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

Sparrow, Alexander J., Sievert, Kolja, Patel, Suketu, Chang, Yu-Fen, Broyles, Connor Neil, Brook, Frances Ann, Watkins, Hugh, Geeves, Michael A., Redwood, Charles S., Robinson, Paul, and others. (2019) Measurement of Myofilament-Localised Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations. Circulation Research, 124 . pp. 1228-1239. ISSN 0009-7330. (doi:10.1161/CIRCRESAHA.118.314600)

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https://doi.org/10.1161/CIRCRESAHA.118.314600

Abstract

Rationale: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators restricted to the myofilament to directly visualise Ca2 changes in the sarcomere.

Objective: To produce and validate myofilament restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil and levosimendan) or following co-transduction of two established hypertrophic cardiomyopathy (HCM) disease causing mutants (cTnT R92Q and cTnI R145G) that alter myofilament Ca2+ handling.

Methods and Results: When expressed in adult ventricular cardiomyocytes RGECO-TnT/TnI sensors localise correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localised Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Co-adenoviral transduction of RGECO-TnT/TnI with HCM causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the two mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+.

Conclusions: RGECO-TnT/TnI are functionally equivalent. They visualise Ca2+ within the myofilament and reveal unrecognised aspects of small molecule and disease associated mutations in living cells.

Item Type: Article
DOI/Identification number: 10.1161/CIRCRESAHA.118.314600
Uncontrolled keywords: Hypertrophic cardiomyopathy, Calcium, Contractility, Genetically encoded Ca2+ indicators
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 15 Feb 2019 16:33 UTC
Last Modified: 07 Aug 2019 23:00 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/72581 (The current URI for this page, for reference purposes)
Geeves, Michael A.: https://orcid.org/0000-0002-9364-8898
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