Systemic sepsis/endotoxaemia is associated with an intrinsic impairment of cardiomyocyte contraction, due in part to a decrease in myofilament calcium (Ca²⁺) sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at serines 23/24. These residues are required for the protein kinase A (PKA)-dependent reduction of myofilament Ca²⁺ sensitivity after b-adrenoceptor stimulation. Such sustained phosphorylation could arise from a sustained activation of the phosphorylating (kinase) pathways or an inhibition of the dephosphorylation pathways. We hypothesis therefore that (i) sustained cTnI phosphorylation is a major mechanism underlying the decreased contractility in septic cardiomyocytes and (ii) a dysregulation of protein phosphatase 2A (PP2A) underlies this increased phosphorylation. To investigate the functional significance of increased cTnI phosphorylation in endotoxaemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) in transgenic mice with cardiac specific expression of slow skeletal TnI (ssTnI, which lacks the N-terminal protein extension containing PKA-sensitive phosphorylation sites in cTnI) and wild type (WT) littermate controls. Studies demonstrated that ssTnI mice showed significant protection against LPS-induced cardiac contractile dysfunction. PP2A is the main phosphatase which dephosphorylates cTnI. Studies revealed that increased cTnI phosphorylation in septic hearts was associated with dysregulation of PP2A in terms of decreased expression of the catalytic and regulatory subunits, an increased percentage of the inactive (demethylated) form of PP2A and a decrease in the specific methyltransferase (PPMT) that catalyses this reaction. Hence we conclude that cTnI phosphorylation is a major mechanism underlying the intrinsic depression of myocyte contractile function in endotoxaemia. Such sustained phosphorylation may arise from a dysregulation of dephosphorylating pathways mediated by PP2A.