Abstract：
OBJECTIVE: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains.
RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia.
CONCLUSIONS: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.
RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia.
CONCLUSIONS: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.
摘要：
目的：最近已经描述了使用心脏磷酸二酯酶（PDE4B）的基因治疗可以有效预防小鼠的心力衰竭。然而,其有益作用的确切分子机制，除了降低心肌细胞环磷酸腺苷（cAMP）水平外，尚未阐明。在这里，我们研究了两种类型的PDE的基因治疗，即PDE2A和PDE4B，可以通过作用于和恢复不同亚细胞微结构域中改变的cAMP区室化来预防小鼠压力超负荷诱导的心力衰竭。
结果：经横主动脉缩窄后，尾静脉注射9型腺相关病毒载体，过表达PDE2A3、PDE4B3或荧光素酶8周，诱发心力衰竭。通过超声心动图和组织学评估心脏形态和功能，显示PDE2A，尤其是PDE4B基因治疗可以减轻心脏肥大，纤维化和收缩功能下降。使用靶向cAMP生物传感器的活细胞成像表明，PDE过表达恢复了与ryanodine受体2型（RyR2）和富含小窝蛋白的质膜相关的微域中改变的cAMP区室化。PDE2A3过表达后，伴随着小窝蛋白-3下降的改善，在过表达PDE4B3的心脏中RyR2磷酸化降低和在异丙肾上腺素刺激下测量的两种PDE在单细胞中的抗心律失常作用。过表达的PDE4B而不是PDE2A与RyR2微域的强关联可以防止人诱导性多能干细胞衍生的心肌细胞中的钙渗漏和心律失常，RyR2中A2254V突变导致儿茶酚胺能多形性室性心动过速。
结论：我们的数据表明，使用磷酸二酯酶的基因治疗可以通过恢复功能相关亚细胞微域中改变的cAMP分隔来预防心力衰竭，包括相关的心脏重塑和心律失常。
结果：经横主动脉缩窄后，尾静脉注射9型腺相关病毒载体，过表达PDE2A3、PDE4B3或荧光素酶8周，诱发心力衰竭。通过超声心动图和组织学评估心脏形态和功能，显示PDE2A，尤其是PDE4B基因治疗可以减轻心脏肥大，纤维化和收缩功能下降。使用靶向cAMP生物传感器的活细胞成像表明，PDE过表达恢复了与ryanodine受体2型（RyR2）和富含小窝蛋白的质膜相关的微域中改变的cAMP区室化。PDE2A3过表达后，伴随着小窝蛋白-3下降的改善，在过表达PDE4B3的心脏中RyR2磷酸化降低和在异丙肾上腺素刺激下测量的两种PDE在单细胞中的抗心律失常作用。过表达的PDE4B而不是PDE2A与RyR2微域的强关联可以防止人诱导性多能干细胞衍生的心肌细胞中的钙渗漏和心律失常，RyR2中A2254V突变导致儿茶酚胺能多形性室性心动过速。
结论：我们的数据表明，使用磷酸二酯酶的基因治疗可以通过恢复功能相关亚细胞微域中改变的cAMP分隔来预防心力衰竭，包括相关的心脏重塑和心律失常。
