To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Tropomyosin (TM), a component of the thin filament of the sarcomere, is encoded by a four-member multigene family: alpha-TM, beta-TM, TPM 3, and TPM 4. The alpha-TM, beta-TM, and TPM 3 genes each utilize an alternative splicing mechanism to encode a striated muscle isoform. Although the alpha-TM and beta-TM striated muscle isoforms are well characterized, relatively little is known about the TPM 3 isoform. We cloned and sequenced the murine TPM 3 cDNA and found that it exhibits a 93% nucleotide homology and 99% amino acid homology to the human sequence. Results show that, unlike humans, TPM 3 is not expressed in any developmental stage of murine hearts. TPM 3 message is expressed in slow-twitch skeletal muscle but is not found in representative fast-twitch musculature. The soleus, a representative slow-twitch muscle, expresses transcript levels of 65% beta-TM, 15% alpha-TM, and 20% TPM 3, but the TPM 3 protein accounts for approximately 31% of the total striated tropomyosin in slow-twitch muscle. In fast-twitch muscle, alpha-TM comprises 71% of the total striated muscle TM protein, and beta-TM comprises 29%. The results demonstrate that a translational mechanism regulates the production of the TM proteins, with beta-TM message not being efficiently translated. The unique distribution pattern of TPM 3 adds to the diversity of the tropomyosin family and strongly suggests functional significance for the different striated muscle TM isoforms.

Tropomyosin (TM), an integral component of the thin filament, is encoded by three striated muscle isoforms: alpha-TM, beta-TM, and TPM 3. Although the alpha-TM and beta-TM isoforms are well characterized, less is known about the function of the TPM 3 isoform, which is predominantly found in the slow-twitch musculature of mammals. To determine its functional significance, we ectopically expressed this isoform in the hearts of transgenic mice. We generated six transgenic mouse lines that produce varying levels of TPM 3 message with ectopic TPM 3 protein accounting for 40-60% of the total striated muscle tropomyosin. The transgenic mice have normal life spans and exhibit no morphological abnormalities in their sarcomeres or hearts. However, there are significant functional alterations in cardiac performance. Physiological assessment of these mice by using closed-chest analyses and a work-performing model reveals a hyperdynamic effect on systolic and diastolic function. Analysis of detergent-extracted fiber bundles demonstrates a decreased sensitivity to Ca(2+) in force generation and a decrease in length-dependent Ca(2+) activation with no detectable change in interfilament spacing as determined by using X-ray diffraction. Our data are the first to demonstrate that TM isoforms can affect sarcomeric performance by decreasing sensitivity to Ca(2+) and influencing the length-dependent Ca(2+) activation.

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