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Transgenic (Tg) mice expressing approximately 95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg-D166V mice were examined using a Guth muscle research system. A large increase in the Ca(2+) sensitivity of force and ATPase (Delta pCa(50)>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg-D166V mice compared with control mice. The cross-bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/force) was slightly increased in Tg-D166V fibers compared with controls. The calculated average force per D166V cross-bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg-D166V fibers compared with control fibers. Histopathological examination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation-dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross-bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg-D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V-positive patients.

SummaryTo study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per crosssectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine ␣-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca 2؉ sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pHinduced shifts in the Ca 2؉ dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca 2؉ from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca 2؉ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca 2؉ transients. release mediated by these two channels (for review see Refs. 1-4). These proteins, including calsequestrin (Csq), calmodulin, triadin, junctin, Junctophilins 1 and 2, MG-29, FKBP12, and others yet to be discovered, along with the RyR and DHPR make up the so-called calcium release units (CRUs) (5).Triadins, a multimember family of proteins that are the product of alternative splicing from a single gene and expressed almost exclusively in striated muscle (3, 6), have generated significant attention in recent years for their involvement in a variety of cellular events in muscle cells, but their precise role in muscle function is mostly unknown. Triadin was first identified in skeletal muscle as a 94-to 95-kDa transmembrane protein (7,8) that is abundantly expressed on the junctional sarcoplasmic reticulum (jSR), were it colocalizes with RyR1 and DHPR (9).Early studies of binding assays of solubilized SR proteins showed that triadin could not only be coimmunoprecipitated with other triadic proteins (10) but also could associate into macromolecular complexes with both the DHPR and RyR1 (7,11,12). Based in this association triadin was proposed as the key molecular linker mediating the DHPR/RyR1 communication during muscle contraction. Although functional interactions between triadin and the DHPR in skeletal muscle have proven difficult to confirm, functional and structural interactions between triadin and RyR1 have been documented by several investigators. In vitro studies have shown that the SR luminal domain of triadin not only interacts with RyR1 but appears to anchor Csq to it, mediating the functional coupling between these two proteins via specific domains (13-17).Several studies have suggested a major role for triadin 95 in modulating RyR channel properties. Both an anti-triadin anti-* This work was supported by American Heart Association Grant 0530250N (to C. F. P.) and National Institute of Health Grant PO1AR47605 (to P. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Together these studies suggest a negative regulatory role for triadin on RyR...