Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

Abstract

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C

“…However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al (2003) Nature 424(6944):35-41]. Domain positioning impacts the effective concentration of cTnI [148][149][150][151][152][153][154][155][156][157][158] presented to cNTnC, and this is how cTnI [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank-Starling law of the heart.…”

“…There are examples in the literature of "fuzzy complexes" (25), some of which involve intrinsically disordered regions (IDRs) that show minimal chemical shift changes upon binding, like the phosphorylated Sic1-Cdc4 protein complex (26) and cystic fibrosis transmembrane conductance regulator (CFTR) R domain complexes (27). Evidence of an interaction can still be found through signal broadening, or relaxation measurements, as turns out to be the case for cTnI [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]-cNTnC.…”

“…, but the most critical NOEs are between cNTnC Ala7 and cTnI Ala42/Ser43. The importance of this contact is underscored by the fact that a double Ser41/43 mutation to aspartate (mimicking phosphorylation) markedly decreases troponin calcium affinity (33). Because cTnI Ala42/ Ser43 are part of a rigid alpha helix bound to cCTnC and Ala7 is part of cNTnC helix N, the interaction fixes the position of cNTnC relative to cCTnC.…”

“…This would be expected if Arg19-21, Arg26, and Lys35-37 are electrostatically tethered, but there is likely transient structuring occurring in the intervening segments as well. [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] could result from fast conformational exchange (microsecond to millisecond timescale) and/or conformational restriction on the nanosecond timescale. Support for the latter comes from accompanying changes in R 1 and heteronuclear NOE values.…”

“…cTnI binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI and a type VIII turn in cTnI [38][39][40][41]. In contrast, cTnI[1-37] remains disordered, although cTnI [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC.…”