Rescue of High Expression β-Tropomyosin Transgenic Mice by 5-Propyl-2-thiouracil: REGULATING THE α-MYOSIN HEAVY CHAIN PROMOTER

Rethinasamy Prabhakar,Greg P Boivin,Brian Hoit,David F Wieczorek

doi:10.1074/jbc.274.41.29558

Rethinasamy Prabhakar, Greg P Boivin + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.41.29558

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Abstract

Tropomyosin is an essential component of the sarcomeric thin filament in striated muscle that participates in the regulation of muscle contraction through Ca(2+)-mediated activation. The two predominant tropomyosin isoforms expressed in striated muscle are alpha- and beta-tropomyosin, which exhibit an 86% amino acid identity between themselves. Previous studies by our laboratory utilized a transgenic mouse system to overexpress beta-tropomyosin in the heart to address the functional differences between these two tropomyosin isoforms. Interestingly, when a high percentage of beta-tropomyosin replaces alpha-tropomyosin in the hearts of transgenic mice, the mice die due to severe cardiac abnormalities. In this study, we have rescued these high expression beta-tropomyosin mice by turning off the alpha-myosin heavy chain promoter, which is driving the beta-tropomyosin transgene. This down-regulation of the alpha-myosin heavy chain promoter was accomplished by the administration of 5-propyl-2-thiouracil, which disrupts thyroid hormone synthesis and inhibits promoter activity through thyroid regulatory elements located in the 5'-flanking region of the promoter. Results show that as beta-tropomyosin expression is down-regulated, alpha-tropomyosin expression is increased. Also, alpha- and beta-myosin heavy chain expression is modified in response to the changes in thyroid hormone expression. Morphological analysis of these rescued mice show a moderate pathological phenotype, characterized by atrial myocytolysis; echocardiographic analyses demonstrate altered ventricular functions, such as peak filling rates and left ventricular fractional shortening. This is the first report demonstrating that transcriptional regulatory elements located within the alpha-myosin heavy chain promoter can be manipulated to rescue potentially lethal phenotypes, such as high expression beta-tropomyosin transgenic mice.

Highlights

Tropomyosin is an essential component of the sarcomeric thin filament in striated muscle that participates in the regulation of muscle contraction through Ca2؉mediated activation
When a high percentage of ␤-tropomyosin replaces ␣-tropomyosin in the hearts of transgenic mice, the mice die due to severe cardiac abnormalities. We have rescued these high expression ␤-tropomyosin mice by turning off the ␣-myosin heavy chain promoter, which is driving the ␤-tropomyosin transgene. This down-regulation of the ␣-myosin heavy chain promoter was accomplished by the administration of 5-propyl-2-thiouracil, which disrupts thyroid hormone synthesis and inhibits promoter activity through thyroid regulatory elements located in the 5؅-flanking region of the promoter
There was no significant difference in fractional shortening between wild type and moderate TG mice when on PTU (45.9 Ϯ 1.2 versus 38.9 Ϯ 3.4); there was a difference in fractional shortening (p Ͻ 0.05) between wild type and moderate TG mice on the switched diet (46.8 Ϯ 1.5 versus 33.9 Ϯ 2.5)

Summary

EXPERIMENTAL PROCEDURES

Animals—The generation of ␤-TM overexpression transgenic mice was described previously [3]. The hypothyroid condition was induced by feeding a diet containing 0.15% PTU (Teklad Premier) to pregnant female ␤-TM TG mice (15 days postcoitus) who were mated with male ␤-TM TG mice. Purified DNA (10 ␮g) was digested with EcoRI, Southern blotted to nylon membrane, and hybridized with a radiolabeled probe corresponding to the SV40 3Ј-untranslated region. Several blots were prepared and hybridized with radiolabeled isoformspecific probes for striated muscle-specific ␣- and ␤-TM, ␣- and ␤-MHC, and glyceraldehyde-3-phosphate dehydrogenase. Two-dimensionally targeted M-mode studies were performed with a 10-MHz intraoperative scan head imaging transducer (ATL HDI 3000) using methods previously described [8]. This transducer has a small offset footprint and has outstanding near field imaging quality. Peak filling rates were measured off line from digitized M-mode using commercial software (Tomtec)

RESULTS

TABLE I Cardiac performance in wild type and TG mice

Histological and Physiological Analyses of the Rescued High Copy Transgenic Mice

DISCUSSION