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

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.