Ca2+ calmodulin dependent protein kinase II (CaMKII) is an important regulator of cardiac excitation-contraction coupling (ECC). Abnormal ECC leads to cardiac dysfunctions in heart failure (HF) that are partly due to changes in CaMKII activity. Exercise training improves ECC parameters in HF, but the role of CaMKII in this change is unknown, however exercise is seen to positively affect CaMKII and thus ECC in healthy individuals. The purpose of this study was to test whether exercise reverses the contractile dysfunction in myocardial infarction (MI)-HF, and whether this is altered by CaMKII. Young adult Wistar rats (male, 250-350g, n=42) were anesthetised (1.5% isoflurane), MI-HF induced via permanent coronary artery ligation, and treadmill running (5 d/week, 6-8 weeks, 90-95% maximal capacity) initiated four weeks post-surgery (MI-TRN, n=14). Sham-operated (SHAM, n=12) and sedentary MI-HF (MI-SED, n=16) rats served as controls. Values are means±SEM, compared by One-Way ANOVA. MI-HF reduced aerobic capacity by 16% (p<0.01); exercise normalised this. Contractile function of twitch-stimulated (1-4Hz at 37C) cardiomyocytes was assessed with edge detection video and Fura-2 fluorescence microscopy with and without the CaMK inhibitor autocamtide-2 related inhibitory peptide (AIP 10uM). MI reduced contractility by 40% (fractional shortening (FS) 13.5±0.5% vs 8.1±0.6% (p<0.01) in SHAM and MI-SED) and increased contraction and relaxation times (SHAM 77±4ms and 195±12ms vs MI-SED 106±4ms and 289±10ms; p<0.01). These effects were explained by reduced Ca2+ transient amplitude (SHAM 0.69±0.04au vs MI-SED 0.33±0.03au; p<0.01) and increased Ca2+ peak and decay times (SHAM 31±4ms and 263±13ms vs MI-SED 44±4ms and 363±9ms; p<0.01). Exercise corrected FS (MI-TRN 11.5±0.5%; p<0.01), tended to correct contraction and relaxation times (100±4ms and 243±10ms; both p<0.1), and improved Ca2+ handling (Ca2+ transient amplitude 0.49±0.04au, time to Ca2+ peak 39±3ms and decay 330±14ms; all p<0.01). CaMKII was then inhibited and experiments repeated, this abolished exercise-induced improvement in FS and Ca2+ transient amplitude (both p<0.01) and impaired relaxation and Ca2+ transient decay times (p<0.01) more in MI-TRN than MI-SED, but had no effect on contraction and systolic Ca2+ release time. MI-HF was associated with increased diastolic Ca2+ (p<0.01), while exercise reduced, but did not normalise this (p<0.01); however, CaMK-inhibition only partly explained this effect. In conclusion, this study confirmed that CaMKII plays an important role in modulating exercise-induced improvements in cardiomyocyte function following MI-HF. In particular, exercise improved sarcoplasmic reticulum (SR) Ca2+-uptake and SR loading parameters by activating CaMKII, whereas Ca2+ release parameters were less affected. This suggests that exercise activated cytoplasmic, but not dyadic CaMKII.