Functional consequences of mutations in the smooth muscle myosin heavy chain at sites implicated in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is frequently associated with mutations in the beta-cardiac myosin heavy chain. Many of the implicated residues are located in highly conserved regions of the myosin II class, suggesting that these mutations may impair the basic functions of the molecular motor. To test this hypothesis, we have prepared recombinant smooth muscle heavy meromyosin with mutations at sites homologous to those associated with FHC by using a baculovirus/insect cell expression system. Several of the heavy meromyosin mutants, in particular R403Q, showed an increase in actin filament velocity in a motility assay and an enhanced actin-activated ATPase activity. Single molecule mechanics, using a laser trap, gave unitary displacements and forces for the mutants that were similar to wild type, but the attachment times to actin following a unitary displacement were markedly reduced. These results suggest that the increases in activity are due to a change in kinetics and not due to a change in the intrinsic mechanical properties of the motor. In contrast to earlier reports, we find that mutations in residues implicated in FHC affect motor function by enhancing myosin activity rather than by a loss of function.     

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln alpha-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure-volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all 'hallmarks' of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.     

The R403Q myosin mutation implicated in familial hypertrophic cardiomyopathy causes disorder at the actomyosin interface

Background

Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the β-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype.

Principal Findings

Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow β-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface.

Significance

These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state.     

Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.     

Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy

A splice donorsite mutation in intron 15 of the cardiac troponin T (TnT) gene hasbeen shown to cause familial hypertrophic cardiomyopathy (HCM). In thisstudy, two truncated human cardiac TnTs expected to be produced by thismutation were expressed in Escherichiacoli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because ofthe replacement of 28 amino acids with 7 novel residues, had beenexchanged generated aCa²⁺-activated maximum force thatwas slightly, but statistically significantly, lower than thatgenerated by fibers into which wild-type TnT had been exchanged whentroponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. Along truncated TnT simply lacking the COOH-terminal 14 amino acids hadno significant effect on the maximum force-generating capability in thefibers with either phosphorylated or dephosphorylated TnI.Both these two truncated TnTs conferred a lower cooperativity and ahigher Ca²⁺ sensitivity on theCa²⁺-activated force generationthan did wild-type TnT, independent of the phosphorylation of TnI bycAMP-dependent protein kinase. The results demonstrate that the splicedonor site mutation in the cardiac TnT gene impairs the regulatoryfunction of the TnT molecule, leading to an increase in theCa²⁺ sensitivity, and a decreasein the cooperativity, of cardiac muscle contraction, which might beinvolved in the pathogenesis of HCM.

     

Functional and spectroscopic studies of a familial hypertrophic cardiomyopathy mutation in Motif X of cardiac myosin binding protein-C

Familial hypertrophic cardiomyopathy is an autosomal dominant genetic disorder caused by mutations in cardiac sarcomeric proteins. One such mutation is a six amino acid duplication of residues 1248-1253 in the C-terminal immunoglobulin domain of cardiac myosin binding protein-C, referred to as Motif X. Motif X binds the myosin rod and titin. Here we investigate the structural and functional alteration in the mutant Motif X protein to understand how sarcomeric dysfunction may occur. The cDNA encoding Motif X was cloned, mutated and expressed as wild-type and mutant proteins in a bacterial expression system. Circular dichroism spectroscopy confirmed that the normal and mutant Motif X exhibited a high beta-content, as predicted for immunoglobulin domains. Thermal denaturation curves showed that Motif X unfolded with at least two structural transitions, with the first transition occurring at 63 degrees C in the wild-type but at 40 degrees C in the mutant, consistent with the mutant being structurally less stable. Sedimentation binding studies with synthetic myosin filaments revealed no significant difference in binding to myosin between the wild-type and the mutant Motif X. Molecular modeling of this duplication mutation onto an homologous IgI structure (telokin) revealed that the duplicated residues lie within the F strand of the immunoglobulin fold, on a surface of Motif X distant from residues previously implicated in myosin binding. Taken together, these data suggest that the Motif X mutation may interfere with other, as yet unidentified, functional interactions.     

Functional analysis of a troponin I (R145G) mutation associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy has been associated with several mutations in the gene encoding human cardiac troponin I (HCTnI). A missense mutation in the inhibitory region of TnI replaces an arginine residue at position 145 with a glycine and cosegregates with the disease. Results from several assays indicate that the inhibitory function of HCTnI(R145G) is significantly reduced. When HCTnI(R145G) was incorporated into whole troponin, Tn(R145G) (HCTnT small middle dotHCTnI(R145G) small middle dotHCTnC), only partial inhibition of the actin-tropomyosin-myosin ATPase activity was observed in the absence of Ca(2+) compared with wild type Tn (HCTnT small middle dotHCTnI small middle dotHCTnC). Maximal activation of actin-tropomyosin-myosin ATPase in the presence of Ca(2+) was also decreased in Tn(R145G) when compared with Tn. Using skinned cardiac muscle fibers, we determined that in comparison with the wild type complex 1) the complex containing HCTnI(R145G) only inhibited 84% of Ca(2+)-unregulated force, 2) the recovery of Ca(2+)-activated force was decreased, and 3) there was a significant increase in the Ca(2+) sensitivity of force development. Computer modeling of troponin C and I variables predicts that the primary defect in TnI caused by these mutations would lead to diastolic dysfunction. These results suggest that severe diastolic dysfunction and somewhat decreased contractility would be prominent clinical features and that hypertrophy could arise as a compensatory mechanism.     

Functional consequences of troponin T mutations found in hypertrophic cardiomyopathy.

Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal truncation weakened the affinity of troponin for the thin filament. Mutation DeltaE160 resulted in thin filaments with increased calcium affinity at the regulatory site of troponin C. Mutations R92Q and F110I resulted in impaired troponin solubility, suggesting abnormal protein folding. Depending upon the mutation, the in vitro unloaded actin-myosin sliding speed showed small increases, showed small decreases, or was unchanged. COOH-terminal truncation mutation resulted in a decreased thin filament-myosin subfragment 1 MgATPase rate. The results indicate that the mutations cause diverse immediate effects, despite similarities in disease manifestations. Separable but repeatedly observed abnormalities resulting from FHC TnT mutations include increased unloaded sliding speed, increased or decreased Ca(2+) affinity, impairment of folding or sarcomeric integrity, and decreased force. Enhancement as well as impairment of contractile protein function is observed, suggesting that TnT, including the troponin tail region, modulates the regulation of cardiac contraction.     

Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy.

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.     

Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in 14 and 15 different disease genes, respectively, in a part of the patients and the disease genes for cardiomyopathy overlap in part with that for limb-girdle muscular dystrophy (LGMD). In this study, we examined an LGMD gene encoding caveolin-3 (CAV3) for mutation in the patients with HCM or DCM. A Thr63Ser mutation was identified in a sibling case of HCM. Because the mutation was found at the residue that is involved in the LGMD-causing mutations, we investigate the functional change due to the Thr63Ser mutation as compared with the LGMD mutations by examining the distribution of GFP-tagged CAV3 proteins. It was observed that the Thr63Ser mutation reduced the cell surface expression of caveolin-3, albeit the change was mild as compared with the LGMD mutations. These observations suggest that HCM is a clinical spectrum of CAV3 mutations.     

A missense mutation in a ubiquitously expressed protein, vinculin, confers susceptibility to hypertrophic cardiomyopathy

The R975W mutation, in the alternatively spliced exon 19 of vinculin (VCL) which yields the isoform metavinculin, was associated previously with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), and shown to alter in vivo organization of intercalated discs. We tested the hypothesis that alterations in the ubiquitously expressed, VCL-encoded protein, vinculin, may provide a pathogenic substrate for HCM. Comprehensive mutational analysis of VCL's 22 translated exons was performed in a cohort of 228 unrelated patients with genotype negative HCM, having no identifiable mutations in 12 HCM-associated myofilament/Z-disc-encoding genes. A novel missense mutation, L277M-VCL, involving a conserved residue was identified in a patient with severely obstructive, mid-ventricular hypertrophy. This mutation was not detected in 400 reference alleles. Immunohistochemical analysis of the proband's myectomy specimen demonstrated markedly reduced vinculin levels in the intercalated discs. We provide the first report of a cardiomyopathy associated mutation in vinculin. Despite its ubiquitous expression, the HCM-associated VCL mutation clinically yielded a cardiac-specific phenotype.     

A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation

A point mutation in exon 13 of the beta cardiac myosin heavy chain (MHC) gene is present in all individuals affected with familial hypertrophic cardiomyopathy (FHC) from a large kindred. This missense mutation converts a highly conserved arginine residue (Arg-403) to a glutamine. Affected individuals from an unrelated family lack this missense mutation, but instead have an alpha/beta cardiac MHC hybrid gene. Identification of two unique mutations within cardiac MHC genes in all individuals with FHC from two unrelated families demonstrates that defects in the cardiac MHC genes can cause this disease. The pathology resulting from a missense mutation at residue 403 further suggests that a critical function of myosin is disrupted by this mutation.     

Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

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.     

Mapping the locus for familial hypertrophic cardiomyopathy to chromosome 11 in a family with a case of apical hypertrophic cardiomyopathy of the Japanese type

To identify the disease locus of familial hypertrophic cardiomyopathy (FHC) in a Chinese family, a genetic linkage study was performed using polymorphisms from various chromosomal regions. This family has eight affected members, including a case with typical features of apical hypertrophic cardiomyopathy of the Japanese type. The results revealed significant evidence of linkage of polymorphisms on chromosome 11p13–q13 and FHC in this family with a maximal lod score of 3.38 at θ = 0.00. Our data suggest that the locus responsible for FHC in this family maps to chromosome 11 and that the molecular basis of FHC in the case of apical hypertrophic cardiomyopathy of the Japanese type might be similar to that of other affected members in the same family. Further studies are needed to elucidate the whole spectrum of the genetic basis of apical hypertrophic cardiomyopathy of the Japanese type. Received: 15 June 1995 / Revised: 22 August 1995     

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.     

Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in alpha-tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.     

心脏肌球蛋白重链基因c.1273G>A突变与肥厚型心肌病的关联分析

为研究中国人家族性肥厚型心肌病(HCM)的致病基因突变位点, 分析基因型与临床表型的相互关系, 文章在1个中国汉族HCM家系中进行心脏肌钙蛋白T (TNNT2) 基因、心脏肌球蛋白结合蛋白C (MYBPC3) 基因和心脏β-肌球蛋白重链 (MYH7) 基因的突变筛查, 聚合酶链式反应(PCR)扩增基因功能区外显子片段并对PCR产物进行测序分析。结果表明: 在该家系接受调查的7名成员中有4名成员携带MYH7基因c.1273G>A杂合突变, 该突变位点位于MYH7基因的14号外显子并使425位的甘氨酸(Gly)转换为精氨酸(Arg)。该突变首次在国内HCM家系中发现, 突变携带者的临床表型在家系内部呈现明显的异质性。该家系成员TNNT2及MYBPC3基因未发现突变且正常对照组相同位置未发现异常。MYH7基因是我国家族性 HCM的致病基因之一, 携带c.1273G>A突变的肥厚型心肌病患者临床表型差异明显, 提示可能有其它因素参与了肥厚型心肌病的发展过程。     

E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.     

Inotropic stimulation induces cardiac dysfunction in transgenic mice expressing a troponin T (I79N) mutation linked to familial hypertrophic cardiomyopathy

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and a high incidence of sudden death, despite causing little or no cardiac hypertrophy. In skinned fibers, I79N increased myofilamental calcium sensitivity (Miller, T., Szczesna, D., Housmans, P. R., Zhao, J., deFreitas, F., Gomes, A. V., Culbreath, L., McCue, J., Wang, Y., Xu, Y., Kerrick, W. G., and Potter, J. D. (2001) J. Biol. Chem. 276, 3743-3755). To further study the functional consequences of this mutation, we compared the cardiac performance of transgenic mice expressing either human TnT-I79N or human wild-type TnT. In isolated hearts, cardiac function was different depending on the Ca(2+) concentration of the perfusate; systolic function was significantly increased in Tg-I79N hearts at 0.5 and 1 mmol/liter. At higher Ca(2+) concentrations, systolic function was not different, but diastolic dysfunction became manifest as increased end-diastolic pressure and time to 90% relaxation. In vivo measurements by echocardiography and Doppler confirmed that base-line systolic function was significantly higher in Tg-I79N mice without evidence for diastolic dysfunction. Inotropic stimulation with isoproterenol resulted only in a modest contractile response but caused significant mortality in Tg-I79N mice. Doppler studies ruled out aortic outflow obstruction and were consistent with increased chamber stiffness. We conclude that in vivo, the increased myofilament Ca(2+) sensitivity due to the I79N mutation enhances base-line contractility but leads to cardiac dysfunction during inotropic stimulation.