Impaired mitochondrial proteostasis underlies cardiomyocyte bioenergetic dysfunction in a murine 3-hit model of heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) predominantly affects older individuals with multiple comorbidities. Although mitochondrial dysfunction appears to be a major contributor to diastolic dysfunction, the mechanisms underlying it during HFpEF remain unclear. We used a clinically relevant two-hit HFpEF model in aged mice to investigate remodeling of cardiomyocyte mitochondrial proteins and to determine whether mitochondrial dysfunction reflects impaired biogenesis or defective mitochondrial proteostasis. HFpEF was induced in aged (15-month-old) male and female mice using a high-fat diet combined with chronic nitric oxide synthase inhibition (L-NAME). This model recapitulated key features of human HFpEF, including preserved ejection fraction with diastolic dysfunction and impaired cardiac reserve in both sexes. Consistent with impaired energy reserve, mitochondrial respiratory capacity measured by high-resolution respirometry (Oroboros) was significantly reduced in left ventricular muscle fibers. To examine cardiomyocyte-intrinsic mechanisms, mitochondria were isolated from cardiomyocytes using Langendorff perfusion and analyzed by proteomics and pathway enrichment analysis. Proteomic profiling revealed broad suppression of mitochondrial proteins that support oxidative metabolism and organelle maintenance, including those involved in oxidative phosphorylation, carbohydrate metabolism, and the TCA cycle. In contrast, key regulators of mitochondrial biogenesis were preserved. Notably, mitochondrial chaperones and proteases for protein quality control were reduced, leading to increased accumulation of ubiquitinated mitochondrial proteins and impaired mitochondrial proteostasis. Together, these findings indicate that mitochondrial dysfunction in a 3-hit HFpEF model is characterized by reduced respiratory capacity and impaired mitochondrial proteostasis, rather than defective biogenesis, suggesting that impaired mitochondrial protein quality control may contribute to reduced cardiac reserve in HFpEF.