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We created a transgenic mouse that expressed the dilated cardiomyopathy (DCM) mutation ACTC E361G at 50% of total cardiac actin. We isolated F-actin from transgenic and non-transgenic (NTG) mice and reconstituted thin filaments using native human cardiac troponin and tropomyosin (from donor heart). In in-vitro motility assays we could observe no differences between E361G and non-transgenic mouse thin filaments; however, when troponin was fully dephosphorylated with acid phosphatase we observed that E361G Ca2+ sensitivity was lower than non-transgenic, as previously observed with the recombinant proteins (EC50 E361G/NTG 2.2±0.1). When we compared natively phosphorylated and dephosphorylated thin filaments we observed that Ca2+ sensitivity did not change in E361G mouse thin filaments (EC50 P/unP=1.0±0.1) but the Ca2+ sensitivity increased 3.0±0.3-fold on dephosphorylation of non-transgenic mice as expected. Thus the only functional change induced by the E361G mutation in cardiac actin was a blunted response to troponin I phosphorylation. We also studied troponin extracted from the explanted heart of a DCM patient carrying the cardiac TnC G159D mutation. In-vitro motility assay investigation of reconstituted thin filaments showed that the cTnC G159D mutation also showed little change in Ca2+ sensitivity when TnI was dephosphorylated (EC50 P/unP=1.2±0.2). In contrast, with donor heart control troponin, Ca2+ sensitivity was increased (EC50 P/unP=4.7±1.9). We conclude that Ca2+ sensitivity per se is not the prime determinant of familial DCM. The causative property shared by mutations in contractile proteins that cause DCM is a blunted response to changes in troponin I phosphorylation that could impair the normal response to adrenergic stimulation.
Funding This research was supported by grants from the British Heart Foundation
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