Forced expression and assembly of rat cardiac troponin T isoforms in cultured muscle and nonmuscle cells

Summary Cardiac troponin T (cTnT), a tropomyosin (TM)-binding subunit of the troponin complex, undergoes a developmentally regulated isoform switch from embryonic form to adult form in the rat heart. To investigate the in vivo assembly of cTnT isoforms, we transiently transfected cDNA clones of either rat cTnT isoform into nonmuscle CHO cells and chick embryo myogenic (CEM) cells. As determined by Western blotting, both isoforms can be expressed in CHO and CEM cells. The expressed proteins had the same mobility as native rat cTnT proteins on SDS polyacrylamide gels and were recognized by anti-TnT antibodies. Conventional and confocal microscopy of transfected cells, double-labelled with antibodies against cTnT and against TM, revealed that neither isoform appears to associate with the nonmuscle TM in CHO cells, although both are able to colocalize with muscle TM-containing microfilament bundles in the myogenic CEM cells. There was no appreciable cTnT isoform-related difference in association with TM, suggesting that the functional significance of isoform variability in rat cTnT does not correspond to an assembly advantage for the maturing cardiac thin filament. To help determine whether cTnT nonassembly in CHO environment is primarily due to the nonmuscle nature of the endogenous TM, or if it involves the absence of other factors specific to muscle, we have isolated several stably-transfected clones of skeletal ßTM-expressing CHO cells which incorporate this muscle TM onto stress fibres. When either isoform of cTnT was transiently expressed in these ßTM-CHO cells, the strictly filamentous ßTM staining pattern was no longer observed. Instead, ßTM codistributed with cTnT in brightly staining aggregates not associated with the intact stress fibres. This suggests that both isoforms of cTnT are interacting with the ßTM in the nonmuscle environment and that other muscle-specific proteins may indeed be required for stable assembly of cTnT onto microfilaments. It also suggests that the interaction between cTnT and muscle TM is stronger than that between muscle TM and nonmuscle microfilaments.