Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations

Journal article

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.

Authors: Sparrow, A; Sievert, K; Patel, S; Chang, Y; Broyles, C

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Heart Association
• Journal: Circulation Research
• Volume: 124
• Issue: 8
• Pages: 1228–1239
• Publication date: 2019-02-08
• Acceptance date: 2019-02-05
• DOI: 10.1161/circresaha.118.314600
• EISSN: 1524-4571
• ISSN: 0009-7330
• Language: English
• Keywords: calcium; genetically encoded Ca2+ indicators; mechanisms; excitationcontraction coupling; hypertrophic cardiomyopathy; fluorescent imaging; contractility