Sepsis-associated cardiac dysfunction represents an intrinsic impairment of cardiomyocyte function due in part to a decrease in myofilament Ca2+ sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at Ser23/24. Dephosphorylation of cTnI is under regulatory control. Thus, muscarinic and adenosine A(1)-receptor agonists antagonize beta-adrenergic stimulation via activation of protein phosphatase 2A (PP2A). The aim of this study was to determine whether modulation of PP2A and thus cTnI phosphorylation could improve sepsis-induced contractile dysfunction. Cardiomyocytes were isolated from control or septic mice 16-18 h after an injection of vehicle or lipopolysaccharide (LPS; 9 mg/kg ip) respectively. Protein expression and phosphatase activity were determined in homogenates of control and septic hearts. Our data showed that LPS significantly increased cTnI phosphorylation at Ser23/24 in cardiomyocytes and reduced contraction amplitude without affecting Ca2+-transients. Treatment of cardiomyocytes with the A(1) agonist cyclopentyladenosine (CPA) or the protein kinase A inhibitor H89 significantly attenuated the LPS-induced contractile dysfunction without effect on Ca2+-transients. Co-treatment with CPA and H89 completely reversed the contractile dysfunction. Increased cTnI phosphorylation in septic hearts was associated with a significant reduction in the protein expression of both the catalytic and regulatory subunits (B56 alpha) of PP2A and a decrease in PP2A activity. CPA treatment of septic hearts increased PP2A activity. An increase in the protein expression of demethylated PP2A and a decrease in the PP2A-methyltransferase (PPMT; the methyltransferase that catalyses this reaction) were also observed. These data support the hypothesis that sustained cTnI phosphorylation underlies the contractile dysfunction seen in sepsis.