RATIONALE

Subcellular Ca indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca sensitivity. Here, we develop and characterize genetically encoded Ca indicators restricted to the myofilament to directly visualize Ca changes in the sarcomere.

OBJECTIVE

To produce and validate myofilament-restricted Ca 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 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 on and increased Ca off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca handling conferred by MYK-461 and levosimendan, including an increase in Ca 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] in systole, lengthen time to peak systolic [Ca], and delay [Ca] release. This contrasts with the effect of the same mutations on cytoplasmic Ca, when measured using unrestricted RGECO where changes to peak systolic Ca are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca] transient amplitude or time to peak Ca.

CONCLUSIONS

RGECO-TnT/TnI are functionally equivalent. They visualize Ca within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.