Abstract Body: Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired contractile reserve and diastolic dysfunction despite preserved systolic cardiac fraction. Myofilament phosphorylation is a key regulator of cardiac performance, yet site-specific sarcomeric phosphorylation changes in HFpEF remain poorly defined.
We performed quantitative proteome and phosphoproteome profiling of left ventricular tissue from a physiologically validated mouse model of HFpEF induced by high-fat diet and nitric oxide synthase inhibition (L-NAME) and matched controls (n = 4 hearts per group). Label-free quantitative mass spectrometry using an Orbitrap Exploris 240 was used to characterize global protein expression and serine/threonine phosphorylation profile.
Myocardium from HFpEF mice exhibited reduced phosphorylation of cardiac troponin I (TNNI3) at N-terminal residues (Ser3/5) (10.5-fold decrease vs control, p = 0.042), phosphorylation of myosin light chain 3 (Myl3) at N-terminal regulatory sites (Ser3, Ser11, Ser21) (11.8-fold decrease, p = 0.03, 9.1-fold decrease, p=0.048; 15.2-fold decrease, p=0.036 respectively), consistent with altered thick-filament regulation. In addition, phosphorylation of titin at Ser23 was reduced (4.6-fold, p = 0.0384). These myofilament changes occurred without proportional reductions in total protein abundance, indicating selective post-translational remodeling. In contrast, phosphorylation of mitochondrial metabolic enzymes, including hydroxyacyl-CoA dehydrogenase (HADH) and pyruvate dehydrogenase (PDH), was increased (8.9 fold change, p=0.001, 10.7 fold change, p=0.017, respectively). Kinase–substrate enrichment analysis indicated increased protein kinase C (PKC), pyruvate dehydrogenase kinase (PDK), and calcium/calmodulin-dependent protein kinase II (CaMKII)activity with reduced protein kinase A (PKA) activity.
These findings reveal a dissociation between myofilament and metabolic phosphorylation programs in HFpEF, suggesting impaired adaptive tuning of the contractile apparatus as a potential mechanism contributing to diastolic dysfunction and reduced stress tolerance.