Background: In HFpEF, elevated cardiomyocyte stiffness stems from hypo-phosphorylated sarcomeric elements, most prominently titin, and dysregulated thin/thick-filament regulators (TnI, MyBPC). Human Relaxin-2, a peptide hormone known for its vasodilatory and antifibrotic properties, has been proposed to improve ventricular compliance; however, its direct effects on human myocardial stiffness remain undefined. We posited that Synthetic Human Relaxin-2 (shRlx-2) acutely improves diastolic mechanics by synchronously re-phosphorylating titin, TnI, and MyBPC, with a supportive shift in titin isoforms toward compliance.
Hypothesis: Acute shRlx-2 treatment enhances cardiomyocyte compliance in HFpEF by increasing phosphorylation of titin (N2B spring), troponin-I (TnI), and myosin-binding protein C (MyBPC), alongside a modest rise in the N2BA/N2B ratio.
Methods and Results: LV and RV myocardium from male and female HFpEF patients and lean donors underwent ex vivo shRlx-2 exposure (4 nmol/L, 60 min), followed by skinned-cell mechanics and sarcomeric biochemistry. ShRlx-2 consistently lowered passive tension across sarcomere lengths in HFpEF LV and RV, shifting Fpassive–SL curves toward donor (non-failing human hearts) (dose/time series 0.2–4 nmol/L; 30–60 min).
Immunoblots revealed increased total titin P-Ser/Thr (notably within N2B), with a small but directionally favorable rise in N2BA/N2B (consistent across sexes and chambers), supporting enhanced molecular spring compliance. Crucially, shRlx-2 also augmented TnI phosphorylation in HFpEF with chamber- and sex-specific significance (multiple LV/RV male and female groups), and increased MyBPC phosphorylation in HFpEF, aligning with observed reduced cardiomyocyte stiffness. These myofilament regulatory changes suggest improved thin-filament Ca²⁺ desensitization (via pTnI) and faster cross-bridge cycling/relaxation (via pMyBPC), acting in concert with titin softening to restore diastolic compliance.
Conclusion: ShRlx-2 rapidly improves diastolic mechanics in human HFpEF through coordinated sarcomeric re-phosphorylation, titin (N2B), TnI, and MyBPC, with a modest N2BA/N2B shift, providing a unifying, sarcomere-centric mechanism for its acute lusitropic effect. These findings nominate shRlx-2 as a mechanism-based therapy that targets both the passive (titin) and regulatory (TnI/MyBPC) determinants of cardiomyocyte stiffness in HFpEF.
