There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca2+) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLBM) that were engineered to compete with the binding of inhibitory wild-type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK293 cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Thus, we propose that PLBM transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca2+ transport and thus contractility in underlying systolic heart failure.

中文翻译：

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SERCA2a 激活 PLB 突变体的病毒表达改善了扩张型心肌病 hiPSC-CM 中的钙循环和同步性。

心力衰竭 (HF) 基因治疗的发展势头越来越大，心力衰竭 (HF) 的定义是钙 (Ca2+) 转运受损和收缩力降低。我们使用荧光标记的 SERCA2a（心脏 Ca2+ 泵）和 PLB（磷蛋白，SERCA 心室肽抑制剂）之间的 FRET（荧光共振能量转移）来直接测试抑制丧失/结合获得的有效性。 LOI/GOB) PLB 突变体 (PLBM)，它们被设计为与抑制性野生型 PLB (PLBWT) 的结合竞争。我们的治疗策略是通过使用由 PLB 介导的储备肾上腺素能能力来增强心脏收缩力来缓解 PLBWT 对 SERCA2a 的抑制。使用 FRET 检测，我们确定 LOI PLB 突变（L31A）和 GOB PLB 突变（I40A）的组合产生了一种新的工程化 LOI/GOB PLBM（L31A/I40A），它有效地与 PLBWT 与 HEK293-6E 细胞中心脏 SERCA2a 的结合竞争。我们证明了 PLBM 的共表达通过增加 PLBWT 抑制的 HEK293 细胞匀浆中的酶 Ca2+ 亲和力 (1/KCa) 来增强 SERCA Ca-ATPase 活性。对于 PLBM 生理有效性的初步评估，我们使用了来自健康个体的人类诱导多能干细胞衍生的心肌细胞 (hiPSC-CM)。在该系统中，我们观察到腺相关病毒 2 (rAAV2) 驱动的 PLBM 表达增强了 SR Ca2+ 释放的幅度和 SR Ca2+ 再摄取的速率。为了评估治疗潜力，我们使用了包含 PLB 突变 R14del 的扩张型心肌病 (DCM) 的 hiPSC-CM 模型，我们观察到 rAAV2 驱动的 PLBM 表达可以挽救心律失常的 Ca2+ 瞬变并减轻 Ca2+ 转运的减少。因此，我们提出 PLBM 转基因表达是一种有前景的基因治疗策略，它直接针对异常 Ca2+ 转运的潜在病理生理学，从而针对潜在收缩性心力衰竭的收缩性。