https://doi.org/10.1007/s00392-024-02526-y
1Universitätsmedizin Göttingen Herzzentrum, Klinik für Kardiologie und Pneumologie Göttingen, Deutschland; 2University Medical Center Göttingen Cellular Biophysics and Translational Cardiology Section Göttingen, Deutschland; 3Universitäts-Herzzentrum Freiburg - Bad Krozingen Institut für Experimentelle Kardiovaskuläre Medizin Freiburg im Breisgau, Deutschland
Introduction: Dysferlin is a Ca2+-sensitive transmembrane protein belonging to the ferlin family. In striated muscle cells, Dysferlin is thought to mediate membrane repair and vesicle fusion events. Human dysferlin mutations cause skeletal muscle dystrophies associated with a progressive form of dilated cardiomyopathy. However, little is known about the role of dysferlin regarding membrane repair and integrity in ventricular myocytes (VMs), expressing highly specific membrane nanodomains like the invaginated transverse-axial tubule (TAT) network. Here, we hypothesized that dysferlin locally stabilizes Ca2+ release units in VMs, and plays a crucial role in the reorganization of the TAT network necessary for the hypertrophic growth of VMs during left-ventricular pressure overload.
Method and Results: Stimulated Emission Depletion (STED) nanoscopy localized small dysferlin signals along the lateral surface membrane, the TAT network and the intercalated disc (ICD) cell-cell contact sites of isolated immunolabeled VMs. BS3 protein crosslinking of intact VMs revealed a baseline dysferlin expression of ~15% on the surface membrane, indicating a predominant storage of dysferlin in a vesicular compartment next to junctions of the TAT network with the sarcoplasmic reticulum as visualized by immunoelectron microscopy. Additionally, co-immunofluorescence STED imaging confirmed dysferlin signals in vicinity to RyR2 Ca2+ release unit clusters, and co-immunoprecipitation experiments demonstrated a protein interaction of dysferlin with RyR2 and the membrane tether and RyR2-binding protein junctophilin-2. In order to provoke membrane remodeling in VMs, we employed transverse aortic constriction (TAC) in wild-type (WT) and dysferlin knockout (KO) mice to mechanistically study the role of dysferlin in pressure overload-induced hypertrophy. Importantly, echocardiography revealed significantly attenuated hypertrophic parameters including left-ventricular anterior and posterior wall thickness in KO compared to WT mice 1 and 4 weeks post-TAC, while cardiac function was unchanged. Volumetry of isolated VMs verified a reduced hypertrophic response in KO mice post-TAC. In left ventricular myocardium of WT animals 1 and 4 weeks post-TAC, dysferlin expression increased up to 192% as shown in immunoblot analyses. While live-membrane imaging of VMs from WT animals showed a profound reorganization of the TAT network in favor of highly proliferating axial tubule components post-TAC, KO VMs failed to show the same degree of TAT network proliferation. Electron tomography of hypertrophied left ventricular myocardium confirmed the proliferation and dilation of axial tubule components in WT VMs induced by pressure overload. These newly shaped axial tubule components were highly decorated by dysferlin signals as shown in STED imaging. Dysferlin-decorated axial tubule components were further observed in left ventricular biopsies from patients with severe aortic stenosis undergoing valve replacement.
Conclusion: Our data suggest that Dysferlin is localized next to and interacts with proteins of the cardiac Ca2+ release unit. In pressure overload-induced hypertrophy, Dysferlin is involved in the proliferation and biogenesis of TAT network components, mediating the hypertrophic growth of VMs. Hence, Dysferlin may serve as a promising target to control membrane remodeling and prevent from excessive cardiac hypertrophy and maladaptation.