Objectives Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic mechanism in ventricular myocytes allowing a faster ventricular relaxation (and diastolic filling) at fast heart rates. Previous studies suggest that CaMKII activity is required for FDAR but the molecular targets remain elusive.

We propose that CaMKII regulates FDAR by a mechanism that involves CaMKII-dependent alteration of myofilament sensitivity to Ca²⁺.

Methods [Ca²⁺]_i and sarcomere length were measured by IonOptix Ca²⁺ image system. Myofilament sensitivity to Ca²⁺ was assessed by measuring the gradient of cell length-fura2 trajectory during contraction and late relaxation. Western blot was used to detect the target proteins.

Results Increasing pacing rate from 0.5Hz to 4Hz in left ventricular (LV) myocytes accelerated Ca²⁺ decline and sarcomere relaxation time constants (from 152 ± 13ms to 60 ± 5ms and from 36 ± 2ms to 18 ± 1ms, respectively, p < 0.05, n = 27) and increased the length-fura2 trajectory gradient (ECa₅₀ increased from 1.62 ± 0.06 to 1.84 ± 0.06, p < 0.05) and shifted the trajectory loop to the right, indicating a consistency of FDAR with the reduction of myofilament sensitivity to Ca²⁺. Inhibition of PKA (H89, 1 μM) or PKC (CHE, 1 μM) had no effect on myofilament Ca²⁺desensitisation and FDAR, whereas CaMKII inhibitor KN93 (1 μM) abolished frequency-dependent myofilament desensitisation to Ca²⁺ and FDAR. Because cardiac troponin I (Tn-I) is the major regulator for myofilament sensitivity to Ca²⁺ and both PKA and PKC share the same phosphorylation sites Ser23/24, we determined the Ser23/24 phosphorylation in ventricular myocytes and found that Ser23/24 phosphorylation was largely reduced by PKA and PKC inhibitors but not by CaMKII inhibition. However, a phospho-Ser-antibody showed that CaMKII inhibitor KN93 significantly reduced Tn-I phosphorylation in the Tn-I immunoprecipitates, indicating that CaMKII phosphorylates Tn-I at sites different from the PKA and PKC sites. Indeed, a co-immunoprecipitation of CaMKII and Tn-I has been detected.

Conclusions Our results suggest that FDAR is regulated by a frequency-dependent desensitisation of myofilament sensitivity to Ca²⁺, in which CaMKII-dependent Tn-I phosphorylation plays a major role.