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Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+
1Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136, USA. dszczesna@med.miami.edu
Abstract:
The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.
Insights
Familial hypertrophic cardiomyopathy mutations in cardiac myosin light chains alter calcium binding. Some mutations disrupt calcium binding, but phosphorylation can restore it, impacting heart muscle function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Familial hypertrophic cardiomyopathy (HCM) is a genetic heart disease.
- Mutations in regulatory light chains of human cardiac myosin are implicated in HCM.
- Understanding these mutations' effects on protein function is crucial for disease mechanism insights.
Purpose of the Study:
- Investigate the impact of specific familial hypertrophic cardiomyopathy mutations on human cardiac myosin regulatory light chains.
- Characterize alterations in calcium (Ca2+) binding and phosphorylation properties.
- Determine how these molecular changes may contribute to hypertrophic cardiomyopathy.
Main Methods:
- Site-directed mutagenesis to introduce familial hypertrophic cardiomyopathy mutations (A13T, F18L, E22K, R58Q, P95A).
- Biochemical assays to measure Ca2+ binding affinity (K(Ca)) and phosphorylation.
- Circular dichroism spectroscopy to assess changes in alpha-helical content.
Main Results:
- Mutations E22K and R58Q significantly altered Ca2+ binding; R58Q lost Ca2+ binding but regained it upon phosphorylation.
- E22K could not be phosphorylated, and its Ca2+ binding affinity was reduced 17-fold.
- Mutation A13T showed reduced Ca2+ affinity, but phosphorylation increased it 6-fold, reversing the wild-type effect.
Conclusions:
- Alterations in Ca2+ binding and phosphorylation of cardiac myosin regulatory light chains due to HCM mutations can disrupt normal heart muscle function.
- Specific mutations like E22K and R58Q directly impair Ca2+ sensitivity and/or phosphorylation.
- These molecular dysregulations are potential contributors to the pathogenesis of hypertrophic cardiomyopathy.