Pseudo-Phosphorylation of Cardiac Troponin I Containing the RCM Troponin T Deletion Glutamic Acid 96 is not an Accurate Indicator of the Phosphorylation Effects by PKA in Skinned Fibers

Abstract

The cardiac TnT deletion (HcTnT-ΔE96) linked to Restrictive Cardiomyopathy (RCM) dramatically increases the Ca2+ sensitivity of force development in skinned fibers (J. Biol. Chem. 283(4):2156–66). This study explores whether HcTnT-ΔE96 interferes with convergent regulation by Protein Kinase A (PKA) which decreases myofilament Ca2+ sensitivity. Native cTnT of porcine cardiac skinned fibers was displaced by HcTnT-WT or HcTnT-ΔE96 and reconstituted with binary complex containing pseudo non-phosphorylatable cTnI (cTnI-SS/AA.cTnC) or pseudo-phosphorylated (cTnI-SS/DD.cTnC). For comparison, skinned fibers were also reconstituted with binary cTnI.cTnC phosphorylated by PKA in vitro (cTnI-2P.cTnC) to assess effects of HcTnT-ΔE96 on Ca2+ sensitivity of force development. As expected, fibers displaced with HcTnT-WT reconstituted with cTnI-SS/DD.cTnC decreased Ca2+ sensitivity of force compared to control reconstituted with cTnI-SS/AA.cTnC. In contrast, fibers displaced with HCTnT-ΔE96 and reconstituted with cTnI-SS/DD.cTnC or cTnI-SS/AA.cTnC had similar Ca2+ sensitivities. When fibers were displaced with HcTnT3-WT or HcTnT3-ΔE96 and reconstituted with cTnI-2P.cTnC, the expected decrease in Ca2+ sensitivity was observed for both. These results suggest that phosphorylation mimetic cTnI-SS/DD may not always mimic phosphorylation by PKA. Furthermore, the mutant HcTnT-ΔE96 protein revealed reduced α-helical content by CD spectroscopy when compared to WT. Thermo-denaturation studies indicated that the mutant protein unfolded earlier than WT and was partially unfolded at physiological temperatures. Fluorescence studies containing CTnC-IAANS demonstrated that a high order of complexity (thin filament + myosin S1) is needed to better approach changes in Ca2+ affinity produced by the RCM cTnT mutation in skinned fibers. The effects of the HcTnT mutation on Ca2+ sensitivity properties were likely due to altered interactions of TnT with other thin filament proteins. Supported AHA 09POST2300030 (MSP), NIH R01-HL42325 (JDP) and J&E King 1KN13-34001 (JRP).