{
  "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.Introduction Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease marked by diastolic dysfunction and left ventricular hypertrophy, often linked with sudden cardiac death (Harris et al., 2006). Genetic studies have associated genetic variants in Z-disk proteins, including Alpha-actinin-2 (ACTN2), with HCM. ACTN2 stabilises the contractile muscle apparatus by anchoring actin filaments (Sjöblom et al., 2008). While a few ACTN2 variants linked to HCM have been identified, research has primarily focused on the actin-binding domain, with no studies examining variants in the full-length protein. Notably, the ACTN2 M228T variant was identified in 11 HCM-affected family members (Girolami et al., 2014), and recent findings from our group indicate that homozygous mice harbouring this mutation are embryonically lethal, possibly due to ACTN2 destabilization (Broadway-Stringer et al., 2023).The precise mechanisms by which ACTN2 variants contribute to HCM remain poorly understood. Herein, we investigated the biophysical implications of ACTN2 variants on protein structure and the functional consequences of the ACTN2-M228T variant to better elucidate potential disease mechanisms.Methods Missense variants in ACTN2 were identified using the Human Mutation Gene Database (HGMD), followed by in silico characterization. Structural modelling was employed to investigate disease mechanisms. Recombinant mutant proteins were generated via mutagenesis, expressed in, and purified from BL21 bacteria. Structural alterations in ACTN2 variants were examined using biophysical techniques, including mass photometry, X-ray crystallography, small-angle X-ray scattering, actin-binding, and thermal assays. Functional and molecular assessments of the ACTN2-M228T homozygous (Hom) variant in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), described in (Cumberland et al., 2023), included contractility analysis, immunofluorescence staining, biochemical fractionation and proteosome and protease inhibitor treatments.Results 1. R327C and R457C ACTN2 variants did not disrupt dimerisation Using the HGMD, 76 ACTN2 variants were identified and further characterized using in silico tools. Of 45 variants predicted to be pathogenic, 20 were associated with HCM. Two of these variants (R327C and R457C) were found to stabilise the ACTN2 dimer structure (figure 1A). However, neither variant disrupted dimer formation after 48 hours of incubation with high-salt concentration (2.5M NaCl) and rigorous shaking (figure 1B). Thermal assays showed that the R327C variant had a lower melting temperature than wild-type (figure 1C) and exhibited increased aggregate formation (figure 1D).Abstract B Figure 1(A) R327 and R457 contribute to stabilizing the ACTN2 dimer interface ( red). (B) Both variants in solution do not disrupt dimer formation after high salt incubation. (C) R327C variant demonstrates decreased thermal stability. (D) R327C variant is prone to aggregate formation based on molecular weight calculations using mass photometry2. G111V, M228T, and T247M variants display impaired thermal stability and actin-binding, with M228T showing aggregate formation at 60 °C Three ACTN2 variants, G111V, M228T and T247M, showed decreased thermal stability indicating impaired function (figure 2A). Actin binding assays revealed increased binding affinity to actin for the M228T and T247M variants, and decreased affinity for G111V (figure 2B). In addition, small angle X-ray scattering (SAXS) analysis of the M228T variant highlighted aggregate formation at 53–60°C (figure 2C-D).Abstract B Figure 2(A) Thermal assays show a 10°C decrease in melting temperature (Tm) in three ACTN2 variants. (B) Actin-binding assay revealing increased actin binding affinity for M228T and T247M variants and decreased affinity in G111V. (C) SAXS log 10 profile of M228T variant demonstrating aggregate formation at 60°C. (D) M228T variant protein profile displaying aggregation3. M228T mutant cardiomyocytes display decreased contractility and developmental delays M228T Hom iPSC-CMs displayed a significant decrease in contraction amplitude and increase in relaxation time relative to wild-type (figure 3A). Mutant cells also indicated developmental delays evident by decreased MYH7 protein and increased MYH6 mRNA (figure 3B). In addition, the cells demonstrated a significant upregulation in fetal-gene programme markers including ANKRD1 and FHL1, collagen markers such as COL1A and COL3A, and cardiac stress markers NPPA and NPPB.Abstract B Figure 3(A) M228T Hom iPSC-CMs display decrease contractility analysed using MUSCLE MOTION software. (B) The Hom cells indicate significant decreased in MYH7 protein and increase in MYH6 mRNA. (C) The Hom cells reveal upregulation of fetal-gene markers, fibrosis, and cardiac stress markers. Unpaired t-test was used. Values are the means ± S.D. * p < 0.05; Total n = 5 (Protein), and n=8 (mRNA)4. M228T cells display protein aggregation, destabilisation and autophagy activation Immunofluorescence staining of mutant cardiomyocytes showed aggregates in ACTN2 and sarcomeric proteins including myomesin, titin, cardiac troponin T, and actin (figure 4A). Mutant cardiomyocytes showed destabilised ACTN2 using biochemical fractionation techniques (figure 4B). In addition, M228T Hom cells showed upregulation in autophagy markers (LC3I and LC3II) (figure 4C).Abstract B Figure 4(A) M228T Hom iPSC-CMs show protein aggregation of ACTN2 (in green), and cardiac proteins (TNNT2: Cardiac-troponin-T, Phalloidin: stain for F-actin, Myomesin; TTN-T12: stain for titin Z-disk portion, in red). (B) Mutant cells highlight significant decrease in soluble ACTN2 upon biochemical fractionation and decrease in myofilament fraction. (C) The cells also show upregulation of autophagy markers. Unpaired t-test was used. Values are the means ± S.D. * p < 0.05; Total n = 5 biological replicatesDiscussion Collectively, this study enhances our understanding of the impact of ACTN2 variants on protein structure and function. Structural modelling elucidated the mechanisms through which these variants contribute to HCM. The biophysical characterisation showed that two variants do not disrupt dimerisation, while three variants affected actin binding. Other variants compromised protein stability, as demonstrated through various assays. Therefore, employing structural analysis before functional studies is an important step in evaluating the pathogenicity of these variants.Moreover, the iPSC-CM model incorporating the M228T variant provided valuable insights into characterizing the HCM phenotype in mutant cardiomyocytes. Notably, the significant upregulation of hypertrophy and fibrosis markers might indicate pathological hypertrophy. Furthermore, the formation of ACTN2 protein aggregates and evidence of protein degradation suggested an impairment in protein function. This was further investigated using specific inhibitors targeting the ubiquitin-protease system and the autophagy lysosomal pathway. Finally, by correlating the structural to functional analysis, this study represents a foundational step toward understanding the disease pathways for Z-disc-associated HCM.Author’s Contribution M. Noureddine, a third year PhD student in basic cardiovascular sciences, and is the first author/presenter and contributor to this work. F. Mohammed, the PhD secondary supervisor, oversaw structural modelling and biophysical studies. H. Mikolajek, N. Coweison, and N. Pintosis contributed to X-ray crystallography and small angle X-ray scattering. P. Robinson assisted with actin-binding assays, while C. Denning and S. Loughna provided guidance on the iPSC-CM model. A. Slater advised on protein expression. K. Gehmlich, the PhD primary supervisor, led the biophysical and functional work.References Broadway-Stringer S, Jiang H, Wadmore K, Hooper C, Douglas G, Steeples V, Azad AJ, Singer E, Reyat JS, Galatik F, Ehler E, Bennett P, Kalisch-Smith JI, Sparrow DB, Davies B, Djinovic-Carugo K, Gautel M, Watkins H, Gehmlich K. Insights into the role of a cardiomyopathy-causing genetic variant in ACTN2. Cells 2023;12(5). https://doi.org/10.3390/cells12050721 Cumberland MJ, Euchner J, Azad AJ, N TNV, Kirchhof P, Holmes AP, Denning C, Gehmlich K. Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model. F1000Res. 2023;12:1224. https://doi.org/10.12688/f1000research.139482.1 Girolami F, Iascone M, Tomberli B, Bardi S, Benelli M, Marseglia G, Pescucci C, Pezzoli L, Sana ME, Basso C, Marziliano N, Merlini PA, Fornaro A, Cecchi F, Torricelli F, Olivotto I. Novel α-actinin 2 variant associated with familial hypertrophic cardiomyopathy and juvenile atrial arrhythmias: a massively parallel sequencing study. Circ Cardiovasc Genet. 2014;7(6):741–750. https://doi.org/10.1161/circgenetics.113.000486 Harris KM, Spirito P, Maron MS, Zenovich AG, Formisano F, Lesser JR, Mackey-Bojack S, Manning WJ, Udelson JE, Maron BJ. Prevalence, clinical profile, and significance of left ventricular remodeling in the end-stage phase of hypertrophic cardiomyopathy. Circulation 2006;114(3):216–225. https://doi.org/10.1161/circulationaha.105.583500 Sjöblom B, Salmazo A, Djinović-Carugo K. Alpha-actinin structure and regulation. Cell Mol Life Sci. 2008;65(17):2688–2701. https://doi.org/10.1007/s00018-008-8080-8",
  "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": "B Biophysical and functional characterisation of alpha-actinin-2 variants in hypertrophic cardiomyopathy",
  "uid": "ae45b6d4-950c-5f0f-abba-a8557dc05a00"
}