Troponin I Ser-150 pseudo-phosphorylation alters cardiac contractile mechanics

Abstract

The cardiac troponin I (cTnI) subunit of the troponin complex is central in the calcium regulation of cardiac muscle contraction. The functional effects of beta-adrenergic induced cTnI phosphorylation at Ser-23/24 are well established, yet other cTnI residues can also be phosphorylated with unknown effects. Cardiac troponin I is phosphorylated by p21-activated kinase and AMP-activated Protein kinase at Ser-150. To determine the effects of cTnI Ser-150 phosphorylation on calcium regulated steady state force development we exchanged skinned cardiac fiber bundles with either wild type or troponin containing cTnI pseudo-phosphorylated at Ser-150 (S150D). Force vs. calcium measurements demonstrate cTnI S150D significantly increases myofilament calcium sensitivity (pCa50 WT=5.90±0.01; S150D=6.12±0.01; p<0.001) in the absence of an effect on maximal force, Hill coefficient or length dependent activation. Troponin structure and biochemical data indicate the cTnI Ser-150 region is localized in close proximity to the cTnI N-terminal region containing the Ser-23/24 phosphorylation sites. To investigate if Ser-150 phosphorylation alters the effect of phosphorylation at Ser-23/24 we measured force development in fibers exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-23/24 (S23/24D) or cTnI pseudo-phosphorylated at Ser-23/24/150 (S23/24/150D). Results demonstrate cTnI S150D similarly increased myofilament calcium sensitivity independent of the Ser-23/24 phosphorylation state (pCa50 S23/24D=5.67±0.03; S23/24/150D=5.85±0.03; p<0.001) resulting in a S23/24/150D calcium sensitivity similar to wild type. This occurs in the absence of an effect on maximal force, Hill coefficient or length dependent activation. Biochemical investigations into the mechanism of cTnI Ser-150 phosphorylation to effect force development are in progress. These data suggest that the increased calcium sensitivity resulting from cTnI phosphorylation at Ser-150 effectively eliminates the desensitizing effect of beta-adrenergic induced cTnI phosphorylation at Ser-23/24 such that in the presence of Ser-23/24/150 phosphorylation myofilament calcium sensitivity is similar to that of un-phosphorylated cTnI.