Cardiac Myosin Binding Protein-C Phosphorylation Modulates Myofilament Length-Dependent Activation

Abstract

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is an important regulator of contractile function, however, its contributions to length-dependent changes in cross-bridge (XB) kinetics is unknown. Therefore, we performed mechanical experiments to quantify contractile function in detergent-skinned ventricular preparations isolated from wild-type (WT) hearts, and hearts expressing non-phosphorylatable cMyBP-C [Ser to Ala substitutions at residues Ser273, Ser282, and Ser302 (i.e., 3SA)], at sarcomere length (SL) 1.9 μm or 2.1μm, prior and following protein kinase A (PKA) treatment. Steady-state force generation measurements revealed a blunting in the length-dependent increase in myofilament Ca(2+)-sensitivity of force generation (pCa50) following an increase in SL in 3SA skinned myocardium compared to WT skinned myocardium. Dynamic XB behavior was assessed at submaximal Ca(2+)-activations by imposing an acute rapid stretch of 2% of initial muscle length, and measuring both the magnitudes and rates of resultant phases of force decay due to strain-induced XB detachment and delayed force rise due to recruitment of additional XBs with increased SL (i.e., stretch activation). The magnitude (P2) and rate of XB detachment (k rel) following stretch was significantly reduced in 3SA skinned myocardium compared to WT skinned myocardium at short and long SL, and prior to and following PKA treatment. Furthermore, the length-dependent acceleration of k rel due to decreased SL that was observed in WT skinned myocardium was abolished in 3SA skinned myocardium. PKA treatment accelerated the rate of XB recruitment (k df) following stretch at both SL's in WT but not in 3SA skinned myocardium. The amplitude of the enhancement in force generation above initial pre-stretch steady-state levels (P3) was not different between WT and 3SA skinned myocardium at any condition measured. However, the magnitude of the entire delayed force phase which can dip below initial pre-stretch steady-state levels (Pdf) was significantly lower in 3SA skinned myocardium under all conditions, in part due to a reduced magnitude of XB detachment (P2) in 3SA skinned myocardium compared to WT skinned myocardium. These findings demonstrate that cMyBP-C phospho-ablation regulates SL- and PKA-mediated effects on XB kinetics in the myocardium, which would be expected to contribute to the regulation of the Frank-Starling mechanism.

Keywords: cardiac myosin binding protein-C; cross-bridge kinetics; phosphorylation; protein kinase A; skinned myocardium; stretch-activation.

Figure 1

Effect of PKA treatment on the stretch activation responses in WT and 3SA skinned myocardium. Traces of representative force responses elicited at ~35% of maximal Ca²⁺ activation level by a sudden 2% stretch in muscle length (ML) in isometrically-contracting (A) WT and (B) cMyBP-C phospho-ablated (i.e., 3SA) myocardial preparations prior to (black) and following incubation with PKA (red). 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). Phase 1 represents the immediate increase in force in response to the sudden increase in ML. P1 is the magnitude of the immediate force response and is measured from the pre-stretch isometric steady-state force to the peak of phase 1, and it represents the magnitude of XB stiffness. Phase 2 represents the rapid decay in force with a dynamic rate constant k_rel and is an index of the rate of XB detachment. P2 represents the minimum force attained at the end of Phase 2 of the stretch-activation response and is an index of the magnitude of XB detachment. Phase 3 represents the delayed force development with a dynamic rate constant k_df and is an index of the rate of XB recruitment. P3 represents the new steady-state force attained in response to the imposed stretch in muscle length and is an index of force enhancement above initial pre-stretch isometric levels. P_df represents the amplitude of the delayed force development and is an index of the overall number of XBs being recruited into the force-bearing state in response to a sudden 2% stretch in ML i.e., it represents the magnitude of XB recruitment. PKA treatment significantly accelerated both k_rel and k_df in WT skinned myocardium but not in 3SA skinned myocardium. AU, arbitrary units.

Figure 2

Western Blot and Pro-Q analysis to assess the phosphorylation status of myofilament filament proteins in WT and 3SA myocardium. (A) Western blots showing cMyBP-C and cTnI phosphorylation prior to and following PKA treatment in WT and 3SA heart samples. cTnI phosphorylation at residues Ser23/24 was similar between WT and 3SA samples under basal conditions (-PKA) and also following PKA treatment. cMyBP-C phosphorylation at residues Ser273, Ser282, and Ser302 was absent in 3SA heart samples, and their phosphorylation levels were enhanced in the WT samples following PKA incubation. (B) Representative Pro-Q Diamond-stained (right) and Coomassie-stained (left) SDS gels showing the expression and phosphorylation status of myofilament proteins prior to and following PKA treatment in WT and 3SA heart samples. (C) Quantification of protein phosphorylation in WT and 3SA hearts as determined by Pro-Q Diamond staining. The intensity of the phosphorylation signal was normalized to the intensity of the total protein signal and the untreated WT myofibril protein phosphorylation was set to 1 as done in our previous studies (Gresham et al., ; Mamidi et al., 2015). cMyBP-C phosphorylation was enhanced following PKA treatment in WT samples but not in 3SA samples. No differences in phosphorylation of other myofilament proteins were observed between WT and 3SA samples under basal conditions and following PKA treatment. cTnI phosphorylation was significantly enhanced following PKA treatment in both WT and 3SA samples. Basal phosphorylation levels of cMyBP-C (no PKA) observed in 3SA hearts was similar to that reported in an earlier study (Tong et al., 2008). Myofibrils were isolated from 5 to 6 hearts for quantification of protein phosphorylation in WT and 3SA groups. Asterisks in (C) indicate statistical differences when compared to the corresponding pre PKA WT group. WT, wild-type; cMyBP-C, cardiac myosin binding protein-C; 3SA, non-phosphorylatable cMyBP-C; cTnT, cardiac troponin T; cTnI, cardiac troponin I; RLC, regulatory light chain.

Figure 3

Effect of PKA treatment on myofilament Ca²⁺ sensitivity (pCa₅₀) in WT and 3SA skinned myocardium. Force-pCa relationships were constructed by plotting normalized forces generated by incubating the myocardial preparations in a range of pCa prior to and following PKA treatment. Effect of PKA treatment on the force-pCa relationships in WT preparations at (A) long SL, and (B) short SL. Effect of PKA treatment on the force-pCa relationships in 3SA preparations at (C) long SL, and (D) short SL. PKA treatment resulted in a significant right-ward shift (decrease in pCa₅₀) in the force-pCa relationships in all the groups (values are shown in Table 1). The number of preparations used for each group are shown in Table 1. A minimum of 4 hearts per group were used with multiple preparations from each heart.

Figure 4

Effect of cMyBP-C phospho-ablation on SL- and PKA-dependent changes in the rate of XB detachment (k_rel). Isometrically-contracting myocardial preparations were subjected to a sudden 2% stretch in muscle length and the elicited force responses at ~35% of maximal Ca²⁺ activation level were used to measure (A) k_rel under basal conditions (-PKA) and (B) k_rel following PKA treatment at short and long SL's in WT and 3SA groups. WT preparations displayed significant accelerations in k_relat short SL compared to long SL under basal conditions, however, no acceleration in k_rel was observed in 3SA preparations—indicating that SL-dependent changes in XB detachment are abolished in cMyBP-C phospho-ablated skinned myocardium. Furthermore, k_relwas significantly slower in the 3SA preparations compared to WT preparations at both short and long SL's under basal conditions and following PKA treatment—indicating that the rate of XB detachment was significantly slowed in cMyBP-C phospho-ablated skinned myocardium. Values are expressed as mean ± S.E.M. The number of preparations used for each group are shown in Table 2. A minimum of 3 hearts per group were used with multiple preparations from each heart. ^*P < 0.05.

Figure 5

Effect of cMyBP-C phospho-ablation on SL- and PKA-dependent changes in the magnitude of sudden-stretch induced increase in the XB stiffness (P1). P1 was calculated from the force responses elicited due to a sudden 2% stretch in muscle length imposed on isometrically-contracting myocardial preparations at ~35% of maximal Ca²⁺ activation level (Stelzer et al., 2006c) in (A) WT, and (B) 3SA myocardial preparations. PKA treatment significantly decreased P1 at both SL's in WT preparations but not in 3SA preparations—indicating that PKA-dependent changes in P1 are abolished in cMyBP-C phospho-ablated skinned myocardium. Values are expressed as mean ± S.E.M. The number of preparations used for each group are shown in Table 2. A minimum of 3 hearts per group were used with multiple preparations from each heart. ^*P < 0.05.

Figure 6

Effect of cMyBP-C phospho-ablation on SL- and PKA-dependent changes in the rate of XB recruitment (k_df). Isometrically-contracting myocardial preparations were subjected to a sudden 2% stretch in muscle length at ~35% of maximal Ca²⁺ activation level and the elicited force responses were used to measure k_df in (A) WT, and (B) 3SA myocardial preparations. Decreased SL accelerated k_df under basal conditions and following PKA treatment in both WT and 3SA preparations. PKA treatment significantly accelerated k_df at both short and long SL's in the WT group, but not in the 3SA group—indicating that PKA-induced accelerations in the rate of XB recruitment are abolished in cMyBP-C phospho-ablated skinned myocardium. Values are expressed as mean ± S.E.M. The number of preparations used for each group are shown in Table 2. A minimum of 3 hearts per group were used with multiple preparations from each heart. ^*P < 0.05.

Figure 7

Effect of cMyBP-C phospho-ablation on SL- and PKA-dependent changes in the magnitude of stretch-induced XB recruitment (P_df). Isometrically-contracting myocardial preparations were subjected to a sudden 2% stretch in their muscle length at ~35% of maximal Ca²⁺ activation level and the elicited force responses were used to measure (A) P_df under basal conditions (-PKA) and (B) P_df following PKA treatment at short and long SL's in WT (white bars) and 3SA (gray bars) groups. P_df was significantly lower at short SL compared to long SL in the WT and 3SA groups under basal conditions and following PKA treatment. However, P_df was significantly lower in the 3SA group compared to WT group at both SL's under basal conditions and following PKA treatment—indicating that the overall number of XBs being recruited into the force-bearing state in response to a stretch in muscle length is significantly decreased in cMyBP-C phospho-ablated skinned myocardium. Values are expressed as mean ± S.E.M. The number of preparations used for each group are shown in Table 2. A minimum of 3 hearts per group were used with multiple preparations from each heart. ^*P < 0.05.