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

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 characterize genetically encoded Ca²⁺ indicators restricted to the myofilament to directly visualize 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 cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca²⁺ 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 Ca²⁺ on and increased Ca²⁺ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized 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. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy 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 2 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 visualize Ca²⁺ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

Keywords: calcium; cardiomyopathies; fluorescence; mutation; sarcomere.