Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

Autor:	Sparrow AJ; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Sievert K; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Patel S; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Chang YF; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Broyles CN; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Brook FA; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Watkins H; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; Department of Cardiology, Oxford University NHS Hospitals Trust, United Kingdom (H.W., M.J.D.)., Geeves MA; Department of Biosciences, University of Kent, Canterbury, United Kingdom (M.A.G.)., Redwood CS; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Robinson P; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom., Daniels MJ; From the Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.J.S., K.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Research Excellence (A.J.S., S.P., Y.-F.C., C.N.B., F.A.B., H.W., C.S.R., P.R., M.J.D.), University of Oxford, United Kingdom.; BHF Centre of Regenerative Medicine (M.J.D.), University of Oxford, United Kingdom.; Department of Cardiology, Oxford University NHS Hospitals Trust, United Kingdom (H.W., M.J.D.).; Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan (M.J.D.).
Jazyk:	angličtina
Zdroj:	Circulation research [Circ Res] 2019 Apr 12; Vol. 124 (8), pp. 1228-1239.
DOI:	10.1161/CIRCRESAHA.118.314600
Abstrakt:	Rationale: Subcellular Ca 2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca 2+ sensitivity. Here, we develop and characterize genetically encoded Ca 2+ indicators restricted to the myofilament to directly visualize Ca 2+ changes in the sarcomere. Objective: To produce and validate myofilament-restricted Ca 2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca 2+ handling. Methods and Results: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca 2+ on and increased Ca 2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca 2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca 2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca 2+ ] in systole, lengthen time to peak systolic [Ca 2+ ], and delay [Ca 2+ ] release. This contrasts with the effect of the same mutations on cytoplasmic Ca 2+ , when measured using unrestricted RGECO where changes to peak systolic Ca 2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca 2+ ] transient amplitude or time to peak Ca 2+ . Conclusions: RGECO-TnT/TnI are functionally equivalent. They visualize Ca 2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.
Databáze:	MEDLINE
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