Molecular and Physiological Effects of Overexpressing Striated Muscle β-Tropomyosin in the Adult Murine Heart

Abstract

Tropomyosins comprise a family of actin-binding proteins that are central to the control of calcium-regulated striated muscle contraction. To understand the functional role of tropomyosin isoform differences in cardiac muscle, we generated transgenic mice that overexpress striated muscle-specific ␤-tropomyosin in the adult heart. Nine transgenic lines show a 150-fold increase in ␤-tropomyosin mRNA expression in the heart, along with a 34-fold increase in the associated protein. This increase in ␤-tropomyosin message and protein causes a concomitant decrease in the level of ␣-tropomyosin transcripts and their associated protein. There is a preferential formation of the ␣␤-heterodimer in the transgenic mouse myofibrils, and there are no detectable alterations in the expression of other contractile protein genes, including the endogenous ␤-tropomyosin isoform. When expression from the ␤-tropomyosin transgene is terminated, ␣-tropomyosin expression returns to normal levels. No structural changes were observed in these transgenic hearts nor in the associated sarcomeres. Interestingly, physiological analyses of these hearts using a work-performing model reveal a significant effect on diastolic function. As such, this study demonstrates that a coordinate regulatory mechanism exists between ␣-and ␤-tropomyosin gene expression in the murine heart, which results in a functional correlation between ␣-and ␤-tropomyosin isoform content and cardiac performance.

Tropomyosin Dephosphorylation Results in Compensated Cardiac Hypertrophy

“…Cardiac ␣-Tm S283A Protein Expression and Phosphorylation in Transgenic Mice-Often, mutations in Tm isoforms lead to differential migration on SDS-PAGE gels (16,18,22,28). However, because serine is only 16 daltons larger than alanine and has nearly an identical isoelectric point, expression levels of TG and endogenous protein cannot be separated using traditional methods.…”

“…Transgenic Protein Quantification and Western Blot AnalysesMyofibrillar proteins were extracted from NTG and TG male mouse ventricles as previously described (22). 30 g of the myofibrillar protein preparations were separated on a 10% SDS-PAGE gel.…”

“…The ␣-Tm S283A construct was then cloned into a vector containing the ␣-myosin heavy chain (␣-MHC) promoter and a human growth hormone 3Ј-UTR and poly-A tail sequence (21). Transgenic mice were generated using the FVB/N strain as previously described (22). Founder mice were identified using PCR.…”

The Insulin-like Growth Factor 1 Receptor Induces Physiological Heart Growth via the Phosphoinositide 3-Kinase(p110α) Pathway

“…The striated muscle form of TPM3 is expressed in the adult human heart (64), and cardiac overexpression of TPM3 in transgenic mice leads to hypercontractility (65). By contrast, TPM2 is usually expressed at low levels during postnatal life (66), and transgenic mice with low overexpression of TPM2 displayed impairment of diastolic function, which was linked to impaired myocyte relaxation and decreased maximal tension in isolated preparations (67,68). Higher overexpression resulted in severe cardiac pathology including dilatation, myolysis, fibrosis, decreased contractility, formation of thrombi, and lethality in early postnatal life (69).…”

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

“…The ␣-TM-D137L cDNA was cloned into a vector (18), which contained the cardiac specific ␣-myosin heavy chain promoter and the human growth hormone poly(A) signal sequence. The transgene construct was purified to generate TG mice (FVB/N strain) as described (19). Founder mice were identified by PCR, and the mutation in the TG lines was verified by nucleotide sequencing of TG mouse genomic DNA.…”