Impaired cardiac mitochondrial bioenergetics contribute to loss of cardiac function, in a porcine multiple risk factor model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch CardioVascular Alliance (an initiative with support of the Dutch Heart Foundation) Grant 2020B008 RECONNEXT, and the German Center for Cardiovascular Research (DZHK; 81Z0600207) Background Heart failure with preserved ejection fraction lacks targeted therapies, due to insufficient understanding of pathogenesis. A multiple comorbidity swine model (MCS) was developed by exposure to three risk factors for six months including diabetes (streptozotocin), chronic kidney disease (renal embolization), and an unhealthy diet (high-fat, high salt diet). An additional risk factor ovariectomy was added on top of the MCS model (MCS+). MCS and MCS+ presented with left ventricular diastolic dysfunction, oxidative stress, coronary microvascular dysfunction and inflammation. Furthermore, myocardial oxygen consumption was increased given the same level of cardiac work (Fig. A), suggesting an impairment in myocardial mitochondrial function. Using a combined-omics approach, the purpose of this study is to unravel alterations in gene expression and proteome abundance regarding mitochondrial function in MCS swine. Methods 15 MCS, 4 MCS+ and 10 healthy control female swine were included in the study. Proteome analysis and single nuclei RNA sequencing, were performed on frozen MCS left ventricle myocardial samples. Cardiac mitochondrial function was measured by an O2k-FlouRespirometer ex vivo in MCS+ swine versus control. Results Proteome analysis showed a reduced abundance of proteins involved in branched-chain amino acid (BCAA) catabolism (Fig. B). Single nuclei RNA sequencing of a subgroup of the same animals demonstrated a downregulation of these genes specifically in cardiomyocyte subpopulations. BCAA can act as a source of ATP via oxidative phosphorylation and can modulate mitochondrial substrate utilisation, and impair mitochondrial function, resulting in increased mitochondrial production of reactive oxygen species (ROS). Mitochondrial function was measured in fresh myocardial tissue, using pyruvate/malate/glutamate as substrate. Low mitochondrial respiratory sensitivity to ADP was observed 26±2.5 in MCS+ vs 43.6±1.5 pmol/(s*mg tissue) in healthy control, reflecting low oxidative capacity. Moreover, the respiratory rate after uncoupling by FCCP was reduced in MCS+ versus healthy control (73.6±11.7 vs 118.4±15.6 pmol/(s*mg tissue). These findings are consistent with our data in the MCS swine and indicate impaired BCAA in the myocardium of animals with comorbidities, which is associated with increased ROS levels (8-isoprostane 12.9±0.8 pg/mg protein in MCS vs 10.3±0.5 in healthy animals) and impaired myocardial efficiency during exercise (Fig. A). Conclusion A combined omics approach suggested an alteration in cardiac mitochondria. Respiratory analyses showed impaired mitochondrial bioenergetics and increased oxidative stress, which can contribute to diastolic dysfunction.