In vivo definition of cardiac myosin-binding protein C’s critical interactions with myosin

Abstract

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C S2− ). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation–Induced Cardiac Proteotoxicity

“…Echocardiograms were performed on isoflurane‐anesthetized mice with a VisualSonics Vevo 2100 Imaging System (Toronto, Ontario, Canada) using a 40‐MHz transducer to assess cardiac functional parameters 20, 21. Briefly, 2‐dimensional directed M‐mode echocardiographic images along the parasternal short axis were recorded by investigators blinded to genotype to determine left ventricular (LV) size and systolic function.…”

“…For electron microscopic examination, D7 Des Tg and non‐transgenic (Ntg) hearts were perfused with 1% paraformaldehyde/2% glutaraldehyde in the cardioplegic buffer, then with 1% paraformaldehyde/2% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2, postfixed in 1% OsO 4 and processed for thin sectioning as described previously 20, 21…”

“…Hearts were collected, fixed in 10% buffered formalin, and embedded in paraffin as described previously 20, 21. Serial 5‐μm heart sections from each group were stained with Masson's trichrome21 and Sirius Red 25.…”

“…Paraffin‐embedded heart tissue sections (5 μm) were stained for immunofluorescent detection of desmin aggregates in D7‐Des Tg mice hearts according to the procedures described previously 10, 20. Briefly, heart tissue sections (5 μm) were deparaffinized through washes in xylene, and graded alcohol series following rehydration with deionized water and 0.1M phosphate‐buffered saline, pH 7.4 (PBS) for 10 minutes each.…”

“…Briefly, isolated NRCs were plated on Lab‐Tek II chamber slides (Thermo Scientific, 154461) at a density of 1×10 5  cells/well. Immunocytochemistry was then performed according to the procedures reported previously 17, 20, 21. Plated cells were first washed with 0.1 mol/L PBS followed by fixation and permeabilization in 4% paraformaldehyde with 0.5% Triton‐X 100 in PBS for 15 minutes.…”