Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone.

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins.

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC(17b)) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

Bovine distal pulmonary arterial media is composed of a uniform population of well-differentiated smooth muscle cells with low proliferative capabilities.

The media of the normal bovine main pulmonary artery (MPA) is composed of phenotypically heterogeneous smooth muscle cells (SMC) with markedly different proliferative capabilities in response to serum, mitogens, and hypoxia. Little, however, is known of the SMC phenotype in distal pulmonary arteries (PA), particularly in arterioles, which regulate the pulmonary circulation. With a panel of muscle-specific antibodies against alpha-smooth muscle (SM)-actin, SM-myosin heavy chains (SM-MHC), SM-MHC-B isoform, desmin, and meta-vinculin, we demonstrate a progressive increase in phenotypic uniformity and level of differentiation of SMC along the proximal-to-distal axis of normal adult bovine pulmonary circulation so that the media of distal PA (1,500- to 100-microm diameter) is composed of a phenotypically uniform population of "well-differentiated" SMC. Similarly, when isolated and assessed in vitro, distal PA-SMC is composed of a single, uniform population of differentiated SMC that exhibited minimal growth responses to a variety of mitogens while their cell size increased substantially in response to serum. Their growth was inhibited by hypoxic exposure under all conditions tested. Distal PA-SMC also differed from MPA-SMC by exhibiting a distinct pattern of DNA synthesis in response to serum and mitogens. Thus, in contrast to the MPA, distal PA media is composed of an apparently uniform population of well-differentiated SMC that are proliferation resistant and have a substantial capacity to hypertrophy in response to growth-promoting stimuli. We thus speculate that distinct SMC phenotypes present in distal vs. proximal PA may confer different response mechanisms during remodeling in conditions such as hypertension.