Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

Abstract

To study the functional consequences of various cardiomyopathic mutations in human cardiac ␣-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 Ca 2؉ -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 Ca 2؉ -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 Ca 2؉ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCMassociated mutations both decreased the Ca 2؉ 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.

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

“…The pCa 50 and cooperativity values from our ATPase and ANS fluorescence assays using RTFs containing WT Tm were consistent with those of previous studies (24,33), but were different between the two assays (Tables 3 and 4). These differences in WT parameters (Fig.…”

Familial Hypertrophic Cardiomyopathy-Related Troponin Mutations and Sudden Cardiac Death

“…This finding implies that L29Q may decrease the Ca 2+ sensitivity and disrupt the signal from the phosphorylated cTnI to cTnC. However, this finding contradicts other reports of FHC mutations generally having higher myofilament Ca 2+ sensitivities (Chang et al, 2005;Karibe et al, 2001). Recent NMR and ultraviolet/visual spectrum titration studies showed that L29Q essentially has the same Ca 2+ affinity as that of wild-type cTnC (Baryshnikova et al, 2008) although this technique presents challenges for measuring Ca 2+ affinity (see below).…”

Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca2+binding and myofilament contractility

“…Several groups have postulated that myofilament Ca 2ϩ sensitivity is the underlying mechanism of these cardiomyopathies. In fact, it has been hypothesized that all FHC-associated tropomyosin and troponin mutations increase myofilament sensitivity, and, conversely, dilated cardiomyopathy-associated mutations are associated with decreased Ca 2ϩ sensitivity (8,19,20), however, these relationships remain controversial. In addition, mutations in other sarcomeric proteins, such as the regulatory myosin light chain (E22K), which are associated with FHC, have frequently been shown to increase the Ca 2ϩ sensitivity of force generation (39).…”