cMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium

Abstract

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Figure 1.

Generation of mice expressing cMyBP-C. (A) Diagrammatic representation of the M-domain residues of interest in tWT, t3SA, and t3SD myocardium. (B) A PCR genotyping strategy was used to identify the transgenic mice. Shown are PCR products amplified from genomic DNA from WT (cMyBP-C^+/+), cMyBP-C null (cMyBP-C^−/−), tWT (cMyBP-C^−/−, TG-WT^+/0), t3SA (cMyBP-C^−/−, TG-3SA^+/0), and t3SD (cMyBP-C^−/−, TG-3SD^+/0) mice.

Figure 2.

Experimental protocol for determining V_o. Inset: Length steps of varying amplitudes (i.e., 8–20% per ML) were imposed and held for 500 ms, followed by rapid reextension. The time from imposition of the slack step to the onset of force redevelopment was measured at the intersection of a horizontal line through the force baseline and a dashed straight line drawn through the initial phase of force redevelopment. Plot: Length change was plotted as a function of time of unloaded shortening, and V_o was obtained from the slope of the line fitted to the data by linear regression analysis.

Figure 3.

Slack test data from WT, cTnI_Ala2, and cMyBP-C–null myocardium. Representative slack test data were obtained from permeabilized myocardium during half-maximal (V_H: filled triangle; V_L: empty triangle) and maximal (●) Ca²⁺ activations. Ca²⁺-independent and maximal Ca²⁺-activated forces were WT (1.7 mN mm⁻² and 21.1 mN mm⁻²; preparation length, 550 µm), cTnI_Ala2 (2.9 mN mm⁻² and 22.7 mN mm⁻²; preparation length, 520 µm), and cMyBP-C null (4.2 mN mm⁻² and 26.0 mN mm⁻²; preparation length, 560 µm).

Figure 4.

PKA phosphorylation of cMyBP-C and cTnI in WT and cTnI_Ala2 myocardium. (A) Myofibrillar proteins isolated from WT and cTnI_Ala2 myocardium were separated via SDS-PAGE and then stained with Pro-Q Diamond and SYPRO-Ruby to estimate the levels of cMyBP-C and cTnI phosphorylation under control (basal) conditions and following treatment with PKA. cTnT, cardiac troponin T. (B) Relative to control conditions, PKA treatment significantly (*, P < 0.05, t test) increased the phosphorylation of cMyBP-C and cTnI in WT myocardium but only cMyBP-C in cTnI_Ala2 myocardium. All values are means ± SEM.

Figure 5.

PKA effects on V_o in WT, cTnI_Ala2, and cMyBP-C–null myocardium at half-maximal Ca²⁺ activation. (A and B) Summary data for the high-velocity (A) and low-velocity (B) phases of unloaded shortening in skinned myocardium isolated from WT, cTnI_Ala2, and cMyBP-C–null myocardium. P/P_o values under control conditions (WT: 0.493 ± 0.017; cTnI_Ala2: 0.585 ± 0.010) and following PKA treatment (WT: 0.551 ± 0.029; cTnI_Ala2: 0.575 ± 0.010). All values are means ± SEM; n = 5 hearts/group. PKA treatment significantly (*, P < 0.05, t test) increased the velocity of shortening in WT and cTnI_Ala2 myocardium.

Figure 6.

Incorporation of cMyBP-C in tWT, t3SA, and t3SD myocardium. (A) Confocal images of WT, tWT, t3SA, and t3SD myocardium. cMyBP-C–null myocardium is included as a negative control. (B) SDS-PAGE of tWT, t3SA, and t3SD myofibrils was used to estimate expression of cMyBP-C. Top: Myosin heavy chain isoform expression. Middle: myofibrillar contractile protein expression. Tm, tropomyosin. Bottom: Densitometric analysis of the expression of cMyBP-C relative to α-actinin (intensity ratio). All values are means ± SEM; n = 5 hearts/group.

Figure 7.

Slack test data from tWT, t3SA, and t3SD myocardium during submaximal and maximal Ca²⁺ activations. (A–C) Representative slack test data were obtained from permeabilized tWT (A), t3SA (B), and t3SD (C) myocardium during half-maximal (V_H: full triangle; V_L: empty triangle) and maximal (●) Ca²⁺ activations. Ca²⁺-independent and maximal Ca²⁺-activated forces were tWT (1.2 mN mm⁻² and 28.1 mN mm⁻²; preparation length, 430 µm), t3SA (4.3 mN mm⁻² and 28.3 mN mm⁻²; preparation length, 450 µm), and t3SD (3.3 mN mm⁻² and 24.0 mN mm⁻²; preparation length, 500 µm).

Figure 8.

Effects of PKA phosphorylation of cMyBP-C on V_o in tWT, t3SA, and t3SD myocardium. (A) PKA phosphorylation of cMyBP-C in tWT, t3SA, and t3SD myocardium. Compared with basal conditions, PKA significantly (*, P < 0.05) increased the phosphorylation of cMyBP-C and cTnI in tWT myocardium but only the phosphorylation of cTnI in t3SA and t3SD myocardium (*, P < 0.05). All values are means ± SEM; n = 5 hearts/group. (B) PKA phosphorylation increased shortening velocity in the high and low velocity phases only in tWT myocardium. P/P_o values control conditions (tWT: 0.481 ± 0.038; t3SA: 0.518 ± 0.036; t3SD: 0.540 ± 0.042) and following PKA treatment (tWT: 0.512 ± 0.036; t3SA: 0.526 ± 0.026; t3SD: 0.542 ± 0.052). All values are means ± SEM; n = 5 hearts/group; *, P < 0.05 indicates significant increases in shortening velocity due to PKA treatment compared with basal conditions.