# Mechanism of regulation of cardiac contraction by phosphorylation of myosin binding protein C

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $680,441

## Abstract

Project Summary/Abstract
Phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates myocardial contraction, but
neither the molecular or structural mechanism nor the in vivo significance of these effects is known. Our
working model is that phosphorylation regulates twitch kinetics by regulating cross-bridge recruitment to the
thin filament. In this dual-PI proposal we will use our complementary expertise in cardiac muscle physiology
and myofilament structure to generate an integrated view of cMyBP-C function. This dual approach includes
strict cross-checks of data over a range of spatial organization, from isolated filaments to working hearts in
vivo. We will test the hypothesis that the mechanism by which phosphorylation of cMyBP-C’s N-terminus
enhances contraction involves disruption of its binding to myosin and increased binding to actin, thereby
increasing the rate of cross-bridge binding to the thin filament. Aim 1 will test the idea that phosphorylation
speeds the cooperative recruitment of cross-bridges in skinned myocardium by measuring rates of force
development (kADP) following photolysis of caged ADP, which activates the cooperative recruitment process.
N-terminal fragments will be used to determine the roles of charged residues within cMyBP-C’s M-domain in
the regulation of force and rate of contraction. Studies will be extended to transgenic mice with the same
residues mutated to disrupt cMyBP-C binding to either myosin or actin to determine the effects of these
mutations on twitch characteristics in vivo; the possibility that phosphorylation of cTnI also contributes to
adrenergic inotropy will be investigated using phosphomimetic cTnI mice. The alternative idea, that
phosphorylation accelerates contraction by increasing the rates of cross-bridge transitions, will be investigated
by characterizing the steps in the cross-bridge cycle corresponding to force development (Pi release) and
relaxation (ADP release). Aim 2 will combine cryo-electron microscopy and 3D reconstruction of thick filaments
with X-ray diffraction of myocardium, to test our model structurally. We will determine whether cMyBP-C
stabilizes the super-relaxed state of myosin heads on the thick filament, whether cMyBP-C phosphorylation
disrupts this, and the role of M-domain charged residues in these effects. We will also test whether
phosphorylation releases the cMyBP-C N-terminus from the thick filament backbone, facilitating its binding to
actin. Since hypertrophic cardiomyopathies (HCM) due to mutations in cMyBP-C are typically associated with
enhanced contraction, Aim 3 explores whether the hypercontractility involves altered interactions of the M-
domain, such that binding to myosin is reduced and/or binding to actin is increased, as we propose with
phosphorylation. These studies will test the idea that HCM mutations weaken cMyBP-C’s stabilization of
myosin heads, accelerating their recruitment to the thin filament. This collaborative project takes a...

## Key facts

- **NIH application ID:** 10223413
- **Project number:** 5R01HL139883-04
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** ROGER W CRAIG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $680,441
- **Award type:** 5
- **Project period:** 2018-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10223413

## Citation

> US National Institutes of Health, RePORTER application 10223413, Mechanism of regulation of cardiac contraction by phosphorylation of myosin binding protein C (5R01HL139883-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10223413. Licensed CC0.

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