Transgenic mice expressing a CaMKII inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr¹⁷ and develop dilated myopathy when stressed by gestation and parturition (Ji, et al., 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of SR CaMKII activity on the contractile characteristics and Ca²⁺ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and re-lengthening under both basal and Iso stimulated conditions. Although twitch Ca²⁺ transient amplitudes were not significantly altered, the rate of twitch [Ca²⁺]_i decline was reduced by ~47% in TG myocytes indicating decreased SR Ca²⁺ uptake function. Caffeine-induced Ca²⁺ transients indicated unaltered SR Ca²⁺ content and Na⁺/Ca²⁺ exchange function. Phosphorylation assays revealed a ~30 % decrease in the phosphorylation of ryanodine receptor Ser²⁸⁰⁹. Iso stimulation increased the phosphorylation of both phospholamban Ser¹⁶ and the ryanodine receptor Ser²⁸⁰⁹ but not phospholamban Thr¹⁷ in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca²⁺ handling in TG hearts.