Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium

Abstract

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during β-adrenergic stimulation and increased stimulus frequency. We propose a model for these effects via a phosphorylation-dependent regulation of the kinetics and extent of cooperative recruitment of cross bridges to the thin filament: phosphorylation of cardiac myosin binding protein-C accelerates cross bridge binding to actin, thereby accelerating recruitment and increasing the amplitude of the cardiac twitch. In contrast, enhanced lusitropy as a result of phosphorylation seems to be caused by a direct effect of phosphorylation to accelerate cross-bridge detachment rate. Depression or elimination of one or both of these processes in a disease, such as end-stage heart failure, seems to contribute to the systolic and diastolic dysfunction that characterizes the disease.

Keywords: cardiac myosin binding protein-C; phosphorylation; regulation.

Figure 1. Salient features of cardiac myosin binding protein-C

Panel A is an electron micrograph of a cardiac sarcomere showing the transverse “stripes” corresponding to the alignment of cMyBP-C in each half-sarcomere. cMyBP-C was visualized by infusion of an N-terminal antibody prior to fixation. Panel B presents a diagram of cMyBP-C, showing its subunit structure and featuring the PKA phosphorylatable serines in the regulatory domain, i.e., M-domain between subunits C1 and C2. cMyBP-C is a phospho-protein that is localized to 9 stripes (the “C” zone) in each half sarcomere. The stripes are 42 nm apart, corresponding to every third myosin crown.

Figure 2. Effects of ablation or phosphorylation of cMyBP-C on the kinetics of force development and relaxation

The rates of delayed force development (rate constant k_df) and relaxation (k_rel) were measured during the force transient resulting from sudden stretch of skinned myocardium activated at different Ca²⁺ concentrations to develop forces of approximately 25%, 50% and 100% of maximum. In panel A, k_df in WT increased nearly 4-fold when activation was increased from 25% to 100% of maximum; homozygous knockout of cMyBP-C accelerated the rate of force development during submaximal activations but not at maximal activation. In panel B, PKA phosphorylation of cMyBP-C in WT myocardium increased k_df during submaximal activations, but there was no effect on k_df at maximal activation. In panel C, knockout of cMyBP-C increased the rate of relaxation (k_rel) at submaximal but not at maximal activation; in panel D, similar to knock of cMyBP-C, PKA phosphorylation of cMyBPC accelerated k_rel in WT myocardium at submaximal but not at maximal activation. Thus, phosphorylation of cMyBP-C had no effect on cross-bridge cycling kinetics at maximal activation but accelerated the rates of force development and relaxation during submaximal activation. These effects were due to phosphorylation of cMyBP-C and not cTnI since WT myocardium expressing non-phosphorylatable TnI (TnI_ala2) exhibited accelerated rates of force development and relaxation.

Figure 2. Effects of ablation or phosphorylation of cMyBP-C on the kinetics of force development and relaxation

The rates of delayed force development (rate constant k_df) and relaxation (k_rel) were measured during the force transient resulting from sudden stretch of skinned myocardium activated at different Ca²⁺ concentrations to develop forces of approximately 25%, 50% and 100% of maximum. In panel A, k_df in WT increased nearly 4-fold when activation was increased from 25% to 100% of maximum; homozygous knockout of cMyBP-C accelerated the rate of force development during submaximal activations but not at maximal activation. In panel B, PKA phosphorylation of cMyBP-C in WT myocardium increased k_df during submaximal activations, but there was no effect on k_df at maximal activation. In panel C, knockout of cMyBP-C increased the rate of relaxation (k_rel) at submaximal but not at maximal activation; in panel D, similar to knock of cMyBP-C, PKA phosphorylation of cMyBPC accelerated k_rel in WT myocardium at submaximal but not at maximal activation. Thus, phosphorylation of cMyBP-C had no effect on cross-bridge cycling kinetics at maximal activation but accelerated the rates of force development and relaxation during submaximal activation. These effects were due to phosphorylation of cMyBP-C and not cTnI since WT myocardium expressing non-phosphorylatable TnI (TnI_ala2) exhibited accelerated rates of force development and relaxation.

Figure 2. Effects of ablation or phosphorylation of cMyBP-C on the kinetics of force development and relaxation

The rates of delayed force development (rate constant k_df) and relaxation (k_rel) were measured during the force transient resulting from sudden stretch of skinned myocardium activated at different Ca²⁺ concentrations to develop forces of approximately 25%, 50% and 100% of maximum. In panel A, k_df in WT increased nearly 4-fold when activation was increased from 25% to 100% of maximum; homozygous knockout of cMyBP-C accelerated the rate of force development during submaximal activations but not at maximal activation. In panel B, PKA phosphorylation of cMyBP-C in WT myocardium increased k_df during submaximal activations, but there was no effect on k_df at maximal activation. In panel C, knockout of cMyBP-C increased the rate of relaxation (k_rel) at submaximal but not at maximal activation; in panel D, similar to knock of cMyBP-C, PKA phosphorylation of cMyBPC accelerated k_rel in WT myocardium at submaximal but not at maximal activation. Thus, phosphorylation of cMyBP-C had no effect on cross-bridge cycling kinetics at maximal activation but accelerated the rates of force development and relaxation during submaximal activation. These effects were due to phosphorylation of cMyBP-C and not cTnI since WT myocardium expressing non-phosphorylatable TnI (TnI_ala2) exhibited accelerated rates of force development and relaxation.

Figure 2. Effects of ablation or phosphorylation of cMyBP-C on the kinetics of force development and relaxation

The rates of delayed force development (rate constant k_df) and relaxation (k_rel) were measured during the force transient resulting from sudden stretch of skinned myocardium activated at different Ca²⁺ concentrations to develop forces of approximately 25%, 50% and 100% of maximum. In panel A, k_df in WT increased nearly 4-fold when activation was increased from 25% to 100% of maximum; homozygous knockout of cMyBP-C accelerated the rate of force development during submaximal activations but not at maximal activation. In panel B, PKA phosphorylation of cMyBP-C in WT myocardium increased k_df during submaximal activations, but there was no effect on k_df at maximal activation. In panel C, knockout of cMyBP-C increased the rate of relaxation (k_rel) at submaximal but not at maximal activation; in panel D, similar to knock of cMyBP-C, PKA phosphorylation of cMyBPC accelerated k_rel in WT myocardium at submaximal but not at maximal activation. Thus, phosphorylation of cMyBP-C had no effect on cross-bridge cycling kinetics at maximal activation but accelerated the rates of force development and relaxation during submaximal activation. These effects were due to phosphorylation of cMyBP-C and not cTnI since WT myocardium expressing non-phosphorylatable TnI (TnI_ala2) exhibited accelerated rates of force development and relaxation.

Figure 3. Modulation of cross-bridge cycling by phosphorylation of cMyBP-C

The diagram shows a plausible mechanism by which phosphorylation of cMyBP-C speeds the rate of force development (©Moss and Fitzsimons, 2010. Originally published in JOURNAL OF GENERAL PHYSIOLOGY. doi:10.1085/jgp.201010471). In the presence of Ca²⁺, nucleotide-bound myosin (M·ADP·P_i) binds actin (A), which is followed by P_i release and force development as cross-bridges populate the primary force generating state (A-M·ADP). Upon release of ADP, the resulting rigor complex rapidly binds ATP, which is followed by dissociation of M·ATP from A, hydrolysis of ATP and re-entry of M·ADP·P_i into the cycle. According to Campbell's model of activation, force-generating cross-bridges bound to the (AM·ADP) cooperatively recruit near-neighbor cross-bridges to bind to actin, shown by the clockwise loop on the left. This cycle of recruitment may involve multiple iterations so that the time required to reach steady force, i.e., a steady population of A-M·ADP cross-bridges, is determined principally by the time taken to complete the iterative recruitment process. In our modification of this model, cMyBP-C slows cooperative recruitment by placing a physical constraint on myosin, thereby reducing the probability of myosin binding to actin and slowing the cooperative spread of cross-bridge binding. Phosphorylation of cMyBP-C by PKA, CaMK2δ or other kinases relieves this constraint either by disrupting the interaction of cMyBP-C with myosin, thereby increasing the probability of myosin binding to actin, or binding to the thin filament, thereby increasing its activation state and accelerating cross-bridge binding. Both of these mechanisms, either singly or together, would be expected to increase the rate of force development.

Figure 4. Maps of the cardiac thin filament showing the activating effects of Ca²⁺ and strongly-bound cross-bridges

The panels in this figure represent the state of activation of the thin filament (A) in the absence of Ca²⁺, (B) when Ca²⁺ binds to a single troponin (yellow structures), and (C) in response to the sequential cooperative binding of cross-bridges in response to Ca²⁺ binding to troponin. Binding of Ca²⁺ alone results in activation that is constrained with respect to amplitude and spread; subsequent binding of one and then another cross-bridge increases the amplitude and spread of activation. We propose that phosphorylation of cMyBP-C increases the rates of transition from the Ca²⁺ activation envelope (shaded area) to the expanded envelope (dashed line) due to binding of one cross-bridge (rate constant k₁) and then the further expanded envelope (dotted line) due to binding of a second cross-bridge (k₂).

Figure 5. Time-courses of force and intracellular [Ca²⁺] during the cardiac twitch

Force and intracellular [Ca²⁺] were recorded during twitches (3 Hz stimulus frequency) from mouse myocardium at room temperature. The traces presented here are the averaged traces from multiple muscle preparations and exhibit the time delay between the peak of the Ca²⁺ transient and the peak of the twitch.