Abstract Body: Background. Dilated cardiomyopathy (DCM) is the most prevalent inherited cardiomyopathy. In about one-third of DCM patients, ventricular arrhythmias are the predominant clinical presentation. These patients are classified as having an arrhythmogenic DCM (aDCM) and are at higher risk of sudden cardiac death (SCD). Notably, carriers of truncating (tv) mutations in FLNC present with intercalated disc disruption, significant interstitial fibrosis and are at high risk of life-threatening arrhythmias in the early stages of the disease, prior to the development of heart failure. To date, there is no effective pharmacological therapy for this deadly cardiac disease, except for defibrillator implantation and heart transplantation. Eps 15 homology domain-containing 2 (EHD2) is involved in endocytosis, vesicle trafficking, and membrane remodeling, but its role in the pathogenesis of FLNC-DCM remains known.
Hypothesis. We hypothesize that loss of FLNC disrupts plasma membrane integrity, activating EHD2-mediated endocytosis, which internalizes membrane protein, subsequently electrical instability in cardiomyocytes
Methods. We generated FLNC knockout (FLNC-KO) human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9 and used adenoviral expressing sh-EHD2 to knockdown EHD2. qPCR, immunoblot (IB) and immunofluorescence (IF) were used to assess candidate protein levels and localization.
Results. Preliminary data from FLNC-KO hiPSC-cardiomyocytes (CMs) show disturbed α-actinin organization and reduced localization of Connexin 43 (Cx43) at the cell membrane, suggesting that FLNC has an important role in regulating cytoskeleton and cell-cell junction. Moreover, EHD2 expression levels, as detected by IB and qPCR, were four-fold higher compared to healthy hiPSC-CMs. Furthermore, we detected increased Cx43 localization to membrane by IF staining upon knockdown of EHD2 using adenoviral particle expressing sh-EHD2. Notably, the IB results in the hiPSC-CMs models are consistent with our RNA sequencing data in human explanted DCM hearts, showing that EHD2 transcripts were upregulated in the explanted hearts of aDCM patients compared to non-failing and classic-DCM hearts.
Conclusion. Taken together, the data suggest that the loss of FLNC dysregulates the integrity of plasma membrane, leading to the mobilization of Cx43 away from the membrane via EHD2. Therefore, downregulating EHD2 to modulate Cx43 localization could be a new strategy for FLNC-DCM and heart failure.