Background: Recent studies suggest aberrant elevation of iNOS (inducible NO synthase) expression and excessive protein s-nitrosylation promote the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, the interplay between NO bioavailability, enzymatic regulation of protein s-nitrosylation by transnitrosylase and denitrosylase, and HFpEF progression remains poorly defined. We investigated the molecular basis of nitrosative stress in HFpEF, focusing on alterations in NO signaling and regulation of protein s-nitrosylation.
Methods: Circulating nitrite (NO bioavailability) and nitrosothiols were quantified in patients with HFpEF. Parallel studies using rodent models of cardiometabolic HFpEF were performed to evaluate cardiac function, NO signaling, and total nitroso species during disease progression. Single-nucleus RNA sequencing and proteomic analysis were conducted to identify regulatory genes and cellular targets of pathological s-nitrosylation.
Results: In patients with HFpEF, circulating nitrosothiols were significantly elevated, indicating heightened nitrosative stress, whereas nitrite levels remained unchanged. In Zucker fatty obese rats, NO bioavailability declined with age, whereas total nitroso species progressively increased as HFpEF worsened. Transcriptomic analysis revealed marked upregulation of a transnitrosylase HBb (hemoglobin-β subunit), validated in both rat and human HFpEF hearts. Enzymatic assays demonstrated aberrant functions of Trx2 (thioredoxin 2) and GSNOR (S-nitrosoglutathione reductase) in Zucker fatty hearts. Cell-based experiments confirmed that altered expression or function of HBb, Trx2, and GSNOR resulted in elevated cellular RxNO. Additionally, similar dysregulation of s-nitrosylation dynamics was observed in the peripheral organs, such as the kidneys and livers, in HFpEF.
Conclusions: These data demonstrate that nitrosative stress, evidenced by dysregulated protein s-nitrosylation occurs in the heart and peripheral organs in cardiometabolic HFpEF. Pathological alterations in NO bioavailability resulting from alterations in NOS expression or function alone do not account for this phenotype. Instead, pathological protein s-nitrosylation results in part from the imbalance between transnitrosylase and denitrosylase function. Restoration of physiological levels of protein s-nitrosylation and NO signaling may represent an effective therapeutic target for HFpEF.