Molecular and Physiological Effects of -Tropomyosin Ablation in the Mouse

From the Department of Molecular Genetics, Biochemistry, and Microbiology (P.R., D.F.W.) and the Department of Pathology and Laboratory Medicine (G.B.), University of Cincinnati (Ohio) Medical Center; the Department of Medical Physiology (M.M.), Texas A&M University Health Science Center, College Station; the Division of Molecular Cardiovascular Biology (T.H.), Children's Hospital Medical Center, Cincinnati, Ohio; and the Department of Physiology and Biophysics (B.M.W., C.E., R.J.S.), University of Illinois at Chicago.

Correspondence to Dr David F. Wieczorek, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Medical Center, Cincinnati, OH 45267-0524. E-mail wieczodf{at}ucbeh.san.uc.edu

	Abstract

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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 ß-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.

Key Words: tropomyosin • knockout mouse • translational regulation

	Introduction

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Tropomyosin, an essential thin filament protein, binds to actin and the troponin complex to regulate the Ca²⁺-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 Ca²⁺ in the myofilament space and binding of Ca²⁺ to troponin, this inhibition is released and leads to muscle contraction.

TM is encoded by a small multigene family consisting of the , ß, 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 ß-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 ß-TMs are expressed at different levels.^{3 6} During fetal development, -TM represents 80% of the total TM in the heart, and 20% is ß-TM. In the adult heart, ß-TM represents 2% of TM expression, with -TM representing 98%.³ However, during pressure-overload hypertrophy, ß-TM is reexpressed in the heart.⁷ The long-term goal of our laboratory is to understand the role of different TM isoforms in muscle contraction. We have chosen a transgenic mouse approach to address this research area. We recently created a transgenic mouse model that overexpresses ß-TM specifically in the heart. These studies show that an increased overexpression of ß-TM results in hearts that contain 45% -TM and 55% ß-TM in their myofibrils; however, there is no change in the total amount of TM protein (+ß-TM) that is produced in these transgenic mouse hearts.⁶ Physiological analyses using a work-performing heart model reveal that diastolic function is significantly altered in these mice by a decreased rate of relaxation, coupled with a delay in the time of relaxation. A possible explanation for this altered diastolic function is that the cardiac myofilaments demonstrate an increase in Ca²⁺ sensitivity of steady-state force.⁸

In the present study, we have addressed whether underexpression of -TM mRNA would alter -TM protein levels and cardiac function. To underexpress -TM transcripts, we ablated the -TM striated muscle–specific exons in murine ES cells by homologous recombination and substitution with an HPRT minigene cassette. Results from heterozygous (+/-) knockout mice show there is a 50% reduction in -TM mRNA in the targeted mouse hearts. There is no compensation at the transcript level for decreased -TM expression by either ß-TM or the slow-twitch -TM isoform (TM-30). Interestingly, the -TM protein level in cardiac myofibrils is not reduced in the heterozygous mutant mice, nor are there functional differences in cardiac performance. Results suggest that normal protein levels are maintained in these heterozygous mice through an increased translatability of -TM transcripts.

	Materials and Methods

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Murine -TMstr Targeting Construct
A targeting vector was designed to replace a 4.4-kb -TM genomic fragment containing the striated muscle–specific exons 12 and 13 with a 2.9-kb HPRT minigene cassette cloned in reverse orientation to the -TM transcription (Fig 1). The rationale behind this targeting scheme incorporated two distinct possibilities: (1) selective removal of only -TMstr or (2) complete ablation of all -TM transcripts. A 2-kb herpes simplex virus–thymidine kinase cassette used as a negative selection marker was cloned downstream from the long homology arm. The 13.6-kb construct was linearized by NotI endonuclease digestion for ES cell electroporation.

View larger version (16K): [in this window] [in a new window]   Figure 1. Genomic map and targeting strategy for the -TM locus. The genomic map of the murine -TM locus is shown at the top. Black boxes represent exons, which are numbered sequentially for the gene. Endonuclease restriction sites are as designated. The locations of the probes and expected wild-type genomic fragment sizes (6 and 9 kb) after endonuclease digestion are illustrated (see Fig 2). The targeting construct used for homologous recombination for exons 12 and 13 of -TM is shown (middle diagram), including the location of the HPRT and tk cassettes. A successfully targeted -TM locus is illustrated at the bottom. Expected genomic fragment sizes (4 and 4.6 kb) after endonuclease restriction in the targeted locus are illustrated.

Generation of -TMstr Mutant Mice
Five nanomoles of the linearized construct was electroporated into 2x10⁷ E14TG2a^(HPRT-) cells. The ES cells were subsequently cultured in the presence of HAT (120 µmol/L hypoxanthine/0.4 µmol/L aminopterin/20 µmol/L thymidine) and ganciclovir (Sigma Chemical Co). Among the 153 positive ES cell clones, homologous recombination was confirmed in 22 ES cell colonies by Southern blot analysis. Three different ES cell clones were selected for blastocyst injection, and successful germ-line transmission was confirmed in one line; chimeric males were bred to Black Swiss females to establish the colony.

Southern Blot Analysis
Genomic DNA from tails and embryos was extracted by overnight lysis at 60°C. Ten micrograms of the genomic DNA was restricted, Southern-blotted, and hybridized with ³²P-radiolabeled -TM probes. The 5' probe consists of the XbaI-SphI fragment surrounding exon 7, and the 3' probe consists of the XhoI-EcoRI fragment located downstream from exon 13. The blots were washed with 0.5x SSC and 0.1% SDS at 65°C and exposed to Kodak x-ray film.

S1 Nuclease Mapping Analysis
Total RNA was isolated from hearts and skeletal muscle of wild-type and heterozygous mice and subjected to S1 nuclease mapping analysis as described.^{2 6} Twenty micrograms of total RNA was hybridized to single-stranded TM and GAPDH DNA probes (4x10⁴ cpm). The murine GAPDH probe was used as an internal control for the reactions and levels of transcripts. Both TM and GAPDH probes were in DNA excess and were subject to S1 analyses in the same reaction tube. The protected fragments were quantified by comparison of the intensities of the TM fragments with the corresponding GAPDH fragment using a PhosphorImager (Molecular Dynamics).

Western Blot Analysis
Total extract and myofibrillar protein fractions were prepared from hearts of wild-type and heterozygous mice as described.⁹ Predetermined concentrations of myofibrillar protein was electrophoresed on 10% SDS-polyacrylamide gels and either stained with Coomassie blue or transferred to nitrocellulose membrane for Western blotting with a monoclonal TM antibody (Sigma).

Quantification of -TM mRNA Bound to Polysomes
The polysome profile analysis was performed according to established procedures.¹⁰ Wild-type and heterozygous hearts were homogenized in 10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L KCl, 10 mmol/L MgCl₂, and 1 mmol/L dithiothreitol. The homogenate was centrifuged at 13 000g for 30 minutes to remove the mitochondria. Ten absorbance units (OD₂₆₀) of the postmitochondrial supernatant were layered onto 12.5 mL of 10% to 45% (wt/vol) sucrose density gradient in the above buffer prepared in a Bio-Rad density gradient maker. The gradients were centrifuged in a Beckman SW40 Ti rotor at 38 000 rpm for 100 minutes at 4°C. Fractions (0.5 mL) were collected by upward displacement, and the OD₂₅₄ was measured and plotted. RNA isolated from the fractions was slot-blotted onto nitrocellulose membrane and hybridized with random primer–labeled ([³²P]dCTP) -TMstr cDNA probe and washed under stringent conditions.

Physiological Analyses
A functional analysis of the heart was ascertained by using the work-performing heart model. The working heart preparations were performed as described previously.^{11 12} Seven wild-type and six heterozygous -TM knockout mice were age- and sex-matched for the analysis. To compare to what extent the hearts could be loaded, cardiac work was varied from 100 to 600 mm HgxmL/min. Mean±SE values were calculated for heart rate, intraventricular pressure, and rates of contraction and relaxation.

Force measurements in bundles of detergent-extracted fibers were measured by a modification of previously described methods.⁸ Adult mice were anesthetized with pentobarbital sodium (50 mg/kg body wt IP), and hearts were quickly removed and put into cold HR solution of the following composition (mmol/L): EGTA 10, MOPS 20, free Mg²⁺ 1, MgATP^2- 5, and creatine phosphate 12, along with 10 IU/mL creatine phosphokinase. The pH of the solution was adjusted to 7.0 with KOH. The ionic strength of all solutions was 150 mmol/L. The papillary muscles from the left ventricle were dissected, and small fiber bundles 150 to 20 µm in width and 4 to 5 mm long were prepared. Fiber bundles were mounted between a micromanipulator and a force transducer with cellulose-acetate glue. Fibers were skinned in the HR solution containing 1% Triton X-100 for 30 minutes. A sarcomere length of 2.0 µm was established from laser diffraction patterns. Isometric tension was recorded on a chart recorder. After they were skinned, the fibers were initially washed in HR solution and then sequentially bathed in LR solution, followed by solutions of varying pCa values (pCa range, from 8.0 to 4.5). The ionic composition of all solutions was computed using a computer program. Compared with HR solution, LR solution contained 0.1 mmol/L EGTA. All solutions also contained the protease inhibitors pepstatin A (2.5 µg/mL), leupeptin (1 µg/mL), and phenylmethylsulfonyl fluoride (50 µmol/L).

	Results

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Targeting of the -TM Genomic Locus
To target the -TM gene, we have isolated the murine TM locus, which spans 24 kb of DNA and contains several alternatively spliced exons (Fig 1).^{4 13} The targeting construct was designed to selectively remove exons 12 and 13, the striated muscle–specific exons that encode amino acids 258 to 284 (exon 12) and the 3' untranslated region (exon 13). The targeting strategy was designed to produce either an -TM striated muscle isoform–specific knockout or a complete ablation of the -TM gene. Fig 1 illustrates the -TM locus, the gene-targeting construct, and the targeted allele after homologous recombination and insertion of the HPRT minigene into the -TM locus. The HPRT minigene cassette was cloned in reverse orientation to the -TM transcription unit; a tk cassette was inserted at the end of the targeting construct to allow for negative selection.

To obtain targeted ES cells, the linearized -TMstr construct was electroporated into E14TG2a^(HPRT-) ES cells. With HAT media used as a positive selection system and ganciclovir as a negative selection agent, 153 ES cell clones were selected. Genomic Southern blot analyses of DNA from ES cell clones were performed to detect the targeting event. The 5' probe consisted of the XbaI-SphI fragment surrounding exon 7, and the 3' probe was the XhoI-EcoRI fragment downstream from exon 13. Hybridization with the 5' probe on HindIII-XhoI–digested DNA from correctly targeted ES cell clones detected 6-kb (wild-type allele) and 4-kb (targeted allele) bands (Fig 2A). Hybridization with the 3' probe detected 9-kb (wild-type allele) and 4.6-kb (targeted allele) bands (Fig 2A). The correct targeting event was confirmed in 22 clones.

View larger version (30K): [in this window] [in a new window]   Figure 2. Genomic Southern blot analysis of wild-type and -TM targeted ES cell and mouse DNA. A, Genomic DNA from wild-type and targeted ES cells was restricted with endonucleases as designated: DNA in the HindIII-XhoI blot was hybridized with probe 1 (see Fig 1). The 6-kb band is the wild-type fragment, and the 4-kb band is the targeted fragment. The DNA in the ScaI–digested panel was hybridized with radiolabeled probe 2 (see Fig 1); the 9-kb band is the wild-type fragment, and the 4.6-kb band is the targeted fragment. B, Southern blot analysis of murine genomic DNA was restricted as designated: DNA in the HindIII-XhoI blot was hybridized with probe 1 (see above). DNA restricted with ScaI was hybridized with probe 2 (see above). C, Southern blot analysis of genomic DNA from homozygous null and heterozygous targeted -TM murine embryos (day 8.5 pc) is shown. DNA was restricted with ScaI and hybridized with probe 2 (see above).

Generation of -TM Knockout Mice
Three correctly targeted ES cell clones were selected for blastocyst-mediated transgenesis. All three clones produced chimeric mice, and successful germ-line transmission was obtained in one line. Southern blot analyses of genomic tail DNA from wild-type and heterozygous knockout mice are shown in Fig 2B. The probes used in these analyses are similar to those shown in the ES cell targeting experiment (see above).

Characterization of Transgenic Mice
Heterozygous -TM mice were distinguished from their littermate controls by genomic Southern blot analyses. The hearts were obtained from heterozygous (+/-) and control mice and examined at both the histological and morphological levels. We did not detect any obvious differences in cardiac morphology between the two groups (data not shown). In addition, these heterozygous mice live a full lifespan and reproduce well.

The generations of homozygous mice through matings from heterozygous crosses do not produce viable offspring. To determine when embryonic and/or fetal loss was occurring, litters from timed pregnant females were analyzed at different gestational periods. When embryos from 8 to 11.5 days pc were isolated, there was an unusually high number of resorptions. Embryos were individually collected (free from contaminating maternal tissue), and genomic DNA was isolated and examined by Southern blot analysis. Results show that the resorbed embryos were homozygous for the targeted -TM loci (Fig 2C). Thus, homozygous null embryos die in utero between day 8 to 11.5 pc, which correlates with the time of formation of the embryonic heart tube. These results demonstrate that -TM protein is essential for embryonic viability.

Determination of TM mRNA Levels in -TM (+/-) Mice
We have previously shown that the striated muscle -TM isoform constitutes 98% of the total TM message in the heart.^{3 6} To determine the level of -TMstr transcripts in heart and skeletal muscle of the -TM heterozygous knockout mice, we performed S1 nuclease mapping analyses. Total RNA, isolated from hearts and skeletal muscle of both heterozygous and wild-type mice, was hybridized with an -TMstr cDNA probe and a GAPDH probe (Fig 3A). The amount of -TMstr message in the heterozygous striated muscle was quantified (using PhosphorImager analyses) by comparing the densities of the protected bands obtained with a 363-bp probe specific for -TMstr with a normalizing control GAPDH probe. Results from several quantitative PhosphorImager analyses using different striated muscle RNA samples show a 52±4.8% reduction in the -TMstr mRNA levels in cardiac and skeletal muscles of heterozygous mice compared with wild-type control mice.

View larger version (41K): [in this window] [in a new window]   Figure 3. S1 Nuclease mapping analysis of wild-type and -TM heterozygous knockout mRNA. A, Single-stranded striated muscle–specific -TM and GAPDH cDNA probes were hybridized in the same reaction to mRNAs from wild-type and -TM heterozygous knockout mice. Lanes are as follows: 1, 3, and 5, wild-type heart, skeletal muscle, and smooth muscle (used as a negative control) mRNAs, respectively; 2 and 4, -TM heterozygous heart and skeletal muscle mRNAs, respectively; 6, tRNA; 7, probes; and 8, size markers. B, Single-stranded striated muscle–specific ß-TM and GAPDH cDNA probes hybridized to the mRNAs from wild-type and heterozygous knockout mice are shown. The mRNA/sample lane order is identical to that in panel A.

In addition to -TM, ß-TM is expressed in the heart during murine embryogenesis and in response to pressure-overload hypertrophy in the adult animal.^{3 7} To determine whether loss of -TM is compensated by ß-TM or by other TM isoforms in the heterozygous knockout mice, we performed S1 nuclease mapping analyses using a ß-TM probe. Results show that the expression of ß-TM levels in heterozygous hearts is similar to that in wild-type hearts (Fig 3B). These data suggest that ß-TM does not compensate for the reduction in -TM transcript levels. Also, ß-TM isoform levels in skeletal muscles of both wild-type and heterozygous mice are similar. Thus, the loss of -TMstr message due to targeted ablation is not compensated by a ß-TM isoform replacement. Also, we did not observe any expression of the -TM slow skeletal (TM-30) isoform in these hearts (data not shown).

-TM Protein Levels Are Not Altered in the Heterozygous (+/-) Knockout Mice
To determine whether the reduction in the mRNA has affected the -TM protein levels, we analyzed -TM protein levels using myofibrillar protein preparations. Analysis of myofibrillar protein fractions purified from total heart homogenates of wild-type and heterozygous mice did not detect any change in -TM levels (Fig 4A). Also, we did not find any significant differences in other contractile proteins. Similarly, results from Western blot analyses showed that -TM protein levels were unaltered between heterozygous and control littermates (Fig 4B). A similar result was found when total protein homogenates were analyzed. These data demonstrate that despite a 50% reduction in the mRNA levels, the -TM protein amount was not altered. These data suggest that a compensatory mechanism may operate at the translational level to maintain normal amounts of -TM production.

View larger version (67K): [in this window] [in a new window]   Figure 4. Protein analysis of wild-type and -TM heterozygous knockout protein. A, Equivalent amounts of cardiac myofibrillar proteins subjected to SDS-PAGE, followed by Coomassie blue staining. The position of -TM protein is labeled. B, Western blot analysis of TM from myofibrillar cardiac muscle preparations using a striated muscle–specific TM antibody followed by peroxidase-conjugated anti-mouse IgG. Lanes 1 to 4 are increasing concentrations of wild-type protein (1, 2, 5, and 10 µg, respectively); lanes 5 to 8 are increasing concentrations of heterozygous -TM protein (1, 2, 5, and 10 µg, respectively). Additional studies show that 10 µg of protein extract produces TM antigen-antibody at saturation levels.

Since normal -TM protein levels are found in heterozygous mice, we sought to determine whether a translational mechanism was acting to compensate for the increase protein production. We isolated polysomal fractions prepared from heterozygous and control littermate cardiac homogenates and analyzed the polysomal profiles and the associated -TM mRNA within these fractions. Polysomal fractions from sucrose gradients were collected according to density from both wild-type and heterozygous cardiac muscle tissue homogenates. The absorption profile of the polysomes was identical between the two genotypes (Fig 5A). We quantified the abundance of -TM transcripts in the polysomal fractions using RNA slot-blot analysis and an -TM striated muscle–specific oligonucleotide probe. Results show that the relative abundance of -TM mRNA bound to polysomes in the +/- mice is similar to that in the control mice (Fig 5B). These polysomal fractionation/RNA analyses were repeated several times and produced similar results. We conclude that despite a reduction in total -TM mRNA levels in heterozygous mice, the amount of polysomal bound -TM mRNA is comparable to that in wild-type mice, thus suggesting that there is an increased translatability for -TM mRNA in the heterozygous knockout mice.

View larger version (46K): [in this window] [in a new window]   Figure 5. Polysome analysis of wild-type and -TM heterozygous knockout hearts. A, Equivalent samples (10 absorbance units at OD260) of the postmitochondrial supernatant of total heart homogenates from wild-type and heterozygous mice fractionated by density gradient centrifugation as described in "Materials and Methods." Fractions (500 µL) were collected by upward displacement and scanned at OD254. The resulting absorbance profiles for both +/+ and +/- samples are shown. B, Slot-blot analysis of the total RNA that was bound to the polysomal fractions and hybridized with a radiolabeled -TM cDNA probe. The RNA was purified from these fractions, and the slot-blot analysis was conducted as described.

Physiological Analysis of -TM Heterozygous Mice
Despite a significant reduction in -TM mRNA, -TM protein levels are normal in the heterozygous cardiac myofibers. Nevertheless, we conducted physiological analyses on these mice to ascertain whether cardiac performance is altered. Using a work-performing heart model, we measured heart rates, intraventricular pressures, and the rates of contraction and relaxation. No significant differences in cardiac function were found between the -TM heterozygous and control hearts (Fig 6A). The mean values for rates of contraction are 5767±279 and 6380±215 mm Hg/ms for the control and -TM knockout mice, respectively. The mean values for rates of relaxation are 4362.1±221.9 and 4299.6±129.0 mm Hg/ms for control and -TM knockout hearts, respectively. Similarly, measurements of heart rates (327±10 and 323±5 bpm for wild-type and knockout mice, respectively) and intraventricular pressure (114±4 and 113±3 mm Hg for wild-type and knockout mice, respectively) demonstrated no significant differences between the two groups.

View larger version (17K): [in this window] [in a new window]   Figure 6. Functional analysis of heterozygous -TM knockout and control hearts. A, Maximal rates of contraction and relaxation determined in working heart preparations. These rates varied similarly with increasing workloads, and the average values for all work levels were similar. B, Comparison of pCa-force relations for detergent-extracted fiber bundles prepared from hearts of wild-type and heterozygous -TM knockout mice. Fiber bundles were extracted with Triton X-100 and studied under conditions described in "Materials and Methods." Both preparations demonstrated the same Ca2+ dependence of force generation (P.05, two-way ANOVA). For wild-type myofilaments, pCa50 was 5.57±0.01 with a Hill n value of 3.21±0.15; for heterozygous -TM myofilaments, pCa50 was 5.56±0.01 with a Hill n value of 3.15±0.18. Values are expressed as mean±SEM for seven preparations from control mice and six preparations from heterozygous -TM mice.

We also directly compared Ca²⁺ activation of myofilaments in skinned fiber bundles prepared from hearts of wild-type and heterozygous -TM knockout mice. Results presented in Fig 6B show that heterozygous -TM knockout and wild-type myofilaments demonstrated the same (P.05, two-way ANOVA) Ca²⁺ dependence of force generation. The pCa₅₀ for the wild-type myofilaments was 5.57±0.01 with a Hill n value of 3.21±0.15. For heterozygote -TM myofilaments, the pCa₅₀ was 5.56±0.01, with a Hill n value of 3.15±0.18. On the basis of the physiological measurements of the work-performing heart analysis and the skinned fiber bundle preparations, we conclude that there are no significant differences in cardiac function between the heterozygous -TM knockout and wild-type mice.

	Discussion

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The goal of the present study was to understand the importance of -TM in muscle development and function. Our studies show that the heterozygous -TM knockout mice are healthy, with no apparent phenotypic effects from the reduced transcript levels. Also, these heterozygous mice exhibit normal myocardial function as measured by the work-performing heart model and pCa-force measurements. However, the -TM homozygous null mice do not survive, dying between day 8 to 11.5 pc, which suggests that TM protein plays an important role in embryonic development. Also, our studies show that ablation of one -TM allele results in a 50% reduction in -TM mRNA. Interestingly, this decreased -TM mRNA expression does not translate into a change in the endogenous -TM protein levels. Furthermore, the present study demonstrates that proper levels of TM are maintained in the heterozygous -TM (+/-) mouse, an essential feature for normal sarcomeric development and function.

Initial studies with the -TM targeted ES cells demonstrated that our targeting construct, designed to eliminate only the striated muscle -TM isoform, disrupted production of all -TM transcripts from the targeted allele (data not shown). Even though the selective removal of the striated muscle exons left the adjoining exon splice sites and branch-point sequences intact, this targeting event essentially disrupted the normal splicing process, resulting in loss of total mRNA synthesis. As such, these results differ from those obtained using a similar approach in Drosophila; in Drosophila, disruption of indirect flight muscle exons allows the generation of other TM isoforms from the same gene.¹⁴ Thus, our targeting strategy to selectively ablate only the muscle-specific -TM mRNA had scientific precedence but generated a complete ablation of all -TM transcripts from the allele.

Our studies show that the -TM homozygous null mice die during fetal development (8 to 11.5 days pc). In the heterozygous mutant mice, there is no evidence of compensation by striated muscle ß-TM or the slow skeletal -TM (TM-30) isoforms. The ß-TM isoform, which is normally expressed during cardiogenesis, does not increase expression after ablation of a single -TM locus. This is quite contrary to what has been observed with another sarcomeric thin filament protein, cardiac -actin, in which homozygous null mice exhibit compensation by other actin isoforms.¹⁵ Therefore, compensatory mechanisms that are activated may differ among various thin filament sarcomeric proteins.

The present study has demonstrated that different regulatory mechanisms exist among contractile protein genes for maintaining protein levels that are necessary for sarcomeric function. Cardiac -actin knockout mice in the heterozygous state exhibit increased expression of various actin isoforms to compensate for reduced expression.¹⁵ Our results show that there is a compensatory increase in expression by neither ß-TM nor TM-30 isoforms in heterozygous -TM knockout mice; however, there is compensation in -TM protein production. Also, recent work in our laboratory has shown that despite a 150-fold overexpression of ß-TM mRNA production in the heart, the total amount of TM protein remains unchanged.⁶ Interestingly, the increased expression of ß-TM causes the endogenous -TM mRNA and protein levels to compensate by decreasing their expression. However, even with this great excess of ß-TM mRNA production, -TM protein still constitutes almost half of the total TM in the heart. A similar situation occurs when wild-type or mutant -TM is overexpressed in the heart.^{16 17} Thus, it appears that wild-type -TM mRNA is translated with significantly greater efficiency than ß-TM or mutant -TM transcripts. These data, coupled with the results from the present study, strongly suggest that translational regulatory mechanisms may play a major role in maintaining TM protein levels in the myocyte. Furthermore, these investigations indicate that a "cross-talk" mechanism also participates in controlling the regulation and expression of the different TM isoforms. Current investigations are directed toward understanding and identifying the trans-acting factors that control the feedback mechanism regulating TM isoform production.

Studies of TM mutations and ablations have demonstrated the essential function of the TM molecule. In yeast, disruption of the TPM1 TM gene leads to disappearance of actin cables supporting cytoskeletal architecture; mutation of the cdc 8 TM gene impairs cytokinesis.^{18 19} Haploinsufficiency of TM in Drosophila leads to disruption of myofibrillar thin filament assembly.^{14 20} Deficiency of TM in axolotl results in severe cardiac abnormalities, which include disorganized myofibrillar structures with an associated lack of heartbeat.²¹ In humans, missense mutations in -TM are associated with FHC.²² The FHC mutations in -TM produce detectable changes in the Ca²⁺ regulation of the sarcomere, presumably by alterations in TM-troponin interactions.^{16 23} On the basis of the transgenic animal models we have generated, we speculate that in humans encoding -TM FHC mutations the total amount of TM would also remain unchanged. The mutant TM mRNA is translated less efficiently than wild-type -TM transcripts, but the mutant protein that is produced is incorporated into the sarcomere and disrupts myofiber function. Thus, the results of these studies strongly indicate that it is unlikely that -TM mutations associated with haploinsufficiency lead to cardiac abnormalities, as can occur with -myosin heavy chain gene ablations.¹²

	Selected Abbreviations and Acronyms

 

-TMstr = striated muscle -TM mRNA isoform ES = embryonic stem FHC = familial hypertrophic cardiomyopathy HR solution = high relaxing solution HPRT = hypoxanthine phosphoribosyl transferase LR solution = low relaxing solution ODx = optical density at x nm pc = post coitum tk = thymidine kinase TM = tropomyosin

	Acknowledgments

 
This study was partially supported by National Institutes of Health grants HL-46826 and HL-54912 awarded to Dr Wieczorek and grant HL-22231 awarded to Dr Solaro and by American Heart Association, Ohio Affiliate, Inc, grant SW-96–28-F awarded to Dr Rethinasamy. Dr Wieczorek is an Established Investigator of the American Heart Association, with funds contributed in part by the American Heart Association, Ohio Affiliate, Inc. The authors gratefully thank Dr J. Duffy and S. Pawlowski for their expertise in producing and maintaining the -TM knockout mice. We acknowledge K. Pieples for data on -TM slow skeletal isoform expression. Also, we acknowledge Drs G. Shull, G. Dean, and especially M. Periasamy for their critical reading of the manuscript.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received August 21, 1997; accepted December 1, 1997.

	References

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