Synthetic Human Relaxin-2 Improves Myofilament Phosphorylation Network and Reverses Cardiomyocyte Stiffness in HFpEF

H. Osman (Bochum)¹, M. Kacmaz (Bochum)², E. Halupka (Bochum )³, M. Herwig (Bochum)², I. Akin (Mannheim)⁴, I. El-Battrawy (Bochum)⁵, M. Khan (Amsterdamm)⁶, L. van Heerebeek (AC Amsterdam)⁷, T. Dschietzig (Bensheim)⁸, N. Hamdani (Bochum)^9
1Ruhr University Bochum Department of Cellular and Translational Physiology Bochum, Deutschland; ²Institut für Physiologie, Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Abteilung für Zelluläre und Translationale Physiologie, Molekulare und Experimentelle Kardiologie Bochum, Deutschland; ³Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Molekulare und Experimentelle Kardiologie Bochum , Deutschland; ⁴Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ⁵Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland; ⁶Department of Cardiology, OLVG NL Amsterdamm, Niederlande; ⁷Onze Lieve Vrouwe Gasthius Amsterdam Department of Cardiology AC Amsterdam, Niederlande; ⁸RELAXERA GmbH & Co. KG Bensheim, Deutschland; ⁹Kath. Klinikum Bochum Cellular Physiology Bochum, Deutschland

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.