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To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G 1 3 A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca 2؉ sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca 2؉ sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca 2؉ sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca 2؉ regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca 2؉ binding site, had dramatically altered Ca 2؉ binding properties. The K Ca value for E22K was decreased by ϳ17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca 2؉ . Interestingly, Ca 2؉binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the ␣-helical content of phosphorylated R58Q greatly increased with Ca 2؉ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca 2؉ affinity 6-fold. Whereas phosphorylation of wildtype light chain decreased its Ca 2؉ affinity, the opposite was true for A13T. The ␣-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca 2؉ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5-7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and t...

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca 2؉ sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher ␣-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca 2؉ sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca 2؉ sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.

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.

The essential light chain of myosin (ELC) is known to be important for structural stability of the ␣-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH 2-terminal ϳ40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH 2-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.cross-bridge kinetics; striated muscle contraction; familial hypertrophic cardiomyopathy; sarcomeric mutations; failing heart; sudden cardiac death STRIATED MUSCLE MYOSIN

2+sensitivity of myofibrillar ATPase activity and force development in Tg-E22K mice compared with Tg-WT or Non-Tg littermates. Our results suggest that E22K-linked FHC is mediated through Ca 2+ -dependent events. The FHC-mediated structural perturbations in RLC that affect Ca 2+ binding properties of the mutated myocardium are responsible for triggering the abnormal function of the heart that in turn might initiate a hypertrophic process and lead to heart failure.

The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca 2؉ sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca 2؉ -Mg 2؉ binding site of HCRLC, which demonstrated no Ca 2؉ binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca 2؉ sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca 2؉ binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC.The regulatory light chain (RLC) 1 of myosin is a major regulatory subunit of smooth-muscle and non-muscle myosins and a modulator of the troponin (Tn) -controlled regulation of the striated muscle contraction. The crystal structures of chicken skeletal S1 (1) and the regulatory domain of scallop myosin, consisting of one RLC, one essential light chain (ELC), and a part of the myosin heavy chain (2), have revealed that the RLC is localized at the head-rod junction of the myosin heavy chain and, together with the ELC, stabilizes the ␣-helical neck of the myosin head. The N terminus of RLC is noncovalently bound to the myosin heavy chain between Asn-825 and Leu-842, whereas its C terminus wraps around the region located between Glu-808 and Val-826 of the myosin heavy chain (1). The N-terminal domain of the RLC contains a divalent cationbinding site, located in the first helix-loop-helix motif, which binds both Ca 2ϩ and Mg 2ϩ (Fig. 1A). The N-terminal region of RLC also contains the myosin light chain kinase-specific phosphorylation site (Ser-15), which is located in the proximity of the cation-binding site (3).Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (4 -6). FHC is an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden cardiac death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (7), myosin-binding protein C (8), ventricular RLC and ELC (4 -6, 9), troponin T (10), troponin I (11), troponin C (TnC) (12), ␣-tropomyosin (13), actin (14), and titin (15). Depending on the affecte...