ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

Abstract

RationaleGenetic variants in ACTN2, encoding α-actinin 2 (ACTN2), are associated with several forms of (cardio)myopathy in the heterozygous state, and can cause progressive, severe cardiomyopathy in the homozygous state. We previously reported a heterozygous missense (c.740C>T) ACTN2 variant, associated with hypertrophic cardiomyopathy (HCM), which induced an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).ObjectiveTo evaluate the impact of heterozygosity and homozygosity of the c.740C>T ACTN2 variant on cardiomyocyte structure and function.Methods and ResultsNext to the previously established hiPSC line carrying the heterozygous ACTN2 variant (ACTN2het), we created a homozygous line (ACTN2hom) with CRISPR/Cas9. Differentiation into cardiomyocytes revealed myofibrillar disarray, increased cell area and volume, multinucleation, and ACTN2 aggregates in mutant hiPSC-CMs. Live cell imaging in hiPSC-CMs showed an inverse correlation between sarcomeric incorporation and aggregation of exogenously expressed mutant ACTN2. RNA-seq and proteomic analyses showed alteration of several canonical pathways involved in metabolism, oxidative and cellular stress, and proteostasis, including the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) in mutant hiPSC-CMs. Detailed evaluation of the UPS and ALP, including a high-content screening with a mWasabi-mTagRFP-hLC3 tandem construct, revealed a global activation of these systems in mutant hiPSC-CMs. The ACTN2 missense variant caused allele dose-dependent and -independent effects on several cardiomyocyte features. ACTN2hom exhibited markedly lower levels of sarcomere-associated proteins, and hypocontractility in engineered heart tissues, resembling a dilated cardiomyopathy phenotype.ConclusionThis study showed allele dose-independent effects of the ACTN2 variant on multinucleation, ACTN2 aggregation, and UPS activation, and allele dose-dependent effects of the variant on cellular hypertrophy, myofibrillar disarray, and ALP activation. Activation of the proteolytic systems is likely to cope with ACTN2 aggregation. Our data indicates proteopathy as an additional cellular feature caused by the c.740C>T ACTN2 variant, which may contribute to human ACTN2-associated inherited cardiomyopathy.

Cardiomyopathy-associated variants alter the structure and function of the α-actinin-2 actin-binding domain

“…A study of an HCM-associated variant, T247M, established in iPSC-derived cardiomyocytes, showed that the variant is disruptive at the cellular level, inducing HCM phenotypes, including Z-disk myofibrillar disarray and disrupted contractility [16]. Moreover, T247M, like A119T, caused α-actinin-2 aggregation and reduced incorporation into Z-disks [29]. How the T247M variant impacts actin binding is not known.…”