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Structural and protein interaction effects of hypertrophic and dilated cardiomyopathic mutations in alpha-tropomyosin
Audrey N Chang1, Norma J Greenfield2, Abhishek Singh3
1Department of Molecular and Cellular Pharmacology, Leonard Miller School of Medicine, University of Miami Miami, FL, USA.
Insights
Dilated and hypertrophic cardiomyopathy mutations in tropomyosin alter its structure and function. These tropomyosin (Tm) protein changes affect muscle contraction by modifying interactions with actin and myosin.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Cardiomyopathies are a group of diseases that affect the heart muscle.
- Tropomyosin (Tm) is a critical protein in muscle contraction, regulating the interaction between actin and myosin.
- Mutations in Tm are associated with inherited cardiomyopathies, including dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM).
Purpose of the Study:
- To investigate structural and functional alterations in tropomyosin (Tm) caused by specific mutations linked to DCM (E40K, E54K) and HCM (E62Q, L185R).
- To elucidate the molecular mechanisms underlying the known functional effects of these Tm mutants.
Main Methods:
- Actomyosin ATPase activity assays to measure muscle contraction dynamics.
- Spectroscopy techniques, including circular dichroism, for thermal denaturation studies.
- Molecular modeling to visualize structural changes in Tm mutants.
Main Results:
- HCM mutants and DCM mutant E54K increased Ca(2+)-induced maximal actomyosin ATPase activity; DCM mutant E40K decreased it.
- HCM mutants showed reduced inhibition of actomyosin ATPase, while DCM mutant E40K showed enhanced inhibition.
- DCM mutants generally destabilized Tm dimers, whereas HCM mutants increased Tm stability.
- E54K demonstrated a complete loss of inhibitory function on actomyosin ATPase.
Conclusions:
- Structural changes in Tm mutants directly impact their regulatory function on actin.
- Altered Tm structure and function modify myosin ATPase rates, contributing to cardiomyopathy pathogenesis.
- These findings highlight the critical role of Tm structure in maintaining normal cardiac muscle function.
Abstract:
The potential alterations to structure and associations with thin filament proteins caused by the dilated cardiomyopathy (DCM) associated tropomyosin (Tm) mutants E40K and E54K, and the hypertrophic cardiomyopathy (HCM) associated Tm mutants E62Q and L185R, were investigated. In order to ascertain what the cause of the known functional effects may be, structural and protein-protein interaction studies were conducted utilizing actomyosin ATPase activity measurements and spectroscopy. In actomyosin ATPase measurements, both HCM mutants and the DCM mutant E54K caused increases in Ca(2+)-induced maximal ATPase activities, while E40K caused a decrease. Investigation of Tm's ability to inhibit actomyosin ATPase in the absence of troponin showed that HCM-associated mutant Tms did not inhibit as well as wildtype, whereas the DCM associated mutant E40K inhibited better. E54K did not inhibit the actomyosin ATPase activity at any concentration of Tm tested. Thermal denaturation studies by circular dichroism and molecular modeling of the mutations in Tm showed that in general, the DCM mutants caused localized destabilization of the Tm dimers, while the HCM mutants resulted in increased stability. These findings demonstrate that the structural alterations in Tm observed here may affect the regulatory function of Tm on actin, thereby directly altering the ATPase rates of myosin.
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