Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is upregulated in congestive heart failure (CHF), contributing to electrical, structural, and functional remodeling. CaMKII inhibition is known to improve CHF, but its direct cardiac effects in CHF remain unclear. We hypothesized that CaMKII inhibition improves cardiomyocyte function, [Ca2+]i regulation, and β-adrenergic reserve, thus improving advanced CHF. In a 16-week study, we compared plasma neurohormonal levels, LV and myocyte functional and [Ca2+]i transient ([Ca2+]iT) responses in male Sprague-Dawley rats (10/group) with CHF induced by isoproterenol (170 mg/kg sq for 2 days). In rats with CHF, we studied the effects of the CaMKII inhibitor KN-93 or its inactive analog KN-92 (n=4) (70 μg/kg/day, mini-pump) for 4 weeks. Compared to controls, isoproterenol-treated rats had severe CHF with 5-fold increased plasma norepinephrine and about 50% decreases in ejection fraction (EF) and LV contractility (EES), but increased . They also showed significantly reduced myocyte contraction (dL/dtmax), relaxation (dL/dtmax), and [Ca2+]iT. Isoproterenol superfusion caused significantly less increases in dL/dtmax and [Ca2+]iT. KN-93 treatment prevented plasma norepinephrine elevation, with increased basal and acute isoproterenol-stimulated increases in EF and EES, and decreased in CHF. KN-93 treatment preserved normal myocyte contraction, relaxation, [Ca2+]iT and β-adrenergic reserve, while KN-92- treatment failed to improve LV and myocyte function and plasma norepinephrine remained high in CHF. Thus, chronic CaMKII inhibition prevented CHFinduced activation of the sympathetic nervous system, restoring normal LV and cardiomyocyte basal and β-adrenergic stimulated contraction, relaxation, and [Ca2+]iT, thereby playing a rescue role in advanced CHF.