ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Abstract

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome

“…Five µg protein was processed for LC-MS/MS analysis, as previously described. 47 Briefly, protein was reduced (2.5 mM dithiothreitol, Sigma-Aldrich; 30 min at 37°C) and alkylated (10 mM iodacetamide, Sigma-Aldrich; 15 min at 37°C) before proteolytic digestion with LysC (enzyme to protein ratio 1:100, Promega) for 3 h and with trypsin (1:25, Promega) for 16 h both at 37°C. The reaction was stopped with 1% acetic acid (Sigma-Aldrich), and the peptide mixtures were desalted on C-18 reverse phase material (ZipTip μ-C18, Millipore).…”

Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor

“…Here we present a lower-cost bioreactor-based cardiac differentiation protocol (~$569 per bioreactor run yielding ~124 million hiPSC-CMs, compared to ~$1075 for ML differentiation with comparable yield, exclusive of manpower; Table S3) with defined benchmarks and controlled freeze/thaw procedures. We extensively characterized the resulting bCMs and demonstrated that they have robust structural and functional properties of cardiomyocytes, reduced batch-to-batch variation, and greater force production in EHTs than previously described 5,[16][17][18][32][33][34]36 . These levels of force were comparable to EHTs that underwent physical conditioning of increasing intensity 35 .…”

Structural and signaling proteins in the Z-disk and their role in cardiomyopathies

“…The results revealed several features of HCM including myofibrillar disarray, cardiomyocyte hypertrophy, impaired relaxation, hypercontractility, increased myofilament calcium sensitivity, and prolonged action potential duration (Prondzynski et al, 2019). A recent study by Zech et al involved used iPSC-CMs heterozygous and homozygous for this ACTN2 variant (p.Thr247Met) and showed multinucleation, cardiomyocyte hypertrophy, and myofibrillar disarray in the cellular models (Zech et al, 2022).…”

“…Global inactivation of alpha-actinin two in mice is not compatible with life, but a cardiac specific mosaic inactivation suggests a crucial role of the protein for cardimyocyte maturation via serum response factor signaling (Guo et al, 2021). Moreover, a role of alpha-actinin two in regulating cardiac mitochondrial function has been proposed recently (Zech et al, 2022), in part by acting as a scaffold for mitochondrial messenger RNAs via binding to RNA-binding proteins (Ladha et al, 2020).…”