Abstract Body: Background: Respiratory symptoms are highly prevalent in heart failure with preserved ejection fraction (HFpEF) and contribute to morbidity and hospitalization, yet the diaphragm remains understudied. Obese ZSF1 rats, a model of HFpEF, exhibit preserved diaphragm function at baseline, suggesting compensatory adaptations that may fail under stress. We hypothesized that acute-on-chronic stress unmasks diaphragm dysfunction driven by intrinsic myofilament alterations.

Methods: Lean (n=6) and obese (n=12) ZSF1 rats (mixed sex, 24 weeks old) underwent in vivo diaphragm echography to assess baseline function. Next, acute-on-chronic stress was induced using an intraperitoneal epinephrine/caffeine mixture (n=6/group). After 45 minutes, diaphragm strips were isolated for ex vivo contractility measurements, and tissue was collected for cellular and molecular analyses.

Results: Baseline diaphragm echography demonstrated preserved diaphragm function in obese rats compared to lean litter mates, characterized by increased end-expiratory thickness (0.56±0.06 vs. 0.40±0.05 mm, p<0.01), reduced length of the zone of apposition (6.6±0.7 vs. 10.7±0.4 mm, p<0.01), and increased respiratory rate (75±9 vs. 59±8 breaths/minute, p<0.01) and contractility (40±11 vs. 18±18% thickening, p<0.01). In contrast, acute stress revealed impaired contractile reserve, with a ~40% reduction in maximal tension (140±45 vs. 231±15 mN/mm², p<0.01) and a trend toward shorter optimal length (2.2±0.1 vs. 2.4±0.2 cm, p=0.052) in obese vs. lean ZSF1 rats. Western blot analyses showed reduced myosin light chain kinase 2 (MYLK2) expression in the obese diaphragm (p=0.03), consistent with reduced regulatory light chain phosphorylation and a shift toward the super-relaxed myosin state, suggesting impaired cross-bridge availability.

Conclusion: Diaphragm function in HFpEF is preserved at baseline but fails under stress due to a reduced contractile reserve. Myofilament alterations, including reduced MYLK2, may contribute to this dysfunction and to respiratory symptoms and exercise intolerance. Targeting diaphragm contractile reserve may represent a novel therapeutic strategy in HFpEF.