Cardiac myosin binding protein-C Ser302 phosphorylation regulates cardiac β-adrenergic reserve

Abstract

Phosphorylation of cardiac myosin binding protein-C (MyBP-C) modulates cardiac contractile function; however, the specific roles of individual serines (Ser) within the M-domain that are targets for β-adrenergic signaling are not known. Recently, we demonstrated that significant accelerations in in vivo pressure development following β-agonist infusion can occur in transgenic (TG) mouse hearts expressing phospho-ablated Ser²⁸² (that is, TG^S282A) but not in hearts expressing phospho-ablation of all three serines [that is, Ser²⁷³, Ser²⁸², and Ser³⁰² (TG^3SA)], suggesting an important modulatory role for other Ser residues. In this regard, there is evidence that Ser³⁰² phosphorylation may be a key contributor to the β-agonist-induced positive inotropic responses in the myocardium, but its precise functional role has not been established. Thus, to determine the in vivo and in vitro functional roles of Ser³⁰² phosphorylation, we generated TG mice expressing nonphosphorylatable Ser³⁰² (that is, TG^S302A). Left ventricular pressure-volume measurements revealed that TG^S302A mice displayed no accelerations in the rate of systolic pressure rise and an inability to maintain systolic pressure following dobutamine infusion similar to TG^3SA mice, implicating Ser³⁰² phosphorylation as a critical regulator of enhanced systolic performance during β-adrenergic stress. Dynamic strain-induced cross-bridge (XB) measurements in skinned myocardium isolated from TG^S302A hearts showed that the molecular basis for impaired β-adrenergic-mediated enhancements in systolic function is due to the absence of protein kinase A-mediated accelerations in the rate of cooperative XB recruitment. These results demonstrate that Ser³⁰² phosphorylation regulates cardiac contractile reserve by enhancing contractile responses during β-adrenergic stress.

Keywords: cardiac contractile function; contractile reserve; cross-bridge kinetics; hemodynamic function.

Fig. 1. Determination of expression, PKA-mediated phosphorylation of MyBP-C, and other sarcomeric proteins.

(A) MyBP-C is composed of eight immunoglobulin (ovals) and three fibronectin III (rectangles) domains labeled C0 to C10 (N to C terminus). The conserved M-domain in the linker between domains C1 and C2 contains three serines (S273, S282, S302; mouse numbering) in the NTG sequence that are targets for PKA phosphorylation. The substitution used to prevent S302 phosphorylation in TG^S302A is shown in red. (B) Representative Western blot showing S273, S282, and S302 phosphorylation, before and after PKA incubation. No Ser³⁰² expression was detected in TG^S302A samples, whereas fully phosphorylatable Ser²⁷³ and Ser²⁸² were observed. (C) Representative 5% tris-HCl gel, stained with silver stain, showing MHC isoform expression in the myocardial samples. (D) Representative gels shown are stained by Pro-Q (left) for protein phosphorylation, and the same gel is shown for total protein (right) stained with Coomassie Blue. cMyBP-C, cardiac myosin binding protein-C; pMyBP-C, phosphorylated cardiac myosin binding protein-C. (E) Relative protein phosphorylation (phosphorylated signal/total protein signal) was calculated for each protein and is expressed as % of PKA-treated NTG values for that protein. Values are expressed as means ± SEM, from three to six hearts in each group. *P < 0.05, different from non–PKA-treated samples from the same line; ^†P < 0.05, different from the corresponding NTG group.

Fig. 2. Analysis of cardiac morphology.

(A) Representative NTG, TG^3SA, and TG^S302A formalin-fixed hearts. TG^S302A hearts showed a similar overall morphology compared to NTG hearts, in contrast to the enlarged heart size observed in TG^3SA hearts. Scale bar, 1 mm. (B) Representative cross sections of NTG, TG^3SA, and TG^S302A hearts from the mid-LV were stained with Masson’s trichrome and imaged at ×100 magnification, demonstrating no observable increase in fibrosis in TG^S302A hearts. Scale bars, 25 μm.

Fig. 3. Analysis of the β-adrenergic acceleration in pressure development.

(A) Developed pressure, where end diastolic pressure was subtracted from instantaneous ventricular pressure, was plotted during early systolic contraction, to yield developed pressure. Dobutamine (DOB) increased the developed pressure in NTG mice starting at 8 ms after the start of contraction compared to baseline (P = 0.015) but did not increase developed pressure in TG^3SA or TG^S302A mice. Following dobutamine treatment, developed pressure in TG^S302A mice was not higher than that in NTG mice before dobutamine treatment at any point examined (that is, NTG at baseline). (B) The rate of pressure development (dP/dt) was measured during early systole. Dobutamine accelerated dP/dt starting at 6 ms after the start of contraction in NTG (P < 0.005). In TG^S302A mice, the acceleration in dP/dt was blunted compared to that in NTG mice and was only faster than baseline after 9 ms (P < 0.05). dP/dt in TG^S302A mice was not faster after dobutamine than NTG dP/dt before dobutamine treatment (that is, NTG at baseline) at any time point examined. *P < 0.05, NTG + dobutamine versus NTG without dobutamine treatment; ^†P < 0.05, TG^S302A + dobutamine versus TG^S302A without dobutamine treatment; ^‡P < 0.05, NTG + dobutamine versus TG^S302A + dobutamine.

Fig. 4. Effect of PKA treatment on the stretch activation responses in NTG, TG^3SA, and TG^S302A skinned myocardium.

Representative force responses elicited by a sudden 2% stretch in muscle length (ML) in isometrically contracting (A) NTG, (B) TG^3SA, and (C) TG^S302A myocardial preparations before (black) and following (red) PKA incubation. (D) Representative stretch activation traces following PKA treatment for NTG (red), TG^3SA (green), and TG^S302A (blue) myocardial preparations. Following PKA treatment, NTG preparations exhibited faster XB contractile behavior, whereas TG^3SA exhibited slower XB contractile behavior and TG^S302A exhibited intermediate XB contractile behavior. In (A), highlighted are the important phases of the force transients and the various stretch activation parameters that are derived from the elicited force response (see Materials and Methods for explanation of each phase and parameters). PKA incubation accelerated both k_rel (P < 0.005) and k_df (P < 0.005) in NTG skinned myocardium. However, in the TG^S302A skinned myocardium, the PKA-mediated acceleration in k_rel is less pronounced (P = 0.02) than that in the NTG coupled with no PKA-mediated accelerations in k_df. There were no PKA-mediated accelerations in k_rel and k_df in TG^3SA skinned myocardium. AU, arbitrary units.

Fig. 5. Effect of PKA on stretch activation parameters.

Measurements in the skinned ventricular preparations were first made under basal conditions (no PKA). Measurements were again made on the same preparations following PKA incubation. The net changes in stretch activation parameters after PKA incubation were calculated and are expressed as % increases from baseline for (A) the magnitude of new steady-state force attained in response to stretch in ML, P3; (B) the magnitude of overall XB recruitment in response to stretch in ML, P_df; and (C) the rate of XB detachment, k_rel in the NTG, TG^3SA, and TG^S302A groups. PKA treatment enhanced the P_df in all the groups, but the PKA-mediated enhancements in P_df were lower in the TG^3SA and TG^S302A groups when compared to the NTG group, indicating that the magnitude of XB recruitment occurs to a lesser extent in the TG^3SA and TG^S302A groups. NTG preparations displayed accelerations in k_rel following PKA incubation; however, this acceleration in k_rel was less pronounced in the TG^S302A group and is completely absent in the TG^3SA group. Similar effects were observed for P3. Values are expressed as means ± SEM. *P < 0.05; 12 preparations isolated from four hearts were used for all the groups.