The intercalated disc (ICD) is unique membrane structure that is indispensable to normal heart function. However, its structural organization is not well understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin 43 (Cx43) localization in cultured mouse neonatal cardiomyocytes. Here, we investigated the role of Tmem65 in ICD organization
in vivo
. A mouse model was established by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 (or scrambled) shRNA. Quantitative polymerase chain reaction (qPCR) and immunoblots confirmed greater than 85% reduction in Tmem65 expression (7.1±0.7% remained for Tmem65 proteins; 14.4±2.5% remained for Tmem65 transcripts,
n
=4) in mouse ventricles compared to control hearts. Tmem65 knockdown (KD) mice exhibited heart failure-like symptoms as early as 3 weeks post viral administration. Specifically, Tmem65 KD mice developed eccentric hypertrophic cardiomyopathy in 3 weeks and dilated cardiomyopathy with severe cardiac fibrosis in 7 weeks, as confirmed by H&E and Masson’s Trichrome staining. Echocardiography and electrocardiography, respectively, showed depressed hemodynamics (19.27±1.46ml/min for cardiac output in control hearts vs. 6.63±0.52ml/min for Tmem65 KD hearts,
n
=6) and impaired conduction, including prolonged PR (22.7±1.85ms in control hearts vs. 28.89±3.85ms in Tmem65 KD hearts, n≥8), QRS intervals (10.47±0.42ms in control hearts vs. 16.35±0.36ms in Tmem65 KD hearts, n≥8), and slowed heart rate (415±10bpm in control hearts vs. 347±16bpm in Tmem65 KD hearts, n≥8) in Tmem65 KD mouse hearts.
Immunoprecipitation and super-resolution microscopy confirmed the physical interaction and localization between Tmem65 and voltage-gated sodium channel β subunit (β1) at the ICD and this interaction was evidently required for the establishment of perinexal nanodomains and voltage-gated sodium channel 1.5 (NaV1.5) localization to the ICD. Disrupting Tmem65 function, thus, impaired perinexal structure, reduced conduction velocity, and ultimately resulted in cardiomyopathy
in vivo
.