Molecular and Physiological Effects of α-Tropomyosin Ablation in the Mouse

Abstract

Abstract-Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of ␣-TM, the murine ␣-TM gene was disrupted by homologous recombination. Homozygous ␣-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for ␣-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle ␣-TM mRNA, with no compensatory increase in transcript levels by striated muscle ␤-TM or TM-30 isoforms. Surprisingly, this reduction in ␣-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle ␣-TM protein are produced and integrated in the myofibril. Quantification of ␣-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of ␣-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous ␣-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression. (Circ Res. 1998;82:116-123.)Key Words: tropomyosin Ⅲ knockout mouse Ⅲ translational regulation T ropomyosin, an essential thin filament protein, binds to actin and the troponin complex to regulate the Ca 2ϩ -sensitive interaction of actin and myosin. TM assembles into an ␣-helical coiled-coil dimer, with each molecule interacting with six or seven actin monomers. The TMs bind to themselves in a head-to-tail manner and wrap around the actin molecule to stabilize thin filament assembly. Although the exact role of TM is still not completely understood, the TM-troponin complex inhibits the actin-myosin interaction in the resting state; with an increase of Ca 2ϩ in the myofilament space and binding of Ca 2ϩ to troponin, this inhibition is released and leads to muscle contraction.TM is encoded by a small multigene family consisting of the ␣, ␤, TM-30, and TM-4 genes. These genes, and associated proteins, exhibit a very high degree of conservation across species ranging from Drosophila to humans. For example, there is 86% amino acid conservation between the striated muscle ␣-and ␤-TM isoforms.1 Previous work conducted by our laboratory 2,3 and others 4,5 has demonstrated that TM isoforms are generated through alternative exon splicing and are regulated in a developmental and tissue-specific manner. The functional significance of the alternatively spliced TM domains and the physiological role of the TM isoforms produced by the various multigene family members remain to be determined.During murine cardiac development, ␣-and ␤-TMs are expressed at different le...

Tropomyosin and Troponin Are Required for Ovarian Contraction in theCaenorhabditis elegansReproductive System

“…In both muscle and nonmuscle cells, TM stabilizes actin filaments by protecting them from disassembly (Cooper, 2002). Null or severe lossof-function mutations of a TM gene are lethal in yeast (Balasubramanian et al, 1992;Drees et al, 1995), mice (Blanchard et al, 1997;Rethinasamy et al, 1998), and C. elegans (Williams and Waterston, 1994;Anyanful et al, 2001). Thus, TM is an important regulator of actin-dependent processes in a variety of cells.…”

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

“…1A) (Blanchard et al, 1997;Rethinasamy et al, 1998). The αTM1 +/− adults were phenotypically normal, with wild-type αTM1 protein levels in muscles and myofibrillar fractions despite a 50% reduction in αTM1 mRNA (Blanchard et al, 1997;Rethinasamy et al, 1998).…”

“…1A) (Blanchard et al, 1997;Rethinasamy et al, 1998). The αTM1 +/− adults were phenotypically normal, with wild-type αTM1 protein levels in muscles and myofibrillar fractions despite a 50% reduction in αTM1 mRNA (Blanchard et al, 1997;Rethinasamy et al, 1998). In contrast, the αTM1 −/− mice were reported to be embryonic lethal sometime between E8 and E11.5 days of gestation, due to unknown causes (Blanchard et al, 1997;Rethinasamy et al, 1998).…”

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

“…Knock-out of ␣TM in transgenic mice has shown that homozygous null mutants are embryonic lethal but that loss of a single allele produces no phenotype, probably due to compensating mechanisms that maintain the quantity of the ␣TM protein (32,33). There have been no reports of isoform-specific knockouts, although this was the intention of at least one previous investigation (33).…”

“…There have been no reports of isoform-specific knockouts, although this was the intention of at least one previous investigation (33). An interesting consequence of the titratability of some of the splicing regulators is that in the highest expressing 3URE mouse line the level of exon 2 inclusion in the aorta was reduced from the usual ϳ90% to less than 10%.…”