The glutamic acid to lysine mutation at the 22^nd amino acid residue (E22K) in the human cardiac myosin regulatory light chain (RLC) gene causes familial hypertrophic cardiomyopathy (FHC) with a phenotype of midventricular obstruction and septal hypertrophy. Our recent histopathology results have shown that the hearts of transgenic E22K mice (Tg-E22K) resemble those of human patients, demonstrating enlarged interventricular septa and papillary muscles. In this study, we show no effect of the E22K mutation on the kinetics of mutated myosin in its ATP-powered interaction with fluorescently labeled single actin filaments compared to nontransgenic or transgenic wild-type (Tg-WT) control mice. Likewise, no change in cross-bridge dissociation rates (g_app) was observed in freshly skinned papillary muscle fibers. In contrast, maximal force and ATPase were decreased 20% in Tg-E22K skinned papillary muscle fibers and intracellular [Ca²⁺] and force transients were significantly decreased in intact papillary muscle fibers from Tg-E22K compared to Tg-WT mice. Moreover, energy metabolism measured in isolated working Tg-E22K mouse hearts perfused under conditions of physiologically relevant levels of metabolic demand was similar in Tg-E22K and control hearts before and after 20 min of no-flow ischemia. Our results suggest that the pathological response observed in the E22K myocardium might be triggered by mutation induced changes in the properties of the RLC Ca²⁺-Mg²⁺ site, the state of the Ca²⁺/Mg²⁺ occupancy and consequently the Ca²⁺ buffering ability of the RLC. By decreasing the affinity of the RLC for Ca²⁺, the E22K mutation most likely promotes a Mg²⁺-saturated RLC producing less force and ATPase than the Ca²⁺-saturated RLC of WT fibers. Decreased Ca²⁺ binding may also lead to faster Ca²⁺ dissociation kinetics in Tg-E22K intact fibers resulting in decreased duration and amplitude of [Ca²⁺] and force transients. These changes when placed in vivo would result in higher workloads and consequently cardiac hypertrophy.--Szczesna-Cordary, D., Jones, M., Moore, J. R., Watt, J., Kerrick, W. G. L., Xu, Y., Wang, Y., Wagg, C., Lopaschuk, G. D. Myosin regulatory light chain E22K mutation results in decreased cardiac intracellular calcium and force transients.