Creatine kinase (CK) catalyses the exchange of inorganic phosphate from phosphocreatine (PCr) to ADP in the myofilament lattice in cardiac myocytes and is essential for normal heart function. CK activity is decreased in heart failure (HF) causing [ADP] to rise, impairing relaxation. We have investigated the role of CK in the diastolic dysfunction of rats with right ventricular (RV) failure. Male Wistar rats (200 g) were injected with 60 mg.kg-1 monocrotaline to induce pulmonary hypertension and RV failure (FAIL; N=14) or an equivalent volume of saline (CON; N=11); FAIL rats were sacrificed upon showing symptoms of HF, CON rats were sacrificed on equivalent days. Homogenates of ventricular tissue were used for Western blotting analysis. Isolated cardiac myocytes were obtained by enzymatic dissociation. Single cell experiments were conducted at 21 or 37 °C. Resting sarcomere length (SL) was measured online by fast Fourier transform of the cell’s video image. Data are presented as mean±SEM; 1 or 2-way ANOVA was used to identify differences and p<0.05 was considered significant. Resting SL was significantly shorter in RV FAIL compared to CON cells (FAIL 1.79±0.01 µm, CON 1.89±0.01 µm; p<0.05, n=79-139). Expression of muscle-isoform CK (normalised to GAPDH) was decreased by 50% in RV FAIL (N=6) compared to RV CON (N=6, p<0.05), there was no difference in CK expression between LV CON and FAIL (N=3) (Fig.1). Compared to Tyrode alone, the CK inhibitor FDNB in Tyrode (20 µM, for 10 min) significantly shortened resting SL in both CON (FDNB 1.78±0.03 µm, Tyrode 1.89±0.01 µm; p<0.05, n=10) and FAIL cells (FDNB 1.67±0.01 µm, Tyrode 1.80±0.01 µm; p<0.05, n=24). CK binds to the myofilaments of skinned muscle fibres. Saponin-skinned cells were incubated (30 min) in solutions containing or lacking bovine CK (4.4 mg.ml-1) and 10 mM PCr; in their absence the resting SL of FAIL RV was significantly shorter than CON RV (CON 1.95±0.01 µm, FAIL 1.83±0.02 µm, p<0.05, n=24) but not in their presence (CON 1.94±0.01µm, FAIL 1.90±0.01µm; p>0.05, n=24). The results suggest that in RV failure the shorter SL is caused by reduced CK expression. Reduced CK expression may reduce resting SL by allowing local [ADP] to increase, with consequent effects on cross-bridge cycling. We are currently investigating the functional consequences of this.