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.

中文翻译：

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在猪多重危险因素模型中，受损的心脏线粒体生物能导致心脏功能丧失


资金致谢 资金来源类型： 公共补助金 – 仅国家预算。主要资金来源：荷兰心血管联盟（荷兰心脏基金会支持的一项倡议）拨款 2020B008 RECONNEXT 和德国心血管研究中心 (DZHK; 81Z0600207) 背景 射血分数保留的心力衰竭缺乏针对性治疗，因为对发病机制认识不足。通过在六个月内暴露于三种危险因素，包括糖尿病（链脲佐菌素）、慢性肾病（肾栓塞）和不健康饮食（高脂肪、高盐饮食），建立了多种合并症猪模型（MCS）。在 MCS 模型 (MCS+) 的基础上添加了额外的危险因素卵巢切除术。 MCS 和 MCS+ 表现为左心室舒张功能障碍、氧化应激、冠状动脉微血管功能障碍和炎症。此外，在相同水平的心脏做功的情况下，心肌耗氧量增加（图A），表明心肌线粒体功能受损。本研究的目的是采用组合组学方法，揭示 MCS 猪线粒体功能相关基因表达和蛋白质组丰度的变化。方法本研究纳入15头MCS、4头MCS+和10头健康对照母猪。对冷冻 MCS 左心室心肌样本进行蛋白质组分析和单核 RNA 测序。通过 O2k-FlouRespirometer 离体测量 MCS+ 猪与对照猪的心脏线粒体功能。结果蛋白质组分析显示参与支链氨基酸 (BCAA) 分解代谢的蛋白质丰度减少（图 B）。 对同一动物亚组的单核 RNA 测序表明，这些基因在心肌细胞亚群中尤其下调。 BCAA 可通过氧化磷酸化作为 ATP 来源，并可调节线粒体底物利用，损害线粒体功能，导致线粒体产生活性氧 (ROS) 增加。使用丙酮酸/苹果酸/谷氨酸作为底物测量新鲜心肌组织中的线粒体功能。 MCS+ 中线粒体对 ADP 的呼吸敏感性较低，为 26±2.5，而健康对照组为 43.6±1.5 pmol/(s*mg 组织)，反映出氧化能力较低。此外，与健康对照相比，MCS+ 中 FCCP 解偶联后的呼吸频率降低（73.6±11.7 vs 118.4±15.6 pmol/（s*mg 组织）。这些结果与我们在 MCS 猪中的数据一致，表明在 MCS 猪中支链氨基酸受损患有合并症的动物的心肌，这与 ROS 水平升高（MCS 中的 8-异前列腺素为 12.9±0.8 pg/mg 蛋白质，而健康动物中为 10.3±0.5）和运动期间心肌效率受损有关（图 A）。该方法表明心脏线粒体发生了变化，呼吸分析显示线粒体生物能量受损和氧化应激增加，这可能导致舒张功能障碍。