The effect of mutations in alpha-tropomyosin (E40K and E54K) that cause familial dilated cardiomyopathy on the regulatory mechanism of cardiac muscle thin filaments.

E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca(2+) sensitivity. E40K also reduces V(max) of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-alpha-Tm, and the two dilated cardiomyopathy-causing mutants. AS-alpha-Tm has a lower cooperative unit size (6.5) than native alpha-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K(T) value was the same for all actin-Tm species; however, the K(T) value of actin-Tm-troponin at pCa 5 was 50% less for AS-alpha-Tm E40K than for AS-alpha-Tm and AS-alpha-Tm E54K. K(b), the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-alpha-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-alpha-Tm E54K, we observed a unique new domain at 55 degrees C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.

Dilated cardiomyopathy mutations in three thin filament regulatory proteins result in a common functional phenotype.

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. Inherited DCM can result from mutations in the genes encoding cardiac troponin T, troponin C, and alpha-tropomyosin; different mutations in the same genes cause hypertrophic cardiomyopathy. To understand how certain mutations lead specifically to DCM, we have investigated their effect on contractile function by comparing wild-type and mutant recombinant proteins. Because initial studies on two troponin T mutations have generated conflicting findings, we analyzed all eight published DCM mutations in troponin T, troponin C, and alpha-tropomyosin in a range of in vitro assays. Thin filaments, reconstituted with a 1:1 ratio of mutant/wild-type proteins (the likely in vivo ratio), all showed reduced Ca(2+) sensitivity of activation in ATPase and motility assays, and except for one alpha-tropomyosin mutant showed lower maximum Ca(2+) activation. Incorporation of either of two troponin T mutants in skinned cardiac trabeculae also decreased Ca(2+) sensitivity of force generation. Structure/function considerations imply that the diverse thin filament DCM mutations affect different aspects of regulatory function yet change contractility in a consistent manner. The DCM mutations depress myofibrillar function, an effect fundamentally opposite to that of hypertrophic cardiomyopathy-causing thin filament mutations, suggesting that decreased contractility may trigger pathways that ultimately lead to the clinical phenotype.

Functional characterisation of a mutant actin (Met132Val) from a patient with nemaline myopathy.

The mutation Met132Val in the ACTA1 gene was identified in a patient with mild nemaline myopathy (NM). We examined actin mRNA and protein from biopsy samples. Sixty-one percent of the mRNA from the biopsy was not cleaved with BstX1, indicating the presence of mutant messenger in vivo. Monomeric actin was extracted from 2.5 mg of mutant muscle and wild type muscle. A proportion of the NM actin did not polymerise in 50 mM KCl, 2.5 mM MgCl2 but all the wild-type actin did. NM actin was fully polymerised by 50 mM KCl, 2.5 mM MgCl2, 150 nM rhodamine-phalloidin. Thin filaments reconstituted with this co-polymer were different from wild-type. The NM actin produces faster sliding of thin filaments at pCa5 and higher relative isometric force. We conclude that the mutant mRNA and protein is expressed and that the mutation reduces polymerisability and alters thin filament function.

Alterations in thin filament regulation induced by a human cardiac troponin T mutant that causes dilated cardiomyopathy are distinct from those induced by troponin T mutants that cause hypertrophic cardiomyopathy.

We have compared the in vitro regulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the DeltaLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing DeltaLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase at pCa4.5. The Ca(2+) sensitivity of ATPase activation was increased (DeltapCa(50) = +0.2 pCa units) and cooperativity of Ca(2+) activation was virtually abolished. Equimolar mixtures of wild type and DeltaLys-210 troponin T gave a lower Ca(2+) sensitivity than with wild type, while maintaining the diminished ATPase activation at pCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca(2+) sensitivity was increased but there was no change in cooperativity. In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the DeltaLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli.