Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse α-cardiac myosin in the laser trap assay

Abstract

Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity (F(max)) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. F(max) was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas F(max) was severely depressed for one of the DCM mutants (65% S532P). Although F(max) was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity (V(actin)) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These F(max) data combined with previous V(actin) measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM.

Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β

“…Compromised coronary flow due to hypertrophy, microvascular dysfunction (42), increased oxidative stress (43), and increased metabolic demands imposed by abnormal biophysical properties of mutant sarcomeres (7,8) are factors that contribute to premature myocyte death and the emergence of focal fibrosis in HCM. Far less is known about mechanisms that expand the extracellular matrix in HCM hearts.…”

“…Activated non-myocyte cells may also be derived from circulating cells (14,(46)(47)(48). Alternatively, the enhanced biomechanical forces resulting from sarcomere protein mutations (7,8) could also provide a local mechanism for activating resident non-myocyte cells. Lineage studies are underway to assess the source of activated non-myocyte cells in HCM hearts.…”

“…Prehypertrophic myocytes had lower levels of Tgf-β transcripts than non-myocyte cells (data not shown) and may reflect some contamination of non-myocyte cells in isolated myocyte preparations (estimated as <10% by immunohistochemical studies). Alternatively, modest expression of myocyte Tgf-β might occur in response to enhanced biophysical properties of mutant sarcomeres (8), similar to increased Tgf-β expression that occurs in cultured WT myocytes subjected to mechanical stretching (53). Biophysical forces of HCM hearts could similarly account for increased Tgf-β expression in nonmyocyte cells, as has been observed when non-myocyte cardiac cells are stretched ex vivo (54,55).…”

“…Mutant myocytes have increased biophysical properties (8) and abnormal Ca 2+ homeostasis (11), factors that trigger mechanical and/or biochemical signals that activate gene transcription, including increased Tgf-β expression. Whether by paracrine and/or autocrine signaling, Tgf-β stimulates non-myocyte proliferation and expression of profibrotic molecules.…”

“…Previous studies of HCM mutations in the myosin heavy chain gene showed that these enhance sarcomere biophysical properties (7)(8)(9) and alter cardiac relaxation (10) and calcium signaling in myocytes (11), events that are associated with widespread transcriptional changes (12). In addition to inducing changes within myocytes, the characteristic histopathology found in HCM hearts implies that sarcomere gene mutations also impact non-myocyte cells.…”