{
  "abstract": "Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by diastolic dysfunction and is frequently associated with sudden cardiac death. HCM is associated with pathogenic variants in Z-disk proteins, a key structural component of the sarcomere. Alpha-actinin 2 (ACTN2), is a crucial Z-disk protein important for cross-linking actin filaments. Few ACTN2 variants have been identified, with a limited focus on the mechanisms by which these variants alter protein structure and function. Thereby, this study investigated the biophysical characterisation of multiple ACTN2 variants, as well as assessing the functional impact of the ACTN2-M228T variant in cellular models. ACTN2 missense variants were identified using the Human Mutation Gene Database and characterized with established in silico tools. Structural modelling approaches were then used to predict the impact of HCM-associated variants on ACTN2. To further assess their structural consequences, ACTN2 variants were recombinantly expressed, purified, and analysed using mass photometry, X-ray crystallography, small-angle X-ray scattering, actin-binding and thermal denaturation assays. Additionally, functional and molecular assessments of the ACTN2-M228T variant were performed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM).Characterization of the identified ACTN2 variants revealed 18 variants distributed among the different domains of the protein. Structural modeling predictions showed that these variants impact the protein structure through distinct mechanisms, including impaired actin-binding, disrupted dimerization, and reduced protein stability. Actin-binding assays of six variants identified that two variants exhibited enhanced binding, two demonstrated reduced binding, and two showed no change. Additionally, the R327C and R457C variants, predicted to disrupt dimerization, were crystalized, with structures revealing no effect on dimerization. Other variants showed reduced protein stability as evidenced by decreased solubility, impaired thermal stability, alterations in structural conformation, and increased aggregation.The functional impact of the ACTN2-M228T variant was evaluated using iPSC-CMs. Cardiomyocytes harbouring the mutant gene displayed developmental delays, decreased contractility, and upregulation of fibrosis and hypertrophy markers. Protein aggregation and destabilization were confirmed by immunofluorescence and biochemical fractionation techniques. Furthermore, protein degradation mechanisms were examined using inhibitors targeting the ubiquitin-proteosome system and the autophagy-lysosomal pathway, revealing that mutant cells demonstrated autophagy upregulation.Taken together, this study provides valuable insights into how ACTN2 variants impact protein structure and function, contributing to HCM pathogenesis. These findings enhance our understanding of disease mechanisms and may inform the development of targeted therapeutic strategies.",
  "authors": [
    {
      "affiliations": [
        "University of Birmingham, Birmingham, UK"
      ],
      "name": "Maya Noureddine"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, UK"
      ],
      "name": "Fiyaz Mohammed"
    },
    {
      "affiliations": [
        "Diamond Light Source, Didcot, UK"
      ],
      "name": "Halina Mikolajek"
    },
    {
      "affiliations": [
        "Diamond Light Source, Didcot, UK"
      ],
      "name": "Nathan Coweison"
    },
    {
      "affiliations": [
        "University of Oxford, Oxford, UK"
      ],
      "name": "Paul Robinson"
    },
    {
      "affiliations": [
        "Birkbeck College, London, UK"
      ],
      "name": "Nikos Pinotsis"
    },
    {
      "affiliations": [
        "University of Nottingham, Nottingham, UK"
      ],
      "name": "Chris Denning"
    },
    {
      "affiliations": [
        "University of Nottingham, Nottingham, UK"
      ],
      "name": "Siobhan Loughna"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, UK"
      ],
      "name": "Alexandre Slater"
    },
    {
      "affiliations": [
        "University of Birmingham, Birmingham, UK"
      ],
      "name": "Katja Gehmlich"
    }
  ],
  "title": "BS32 Biophysical and functional characterisation of alpha-actinin-2 variants in hypertrophic cardiomyopathy",
  "uid": "800b0650-2e75-5b52-8991-ec362c0cdf96"
}