Deciphering the Molecular Mechanisms of ALPK3 and FLNC Cardiomyopathy
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CitationAgarwal, Radhika. 2020. Deciphering the Molecular Mechanisms of ALPK3 and FLNC Cardiomyopathy. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractCardiomyopathies represent a spectrum of diseases characterized by pathologic cardiac enlargement that can lead to heart failure. A significant proportion of non-ischemic cardiomyopathies occur as a consequence of rare damaging genetic variants. Understanding how these variants induce cardiomyocyte dysfunction can provide insight into the pathogenesis of heart failure – a disease for which few therapies exist. CRISPR/Cas9-mediated genome editing in human induced pluripotent stem cells (hiPSCs), coupled with methods to differentiate hiPSCs into cardiomyocytes (hiPSC-CMs), allow the effects of mutated cell lines to be compared directly to isogenic non-edited parent lines. Harnessing these technologies, we characterized the molecular mechanisms by which damaging variants in two genes – FLNC (filamin C) and ALPK3 (alpha-kinase-3) – cause cardiomyopathy.
To investigate FLNC pathogenesis, we engineered four mutant hiPSC lines – one with homozygous null alleles, two with different heterozygote variants, and one with a homozygous missense variant of unknown significance. We found that some FLNC expression was necessary for normal sarcomere thin filament gene expression and assembly – these processes were deficient in FLNC homozygous null hiPSC-CMs. However, hiPSC-CMs carrying heterozygote variants, modeling human disease, displayed an additional, distinct mechanism of pathogenicity characterized by toxic protein accumulation of FLNC-binding partners and increased lysosome abundance. We suggest that strategies to ameliorate protein accumulation may be therapeutic in FLNC-cardiomyopathy.
To investigate ALPK3 pathogenesis, we engineered two mutant hiPSC lines –one containing biallelic ALPK3-null alleles (modeling human disease variants), and the other containing homozygote missense variants predicted to conformationally alter the ALPK3 kinase domain. Both perturbations increased the protein levels of myomesins (MYOM1, MYOM2) and sarcomere thick filament proteins in hiPSC-CMs, effects that we also observed in Alpk3-/- mice. Furthermore, ALPK3 co-localized with MYOM1 and MYOM2 at the sarcomere M-band and the nuclear envelope respectively, and myomesin assembly into these structures was deficient in ALPK3-null hiPSC-CMs. Surprisingly, genetic inhibition of the ALPK3 kinase domain did not detectably alter phosphorylation across the cardiomyocyte phosphoproteome, thus defining this protein as a pseudokinase. Given these results, we propose a model in which ALPK3 serves as a critical scaffold for myomesin incorporation and thick filament turnover, the dysfunction of which results in cardiomyopathy.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365925
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