To evaluate nebulette's role in cardiac myofibrils, cardiomyocytes expressing green fluorescent protein (GFP)-nebulette constructs were monitored for their ability to contract and myofilament protein distribution was analyzed. Cells expressing full-length GFP-nebulette appear unaffected and exhibit normal beating frequencies. Expression of the GFP linker and SH3 results in loss of the endogenous nebulette and tropomyosin; however, Z-line and thick filaments are undisturbed. Cells expressing either of these domains have dramatically reduced beating frequencies, consistent with the loss of thin filament proteins. This loss was inhibited by the addition of protease inhibitors during culturing. The GFP repeat domain disrupts both myofibrillogenesis and contraction in spreading cardiomyocytes, whereas introduction of this protein into well-spread cardiomyocytes results in localization at the Z-line and a 50% reduction in beating frequency. Ultimately, these cells form bundles containing the GFP repeat and many myofilament proteins. Interestingly, butanedione monoxime inhibition of contraction inhibited the formation of these bundles. These results show that the GFP-nebulette domains have a dominant-negative effect on the distribution and function of the sarcomeric proteins. Taken together with the observation that nebulette colocalizes with a-actinin in the pre-, nascent, and mature myofibrils, our data demonstrate the importance of this cardiac-specific nebulin isoform in myofibril organization and function.
ASJC Scopus subject areas
- Cell Biology