Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Abstract

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse ␣-tropomyosin cDNA under the control of a cardiac-specific ␣-myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well-organized sarcomeric myofibrils. Transfection of a ␤-tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a ␤-galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of ␣-tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412-420.1998 Wiley-Liss, Inc.

Tropomyosin is required for cardiac morphogenesis, myofibril assembly, and formation of adherens junctions in the developing mouse embryo

“…Similar observations demonstrating essential functions for troponins in sarcomere formation, as well as exacerbation of defects by unregulated actomyosin activity during myofibril assembly, have been made in studies of zebrafish striated muscles (Sehnert et al, 2002;Huang et al, 2009;Ferrante et al, 2011). The primary direct evidence for a role of TM in myofibril assembly comes from the Mexican axolotl, in which the naturally occurring cardiac mutation eliminates TM expression and disrupts cardiac myofibril assembly via aberrant intracellular targeting of a host of thin filament and sarcomere-associated proteins and lack of coalescence of organized sarcomeres (Lemanski, 1973;Lemanski, 1979;Lemanski et al, 1980;Fuldner et al, 1984;Starr et al, 1989;Erginel-Unaltuna and Lemanski, 1994;La France and Lemanski, 1994;Zajdel et al, 1998;Zajdel et al, 1999;McLean et al, 2006;Zajdel et al, 2007). A heart-specific TM isoform (tpm4) is also essential for myofibril assembly and heartbeat in zebrafish (Zhao et al, 2008), but the lack of confirmed association of tpm4 with thin filaments makes it unclear whether the myofibril assembly defects observed in tpm4-deficient zebrafish represent primary or indirect events.…”

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

“…Their studies using the peptide model system also show that a peptide from human cTnT, hcTn70-170, promotes the affinity of striated muscle tropomyosin for actin up to 15 folds [Palm et al, 2003]. Tropomyosin expression has been shown before to be dramatically decreased in the gene c mutant animals [LaFrance and Zajdel et al, 1998;Zhang et al, 2003]. Decreased expression of cTnT protein in the mutant hearts could possibly be the reason for the failure of incorporation of tropomyosin protein to the myofibril structure, thus abnormally high degradation of tropomyosin protein in the mutant hearts may occur.…”

“…Alternatively, myocytes from the different regions could be under different transcriptional or translational control. Interestingly, it has been reported that the mutant conus region also contains significant amounts of tropomyosin although the ventricular areas are virtually devoid of this protein [Zajdel et al, 1998]. Very likely, cTnT isoforms are partially regulated by common factors shared with tropomyosin, which influences their expression or RNA processing in the mutant condition.…”

“…Among myofibril structural proteins, tropomyosin has been shown by SDS-Page [Lemanski et al, 1979], radio-immunoassay [Moore and Lemanski, 1982], 2-D gel electrophoresis [Starr et al, 1989] and whole heart confocal microscopic analysis to be drastically reduced in mutant hearts [LaFrance and Zajdel et al, 1998;Zhang et al, 2003]. Interestingly, actin, myosin, titin, and troponin proteins were found to be at near normal levels with SDS gel electrophoresis analyses [Lemanski, 1976b;Fuldner et al, 1984;Erginel-Unaltuna and Lemanski, 1994].…”

Reduction of caldesmon expression induces apoptosis and causes disassembly of the sarcomeric protein complex in cardiomyocytes in vivo

“…Actin and tropomyosin are essential components of the thin filaments. 8,9 Alpha-actinin is an actin-binding protein and one of the major structural elements of the Z-disc, interacting with tropomyosin. 10 All of these proteins are mutually bound as a complex and reduced expression of one of them causes instability or degradation of the others.…”