Institute for Experimental Medical Research (J.S., I.A., K.H., M.L., O.S.E., I.V., M.B., S.M., X.S., W.E.L., P.K.L., I.S., C.R.C., J.M.A.), Oslo University Hospital and University of Oslo, Norway.
Department of Pharmacology, Oslo University Hospital, Norway (G.B.L.).
Circulation. 2023 Apr 18;147(16):1221-1236. doi: 10.1161/CIRCULATIONAHA.121.054168. Epub 2023 Mar 6.
Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators.
Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays.
PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding.
Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.
增加 SERCA2（肌浆网-内质网 Ca2+-ATP 酶 2）的活性被认为对慢性心力衰竭有益，但目前尚无选择性 SERCA2 激活药物。PDE3A（磷酸二酯酶 3A）被认为存在于 SERCA2 相互作用组中，并限制 SERCA2 的活性。因此，破坏 PDE3A 与 SERCA2 的结合可能是开发 SERCA2 激活剂的一种策略。
使用共焦显微镜、双色直接随机光学重建显微镜、接近连接测定、免疫沉淀、肽阵列和表面等离子体共振来研究心肌细胞中 SERCA2 和 PDE3A 之间的共定位，绘制 SERCA2/PDE3A 相互作用位点图，并优化释放 PDE3A 与 SERCA2 结合的破坏肽。在心肌细胞和 HEK293 囊泡中进行评估 PDE3A 与 SERCA2 结合对功能影响的功能实验。在两项连续的、随机的、双盲的、对照的临床前试验中，使用共聚焦显微镜评估了在总共 148 只接受重组腺相关病毒 9（rAAV9）-OptF、rAAV9-对照（Ctrl）或 PBS 注射的小鼠中，在主动脉缩窄（AB）或假手术后 20 周内，使用连续超声心动图、心脏磁共振成像、组织学以及功能和分子检测进行心脏死亡率和功能的评估。OptF（优化肽 F）对心脏死亡率和功能的影响。在接受 rAAV9-OptF、rAAV9-Ctrl 或 PBS 注射的小鼠中，SERCA2/PDE3A 破坏肽增加了 SERCA2 活性，即使在蛋白激酶 A 抑制剂存在的情况下，以及在磷蛋白缺陷型小鼠中，SERCA2 活性也没有影响，并且对具有心肌细胞特异性 SERCA2 失活的小鼠没有影响。PDE3A 在心肌细胞中的共转染降低了 HEK293 囊泡中 SERCA2 的活性。与 rAAV9-Ctrl（危险比，0.26[95%CI，0.11 至 0.63]）和 PBS（危险比，0.28[95%CI，0.09 至 0.90]）相比，rAAV9-OptF 治疗可降低 AB 后 20 周的心脏死亡率。与 rAAV9-Ctrl 相比，接受 rAAV9-OptF 治疗的小鼠在主动脉缩窄后收缩力提高，心脏重塑无差异。
我们的结果表明，PDE3A 通过直接结合调节 SERCA2 活性，而不依赖于 PDE3A 的催化活性。靶向 SERCA2/PDE3A 相互作用可预防 AB 后心脏死亡率的增加，这可能是通过改善心脏收缩力来实现的。
