Department of Physiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, M5S 1A8, Canada.
Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Toronto, ON, M5G 1M1, Canada.
Nat Commun. 2022 Oct 18;13(1):6166. doi: 10.1038/s41467-022-33303-y.
The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel β subunit (β1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na currents but did show reduced Ca and K currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.
闰盘（ICD）是一种独特的膜结构，对正常心脏功能不可或缺，但它的结构组织尚不完全清楚。以前，我们表明 ICD 结合跨膜蛋白 65（Tmem65）是连接蛋白 43（Cx43）在培养的小鼠新生心肌细胞中的定位和功能所必需的。在这里，我们通过注射携带 Tmem65 shRNA 的重组腺相关病毒 9（rAAV9）来研究 Tmem65 减少对小鼠模型心肌的功能和细胞影响，与对照 shRNA 注射相比，该病毒可使小鼠心室中的 Tmem65 表达降低 90%。Tmem65 敲低（KD）导致死亡率增加，这伴随着注射后 3 周内偏心性肥大性心肌病的发生，并在注射后 7 周进展为扩张型心肌病伴严重心脏纤维化。Tmem65 KD 心脏表现出心动超声心动图测量的血液动力学降低，以及光学记录中的传导减慢，伴有心电图中 PR 间隔和 QRS 持续时间延长。免疫沉淀和超分辨率显微镜显示 Tmem65 和钠通道 β 亚基（β1）在小鼠心脏中的物理相互作用，这种相互作用似乎是建立周边纳米域结构和定位电压门控钠通道 1.5（NaV1.5）和 Cx43 到 ICDs 所必需的。尽管 ICD 上的 NaV1.5 丢失，但全细胞膜片钳电生理学并未显示 Na 电流减少，但与对照细胞相比，Tmem65 KD 心肌细胞中的 Ca 和 K 电流减少。我们得出结论，破坏 Tmem65 功能会导致 ICD 结构异常、心脏电生理学异常，最终导致心肌病。
