Molecular and Integrated Biology of Thin Filament Protein Phosphorylation in Heart Muscle

Abstract

An increasing body of evidence points to posttranslational modifications of the thin filament regulatory proteins, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) by protein kinase C (PKC) phosphorylation as important in both long- and short-term regulation of cardiac function and potentially implicated in the transition between compensated hypertrophy and decompensation. The main sites for PKC-dependent phosphorylation on cTnI are Ser43, Ser45, and Thr144 and on cTnT are Thr197, Ser201, Thr206, and Thr287 (mouse sequence). We analyzed the function of each phosphorylation residue using a phosphorylation mimic approach introducing glutamates (E) at PKC phosphorylation sites and then measuring the isometric tension of fiber bundles exchanged with these mutants. We also directly phosphorylated cTnI and cTnT by PKC, incorporated the phosphorylated troponins in the myofilament lattice, and determined the isometric tension at varying Ca(2+) concentrations. We followed the experimental data with computational analysis prediction of helical content of cTnI and cTnT peptides that undergo phosphorylation. Here we summarize our recent data on the specific functional role of PKC phosphorylation sites of cTnI and cTnT.

Cardiac thin filament regulation

“…The very Nterminal end of this C-domain-interacting site of cTnI contains the other PKC-specific phosphorylation sites, Ser-43 and Ser-45. Despite the recent finding that these sites are poor substrates for PKC, phosphorylation of these sites has a strong impact on myofilament activity, in particular Ser-45 [72]. For example, substitution of Ser-43/Ser-45 with the charged residue Glu induces a marked depression of actin-activated S1 ATPase activity in solution and a reduction in myofilament Ca 2+ sensitivity [11,48].…”

“…In contrast, in the heart, electrical stimulation readily spreads via low resistance gap junctions such that all myocytes contract each beat, and the strength of the heart is regulated by variation of the contractile strength of the cardiac myocytes. This is accomplished by two main cellular excitation-contractions mechanisms: (a) variation of the amount of Ca 2+ released from the sarcoplasmic reticulum and (b) variation in the response of the myofilaments to activator Ca 2+ [7,16,37,72]. For a discussion of factors that regulate myocardial Ca 2+ fluxes, the reader is referred to [7]; it is important to note that physiologically, the level of Ca 2+ activation in the heart is always less than saturated.…”

“…For a discussion of factors that regulate myocardial Ca 2+ fluxes, the reader is referred to [7]; it is important to note that physiologically, the level of Ca 2+ activation in the heart is always less than saturated. The main modulators of myofilament Ca 2+ sensitivity are sarcomere length (Frank-Starling relation) [40] and posttranslational modification of sarcomere proteins, most notably by kinase-mediated phosphorylation [37,72].…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Sites at Ser 42 and 44 of TnI are substrates for protein kinase C (PKC) and when phosphorylated, there is a reduction of maximum tension and myofilament Ca 21 sensitivity and a depression in cross-bridge kinetics [1,2,4]. Another site of phosphorylation is at Thr 143, in the inhibitory peptide of TnI, which modifies Ca 21 sensitivity and cross-bridge kinetics [9,10]. Phosphorylation of TnT occurs in its C-terminal half in a region that interacts with both the C-lobe of TnC and with TnI.…”

“…Phosphorylation of TnT occurs in its C-terminal half in a region that interacts with both the C-lobe of TnC and with TnI. Phosphorylation of these sites by PKC [10] and potentially Rho kinase [11] induces a severe depression in maximum tension. There is also a phosphorylation site at Tm-Ser283 located in the region of overlap between contiguous Tm along the thin filament.…”