Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy

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

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.     

Prolonged Ca2+ and force transients in myosin RLC transgenic mouse fibers expressing malignant and benign FHC mutations

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force.     

Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy

Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity.     

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation

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

Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

Effects of troponin T mutations in familial hypertrophic cardiomyopathy on regulatory functions of other troponin subunits.

We have previously shown that mutations in troponin T (TnT), which is associated with familial hypertrophic cardiomyopathy (HCM), cause an increase in the Ca(2+) sensitivity and a potentiation of cardiac muscle contraction. To gain further insight into the patho-physiological role of these mutations, four mutations (Arg92Gln, Phe110Ile, Glu244Asp, Arg278Cys) were introduced into recombinant human cardiac TnT, and the mutants were exchanged into isolated porcine cardiac myofibrils. The effects of mutations were tested on maximal ATPase activity, the inhibitory function of troponin I (TnI) in the absence of troponin C (TnC), and the neutralizing function of TnC. Arg92Gln, Phe110Ile, and Glu244Asp markedly impaired the inhibitory function of TnI. Arg278Cys also impaired the inhibitory function of TnI, but the effect was much smaller. Phe110Ile and Glu244Asp markedly enhanced the neutralizing function of TnC and potentiated the maximum ATPase activity. Arg92Gln and Arg278Cys only slightly enhanced the neutralizing function of TnC, and they conferred no potentiation on the maximum ATPase activity. These results indicate that mutations in TnT impair multiple processes of Ca(2+) regulation by troponin, and there are marked differences in the degree of impairment from mutation to mutation.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.     

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.     

Differences in myofilament calcium sensitivity in rat psoas fibers reconstituted with troponin T isoforms containing the alpha- and beta-exons

The carboxy terminus of fast skeletal muscle troponin T (fsTnT) is highly conserved. However, mutually exclusive splicing of exons 16 and 17 in the fsTnT gene results in the expression of either the alpha- or beta-fsTnT isoform. The alpha-isoform is expressed only in adult fast skeletal muscle, whereas the beta-isoform is expressed in varying quantities throughout muscle development. Reconstitution of detergent-skinned adult rat psoas muscle fibers with rat fast skeletal troponin complexes containing either fsTnT isoform demonstrated that reconstitution with alpha-fsTnT resulted in greater myofilament Ca(2+) sensitivity than reconstitution with beta-fsTnT, without changes to Ca(2+)-activated maximal tension, ATPase activity or tension cost. The observed isoform-specific differences in myofilament Ca(2+) sensitivity may be due to changes in the transition of the thin-filament regulatory unit from the off to the on state, possibly due to altered interactions of the C-terminus of fsTnT with troponins I and/or C.     

The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts

Phosphorylation of the myosin regulatory light chain (RLC) by Ca(2+)-calmodulin-activated myosin light chain kinase (MLCK) is known to be essential for the inotropic function of the heart. In this study, we have examined the effects of MLCK-phosphorylation of transgenic (Tg) mouse cardiac muscle preparations expressing the D166V (aspartic acid to valine)-RLC mutation, identified to cause familial hypertrophic cardiomyopathy with malignant outcomes. Our previous work with Tg-D166V mice demonstrated a large increase in the Ca(2+) sensitivity of contraction, reduced maximal ATPase and force and a decreased level of endogenous RLC phosphorylation. Based on studies demonstrating the beneficial and/or protective effects of cardiac myosin phosphorylation for heart function, we hypothesized that an ex vivo phosphorylation of Tg-D166V cardiac muscle may rescue the detrimental contractile phenotypes observed earlier at the level of single myosin molecules and in Tg-D166V papillary muscle fibres. We showed that MLCK-induced phosphorylation of Tg-D166V cardiac myofibrils and muscle fibres was able to increase the reduced myofibrillar ATPase and reverse an abnormally increased Ca(2+) sensitivity of force to the level observed for Tg-wild-type (WT) muscle. However, in contrast to Tg-WT, which displayed a phosphorylation-induced increase in steady-state force, the maximal tension in Tg-D166V papillary muscle fibres decreased upon phosphorylation. With the exception of force generation data, our results support the notion that RLC phosphorylation works as a rescue mechanism alleviating detrimental functional effects of a disease causing mutation. Further studies are necessary to elucidate the mechanism of this unexpected phosphorylation-induced decrease in maximal tension in Tg-D166V-skinned muscle fibres.     

Familial hypertrophic cardiomyopathy mutations from different functional regions of troponin T result in different effects on the pH and Ca2+ sensitivity of cardiac muscle contraction

To understand the molecular function of troponin T (TnT) in the Ca(2+) regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca(2+) dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca(2+) sensitivity. In contrast, the other seven mutants increased the Ca(2+) sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca(2+) sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91-94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms.     

Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.     

The off rate of Ca(2+) from troponin C is regulated by force-generating cross bridges in skeletal muscle.

The effects of dissociation of force-generating cross bridges on intracellular Ca(2+), pCa-force, and pCa-ATPase relationships were investigated in mouse skeletal muscle. Mechanical length perturbations were used to dissociate force-generating cross bridges in either intact or skinned fibers. In intact muscle, an impulse stretch or release, a continuous length vibration, a nonoverlap stretch, or an unloaded shortening during a twitch caused a transient increase in intracellular Ca(2+) compared with that in isometric controls and resulted in deactivation of the muscle. In skinned fibers, sinusoidal length vibrations shifted pCa-force and pCa-actomyosin ATPase rate relationships to higher Ca(2+) concentrations and caused actomyosin ATPase rate to decrease at submaximal Ca(2+) and increase at maximal Ca(2+) activation. These results suggest that dissociation of force-generating cross bridges during a twitch causes the off rate of Ca(2+) from troponin C to increase (a decrease in the Ca(2+) affinity of troponin C), thus decreasing the Ca(2+) sensitivity and resulting in the deactivation of the muscle. The results also suggest that the Fenn effect only exists at maximal but not submaximal force-activating Ca(2+) concentrations.     

Different functional properties of troponin T mutants that cause dilated cardiomyopathy

The effects of Troponin T (TnT) mutants R141W and DeltaK210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca2+ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-DeltaK210 caused a significant decrease in Ca2+ sensitivity, whereas the TnT-R141W did not result in any change in Ca2+ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-DeltaK210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-DeltaK210 mutant decreased maximal ATPase activity in the presence of Ca2+. However, the TnT-K273E mutation caused a significant increase in Ca2+ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca2+ sensitivity is not the only determinant for the phenotype of DCM.     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

Troponin I serines 43/45 and regulation of cardiac myofilament function

We studied Ca(2+) dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.     

Mutant presenilins disturb neuronal calcium homeostasis in the brain of transgenic mice, decreasing the threshold for excitotoxicity and facilitating long-term potentiation

Mutant human presenilin-1 (PS1) causes an Alzheimer's-related phenotype in the brain of transgenic mice in combination with mutant human amyloid precursor protein by means of increased production of amyloid peptides (Dewachter, I., Van Dorpe, J., Smeijers, L., Gilis, M., Kuiperi, C., Laenen, I., Caluwaerts, N., Moechars, D., Checler, F., Vanderstichele, H. & Van Leuven, F. (2000) J. Neurosci. 20, 6452-6458) that aggravate plaques and cerebrovascular amyloid (Van Dorpe, J., Smeijers, L., Dewachter, I., Nuyens, D., Spittaels, K., van den Haute, C., Mercken, M., Moechars, D., Laenen, I., Kuipéri, C., Bruynseels, K., Tesseur, I., Loos, R., Vanderstichele, H., Checler, F., Sciot, R. & Van Leuven, F. (2000) J. Am. Pathol. 157, 1283-1298). This gain of function of mutant PS1 is approached here in three paradigms that relate to glutamate neurotransmission. Mutant but not wild-type human PS1 (i) lowered the excitotoxic threshold for kainic acid in vivo, (ii) facilitated hippocampal long-term potentiation in brain slices, and (iii) increased glutamate-induced intracellular calcium levels in isolated neurons. Prominent higher calcium responses were triggered by thapsigargin and bradykinin, indicating that mutant PS modulates the dynamic release and storage of calcium ions in the endoplasmatic reticulum. In reaction to glutamate, overfilled Ca(2+) stores resulted in higher than normal cytosolic Ca(2+) levels, explaining the facilitated long-term potentiation and enhanced excitotoxicity. The lowered excitotoxic threshold for kainic acid was also observed in mice transgenic for mutant human PS2[N141I] and was prevented by dantrolene, an inhibitor of Ca(2+) release from the endoplasmic reticulum.     

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.     

Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation of force and ATPase. The dissociation constant (K(d)) of calcium green-2 for Ca(2+) was determined for three different Mg(2+) concentrations in solutions similar to those used in the experiment. Increasing Mg(2+) concentration from 1.0 to 8.0 mM had no significant effect on the Ca(2+) sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca(2+) concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca(2+) activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca(2+)-specific binding site on troponin C.