Introduction: Among the group of Ferlin transmembrane proteins with multiple Ca2+-sensitive C2 domains, Dysferlin is the prominent isoform expressed in ventricular cardiomyocytes (VMs), thought to be involved in Ca2+-activated vesicle fusion, membrane repair and trafficking. Dysferlin patient mutations cause progressive forms of dilated cardiomyopathy that were linked to insufficient membrane repair mechanisms. Here we hypothesize that Dysferlin is involved in tubular membrane invagination biogenesis and remodeling in VMs and protects from cell death in case of myocardial infarction (MI). 

Methods and Results: RNA sequencing from isolated mouse VMs showed that among the Ferlin proteins only Dysferlin and Myoferlin are substantially expressed in the heart. Dysferlin is the prominent isoform in VMs, whereas Myoferlin is predominantly expressed in atrial myocytes. Immunofluorescence and super-resolution STimulated Emission Depletion (STED) imaging revealed punctate Dysferlin clusters a) at the transverse-axial tubule system (TATS), b) underneath the lateral surface, and c) at the intercalated discs. To quantify the amount of Dysferlin surface membrane expression, we established a novel Dysferlin crosslinking protocol for isolated VMs using the membrane-impermeable crosslinker BS3. Under baseline conditions, only 15.1% of the Dysferlin protein was shown to be expressed on the surface membrane, whereas the majority of Dysferlin signals is stored in vesicular compartments underneath the tubular/lateral membrane. For both Dysferlin and Myoferlin, Ca2+-activated cleavage by the non-lysosomal cysteine proteases Calpain-1/2 has been shown previously. However, enzymatic treatment of mouse ventricular lysate with Calpain-1/2 demonstrate that Dysferlin is only cleaved at high enzymatic concentrations in contrast to constitutively cleaved Myoferlin. However, protein lysate from human-derived iPSC-cardiomyocytes showed N- and C-terminal Dysferlin cleavage products, presenting a role for Dysferlin cleavage in human VMs. To test the relevance of Dysferlin in TATS membrane proliferation and remodeling, we investigated a mouse model of pressure-overload induced cardiac hypertrophy caused by transaortic constriction (TAC). Interestingly, VMs four weeks post TAC showed a marked proliferation of axial tubule membranes in live-cell membrane imaging. Importantly, quantitative SDS-PAGE revealed an increase of Dysferlin protein expression up to 192%, and STED nanoscopy showed Dysferlin clusters decorating newly shaped axial tubules in hypertrophied VMs. In sharp contrast, Dysferlin-knockout (KO) VMs failed to hypertrophy post-TAC. Echocardiographic measurements of Dysferlin-KO mice four weeks post TAC further presented a significantly reduced hypertrophy compared to WT mice (anterior wall thickness (mm): WT 1.21 ± 0.06 vs. KO 1.02 ± 0.10, n=12 mice, p<0.0001). Post MI, STED imaging revealed a critical role for Dysferlin in stabilizing VMs at the infarct border zone, preventing the myocardium from additional loss of VMs. 

Conclusion: Our data suggest an important role of Dysferlin in maintaining the integrity of the tubular and lateral surface membrane in VMs. In pressure overload, VM hypertrophy depends on the TATS proliferation mediated by Dysferlin. In MI, Dysferlin protects from loss of VMs at the infarct border zone. Hence, Dysferlin may emerge as potential therapeutic approach to control the subcellular membrane remodeling and repair in cardiac diseases.