at 1 year.
These results identify RIα as a key negative regulator of cardiac contractile function, arrhythmia, and pathological remodeling.
心脏表达两种主要的环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA；I型和II型）亚型，它们的调节亚基RIα和RIIα有所不同。RIα基因敲除小鼠的胚胎致死性限制了目前对I型PKA在心肌中功能的理解。本研究的目的是测试RIα在成年心脏收缩性和病理重塑中的作用。
我们检测了人心脏中PKA亚基的表达，并构建了一种条件性小鼠模型，其心肌细胞特异性敲除RIα（RIα-icKO）。通过超声心动图、组织学、心电图以及Langendorff灌注心脏评估心肌结构和功能。通过免疫测定法测定PKA活性和cAMP水平，并通过蛋白质印迹法评估PKA靶点的磷酸化情况。在分离的心室肌细胞（VMs）中记录L型钙电流（）、肌节缩短、钙瞬变、钙火花和钙波以及亚细胞cAMP。
在患有缺血性心肌病的衰竭人心脏中，RIα蛋白减少了50%，在RIα-icKO小鼠的心室和VMs中减少了75%，但在心房或窦房结中未减少。RIα-icKO VMs中的基础PKA活性增加了约3倍。在年轻的RIα-icKO小鼠中，左心室射血分数增加，普萘洛尔的负性肌力作用被阻止，而心率和普萘洛尔的负性变时作用未改变。在普萘洛尔治疗的RIα-icKO小鼠中，受PKA作用位点的受磷蛋白、兰尼碱受体、肌钙蛋白I和心肌肌球蛋白结合蛋白C的磷酸化增加。RIα-icKO小鼠的心脏收缩过强，与VMs中增加的和[Ca]瞬变以及肌节缩短有关。这些作用被PKA抑制剂H89抑制。RIα-icKO心脏中的总体cAMP含量降低，而在RIα-icKO VMs中，受磷蛋白/肌浆网Ca ATP酶复合物处的局部cAMP未改变。RIα-icKO VMs中钙火花和促心律失常性钙波的频率增加，RIα-icKO小鼠对室性心动过速的易感性增加。随着年龄增长，RIα-icKO小鼠出现进行性收缩功能障碍、心脏肥大和纤维化，最终导致射血分数降低的充血性心力衰竭，1年内死亡率达50%。
这些结果表明RIα是心脏收缩功能、心律失常和病理重塑的关键负调节因子。
