Abstract Body (Do not enter title and authors here): Background: Reduced H₂S levels in HFpEF patients suggest that restoring its bioavailability could improve disease pathology. Previous studies suggest that some of the acute cardioprotective effects of H₂S therapy are mediated through modulation of calcium ion channels and associated transporters. We hypothesized that H₂S donor therapy via oral polysulfide (SG1002), which has previously been tested in HFrEF patients, would improve HFpEF outcomes by modulating myocardial calcium signaling pathways.

Methods: 9-week-old male C57BL6/N mice were fed a combination of high-fat diet and L-NAME (0.5 g/L) in drinking water to induce HFpEF. Five weeks after the onset of HFpEF (n=12 per group) were administered oral H₂S donor SG-1002 (95 mg/kg/day) for 5 weeks and compared to HFD + L-NAME alone mice (i.e., control). Exercise capacity was measured using forced treadmill running. Diastolic function was evaluated via transthoracic echocardiography and invasive hemodynamics. Bulk RNA Seq was performed on cardiac muscle to determine the transcriptional response to SG1002 therapy. Pathway enrichment analysis was performed to generate a natural enrichment score (NES) using globaltest software in R (version 4.3.2, R Foundation).

Results: SG1002 treatment significantly (150%) elevated plasma H₂S levels (p=0.0001), and enhanced exercise capacity (p=0.0360). SG1002 therapy reduced E/E’ ratio (-7.5±6.8, p=0.0016)) and markedly decreased left ventricular (LV) end-diastolic pressure (-12.3±5.1 mmHg, p<0.0001) when compared to HFpEF control mice. Histological analysis demonstrated attenuated cardiac hypertrophy in SG1002-treated HFpEF mice, with reductions in cardiomyocyte size (10.6% reduction, p<0.01) and an increase in LV lumen area (68.6% increase, p<0.001). RNA sequencing revealed significant downregulation of genes associated with dilated cardiomyopathy (Tnnt2 and Mhy6), and pathological calcium signaling pathways (Pln and Atp2a2) in SG1002-treated HFpEF mice compared to HFpEF mice (p<0.001, z-score=-2.0).

Conclusion: Oral H₂S donor therapy improved LV diastolic function, increased treadmill exercise capacity, and reduced cardiac hypertrophy in cardiometabolic HFpEF mice. The downregulation of calcium signaling and dilated cardiomyopathy gene expression pathway supports the therapeutic potential of H₂S therapy in HFpEF.