Cardiac troponin T (cTnT) is a central component of the regulatory thin filament. Mutations in cTnT have been linked to severe forms of familial hypertrophic cardiomyopathy. A mutational ''hotspot'' that leads to distinct clinical phenotypes has been identified at codon 92. Although the basic functional and structural roles of cTnT in modulating contractility are relatively well understood, the mechanisms that link point mutations in cTnT to the development of this complex cardiomyopathy are unknown. To address this question, we have taken a highly interdisciplinary approach by first determining the effects of the residue 92 mutations on the molecular flexibility and stability of cTnT by means of molecular dynamics simulations. To test whether the predicted alterations in thin filament structure could lead to distinct cardiomyopathies in vivo, we developed transgenic mouse models expressing either the Arg-92-Trp or Arg-92-Leu cTnT proteins in the heart. Characterization of these models at the cellular and whole-heart levels has revealed mutation-specific early alterations in transcriptional activation that result in distinct pathways of ventricular remodeling and contractile performance. Thus, our computational and experimental results show that changes in thin filament structure caused by single amino acid substitutions lead to differences in the biophysical properties of cTnT and alter disease pathogenesis.contractility ͉ molecular dynamics ͉ thin filament ͉ familial hypertrophic cardiomyopathy T he regulatory function of the cardiac sarcomere resides in the thin filament. Muscle contraction depends on the access of the myosin head to the actin filament, which is regulated by a cascade of allosteric changes in the interactions of the proteins within the troponin [cardiac troponin T (cTnT), cTnI, and cTnC] and tropomyosin-actin complexes upon the binding of Ca 2ϩ (1, 2). Disruption of these important protein-protein interactions by many naturally occurring thin filament mutations is poorly tolerated. Many mutations in cTnT result in a severe form of genetic cardiomyopathy, familial hypertrophic cardiomyopathy (FHC). FHC caused by cTnT mutations is characterized by a high frequency of early sudden cardiac death, often in the absence of overt ventricular hypertrophy (3). The direct link between mutations in the structural components of the cardiac sarcomere and the resultant complex clinical phenotype remains unknown.cTnT is a highly elongated protein that interacts with all other components of the thin filament and has been described as the ''glue'' of the contractile regulatory system (1). Codon 92 in cTnT is a mutational ''hotspot,'' and patients carrying each of the three predicted missense mutations have been identified and exhibit distinct clinical phenotypes (4-6). Patients carrying the Arg-92-Trp (R92W) missense mutation in cTnT develop mild or no ventricular hypertrophy, yet they experience a high frequency of early cardiac sudden death (7). In contrast, although carriers of the Arg-92-Leu (R92L) mutatio...

Naturally occurring mutations in cardiac troponin T (cTnT) result in a clinical subset of familial hypertrophic cardiomyopathy. To determine the mechanistic links between thin-filament mutations and cardiovascular phenotypes, we have generated and characterized several transgenic mouse models carrying cTnT mutations. We address two central questions regarding the previously observed changes in myocellular mechanics and Ca(2+) homeostasis: 1) are they characteristic of all severe cTnT mutations, and 2) are they primary (early) or secondary (late) components of the myocellular response? Adult left ventricular myocytes were isolated from 2- and 6-mo-old transgenic mice carrying missense mutations at residue 92, flanking the TNT1 NH(2)-terminal tail domain. Results from R92L and R92W myocytes showed mutation-specific alterations in contraction and relaxation indexes at 2 mo with improvements by 6 mo. Alterations in Ca(2+) kinetics remained consistent with mechanical data in which R92L and R92W exhibited severe diastolic impairments at the early time point that improved with increasing age. A normal regulation of Ca(2+) kinetics in the context of an altered baseline cTnI phosphorylation suggested a pathogenic mechanism at the myofilament level taking precedence for R92L. The quantitation of Ca(2+)-handling proteins in R92W mice revealed a synergistic compensatory mechanism involving an increased Ser16 and Thr17 phosphorylation of phospholamban, contributing to the temporal onset of improved cellular mechanics and Ca(2+) homeostasis. Therefore, independent cTnT mutations in the TNT1 domain result in primary mutation-specific effects and a differential temporal onset of altered myocellular mechanics, Ca(2+) kinetics, and Ca(2+) homeostasis, complex mechanisms which may contribute to the clinical variability in cTnT-related familial hypertrophic cardiomyopathy mutations.

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The goal of this prospective longitudinal study was to determine the serological profile of early rheumatoid arthritis (RA), and to test whether antikeratin antibody (AKA), antiperinuclear factor (APF), anti-RA33 antibody and antinuclear antibodies (ANA) had an additional diagnostic value when prescribed after rheumatoid factor (RF)-detecting methods. Sixty-nine patients with early polyarthritis suggestive of RA, seen between 1991 and 1993, were included. Five autoantibodies (i.e. RF, AKA, APF, RA33, ANA) were looked for at regular intervals. After 24 months follow-up, patients were classified as having RA (n = 49), unclassified polyarthritis (UP; n = 15) or other rheumatic diseases. Among patients with early RA, the sensitivity of these markers was 40.8% for RF, 36.7% for AKA, 28.6% for APF and 28.6% for anti-RA33. Among RF-negative RA patients, 51.7% were positive for AKA, APF, anti-RA33 antibodies and/or ANA. Positivity of the three recent markers usually persisted throughout follow-up, whereas RF was lost by 58% of patients with early, RF-positive, treated RA. Using multivariate analysis, only latex, RF test and AKA or APF had an independent and statistically significant diagnostic value for early RA. Our data suggest that RF and AKA (or APF) should be concomitantly determined for diagnosis in patients with suspected early RA.

Abstract-Previous studies demonstrated increased fatty acid uptake and metabolism in MHC-FATP transgenic mice that overexpress fatty acid transport protein (FATP)1 in the heart under the control of the ␣-myosin heavy chain (␣-MHC) promoter. Doppler tissue imaging and hemodynamic measurements revealed diastolic dysfunction, in the absence of changes in systolic function. The experiments here directly test the hypothesis that the diastolic dysfunction in MHC-FATP mice reflects impaired ventricular myocyte contractile function. In vitro imaging of isolated adult MHC-FATP ventricular myocytes revealed that mean diastolic sarcomere length is significantly (PϽ0.01) shorter than in wild-type (WT) cells (1.79Ϯ0.01 versus 1.84Ϯ0.01 m). In addition, the relaxation rate (dL/dt) is significantly (PϽ0.05) slower in MHC-FATP than WT myocytes (1.58Ϯ0.09 versus 1.92Ϯ0.13 m/s), whereas both fractional shortening and contraction rates are not different. Application of 40 mmol/L 2,3-butadionemonoxime (a nonspecific ATPase inhibitor that relaxes actin-myosin interactions) increased diastolic sarcomere length in both WT and MHC-FATP myocytes to the same length, suggesting that MHC-FATP myocytes are partially activated at rest. Direct measurements of intracellular Ca 2ϩ revealed that diastolic [Ca 2ϩ ] i is unchanged in MHC-FATP myocytes and the rate of calcium removal is unexpectedly faster in MHC-FATP than WT myocytes. Moreover, diastolic sarcomere length in MHC-FATP and WT myocytes was unaffected by removal of extracellular Ca 2ϩ or by buffering of intracellular Ca 2ϩ with the Ca 2ϩ chelator BAPTA (100 mol/L), indicating that elevated intracellular Ca 2ϩ does not underlie impaired diastolic function in MHC-FATP ventricular myocytes. Functional assessment of skinned myocytes, however, revealed that myofilament Ca 2ϩ sensitivity is markedly increased in MHC-FATP, compared with WT, ventricular cells. In addition, biochemical experiments demonstrated increased expression of the ␤-MHC isoform in MHC-FATP, compared with WT ventricles, which likely contributes to the slower relaxation rate observed in MHC-FATP myocytes.Collectively, these data demonstrate that derangements in lipid metabolism in MHC-FATP ventricles, which are similar to those observed in the diabetic heart, result in impaired diastolic function that primarily reflects changes in myofilament function, rather than altered Ca Key Words: metabolism Ⅲ diabetes Ⅲ myofilaments Ⅲ remodeling M ounting evidence indicates that cardiac metabolism and disease are intimately related. In this regard, altered energy metabolism is a prominent feature of and, in some instances, may cause heart failure. 1,2 It is also now well recognized that patients with diabetes mellitus have an increased risk of cardiac disease that is independent of the presence of secondary risk factors such as coronary artery disease. 2 These observations suggest that derangements of cardiac metabolism have a direct consequence on cardiac function. The molecular mechanisms potentially linking alterations in metab...

There is considerable evidence to support a role for lipotoxicity in the development of diabetic cardiomyopathy, although the molecular links between enhanced saturated fatty acid uptake/ metabolism and impaired cardiac function are poorly understood. In the present study, the effects of acute exposure to the saturated fatty acid, palmitate, on myocardial contractility and excitability were examined directly. Exposure of isolated (adult mouse) ventricular myocytes to palmitate, complexed to bovine serum albumin (palmitate:BSA) as in blood, rapidly reduced (by 54±4%) mean (± SEM) unloaded fractional cell shortening. The amplitudes of intracellular Ca 2+ transients decreased in parallel. Current-clamp recordings revealed that exposure to palmitate:BSA markedly shortened action potential durations at 20, 50 and 90% repolarization. These effects were reversible and were occluded when the K + in the recording pipettes was replaced with Cs + , suggesting a direct effect on repolarizing K + currents. Indeed, voltage-clamp recordings revealed that palmitate:BSA reversibly and selectively increased peak outward voltage-gated K + (Kv) current amplitudes by 20 ± 2%, whereas inwardly rectifying K + (Kir) currents and voltage-gated Ca 2+ currents were unaffected. Further analyses revealed that the individual Kv current components I to,f , I K,slow and I ss , were all increased (by 12 ± 2 %, 37 ± 4 % and 34 ± 4, respectively) in cells exposed to palmitate:BSA. Consistent with effects on both components of I K,slow (I K,slow1 and I K,slow2 ) the magnitude of the palmitate-induced increase was attenuated in ventricular myocytes isolated from animals in which the Kv1.5 (I K,slow1 ) or the Kv2.1 (I K,slow2 ) locus was disrupted and I K,slow1 or I K,slow2 is eliminated. Both the enhancement of I K,slow and the negative inotropic effect of palmitate:BSA were reduced in the presence of the Kv1.5 selective channel blocker, diphenyl phosphine oxide-1 (DPO-1). Taken together, these results suggest that elevations in circulating saturated free fatty acids, as occurs in diabetes, can directly augment repolarizing myocardial Kv currents and impair excitation-contraction coupling.

(normal 5-45 ,ug/ml (5-45 mg/i)), total protein 6-3 g/dl (63 g/l) (normal 6-5-8-5 g/dl (65-85 g/l)), albumin 3X7 g/dl (37 g/l) (normal 3-2-5-0 g/dl (32-50 g/l)), globulin 2-6 g/dl (26 g/l) (normal 2-5-4-5 g/dl (25-45 g/l)), uric acid 7-0 mg/dl (1P2 mmol/l) (normal 3-0-7 5 mg/dl (0-5-1P2 mmol/l)), creatinine 1-2 mg/dl (106 ,umol/l) (normal 0-7-15

The prevalence of pseudoexfoliation among patients of Sephardic origin was more than twice the expected when compared with the ethnic distribution of the population consulting the Rambam Medical Center eye clinic. A strong association between brown irises and pseudoexfoliation was noted. No uniform scanning electron microscope pattern was seen of the pseudoexfoliation.