{
  "abstract": "Inherited cardiomyopathies are a group of genetic diseases that affect cardiac structure and function. One of the most common forms is hypertrophic cardiomyopathy (HCM), a condition characterised by diastolic dysfunction and arrhythmias which are often linked to sudden cardiac death. HCM has been associated with genetic variants in several cardiac proteins, including a key Z-disk protein Alpha-actinin-2 (ACTN2). A 2014 study identified a novel ACTN2 missense variant, M228T, in 11 family members affected with HCM and arrhythmias. Our previous in vivo work further demonstrated embryonic lethality in mice homozygous for the M228T variant, as well as atrial electrical alterations in the heterozygous model.The mechanism by which the ACTN2 M228T variant impacts protein function in cardiomyocytes remains poorly understood. This study therefore aims to assess the changes occurring at the functional level of the mutant protein using an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model incorporating the homozygous copy of the M228T variant.Mutant cardiomyocytes showed an upregulation in markers of hypertrophy, stress-response and fibrosis. Immunostaining revealed protein aggregates positive for ACTN2 and other thin- and thick-filament proteins in the iPSC-CM model. In addition, mutant cardiomyocytes showed reduced ACTN2 protein levels, a finding further confirmed by biochemical fractionation assays. To investigate whether protein degradation mechanisms contributed to ACTN2 loss, the ubiquitin proteosome system and the autophagy lysosomal pathway were pharmacologically inhibited. These experiments did not support a clear role for either pathway in driving ACTN2 degradation, however, mutant cardiomyocytes revealed autophagy dysregulation.Further analysis demonstrated impaired maturation in mutant cardiomyocytes, with contractility measurements showing prolonged relaxation times and a reduction in contraction duration. Optical mapping using voltage- and calcium- sensitive dyes revealed shortened action potential duration and delayed calcium transient kinetics. Analysis of transcriptional ion channels showed upregulation of several potassium and sodium channel genes, with no changes in calcium channel expression. Additional analysis using Seahorse metabolic flux analysis demonstrated reduced energetic demand in mutant cardiomyocytes under both baseline and maximal conditions.Collectively, this study provides important insights into the functional impact of ACTN2M228T variant in cardiomyocytes. Our analysis represents a crucial step towards understanding disease pathways for cardiomyopathy-linked ACTN2 variants.",
  "authors": [
    {
      "affiliations": [
        "University of Birmingham, Birmingham, United Kingdom"
      ],
      "name": "Maya Noureddine"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, United Kingdom"
      ],
      "name": "Fiyaz Mohammed"
    },
    {
      "affiliations": [
        "University of Warwick, Warwick, United Kingdom"
      ],
      "name": "Christopher O’Shea"
    },
    {
      "affiliations": [
        "Diamond Light Source, Harwell, United Kingdom"
      ],
      "name": "Halina Mikolajek"
    },
    {
      "affiliations": [
        "University of Nottingham, Nottingham, United Kingdom"
      ],
      "name": "Chris Denning"
    },
    {
      "affiliations": [
        "University of Nottingham, Nottingham, United Kingdom"
      ],
      "name": "Siobhan Loughna"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, United Kingdom"
      ],
      "name": "Sophie Broadway-Stringer"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, United Kingdom",
        "University of Oxford, Oxford, United Kingdom"
      ],
      "name": "Katja Gehmlich"
    }
  ],
  "title": "402 Understanding the impact of an alpha-actinin-2 variant on protein function in induced pluripotent stem cell-derived cardiomyocytes",
  "uid": "f01e54fc-eb47-5130-bac8-155dae3f6339"
}