Abstract Body: Sinus node dysfunction (SND), also called sinoatrial node dysfunction, is a disorder characterized by impaired pacemaker activity of the sinoatrial node (SAN), leading to manifestations such as arrhythmias, sinus arrest, and even sudden death. Fibrosis represents a major cause of SND. To date, there is no approved pharmacologic therapy to treat SND, which causes a therapeutic gap. ROCK2 (Rho-associated coiled-coil-containing protein kinase 2) is a serine/threonine kinase that functions as a key regulator of the cytoskeleton and has been implicated in cardiac fibrosis and hypertrophy. Recently, the FDA approved ROCK2 inhibitor KD025 to treat chronic graft versus host disease. Meanwhile, it is under clinical trial for several fibrosis-related diseases, such as idiopathic pulmonary fibrosis. However, ROCK2 functions in SAN are largely unknown, and whether KD025 may represent a viable therapeutic strategy remains unknown. We found that KD025 can function as a new drug to treat SND and reduce SAN fibrosis.

We treated 2-month-old control and Lats1/2 CKO mice with KD025 and vehicles by intraperitoneal injections and performed telemetry ECG recording. Male and female C57BL/6 mice were used in this study. No significant sex-linked differences were observed in the preliminary analysis; therefore, data from both sexes were pooled for subsequent experiments. Data are presented as mean ± SEM. Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant. All analyses were performed using Graphpad Prism 10. A minimum of 3 mice per group was used for all experiments to ensure reproducibility. Compared to controls, Lats1/2 CKO mice treated with vehicles exhibited hallmark features of SND , yet KD025 treatment (100 mg/kg) ameliorated SND. KD025-treated Lats1/2 CKO mice showed a significant restoration of PR and RR intervals, along with a substantial reduction in arrhythmia burden. We further found that KD025 alleviates fibrosis by inhibiting the CTGF and TGF-β pathways. We also confirmed these findings using human iPSC-derived pacemaker cells and human cardiac fibroblasts. In summary, our study identifies increased ROCK2 as a central pathogenic mediator of SND, and demonstrates that pharmacologic inhibition with the FDA-approved compound KD025 can ameliorate arrhythmia and fibrosis in a SND mouse model.