Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium

Abstract

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Fig. 1

(A) Representative x-ray pattern from skinned cTnI_Ala2 mouse myocardium with labeled 1,0 (inner two spots, arising from thick filament-associated mass) and 1,1 (outer two spots, arising from thick and thin filament-associated mass) equatorial spots [32, 34]. (B) Bar graph representations of the ratio of intensities of the 1,1 and 1,0 equatorial x-ray reflections (I_1,1/I_1,0) from skinned WT and cTnI_Ala2 myocardium untreated (control) and treated with PKA in relaxing solution (pCa 9.0). *Significant differences between untreated (i.e., unphosphorylated) and PKA treatment (i.e., phosphorylated) (p<0.05). The measurements in WT myocardium were collected during the same experimental sessions as cTnI_Ala2, but were reported earlier [32]. (C) Bar graph representations of I_1,1/I_1,0 from cMyBP-C(t3SA), cMyBP-C(tWT), and cMyBP-C(t3SD) skinned myocardium in pCa 9.0. For reference, I_1,1/I_1,0 from cMyBP-C^-/- null myocardium reported earlier using the same experimental conditions was greater than WT myocardium at 0.40 ± 0.06 and did not change with PKA treatment [27].

Fig. 2

(A) Bar graph representations of the inter-thick filament spacing (IFS) determined from the d_1,0 lattice spacing [32, 34] in untreated (control) and PKA-treated (phosphorylated) WT and cTnI_Ala2 myocardium. #Significant differences between untreated WT and cTnI_Ala2 (p<0.05). The measurements in WT myocardium were collected during the same experimental sessions as cTnI_Ala2, but were reported earlier [32]. (B) Bar graph representations of IFS in cMyBP-C(t3SA), cMyBP-C(tWT), and cMyBP-C(t3SD) skinned myocardium. *Significant differences between cMyBP-C(t3SA), cMyBP-C(tWT), and cMyBP-C(t3SD) myocardium (p<0.05). For reference, IFS from cMyBP-C^-/- null myocardium reported earlier using the same experimental conditions was not different from WT myocardium prior to PKA treatment (54 ± 1 nm) and increased with PKA treatment (57 ± 1 nm) [27].

Fig. 3

(A) Force-pCa relationships established in WT skinned myocardium untreated (closed circle, solid line fit) and treated with PKA (closed downward triangle, dotted line). Fitting the mean data with the Hill equation yield pCa₅₀. (B) Force-pCa relationships in cTnI_Ala2 skinned myocardium untreated (closed circles, solid line fit) and treated with PKA (closed downward triangle, dotted line). The measurements in WT myocardium were collected during the same experimental sessions as cTnI_Ala2, but were reported earlier [32]. For reference, the Ca²⁺-sensitivity of force in cMyBP-C^-/- null myocardium is similar to WT myocardium [10].

Fig. 4

The rate of force redevelopment (k_tr) was measured as described [32] at sub-maximal (pCa 6.0 - 5.5) and maximum (pCa 4.5) [Ca²⁺]_free in untreated and PKA-treated WT and cTnI_Ala2 skinned myocardial preparations. Effects of cTnI_Ala2 mutation on (A) k_tr-pCa and (B) k_tr-relative force relationships were established by comparison of unphosphorylated WT (closed circle) and cTnI_Ala2 (open circle) myocardium. (C) Effects of PKA phosphorylation on k_tr-force relationships were established in unphosphorylated (closed circle) and phosphorylated (closed downward triangle) WT skinned myocardial preparations. (D) Effects of PKA phosphorylation on k_tr-force relationships in cTnI_Ala2 skinned myocardium. The measurements in WT myocardium were collected during the same experimental sessions as cTnI_Ala2, but were reported earlier [32]. For reference, the rate of force development in cMyBP-C^-/- null myocardium was accelerated compared to WT myocardium and PKA did not further accelerate k_tr [10].