Functional and structural changes in isolated atrial cardiomyocytes of an aged multi-Hit HFpEF model

Background Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous, systemic syndrome driven by multiple cardiometabolic stressors. Appropriate preclinical models that recapitulate this complexity are required to dissect cellular mechanisms. The established “two-hit” model (high-fat diet [HFD] + L-NAME) reproduces key risk factors such as obesity and hypertension. However, HFpEF frequently develops in the context of multiple cumulative stressors and is associated with atrial remodeling and atrial fibrillation. We therefore investigated whether additional hits aggravate atrial cardiomyocyte dysfunction in aged HFpEF mice. Methods HFpEF was induced in aged mice using the classical two-hit protocol (HFD + L-NAME) or an intensified five-hit protocol (additional DOCA and NaCl). Untreated aged mice served as controls. Left atrial cardiomyocytes were enzymatically isolated, loaded with Fluo-4 AM and MitoTracker, and analyzed by confocal line-scan imaging during 1 Hz stimulation. Calcium transients (F/F₀), caffeine-induced decay kinetics (NCX activity), and mitochondrial density were quantified. Results Atrial cardiomyocytes (ACMs) from aged five-hit mice exhibited significant alterations in Ca²⁺ handling compared with aged naïve controls. Time-to-peak of the Ca²⁺ transient was prolonged (67±19 vs. 52±17 ms), as was time to 50% (TF₅₀: 23.6±11.3 vs. 16.7±5.5 ms), indicating slowed Ca²⁺ release kinetics. In addition to temporal slowing, spatial organization of Ca²⁺ release was disrupted. Five-hit ACMs displayed heterogeneous and patchy subcellular Ca²⁺ signals, reflected by an increased standard deviation of local Ca²⁺ peak amplitude (0.45±0.38 vs. 0.23±0.12), consistent with impaired release synchrony. Caffeine-induced Ca²⁺ decay was significantly prolonged in five-hit mice (tau: 2764±683 vs. 1874±353 ms), suggesting reduced NCX-dependent Ca²⁺ extrusion and impaired cytosolic Ca²⁺ clearance. Moreover, mitochondrial density was modestly but significantly increased in five-hit ACMs (40.8±1.5% vs. 38.7±1.9%), indicating metabolic remodeling that may influence Ca²⁺ buffering and excitation–metabolism coupling. Importantly, ACMs from aged two-hit HFpEF mice did not show significant differences compared with aged naïve controls. Conclusion The five-hit HFpEF model induces marked atrial Ca²⁺ handling and metabolic remodeling in aged mice, whereas the classical two-hit model does not. This intensified model may better reflect the multifactorial atrial phenotype of human HFpEF and provide a platform for mechanistic and therapeutic studies.