The Influence of Troponin C, Isoform 4 on Drosophila Development, Stretch Activation, and Power Generation

Abstract

Drosophila indirect flight muscle (IFM) is one of the most rapidly contracting muscle types known. To increase IFM efficiently and power generation many insect species have evolved high degrees of stretch activation and shortening deactivation in their IFMs. In addition to expressing TnC1, a typical calcium binding troponin C, these IFMs express an unusual unique, additional isoform, TnC4. TnC1 has two calcium binding sites, a high-affinity structural site and a lower-affinity regulatory site. TnC4 has only the high-affinity structural binding site. It is hypothesized to respond to stretch rather than calcium concentration to further activate the thin filament. We are investigating the roles of TnC4 and TnC1 in Drosophila using RNAi and by creating a TnC4 null mutant. Using RNAi, we eliminated TnC1 expression, which resulted in upregulation of TnC4. Power generation and stretch activation of IFMs without TnC1 were statistically identical to wild-type. IFMs with undetectable levels of TnC4 expression produced by RNAi do not generate power or display stretch-activation, suggesting that only TnC4 is necessary for normal muscle function. We have also created a TnC4 null, which will allow us to mechanically evaluate transgenically expressed TnC isoforms and mutants in IFM. Preliminary characterization of the TnC4 null indicates that it is homozygous lethal, but viable as a heterozygote. Flight ability of the heterozygotes is reduced, displaying a 10% reduction in wing-beat frequency than wild-type. The homozygous nulls die at the first molt suggesting that TnC4 is also expressed in some larval muscle types and that it plays a vital role in fly development.