Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation

Abstract

cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac-specific N-terminus (cTnI(R21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions. Here, we tested whether this also occurs for cTnI(P83S). cTnI(P83S) increased both Ca(2+) binding affinity to cTn (KCa) and affinity of cTnC for cTnI (KC-I), and eliminated the reduction of KCa and KC-I observed for phosphorylated-cTnI(WT). In isolated myofibrils, cTnI(P83S) maintained maximal tension (TMAX) and Ca(2+) sensitivity of tension (pCa50). For cTnI(WT) myofibrils, PKA-mediated phosphorylation decreased pCa50 and sped up the slow-phase relaxation (especially for those Ca(2+) conditions that heart performs in vivo). Those effects were blunted for cTnI(P83S) myofibrils. Molecular-dynamics simulations suggested cTnI(P83S) moderately inhibited an intrasubunit interaction formation between inhibitory-peptide and N-terminus, but this "blunting" effect was weaker than that with cTnI(R146G) or cTnI(R21C). In summary, cTnI(P83S) has similar effects as other HCM-associated cTnI mutations on troponin and myofibril function even though it is in the I-T arm of cTnI.

Figure 1

Human cTnI sequence (A) and ternary structure (B) with HCM-related mutation site (P82S) and PKA-mediated phosphorylation sites (Ser-23/Ser-24). Here, in the ternary structure, cTnC (1–161) is shown in blue; cTnI (1–172) is in red, and cTnT (236–285) is in yellow. The asterisks indicate the key positions in cTnI, in which residues 137–146 form the inhibitory-peptide of cTnI and residues 147–163 belong to the switch-peptide of cTnI.

Figure 2

IANBD fluorescence emission intensity changes of (A) cTnC^C35S in complex with cTnT^WT and cTnI variants with titration of Ca²⁺ and (B) of cTnC^C35S alone with titration of cTnI variants.

Figure 3

Raw (A) and normalized (B) PKA phosphorylation profile of cTnI for myofibrils exchanged with cTn containing cTnI^WT or cTnI^P83S before and after PKA treatment.

Figure 4

Representative tension trace (at pCa 5.4) for isolated rat cardiac myofibril after exchanging with recombinant cTn complexes containing cTnI^WT and cTnI^P83S. The inset is a close up of slow-phase of relaxation demonstrating how k_REL,slow and t_REL,slow are measured.

Figure 5

Tension (A), pCa-tension relationship (B), the kinetics of activation (C; k_ACT), and the k_ACT/k_TR ratio (D) for cTnI^WT vs cTnI^P83S exchanged myofibrils prior to and after PKA treatment. *p < 0.05.

Figure 6

Slow-phase relaxation at submaximal Ca²⁺ level for (A) WT-cTn and (B) cTnI^P83S cTn exchanged rat cardiac myofibrils before (black) and after (red) PKA treatment. The kinetics (C; k_REL,slow) and duration (D; t_REL,slow) of slow-phase relaxation for cTnI^WT vs cTnI^P83S exchanged myofibrils prior to and after PKA treatment. *p < 0.05.

Figure 7

(A–D) Comparison of average (±SD) RMSF values of cTnC and cTnI for WT and cTnI^P83S and cTnI^{P83S/S23D/S24D} cTn systems in triplicate rounds of MD simulations. Here, site I and site II (the Ca²⁺ binding loop) of cTnC are highlighted in green and blue, respectively, and the inhibitory-peptide and switch-peptide regions of cTnI are highlighted in orange and pink, respectively. (E, F) The superposition of snapshots in cartoon representation extracted every 10 ns during 150 ns MD simulations for both complexes. The cTnC is shown in blue, cTnI is in red, cTnT is in gold, and all the key regions are highlighted with dashed circles.

Figure 8

Distances between Ca²⁺ and its coordinating residue Ser-69 (left) and Thr-71 (right) of cTnC site II over the course of each MD simulation for WT, cTnI^P83S, and cTnI^{P83S/S23D/S24D} cTn systems. Here, the 1st run result is shown in black, the 2nd run result is in red, and the 3rd run result is in blue.

Figure 9

Average contact map of residue–residue pairs between the N-terminus (residues 1–41) and inhibitory-peptide region (residues 138–147) of cTnI for the (A) WT, (B) cTnI-S23D/S24D, (C) cTnI-P83S, and (D) cTnI-P83S/S23D/S24D cTn models. The blue end of the spectrum (value 0) reflects no contact within the residue–residue pair, while the red end of the spectrum (value 1) represents 100% contact within the residue–residue pair.

Figure 10

(A–C) Difference contact map of residue–residue pairs between 14 hydrophbic NcTnC residues and the switch-peptide of cTnI mostly affected upon introduction of mutation or the bisphosphomimic substitutions. The 14 hydrophobic residues of NcTnC are the following (from left to right): Phe-20, Ala-23, Phe-24, Ile-26, Phe-27, Ile-36, Leu-41, Val-44, Leu-48, Leu-57, Met-60, Phe-77, Met-80, and Met-81. Color green (value 0) reflects no difference between the two systems; the red end of the spectrum (values above 0) reflects more contacts in the P83S (A), S23D/S24D (B), or P83S/S23D/ S24D (C) cTn system, and the blue of the spectrum (values below 0) indicates more contacts in the WT (A), WT (B), or P83S (C) model.