BS02 Alpha-actinin-2 M228T missesnse variant and hypertrophic cardiomyopathy: characterising a phenotype

Alpha-actinin 2 (ACTN2) is a key protein at the Z-disk of the sarcomere. It is critical for stabilizing the contractile muscle apparatus and organizing actin filaments. Genetic missense variants of ACTN2 are associated with hypertrophic cardiomyopathy (HCM), a disease characterized by sudden cardiac death. In 2014, a study identified an ACTN2 missense variant, M228T, in a family of 11 patients with HCM. In addition, our previous in vivo study showed that mice homozygous for M228T were embryonically lethal, whereas mice heterozygous for M228T exhibited little to no cardiac phenotype. The mechanism by which the M228T variant impacts ACTN2 is poorly understood. Herein, we examine the changes occurring at the structural level of the mutant protein using structural modelling and biophysical characterization techniques. Molecular changes were assessed using an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model incorporating the M228T variant. Structural predictions showed that the M228T variant may destabilize the interface between the two calponin homology domains (CH1 and CH2) of ACTN2, thus impacting its binding to actin. Purification of mutant ACTN2 from E. coli showed a significant decrease in protein solubility. Further biophysical characterization techniques, including size-exclusion chromatography SAXS, mass photometry, and dynamic light scattering, were used. Furthermore, protein aggregates were identified in the iPSC-CM model homozygous for the M228T variant. Mutant cardiomyocytes showed upregulation in the fetal gene programme, as well as in markers of hypertrophy and fibrosis. Molecular analysis revealed significant degradation of ACTN2 protein. This was further analyzed using biochemical fractionation techniques. The role of the ubiquitin-proteasome system in protein degradation was assessed using a proteasome inhibitor, MG-132. Mutant cardiomyocytes also showed upregulation of autophagy markers, indicating a potential role for the autophagy-lysosomal pathway in protein degradation. Collectively, this study provides a better understanding of the impact of M228T missense variant on ACTN2 structure and function. Therefore, this may be the first step towards understanding disease pathways for Z-disc-associated HCM. Conflict of Interest None