HIGHLIGHTED TOPICS
Plasticity in Skeletal, Cardiac, and Smooth Muscle
Invited Review: Pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation

¹ Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136; and ² Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905

	ABSTRACT

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

Cardiac muscle contraction depends on the tightly regulated interactions of thin and thick filament proteins of the contractile apparatus. Mutations of thin filament proteins (actin, tropomyosin, and troponin), causing familial hypertrophic cardiomyopathy (FHC), occur predominantly in evolutionarily conserved regions and induce various functional defects that impair the normal contractile mechanism. Dysfunctional properties observed with the FHC mutants include altered Ca²⁺ sensitivity, changes in ATPase activity, changes in the force and velocity of contraction, and destabilization of the contractile complex. One apparent tendency observed in these thin filament mutations is an increase in the Ca²⁺ sensitivity of force development. This trend in Ca²⁺ sensitivity is probably induced by altering the cross-bridge kinetics and the Ca²⁺ affinity of troponin C. These in vitro defects lead to a wide variety of in vivo cardiac abnormalities and phenotypes, some more severe than others and some resulting in sudden cardiac death.

familial hypertrophic cardiomyopathy; troponin; tropomyosin; actin; myocardium

	INTRODUCTION

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

THIS BRIEF REVIEW WILL SUMMARIZE some recent insights into thin filament regulation of cardiac muscle contraction and relaxation. Several mutations in regulatory proteins of the thin filament have been described that change the regulation of contraction and relaxation, and, recently, their impact on in vivo and in vitro contractility was studied. Within the framework of current knowledge, we will also offer some suggestions for directions for further research.

	REGULATION OF CONTRACTION BY THIN FILAMENT PROTEINS

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

Contraction of vertebrate striated (skeletal and cardiac) muscle is activated by the binding of Ca²⁺ to the Ca²⁺-binding subunit troponin C (TnC) of the troponin complex, which, together with troponin I (TnI), troponin T (TnT), and tropomyosin (Tm), forms the regulatory system of the contractile apparatus (21, 72, 79, 96). Contraction occurs when the myosin head in the thick filament interacts with actin in the thin filament causing the two filaments to slide past each other. The troponin complex in the thin filament regulates the actin-myosin interaction. Figure 1 summarizes the changes in the thin filament during activation of contraction. In the absence of Ca²⁺, the troponin complex exists in a closely held conformation. TnI inhibits actin-myosin binding by interacting with actin-Tm and fixing the troponin complex on actin through TnI's interaction with TnT (14, 15, 26, 68). This inhibitory interaction of TnI with actin is completely eliminated when Ca²⁺ binds to the amino-terminal Ca²⁺-specific sites (only site II in cardiac muscle) of TnC and the carboxyl terminus of TnI dissociates from the actin-Tm complex, which is then followed by the movement of Tm into a nonblocking position, allowing myosin to bind to actin (15, 26, 68). The exact function of TnT is still somewhat controversial, but it is thought to stabilize the troponin complex and to affect the Ca²⁺ sensitivity of actomyosin ATPase activity and the level of ATPase activation and/or force development (23, 44, 66, 68). The extended amino acid terminus of TnT may interact with actin and with the overlapping regions of adjacent Tm molecules (28). TnC, an EF-hand protein, transfers the Ca²⁺ signal, eliciting contraction to the fiber, whereas the main function of TnI is to inhibit the actomyosin ATPase activity (40). TnT directly interacts with Tm (66), TnI (66), and TnC (68). Conformational changes in TnC affected by Ca²⁺ binding (reviewed in Ref. 16) ultimately evoke the interactions between the thin filament proteins. In addition, TnI interaction with the other troponin subunits is affected by TnI phosphorylation (1, 32, 62) and by the oxidation state of Cys48 and Cys64 at the carboxyl terminus of TnI when interacting with TnT (9).

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of the regulation of muscle contraction by troponin. TnC, TnI, and TnT, tropomyosin C, I, and T, respectively. N, amino terminus; C, carboxyl terminus (78a).

	MUTATIONS OF THIN FILAMENT PROTEINS IN FAMILIAL HYPERTROPHIC CARDIOMYOPATHY

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibril disarray, and sudden cardiac death (SCD). Numerous studies have shown that FHC is caused by one of many missense or deletion mutations in various genes that encode for -myosin heavy chain (3, 13, 20, 24, 92, 93), ventricular myosin light chains 1 and 2 (12, 17, 67), myosin binding protein C (88), titin (75), actin (50, 65), -Tm (10, 33, 57, 59, 71, 84, 87, 90), TnT (2, 11, 36, 43, 51, 84-86, 89, 90), and TnI (34, 37, 55, 77). Whereas individuals with -myosin heavy chain mutations, in general, have a higher level of cardiac hypertrophy, those with TnT mutations have less hypertrophy but a higher incidence of SCD in young adults (90). To date, 15 human cardiac TnT mutations have been associated with FHC: I79N, R92Q/W/L, R94L, A104V, F110I, R130C, E160, E163K/R, S179F, E244D, R278C, and a mutation that arises from abnormal splicing of intron 16 (G₁A) (2, 18, 27, 36, 51, 58, 84, 86, 90, 91). Other mutations associated with FHC found in thin filament proteins are the cardiac Tm mutations A63V, K70T, V95A, D175N, and E180G/V (10, 33, 57, 59, 71, 84, 87, 90); the TnI mutations R145G/Q, R162W, K183, S199N, G203S, K206Q (34, 37), and an exon 8 deletion mutant (55); and four missense mutations in actin (50, 65). Table 1 summarizes the clinical manifestations of each of these mutations in -Tm, actin, TnI, and TnT. The mechanism by which these mutations alter cardiac contraction and relaxation is still largely unknown, although some studies suggest that changes in myofibrillar Ca²⁺ sensitivity can lead to diastolic dysfunction and sensitivity to dysrhythmias, which at times cause sudden death. Tables 2 and 3 summarize the results from in vitro studies and from studies on transgenic animals, respectively, that examine the mechanisms of these mutations and pathologies associated with FHC. We will now highlight several key observations that relate to the mechanisms of FHC, in particular those caused by TnT mutations.

View this table: [in this window] [in a new window]   Table 1.   Clinical studies of FHC associated with thin filament mutations

View this table: [in this window] [in a new window]   Table 2.   In vitro analysis of thin filament mutations associated with FHC

View this table: [in this window] [in a new window]   Table 3.   Transgenic models of FHC associated with thin filament mutations

TnT. Several mutations in TnT are responsible for mechanical abnormalities in the sarcomeric complex. Replication-defective recombinant adenovirus vectors for gene transfer of I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac cells in primary culture show a regular periodic pattern of immunolabeled TnT localization (74). In this system, unlike others mentioned below, direct force measurements demonstrated marked desensitization of submaximal Ca²⁺-activated tension (74). It was suggested that the decrease in Ca²⁺ sensitivity may be attributed to a change in the secondary structure caused by the mutation of the incorporated protein (85) and that this change in structure could subsequently modify the actin-Tm interaction of the myofibril (28).

The TnT mutation in the intron 16 splice donor site is associated with pronounced left ventricular thickening and a high risk of sudden death (56, 91). A glycine to alanine mutation in the intron 16 splice donor site of TnT was identified in all affected and three clinically unaffected adults of a family with a history of FHC (84). This mutation results in two atypical truncated transcripts that translate into a short and a long peptide with a loss of 28 or 14 amino acids residues in the carboxyl end plus 7 new amino acids in the shorter protein (90, 91). Actomyosin ATPase assays of troponin complexes reconstituted with the truncated proteins show that activation of the ATPase activity is greatly reduced for both mutants in the presence of Ca²⁺ (56). The carboxyl terminus of TnT interacts with both TnC and TnI, and, predictably, these truncated proteins have a lower affinity for TnI (56) and, when reconstituted in a troponin complex, for actin-Tm (85). This change in the affinity of the truncated TnTs for particular proteins or filament complexes may account for the inadequate regulatory function. Transgenic mice expressing low levels of truncated TnT (~5%) have myocyte disarray, atrial hypertrophy, reduced ventricular mass, and overall fewer and smaller cardiomyocytes (resulting in a smaller heart) than their wild-type litter mates, whereas homozygous mice (twice the expression of truncated TnT) died within 24 h of birth (81). The surviving transgenic mice showed diastolic dysfunction and cardiac arrhythmias postexercise consistent with impaired contractility.

Some TnT mutations cause an increase in unloaded myofilament sliding speed indicative of defective actomyosin cross-bridge activity and an increased or decreased Ca²⁺ affinity (85). A faster myofilament sliding speed was observed for I79N (42), R92Q (78), E244D, and the intron 16 (G₁A) truncated TnT mutant, whereas E160 appears to decrease unloaded sliding speed of the filament (85). The shift in sliding speed of the I79N, R92Q, and E160 TnT mutants is also accompanied by changes in Ca²⁺ sensitivity of force or ATPase activity (reviewed in Ref. 85).

The TnT-I79N mutation is of special interest because it poses the highest risk of SCD in young adults (90). At present, there is no clear understanding of why this TnT-I79N mutation is associated with increased SCD. Several investigators have demonstrated an in vitro effect of the TnT-I79N mutation on the contractile properties of cardiac and skeletal muscle with conflicting results. Lin et al. (42), using rat cardiac TnT containing a mutation in an equivalent position to the TnT-I79N mutation in humans, showed that this mutant TnT had a normal affinity for actin-Tm and conferred normal Ca²⁺ sensitivity to actomyosin ATPase activity. The regulated thin filaments, however, moved 50% faster over heavy meromyosin (HMM) than control filaments in an in vitro motility assay. Additional measurements utilizing the same system revealed that HMM exerted reduced isometric force on single thin filaments reconstituted with the TnT-I79N mutant (28). Sweeney et al. (78) reported that TnT-I79N transfected quail skeletal muscle myotubules had decreased Ca²⁺ sensitivity of force production, whereas the unloaded shortening velocity was increased by about twofold. An embryonic isoform of rat TnT-I79N expressed in adult rat cardiac myocytes induced a decreased Ca²⁺ sensitivity of isometric force (74). Our results on TnT-I79N reconstituted porcine fibers (79) are in accord with Morimoto et al. (54), who demonstrated that TnT-I79N reconstituted skinned rabbit trabeculae increased the Ca²⁺ sensitivity of contraction. A recent study confirmed increased Ca²⁺ sensitivity of myofibrillar ATPase activity of TnT-I79N reconstituted rabbit cardiac myofibrils (95). Part of the disparity is likely due to the different in vitro assays used by these investigators, illustrating the need to study the effect of the mutations in an in vivo system.

Until now, a transgenic model for the TnT-I79N has not been reported, although other mutant TnT transgenic mice have been described (Table 3). Miller et al. (49) examined a wild-type (Tg-WT) and two I79N-transgenic mouse lines (Tg-I79N) of human cardiac TnT (hcTnT) driven by a murine -myosin heavy chain promoter. The levels of expression of either Tg-WT or Tg-I79N, relative to mouse cTnT, were 71% (WT) or 35% and 52% in the two I79N lines. Extensive characterization of the Tg-I79N lines compared with Tg-WT and/or non-Tg mice demonstrated the following: normal survival and no cardiac hypertrophy even with chronic exercise in all groups, large increases in the Ca²⁺ sensitivity of ATPase activity and force in skinned fibers, a substantial increase in the rate of force activation and a small increase in the rate of force relaxation in flash photolysis experiments, significantly lower maximal force/cross-sectional area and ATPase activity, and a loss of sensitivity to pH-induced shifts in the Ca²⁺ dependence of force correlated with hcTnT-I79N expression levels (49).

When native TnT is replaced with mutant I79N TnT, an increased filament speed is not unexpected. Alterations in the actin interaction surface may allosterically change actomyosin binding and cross-bridge kinetics. If changes in actomyosin interactions increase the rate of cross-bridge dissociation from actin, peak isometric force will be reduced and maximal filament sliding velocity increases. The increased Ca²⁺ sensitivity of force in reconstituted skinned cardiac fibers in which native TnT was replaced with TnT-I79N (79) and in skinned cardiac fibers of transgenic mice (49) is not readily explained by changes in cross-bridge kinetics alone. The results from skinned cardiac fiber studies, flash photolysis force transients, and force-pCa relations (49) were reproduced by computer simulations that integrate Ca²⁺ binding to intracellular Ca²⁺ buffers and predict force generation based on a modification of the model of Robertson et al. (73) and a two-state cross-bridge model (48) that utilized an exponential dependence of the TnC off-rate for Ca²⁺ (Ca²⁺-specific site II) on force (29, 38). Simulations postulate that an increase in the apparent rate of actomyosin cross-bridge detachment (g) was not sufficient to account for all experimental observations. The combination of an increased affinity of TnC for Ca²⁺ and an increase in g reproduced all experimental observations in skinned fibers that contain the human TnT-I79N. An increased g was also inferred by others from their in vitro motility assays (28, 42) or TnT-I79N transfected myotubes (28), and a consistent picture regarding this mutation is emerging based on results from several different approaches. Moreover, the cross-bridge effects brought about by this mutation are distinct from the calcium effects, and it is possible that the former arise from an alteration in the interactions between TnT, Tm, and actin, whereas the latter arise from altered TnT and TnC interactions. The predicted increased affinity of TnC for Ca²⁺ still requires experimental verification.

Compared with wild-type TnT, relaxation of force in TnT-I79N containing myocardium is slowed by a decreased affinity of TnC for Ca²⁺; however, this effect is somewhat attenuated by an increase in g. The twitch contraction operates from a pCa range of ~7.7 at rest to a peak systolic value of 5.7. With the use of these pCa values, simulated peak twitch force in steady-state conditions in fibers containing Tg-I79N was shown to be higher than in fibers that contained Tg-WT (Fig. 2). Simulations for intact living myocardium further predict a faster rise of force, a slower isometric relaxation, and an increased residual force or resting tension at the onset of the next contraction. If twitch amplitudes in both Tg-WT and Tg-I79N are the same or are normalized, isometric relaxation of Tg-I79N myocardium is slower than in the WT, as observed in isovolumetrically contracting isolated heart (Fig. 2) (35). The higher basal contractile state, the increased rate of contraction, and the slower relaxation (35) in TnT-I79N myocardium would have the following consequences. First, the contractile reserve is decreased, and an increase in heart rate and/or of contractility, such as after isoproterenol administration, would jeopardize relaxation and cause diastolic dysfunction. An increased contractility and heart rate would increase diastolic intracellular Ca²⁺ concentration, cause intracellular Ca²⁺ overload, and dysrhythmias. These predictions seem to hold true for Tg-I79N mice challenged with isoproterenol (35), which demonstrated an impaired inotropic response, relaxation impairment, and fatal dysrhythmias. It is likely that these mechanisms contribute to the mortality observed in patients with a TnT-I79N-based FHC, especially in stimulated states of contractility such as seen during vigorous exercise or during inotropic interventions.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Simulation of Ca2+ transients and force during twitches. Time courses are shown of the intracellular Ca2+ concentration transient (top) and of the corresponding force (middle) for isometric twitches of intact ventricular myocardium during repetitive stimulation at 400-ms intervals in steady-state control conditions for the TnT transgenic wild-type (WT; solid lines) and for the TnT transgenic I79N mutation (dashed lines). Bottom: normalized force traces for the same twitches, demonstrating a slower isometric relaxation in I79N myocardium than in WT. The TnT-I79N mutant data were obtained by decreasing the apparent dissociation rate of Ca2+ from TnC from 300 to 88 s1 and increasing the cross-bridge apparent dissociation constant from 10 to 20 s1.

The E244D mutation of TnT also appears to increase Ca²⁺ sensitivity (61). The TnT-E244D mutation has a shorter side chain due to the absence of a methyl group in the aspartic acid residue, yet the charge is conserved. The spatial difference due to the lack of a methyl group becomes significant if it 1) alters TnT secondary structure, 2) alters protein-protein interaction within the troponin complex, 3) induces or eliminates structural changes in the troponin complex required for its regulatory properties, or 4) produces other effects. At least one of these conditions appears to have an impact on thin filament regulation. Skinned cardiac fiber reconstituted with the E244D TnT increased the maximum force and increased the Ca²⁺ sensitivity of the force generated compared with wild-type TnT (61). The E244D mutation is located in a region of the carboxyl terminus of TnT that interacts with Tm, and changes in TnT-Tm interaction may alter actomyosin regulation. Indeed, the E224D mutation was found to have a lower affinity for actin-Tm than wild-type TnT (85). This mutation has been found in affected individuals of families with FHC having poor prognosis (90).

The F110I TnT mutation is associated with variable cardiac morphologies and prognosis (2, 36, 43, 90). This mutation, located in a region of TnT that interacts with actin and Tm (16), also increased the maximum force in skinned muscle fibers but did not influence the Ca²⁺ sensitivity of force generation (61).

Another TnT transgenic animal model reported by Oberst et al. (63) was generated for the human cardiac TnT-R92Q mutation using a murine cTnT promoter. The level of expression in transgenic lines (wild-type and R92Q) varied from 1 to 10% of the total cTnT pool. Diastolic dysfunction and myocyte disarray were observed in the mutant mice but not in wild-type mice (63). The same TnT-R92Q mutant was expressed in transgenic mice, in which the level of R92Q expression varied from 30 to 92% (82). A murine cTnT cDNA and a rat -myosin heavy chain promoter were used in the latter study. The R92Q hearts had a significant induction of atrial natriuretic factor and -myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from these R92Q transgenic mice had increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working hearts showed hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC (82). Transgenic mice expressing 30%, 67%, and 92% R92Q TnT show a dose-related induction and progression of atrial hypertrophy but a constant and age-independent decrease in ventricular mass. Analogous to the cardiomyocytes and hearts of the truncated TnT transgenic mice (above), the R92Q myocytes and hearts are smaller than their wild-type counterparts. The left ventricle contains both hypertrophied and dying myocytes with abnormal histopathologies, which are particularly prevalent in the higher expressing mice (82). The mice expressing 67% R92Q displayed higher heart rates and diastolic dysfunction, prolonged contraction and relaxation, and possible early activation of ATPase activity. The diastolic dysfunction is attributed to an increase in Ca²⁺ sensitivity, as demonstrated in in vitro studies by Morimoto et al. (54), Szczesna et al. (79), and Yanaga et al. (95) or a deregulation of intracellular Ca²⁺. It is reasonable to assume that a change to a more polar amino acid residue in the Tm-interacting domain of TnT will affect troponin interactions as shown by a reduction of the stability of troponin complex formation containing R92Q (74, 79). In a recent paper (41), a murine -myosin heavy chain promoter was used to produce a transgenic mouse expressing human cardiac TnT-R92Q. The level of protein expression was relatively low, and the mutant mice demonstrated myocyte disarray and excess interstitial collagen. Interestingly, none of these transgenic mice demonstrated significant cardiac hypertrophy.

Exchange studies of TnT-R278C vastly increased Ca²⁺ sensitivity, more than any other TnT mutation known hitherto (53, 79). Reconstituted skinned cardiac fibers containing the R278C mutation in the globular carboxyl terminal domain of TnT greatly increases the Ca²⁺ sensitivity of force development (79). TnT residues 188-288, characterized as the T2 proteolytic fragment having an unusually large number of positively charged residues, have been reported to interact with Tm, TnI, and TnC (reviewed in Refs. 16 and 70). The interaction of the TnT carboxyl terminus with Tm appears to be Ca²⁺ dependent (76). Mutation of a basic amino acid to a neutral in the carboxyl end of TnT may induce steric changes within the troponin complex, altering the contacts between the regulatory subunits in response to Ca²⁺. Ohtsuki's group (53) showed that this TnT mutation induced a higher level of sub-half-maximum force as a result of a decrease in maximum force and cooperativity, although only a slight increase in Ca²⁺ sensitivity was observed.

TnI. The R145G TnI mutation identified in patients with FHC constitutes a change in charge in an evolutionarily conserved residue (34). We observed impairment in both ATPase assays and in the relaxation of force in skinned cardiac muscle preparations incorporating the R145G mutation (Lang R, Zhao J, Housmans PR, and Potter JD, unpublished observations). Another group reported that actin-Tm-activated myosin S1 ATPase assays using troponin complexes reconstituted with R145G TnI revealed no change in activation but induced minimal inhibition and increased the Ca²⁺ sensitivity of regulation compared with wild-type TnI complexes (11). The switch from a positively charged to a neutral residue in the R145G TnI mutation occurs in a region that interacts with the amino-terminal domain of TnC and possibly actin (reviewed in Ref. 16) and, consequentially, may alter the dynamics or influence the regulatory mechanism of the troponin complex. We detected a slight increase in the -helical content in circular dichroism studies of the R145G mutant protein in addition to an increase in the affinity between TnC and R145G TnI in the presence of Ca²⁺ and Mg²⁺ vs. Mg²⁺ alone (Lang R, Zhao J, Housmans PR, and Potter JD, unpublished observations). The large change in the ability of this mutant TnI to inhibit contraction, combined with the increased Ca²⁺ sensitivity of contraction, would likely lead to severely impaired diastolic function.

-Tm. Six missense mutations in the cardiac -Tm have been linked to FHC (Table 1). In vitro analysis of several missense mutations in the cardiac -Tm gene have also shown increased Ca²⁺ sensitivity (see Table 2). The A63V mutation, associated with poor prognosis and a risk of sudden death in individuals with FHC (59, 94), was found to increase the Ca²⁺ sensitivity of force production in the isometric force recordings of single adult rat myocytes that expressed this mutant -Tm by means of an adenovirus vector (47). Three other mutations associated with FHC, K70T, E180G, and D175N, showed a smaller increase in Ca²⁺ sensitivity of force production, with the smallest increase reported for the D175N mutation (4, 47). The D175N Tm mutation is associated with variable prognosis and pathophysiologies; however, favorable prognosis in some cases have been documented (10, 59, 87). The D175N -Tm mutation in transgenic mice causes impaired contractility and relaxation and enhances Ca²⁺ sensitivity (57). The increases in Ca²⁺ sensitivity of force production, as reported by Michele et al. (47) (A63V > K70T > E180G D175N = wild type), appear to be directly related to the severity of the disease. Several lines of transgenic mice overexpressing the D175N Tm mutation at <40% and 60% of total Tm myofibrillar content suggest that the degree of physiological abnormalities associated with the mutation is dependent on the amount of mutant -Tm incorporated in the filaments (57). Mice expressing 60% of the mutant protein had reduced contraction and relaxation rates during exercise compared with the mice expressing <40% of the D175N protein or compared with the wild-type littermates (57). The abnormal cardiac performance of the D175N transgenic mice was attributed to an increase in Ca²⁺ sensitivity, which was observed in skinned fiber preparations. Protein-protein interactions within the troponin-Tm complex may mediate the regulatory dysfunction because both D175N and E180G are within a region considered to interact with TnT, whereas A63V and K70T are in repeat sequences and may alter Tm-actin interaction (reviewed in Refs. 6, 16, 70).

-Actin. In 1999, the first -actin mutation linked to FHC was reported in a Danish family. The A295S -actin mutation was found in 13 individuals with different disease phenotypes and a variable age of disease onset (as early as 4 yr old) (50). Only one individual was asymptomatic, whereas 3 of the 13 with the mutation showed more severe phenotypes, which included pronounced hypertrophy, ventricular tachycardia, and diastolic dysfunction (50). The only other report identifying cardiac actin mutations linked to FHC is a recent study describing the phenotypes of family members of three individuals with different actin mutations. In this study, two de novo mutations (P164A and A331P) were associated with early-onset (8 yr and 17 mo of age, respectively) left ventricular hypertrophy and repolarization abnormalities (65). The third actin mutation, E99K, was found to be autosomal dominant, with affected individuals having a later onset of the disease, variable hypertrophy, and diastolic dysfunction (65). FHC-linked mutations in other genes were excluded in both studies. The four missense cardiac actin mutations are located near or within regions that interact with other actin molecules, troponin components, or the myosin head and may either destabilize the sarcomeric structure or alter the force generation during the cross-bridge cycle of contraction. Defective myofilament regulation or function may be due to reduced or enhanced filament protein-protein interaction caused by replacement of a neutral or acidic amino acid with a polar or basic amino acid (A295S and E99K missense mutations); a faulty actin-actin interaction may affect calcium signal transduction by altering the Tn-Tm-actin interaction. A change in the secondary structure of actin, possibly induced by replacement or addition of the -helical-destabilizing amino acid proline (as in the case of P164A and A331P), could also conceivably alter the native environment in actin that interacts with itself or other filament components and ultimately affect force generation and regulation. These cardiac actin mutations were only recently identified; therefore, little is known about their functional consequences.

	SUMMARY AND CONCLUSIONS

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

Thin filament proteins provide for a very tight regulation of cardiac contraction and relaxation. Each of several mutations profoundly impacted basal contractility and the time course of relaxation. In most mutations, Ca²⁺ sensitivity of force development was increased; in some cases, filament sliding velocity was also increased (Table 2). The myofilament is a highly interacting system in which even a slight change in interaction between any of the filament components may alter the Ca²⁺ sensitivity of force generation. TnT plays a key role in maintaining the Ca²⁺-dependent competition of TnC and actin-Tm for the inhibitory region of TnI (64). The biophysical consequences of one amino acid substitution in a critical part of TnT will alter the balance of power between the competing actin-Tm and TnC sites for the inhibitory region of TnI. McKay et al. (46) illustrated the precision of filament protein interactions by describing a skeletal TnC mutant (without Ca²⁺-binding site I, similar to the cardiac TnC in that cardiac TnC has no functional Ca²⁺ site I), with a "delicate energetic balance" between Ca²⁺ and TnI, binding to the TnC regulatory domain in the "open" state. It is therefore not surprising that several TnT mutations, and mutations of other thin filament proteins, manifest themselves by altering both cross-bridge kinetics (actomyosin dependent), and the Ca²⁺ affinity of TnC, as recently proposed (49). Impaired contractile function due to thin filament mutations would most likely contribute to the progression of compensatory hypertrophy (FHC).

Mutations in thin filament proteins give rise to varying degrees of hypertrophy and incidences of SCD (Table 1). One of the most puzzling questions regarding the pathogenesis of TnT-related FHC is the mechanism of SCD. Individuals with TnT-related FHC tend to exhibit mild or no ventricular hypertrophy, yet experience a high frequency of sudden death at an early age. These events could result from diastolic dysfunction, from the lack of pH sensitivity of Ca²⁺ sensitivity of force, from alterations in intracellular Ca²⁺ homeostasis, and/or from other processes. It remains to be determined whether the occurrence of SCD results from an altered thin filament environment or whether additional primary affects of the point mutation act at the level of the cardiac myocyte. Future work in this area should provide insights into the molecular mechanisms responsible for the induction and progression of FHC.

	ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AR-45391 (to J. D. Potter), HL-42325 (to J. D. Potter), and GM-36365 (to P. R. Housmans).

	FOOTNOTES

Address for reprint requests and other correspondence: J. D. Potter, Dept. of Molecular and Cellular Pharmacology, Univ. of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136 (E-mail: jdpotter{at}miami.edu).

	REFERENCES

TOP ABSTRACT INTRODUCTION REGULATION OF CONTRACTION BY... MUTATIONS OF THIN FILAMENT... SUMMARY AND CONCLUSIONS REFERENCES

1.   Al-Hillawi, E, Bhandari DG, Trayer HR, and Trayer IP. The effects of phosphorylation of cardiac troponin-I on its interactions with actin and cardiac troponin-C. Eur J Biochem 228: 962-970, 1995[Web of Science][Medline].

2.   Anan, R, Shono H, Kisanuki A, Arima S, Nakao S, and Tanaka H. Patients with familial hypertrophic cardiomyopathy caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis. Circulation 98: 391-397, 1998[Abstract/Free Full Text].

3.   Anan, R, Shono H, and Tei C. Novel cardiac beta-myosin heavy chain gene missense mutations (R869C and R870C) that cause familial hypertrophic cardiomyopathy. Hum Mutat 15: 584, 2000[Medline].

4.   Bing, W, Knott A, Redwood C, Esposito G, Purcell I, Watkins H, and Marston S. Effect of hypertrophic cardiomyopathy mutations in human cardiac muscle -Tm (Asp175Asn and Glu180Gly) on the regulatory properties of human cardiac troponin determined by in vitro motility assay. J Mol Cell Cardiol 32: 1489-1498, 2000[Web of Science][Medline].

5.   Bing, W, Redwood CS, Purcell IF, Esposito G, Watkins H, and Marston SB. Effects of two hypertrophic cardiomyopathy mutations in alpha-Tm, Asp175Asn and Glu180Gly, on Ca²⁺ regulation of thin filament motility. Biochem Biophys Res Commun 236: 760-764, 1997[Web of Science][Medline].

6.   Bonne, G, Carrier L, Richard P, Hainque B, and Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res 83: 580-593, 1998[Abstract/Free Full Text].

7.   Bottinelli, R, Coviello DA, Redwood CS, Pellegrino MA, Maron BJ, Spirito P, Watkins H, and Reggiani C. A mutant Tm that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. Circ Res 82: 106-115, 1998[Abstract/Free Full Text].

8.   Charron, P, Dubourg O, Desnos M, Isnard R, Hagege A, Millaire A, Carrier L, Bonne G, Tesson F, Richard P, Bouhour JB, Schwartz K, and Komajda M. Diagnostic value of electrocardiography and echocardiography for familial hypertrophic cardiomyopathy in a genotyped adult population. Circulation 96: 214-219, 1997[Abstract/Free Full Text].

9.   Chong, PC, and Hodges RS. Photochemical cross-linking between rabbit skeletal troponin subunits. Troponin I-troponin T interactions. J Biol Chem 257: 11667-11672, 1982[Abstract/Free Full Text].

10.   Coviello, DA, Maron BJ, Spirito P, Watkins H, Vosberg HP, Thierfelder L, Schoen FJ, Seidman JG, and Seidman CE. Clinical features of hypertrophic cardiomyopathy caused by mutation of a "hot spot" in the -tropomyosin gene. J Am Coll Cardiol 29: 635-640, 1997[Abstract].

10a.   Elliott, PM, D'Cruz L, and McKenna WJ. Late-onset hypertrophic cardiomyopathy caused by a mutation in the cardiac troponin T gene. N Engl J Med 341: 1855-1856, 1999[Free Full Text].

11.   Elliott, K, Watkins H, and Redwood CS. Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem 275: 22069-22074, 2000[Abstract/Free Full Text].

12.   Epstein, ND. The molecular biology and pathophysiology of hypertrophic cardiomyopathy due to mutations in the  myosin heavy chains and the essential and regulatory light chains. Adv Exp Med Biol 453: 105-114, 1998[Web of Science][Medline].

13.   Epstein, ND, Cohn GM, Cyran F, and Fananapazir L. Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908LeuVal mutation and a 403ArgGln mutation. Circulation 86: 345-352, 1992[Abstract/Free Full Text].

14.   Farah, CS, Miyamoto CA, Ramos CH, da Silva AC, Quaggio RB, Fujimori K, Smillie LB, and Reinach FC. Structural and regulatory functions of the NH₂- and COOH-terminal regions of skeletal muscle troponin I. J Biol Chem 269: 5230-5240, 1994[Abstract/Free Full Text].

15.   Farah, CS, and Reinach FC. The troponin complex and regulation of muscle contraction. FASEB J 9: 755-767, 1995[Abstract].

16.   Filatov, VL, Katrukha AG, Bulargina TV, and Gusev NB. Troponin: structure, properties, and mechanism of functioning. Biochemistry (Mosc) 64: 969-985, 1999[Medline].

17.   Flavigny, J, Richard P, Isnard R, Carrier L, Charron P, Bonne G, Forissier JF, Desnos M, Dubourg O, Komajda M, Schwartz K, and Hainque B. Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy. J Mol Med 76: 208-214, 1998[Web of Science][Medline].

18.   Forissier, JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, Hainque B, Townsend PJ, Yacoub MH, Faure S, Dubourg O, Millaire A, Hagege AA, Desnos M, Komajda M, and Schwartz K. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation 94: 3069-3073, 1996[Abstract/Free Full Text].

19.   Frey, N, Franz WM, Gloeckner K, Degenhardt M, Muller M, Muller O, Merz H, and Katus HA. Transgenic rat hearts expressing a human cardiac troponin T deletion reveal diastolic dysfunction and ventricular arrhythmias. Cardiovasc Res 47: 254-264, 2000[Abstract/Free Full Text].

20.   Geisterfer-Lowrance, AA, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, and Seidman JG. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell 62: 999-1006, 1990[Web of Science][Medline].

21.   Gergely, J. Molecular switches in troponin. Adv Exp Med Biol 453: 169-176, 1998[Web of Science][Medline].

22.   Golitsina, N, An Y, Greenfield NJ, Thierfelder L, Iizuka K, Seidman JG, Seidman CE, Lehrer SS, and Hitchcock-DeGregori SE. Effects of two familial hypertrophic cardiomyopathy-causing mutations on -tropomyosin structure and function. Biochemistry 36: 4637-4642, 1997[Medline]. [Corrigenda. Biochemistry 38: March 23, 1999, p. 3850.]

23.   Greaser, ML, and Gergely J. Reconstitution of troponin activity from three protein components. J Biol Chem 246: 4226-4233, 1971[Abstract/Free Full Text].

24.   Gruver, EJ, Fatkin D, Dodds GA, Kisslo J, Maron BJ, Seidman JG, and Seidman CE. Familial hypertrophic cardiomyopathy and atrial fibrillation caused by Arg663His -cardiac myosin heavy chain mutation. Am J Cardiol 83: 13H-18H, 1999[Web of Science][Medline].

25.   Harada, K, Takahashi-Yanaga F, Minakami R, Morimoto S, and Ohtsuki I. Functional consequences of the deletion mutation Glu160 in human cardiac troponin T. J Biochem (Tokyo) 127: 263-268, 2000[Abstract/Free Full Text].

26.   Hinkle, A, Goranson A, Butters CA, and Tobacman LS. Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T. J Biol Chem 274: 7157-7164, 1999[Abstract/Free Full Text]. [Corrigenda. J Biol Chem 274: October, 29 1999, p. 31750.]

27.   Ho, CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, and Seidman CE. Homozygous mutation in cardiac troponin T: implications for hypertrophic cardiomyopathy. Circulation 102: 1950-1955, 2000[Abstract/Free Full Text].

28.   Homsher, E, Lee DM, Morris C, Pavlov D, and Tobacman LS. Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium. J Physiol (Lond) 524: 233-243, 2000[Abstract/Free Full Text].

29.   Housmans, PR, and Wanek LA. Effects of halothane, enflurane, and isoflurane on measurements of Ca²⁺ by calcium electrode and aequorin luminescence. Anal Biochem 284: 60-64, 2000[Web of Science][Medline].

30.   Jaaskelainen, P, Soranta M, Miettinen R, Saarinen L, Pihlajamaki J, Silvennoinen K, Tikanoja T, Laakso M, and Kuusisto J. The cardiac -myosin heavy chain gene is not the predominant gene for hypertrophic cardiomyopathy in the Finnish population. J Am Coll Cardiol 32: 1709-1716, 1998[Abstract/Free Full Text].

31.   James, J, Zhang Y, Osinska H, Sanbe A, Klevitsky R, Hewett TE, and Robbins J. Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy. Circ Res 87: 805-811, 2000[Abstract/Free Full Text].

32.   Jaquet, K, Lohmann K, Czisch M, Holak T, Gulati J, and Jaquet R. A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus. J Muscle Res Cell Motil 19: 647-659, 1998[Web of Science][Medline].

33.   Karibe, A, Bachinski LL, Arai AE, Tripodi D, Roberts R, and Fananapazir L. Familial hypertrophic cardiomyopathy caused by a novel -tropomyosin mutation (Val95Ala) is associated with mild cardiac hypertrophy but a high incidence of sudden death (Abstract). Circulation 100: I-619, 1999.

34.   Kimura, A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, and Sasazuki T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 16: 379-382, 1997[Web of Science][Medline].

35.  Knollmann BC, Blatt SA, Horton K, deFreitas F, Miller T, Bell M, Housmans PR, Weissman NJ, Morand M, and Potter JD. Inotropic stimulation induces cardiac dysfunction in transgenic mice expressing a troponin T (I79N) mutation linked to familial hypertrophic cardiomyopathy. J Biol Chem In press.

36.   Koga, Y, Toshima H, Kimura A, Harada H, Koyanagi T, Nishi H, Nakata M, and Imaizumi T. Clinical manifestations of hypertrophic cardiomyopathy with mutations in the cardiac -myosin heavy chain gene or cardiac troponin T gene. J Card Fail 2: S97-103, 1996[Medline].

37.   Kokado, H, Shimizu M, Yoshio H, Ino H, Okeie K, Emoto Y, Matsuyama T, Yamaguchi M, Yasuda T, Fujino N, Ito H, and Mabuchi H. Clinical features of hypertrophic cardiomyopathy caused by a Lys183 deletion mutation in the cardiac troponin I gene. Circulation 102: 663-669, 2000[Abstract/Free Full Text].

38.   Landesberg, A, and Sideman S. Coupling calcium binding to troponin C and cross-bridge cycling in skinned cardiac cells. Am J Physiol Heart Circ Physiol 266: H1260-H1271, 1994[Abstract/Free Full Text].

40.   Leavis, PC, and Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem 16: 235-305, 1984[Web of Science][Medline].

41.   Lim, DS, Oberst L, McCluggage M, Youker K, Lacy J, DeMayo F, Entman ML, Roberts R, Michael LH, and Marian AJ. Decreased left ventricular ejection fraction in transgenic mice expressing mutant cardiac troponin T-Q(92), responsible for human hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 365-374, 2000[Web of Science][Medline].

42.   Lin, D, Bobkova A, Homsher E, and Tobacman LS. Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy. J Clin Invest 97: 2842-2848, 1996[Web of Science][Medline].

43.   Lin, T, Ichihara S, Yamada Y, Nagasaka T, Ishihara H, Nakashima N, and Yokota M. Phenotypic variation of familial hypertrophic cardiomyopathy caused by the Phe(110)Ile mutation in cardiac troponin T. Cardiology 93: 155-162, 2000[Web of Science][Medline].

44.   Malnic, B, Farah CS, and Reinach FC. Regulatory properties of the NH₂- and COOH-terminal domains of troponin T. ATPase activation and binding to troponin I and troponin C. J Biol Chem 273: 10594-10601, 1998[Abstract/Free Full Text].

45.   Marian, AJ, Zhao G, Seta Y, Roberts R, and Yu QT. Expression of a mutant (Arg92Gln) human cardiac troponin T, known to cause hypertrophic cardiomyopathy, impairs adult cardiac myocyte contractility. Circ Res 81: 76-85, 1997[Abstract/Free Full Text].

46.   McKay, RT, Saltibus LF, Li MX, and Sykes BD. Energetics of the induced structural change in a Ca²⁺ regulatory protein: Ca²⁺ and troponin I peptide binding to the E41A mutant of the N-domain of skeletal troponin C. Biochemistry 39: 12731-12738, 2000[Medline].

47.   Michele, DE, Albayya FP, and Metzger JM. Direct, convergent hypersensitivity of calcium-activated force generation produced by hypertrophic cardiomyopathy mutant -tropomyosins in adult cardiac myocytes. Nat Med 5: 1413-1417, 1999[Web of Science][Medline].

48.   Mikane, T, Araki J, Kohno K, Nakayama Y, Suzuki S, Shimizu J, Matsubara H, Hirakawa M, Takaki M, and Suga H. Mechanism of constant contractile efficiency under cooling inotropy of myocardium: simulation. Am J Physiol Heart Circ Physiol 273: H2891-H2898, 1997[Abstract/Free Full Text].

49.  Miller T, Szczesna D, Housmans PR, Zhao J, deFreitas F, Gomes AV, Culbreath L, McCue J, Wang Y, Xu Y, Kerrick WG, and Potter JD. Abnormal contractile function in transgenic mice expressing an FHC-linked troponin T (179N) mutation. J Biol Chem In press.

49a.   Mogensen, J, Klausen IC, Egebald H, Baandrup U, and Borglum AD. Sudden cardiac death in familial hypertrophic cardiomyopathy is associated with a novel mutation in the troponin I gene. Circulation 100: I-618, 1999.

50.   Mogensen, J, Klausen IC, Pedersen AK, Egeblad H, Bross P, Kruse TA, Gregersen N, Hansen PS, Baandrup U, and Borglum AD. Alpha-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. J Clin Invest 103: R39-R43, 1999[Medline].

51.   Moolman, JC, Corfield VA, Posen B, Ngumbela K, Seidman C, Brink PA, and Watkins H. Sudden death due to troponin T mutations. J Am Coll Cardiol 29: 549-555, 1997[Abstract].

52.   Moolman-Smook, JC, De Lange WJ, Bruwer EC, Brink PA, and Corfield VA. The origins of hypertrophic cardiomyopathy-causing mutations in two South African subpopulations: a unique profile of both independent and founder events. Am J Hum Genet 65: 1308-1320, 1999[Web of Science][Medline].

53.   Morimoto, S, Nakaura H, Yanaga F, and Ohtsuki I. Functional consequences of a carboxyl terminal missense mutation Arg278Cys in human cardiac troponin T. Biochem Biophys Res Commun 261: 79-82, 1999[Web of Science][Medline].

54.   Morimoto, S, Yanaga F, Minakami R, and Ohtsuki I. Ca²⁺-sensitizing effects of the mutations at Ile-79 and Arg-92 of troponin T in hypertrophic cardiomyopathy. Am J Physiol Cell Physiol 275: C200-C207, 1998[Abstract/Free Full Text].

55.   Morner, S, Richard P, Kazzam E, Hainque B, Schwartz K, and Waldenstrom A. Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 521-525, 2000[Web of Science][Medline].

56.   Mukherjea, P, Tong L, Seidman JG, Seidman CE, and Hitchcock-DeGregori SE. Altered regulatory function of two familial hypertrophic cardiomyopathy troponin T mutants. Biochemistry 38: 13296-13301, 1999[Medline].

57.   Muthuchamy, M, Pieples K, Rethinasamy P, Hoit B, Grupp IL, Boivin GP, Wolska B, Evans C, Solaro RJ, and Wieczorek DF. Mouse model of a familial hypertrophic cardiomyopathy mutation in -tropomyosin manifests cardiac dysfunction. Circ Res 85: 47-56, 1999[Abstract/Free Full Text].

58.   Nakajima-Taniguchi, C, Matsui H, Fujio Y, Nagata S, Kishimoto T, and Yamauchi-Takihara K. Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol 29: 839-843, 1997[Web of Science][Medline].

59.   Nakajima-Taniguchi, C, Matsui H, Nagata S, Kishimoto T, and Yamauchi-Takihara K. Novel missense mutation in -tropomyosin gene found in Japanese patients with hypertrophic cardiomyopathy. J Mol Cell Cardiol 27: 2053-2058, 1995[Web of Science][Medline].

60.   Nakaura, H, Morimoto S, Yanaga F, Nakata M, Nishi H, Imaizumi T, and Ohtsuki I. Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy. Am J Physiol Cell Physiol 277: C225-C232, 1999[Abstract/Free Full Text].

61.   Nakaura, H, Yanaga F, Ohtsuki I, and Morimoto S. Effects of missense mutations Phe110Ile and Glu244Asp in human cardiac troponin T on force generation in skinned cardiac muscle fibers. J Biochem (Tokyo) 126: 457-460, 1999[Abstract/Free Full Text].

62.   Noland, TA, Jr, Guo X, Raynor RL, Jideama NM, Averyhart-Fullard V, Solaro RJ, and Kuo JF. Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca²⁺-stimulated MgATPase of reconstituted actomyosin S-1. J Biol Chem 270: 25445-25454, 1995[Abstract/Free Full Text].

63.   Oberst, L, Zhao G, Park JT, Brugada R, Michael LH, Entman ML, Roberts R, and Marian AJ. Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice. J Clin Invest 102: 1498-1505, 1998[Web of Science][Medline].

64.   Ohtsuki, I. Calcium ion regulation of muscle contraction: the regulatory role of troponin T. Mol Cell Biochem 190: 33-38, 1999[Web of Science][Medline].

65.   Olson, TM, Doan TP, Kishimoto NY, Whitby FG, Ackerman MJ, and Fananapazir L. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 1687-1694, 2000[Web of Science][Medline].

66.   Perry, SV. Troponin T: genetics, properties and function. J Muscle Res Cell Motil 19: 575-602, 1998[Web of Science][Medline].

67.   Poetter, K, Jiang H, Hassanzadeh S, Master SR, Chang A, Dalakas MC, Rayment I, Sellers JR, Fananapazir L, and Epstein ND. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet 13: 63-69, 1996[Web of Science][Medline].

68.   Potter, JD, Sheng Z, Pan BS, and Zhao J. A direct regulatory role for troponin T and a dual role for troponin C in the Ca²⁺ regulation of muscle contraction. J Biol Chem 270: 2557-2562, 1995[Abstract/Free Full Text].

69.   Redwood, C, Lohmann K, Bing W, Esposito GM, Elliott K, Abdulrazzak H, Knott A, Purcell I, Marston S, and Watkins H. Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca²⁺ regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein. Circ Res 86: 1146-1152, 2000[Abstract/Free Full Text].

70.   Redwood, CS, Moolman-Smook JC, and Watkins H. Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy. Cardiovasc Res 44: 20-36, 1999[Abstract/Free Full Text].

71.   Regitz-Zagrosek, V, Erdmann J, Wellnhofer E, Raible J, and Fleck E. Novel mutation in the -tropomyosin gene and transition from hypertrophic to hypocontractile dilated cardiomyopathy. Circulation 102: E112-E116, 2000.

72.   Reinach, FC, Farah CS, Monteiro PB, and Malnic B. Structural interactions responsible for the assembly of the troponin complex on the muscle thin filament. Cell Struct Funct 22: 219-223, 1997[Web of Science][Medline].

73.   Robertson, SP, Johnson JD, and Potter JD. The time-course of Ca²⁺ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca²⁺. Biophys J 34: 559-569, 1981[Web of Science][Medline].

74.   Rust, EM, Albayya FP, and Metzger JM. Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins. J Clin Invest 103: 1459-1467, 1999[Web of Science][Medline].

75.   Satoh, M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, and Kimura A. Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene. Biochem Biophys Res Commun 262: 411-417, 1999[Web of Science][Medline].

76.   Schaertl, S, Lehrer SS, and Geeves MA. Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation. Biochemistry 34: 15890-15894, 1995[Medline].

77.   Solaro, RJ. Troponin I, stunning, hypertrophy, and failure of the heart. Circ Res 84: 122-124, 1999[Free Full Text].

78.   Sweeney, HL, Feng HS, Yang Z, and Watkins H. Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: insights into disease pathogenesis and troponin function. Proc Natl Acad Sci USA 95: 14406-14410, 1998[Abstract/Free Full Text].

78a.  Szczesna D and Potter JD. The role of troponin in the Ca²⁺-regulation of skeletal muscle contraction. In: Molecular Interactions of Actin, edited by dasRemedios CG and Thomas DD. Heidelberg, Germany. In press.

79.   Szczesna, D, Zhang R, Zhao J, Jones M, Guzman G, and Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. J Biol Chem 275: 624-630, 2000[Abstract/Free Full Text].

80.   Takahashi-Yanaga, F, Morimoto S, and Ohtsuki I. Effect of Arg145Gly mutation in human cardiac troponin I on the ATPase activity of cardiac myofibrils. J Biochem (Tokyo) 127: 355-357, 2000[Abstract/Free Full Text].

81.   Tardiff, JC, Factor SM, Tompkins BD, Hewett TE, Palmer BM, Moore RL, Schwartz S, Robbins J, and Leinwand LA. A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy. J Clin Invest 101: 2800-2811, 1998[Web of Science][Medline].

82.   Tardiff, JC, Hewett TE, Palmer BM, Olsson C, Factor SM, Moore RL, Robbins J, and Leinwand LA. Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy. J Clin Invest 104: 469-481, 1999[Web of Science][Medline].

83.   Thierfelder, L, MacRae C, Watkins H, Tomfohrde J, Williams M, McKenna W, Bohm K, Noeske G, Schlepper M, Bowcock A, A familial hypertrophic cardiomyopathy locus maps to chromosome 15q2. Proc Natl Acad Sci USA 90: 6270-6274, 1993[Abstract/Free Full Text].

84.   Thierfelder, L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, and Seidman CE. -Tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 77: 701-712, 1994[Web of Science][Medline].

85.   Tobacman, LS, Lin D, Butters C, Landis C, Back N, Pavlov D, and Homsher E. Functional consequences of troponin T mutations found in hypertrophic cardiomyopathy. J Biol Chem 274: 28363-28370, 1999[Abstract/Free Full Text].

86.   Varnava, A, Baboonian C, Davison F, de Cruz L, Elliott PM, Davies MJ, and McKenna WJ. A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart 82: 621-624, 1999[Abstract/Free Full Text].

87.   Watkins, H, Anan R, Coviello DA, Spirito P, Seidman JG, and Seidman CE. A de novo mutation in -tropomyosin that causes hypertrophic cardiomyopathy. Circulation 91: 2302-2305, 1995[Abstract/Free Full Text].

88.   Watkins, H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, Maron BJ, Seidman JG, and Seidman CE. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet 11: 434-437, 1995[Web of Science][Medline].

89.   Watkins, H, MacRae C, Thierfelder L, Chou YH, Frenneaux M, McKenna W, Seidman JG, and Seidman CE. A disease locus for familial hypertrophic cardiomyopathy maps to chromosome 1q3. Nat Genet 3: 333-337, 1993[Web of Science][Medline].

90.   Watkins, H, McKenna WJ, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, Spirito P, Matsumori A, Moravec CS, Seidman JG, Mutations in the genes for cardiac troponin T and -tropomyosin in hypertrophic cardiomyopathy. N Engl J Med 332: 1058-1064, 1995[Abstract/Free Full Text].

91.   Watkins, H, Seidman CE, Seidman JG, Feng HS, and Sweeney HL. Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action. J Clin Invest 98: 2456-2461, 1996[Web of Science][Medline].

92.   Watkins, H, Thierfelder L, Anan R, Jarcho J, Matsumori A, McKenna W, Seidman JG, and Seidman CE. Independent origin of identical beta cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy. Am J Hum Genet 53: 1180-1185, 1993[Web of Science][Medline].

93.   Watkins, H, Thierfelder L, Hwang DS, McKenna W, Seidman JG, and Seidman CE. Sporadic hypertrophic cardiomyopathy due to de novo myosin mutations. J Clin Invest 90: 1666-1671, 1992.

94.   Yamauchi-Takihara, K, Nakajima-Taniguchi C, Matsui H, Fujio Y, Kunisada K, Nagata S, and Kishimoto T. Clinical implications of hypertrophic cardiomyopathy associated with mutations in the -tropomyosin gene. Heart 76: 63-65, 1996[Abstract/Free Full Text].

95.   Yanaga, F, Morimoto S, and Ohtsuki I. Ca²⁺ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy. J Biol Chem 274: 8806-8812, 1999[Abstract/Free Full Text].

96.   Zot, AS, and Potter JD. The effect of [Mg²⁺] on the Ca²⁺ dependence of ATPase and tension development of fast skeletal muscle. The role of the Ca²⁺-specific sites of troponin C. J Biol Chem 262: 1966-1969, 1987[Abstract/Free Full Text].