Background: Heart failure (HF) with preserved ejection fraction (HFpEF) is clinically related to a reduced ability to increase cardiac function with physical stress. Rate-dependent adaptation of excitation-contraction coupling is modulated by calcium-dependent calmodulin kinase II (CaMKII). Chronical activation of CaMKII as observed in HFrEF leads to pathological remodeling and dysregulated cardiomyocyte calcium handling. We investigated in a metabolic HFpEF model whether the functional response to increased stimulation frequency is impaired in left ventricular cardiomyocytes. Additionally we explored the effects of RA306, a novel orally available inhibitor of the CaMKII (CaMKIIi) delta isoform in our HFpEF model.

Methods: We used a previously described murine two-hit metabolic HFpEF model. To induce HFpEF, adult male, 12 weeks old C57BL/6J mice received a high fat diet (D12492, Research Diet) and L-NAME (1g/l, via the drinking water). Mice received once daily the orally available CaMKIIi RA306 (30mg/KG body weight, HFpEF+CaMKIIi group) or vehicle (PBS+captisol 10%, HFpEF group) by gavage for 4 weeks from week 12-15 during the HFD+L-NAME feeding. The sham group were fed with regular chow. Mice of all groups underwent echocardiography and a treadmill test to evaluate running distance. In final experiments adult murine ventricular myocytes (AMVM) were isolated from the left ventricle and sarcomere shortening and calcium handling (Fura-2 loaded) were measured. Cardiomyocytes were exposed to a frequency stair with 1Hz, 3Hz and 5Hz. Measurements were analysed as repeated measurements at 1Hz, 3Hz and 5Hz for every cell and averaged by animal for statistical analyses. ANOVA and post-hoc pairwise comparison were used with P<0.05 as significance level.

Results: HFpEF mice (N=12) showed a lung edema (increased wet lung/tibia length ratio), higher body weight, increased E/e’, preserved LVEF and significantly reduced exercise tolerance in the treadmill test (39,2m in HFpEF vs. 158m in Sham , N= 10 HFpEF, N=7 sham). CAMKII inhibition via RA306 did not alter these HFpEF characteristics. In isolated AMVMs the amplitude of sarcomere shortening at 1Hz was significantly higher in HFpEF (7,25% of baseline) and in HFpEF+CAMKIIi (6,69% of baseline) group as to sham (4,37% of baseline). With increased pacing frequency from 1Hz to 5Hz sarcomere shortening decreased in HFpEF (negative shortening-frequency stair), whereas it remained stable in Sham, so that as 5Hz there were no significant differences in sarcomere shortening (5,38% HFpEF vs. 4,26% HFpEF+CAMKIIi vs. 4,55% sham, of baseline). Sham AMVMs exhibited a significant increase in calcium transient amplitude from 1Hz to 5Hz, whereas no change was seen in HFpEF and HFpEF+CAMKIIi. Sarcomere relaxation and diastolic Ca2+ decay accelerated with increasing stimulation frequency in HFpEF and Sham, but were significantly faster in HFpEF at baseline (1 Hz) as compared to Sham. Chronic CaMKII inhibition had no effect on the sarcomere or Ca2+ frequency response in HFpEF.

Conclusion: In this metabolic two-hit model of HFpEF baseline cardiomyocyte function at 1 Hz was enhanced, but the functional response of sarcomere shortening and Ca²⁺ transient amplitude with increased stimulation frequency were reduced in HFpEF as compared to sham. Treatment of HFpEF mice with orally available CAMKIIi did not restore the functional reserve.