Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

Abstract

β-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 μm and SL = 2.3 μm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

Keywords: Frank-Starling Mechanism; contractile protein; heart; mouse; protein kinase A (PKA); signaling; β-adrenergic stimulation.

FIGURE 1.

Impact of PKA treatment. Force-[Ca²⁺] relationships recorded on myocytes from NTG, cMyBP-C phospho-null (AAA) and phospho-mimetic (DDD) at short (red) and long (green) SL, both before (open symbols, solid lines) and after PKA treatment (closed symbols, dashed lines). Hill fit parameters are summarized in Table 1.

FIGURE 2.

PKA impact on LDA. Myofilament LDA was indexed as ΔEC₅₀, the difference in myofilament Ca²⁺ sensitivity between long and short sarcomere length. *, p < 0.05 (−) PKA versus (+) PKA.

FIGURE 3.

Phosphorylation status of cMyBP-C and cTnI. Panel A, Western blot using phospho-specific and pan nonspecific antibodies was employed to determine the phosphorylation status of cMyBP-C (residue Ser-302) and cTnI (residues Ser-22, Ser-23). Actin was probed with an actin antibody. The intensities of both phospho-specific and pan nonspecific blot intensities were determined using ImageJ. Panel B, average cTnI phosphorylation, normalized to cTnI-P post-PKA.

FIGURE 4.

LDA after recombinant troponin exchange. Force-[Ca²⁺] relationships recorded on myocytes from AAA (panel A) and DDD hearts (panel B) at two SLs (cf. Fig. 1) after troponin exchange containing either phospho-null (open symbols, solid lines) or phospho-mimetic (closed symbols, dashed lines) cTnI. Insets: Western blot analysis of troponin exchange. Average fit parameters are summarized in Table 2. C = non-exchanged controls. Panel C, average LDA and estimate of the relative contribution of cMyBP-C and cTnI phosphorylation. *, p < 0.05 AAA versus DDD; and #, p < 0.05 AA versus DD using 2-way analysis of variance; this analysis furthermore revealed that the average impact on ΔEC₅₀ of cMyBP-C-DDD versus cMyBP-C-AAA was −0.48 μm, and that of cTnI-DD versus cTnI-AA was −0.24 μm.

FIGURE 5.

Proposed model for cMyBP-C and cTnI PKA phosphorylation impacts on LDA. Four states of phosphorylation are shown: cMyBP-C and cTnI both dephosphorylated (top left), cTnI phosphorylated (top right; horizontal), cMyBP-C phosphorylated (bottom left; vertical), and both proteins phosphorylated (bottom right). Myosin is in blue, with the S1 head domain; cMyBP-C is in red, with the N terminus (N_t) containing the phospho sites (AAA or DDD); cTnI is in yellow, containing the inhibitory peptide (IP) and switch peptide (SW) domains, and the N-terminal phospho sites (AA or DD). See “Discussion” for details.