Though there is intense interest in left ventricular (LV) heart failure, less is known about the etiology of diastolic dysfunction in right ventricular (RV) failure caused by pulmonary arterial hypertension. Diastolic dysfunction can be caused by increased fibrosis but also by dysregulation of myofilament interactions. In this study we are investigating the diastolic properties of myocytes from a rodent model of RV failure induced by pulmonary arterial hypertension. Male Wistar rats (200g) were injected with monocrotaline (60mg/kg i.p.) to induce RV failure (FAIL, n=16) or a saline equivalent (CON, n=14). Within 4 weeks FAIL animals developed RV hypertrophy and symptoms of heart failure. Rats were humanely killed by stunning and cervical dislocation then single LV and RV myocytes were obtained by enzymatic dissociation. Sarcomere length was measured online by fast Fourier transform of the video image of cells using an inverted microscope. Experiments were carried out at 22 and 37C in a physiological HEPES buffered solution. Data are presented as mean ± SEM. Compared to CON, resting SL was significantly shorter in the RV of FAIL animals (FAIL, 1.77±0.01 µm; CON, 1.91±0.01 µm; p<0.001) but not in the LV (FAIL, 1.89±0.01 µm; CON, 1.89±0.01 µm) (n=18-22; 2 way ANOVA). There was no difference in resting [Ca2+] between CON and FAIL myocytes loaded with the Ca2+-indicator Fura-2AM (p>0.05, n=14-18). Myofilament Ca2+-sensitivity was estimated from the SL-Ca2+ relationship in intact myocytes loaded with Fura-2AM and exposed to the SERCA inhibitor thapsigargin (1µM, 15min) during relaxation from 10s field stimulation at 10Hz. Myofilament Ca2+ sensitivity (indexed by the slope of the SL-Ca2+ relationship) was significantly decreased in FAIL RV and LV cells compared to CON (RV, FAIL -1.20±0.09, CON -1.70±0.08 µm/ratio unit, p<0.001; LV, FAIL -0.98±0.10, CON -1.43±0.1 µm/ratio unit, p=0.002; n=16-26). Incubating intact myocytes with the reactive oxygen species (ROS) scavenger n-acetylcysteine (20mM for at least 1h) did not lengthen SL in RV FAIL myocytes (p>0.05 vs. untreated FAIL, n=53-61). Chemically skinning myocytes by exposure to 0.01 mg/ml saponin did not lengthen SL in RV FAIL myocytes (p>0.05 vs. untreated FAIL, n=16-27). Decreased resting SL is likely to contribute to diastolic dysfunction in failing hearts. We conclude that the shorter resting SL in failing RV myocytes is not caused by changes in either resting Ca2+ levels or myofilament Ca2+ sensitivity. Even though ROS levels are reported to be enhanced in MCT hearts (Redout et al, 2007) acute antioxidant treatment was ineffective and the lack of effect of skinning suggests alteration of the intracellular milieu is not responsible for the shortened SL. Mechanisms associated with local regulation of cross-bridges are under investigation.