-Tropomyosin Knockouts

A Blow Against Transcriptional Chauvinism

Correspondence to J. Robbins, PhD, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}chmcc.org

Key Words: tropomyosin • transgenic • muscle • cardiomyopathy • gene

The pace at which the protein complement of the heart is precisely remodeled by either gene targeting or transgenesis in animal models continues to accelerate.¹ A logical focus of these approaches has been to establish structure-function relationships of the proteins present in the terminally differentiated cardiomyocyte. Rather unexpectedly, these experiments have also highlighted the importance of multiple control points, both at the transcriptional and posttranscriptional levels, which are able to regulate the overall stoichiometry of the cardiac thick and thin filament proteins. Several recent articles in Circulation Research illustrate these points for the thin filament protein, -tropomyosin (-TM).^{2 3 4} In striated muscle, the thin filament consists largely of actin, tropomyosin (TM), and the troponin complex (troponins I, C, and T) and is responsible for mediating Ca²⁺ control of contraction and relaxation. TM is a rigid rod-shaped protein that binds along the length of the actin filament and is intimately associated with troponin. It both stabilizes and stiffens the filament.⁵ In the absence of Ca²⁺ binding to troponin C, TM blocks the myosin binding site of actin. TM is a small polypeptide, 284 amino acids, depending on the particular isoform, and dimerizes to form a head-to-tail coiled-coil structure that lies in the major groove of the actin filament. The placement of the TM dimer is consistent with one of its important roles, which is to help mediate cooperativity of Ca²⁺ activation along the length of the myofilament.^{6 7}

Like other sarcomeric proteins, TM exists as a set of isoforms; these are generated as a result of both differential transcription of the four members of the gene family (-TM, ß-TM, TM-4, and TM-30) and by alternative splicing patterns from the primary transcripts. The - and ß-TMs are the two striated muscle isoforms and are expressed at varying levels in the different muscle types, with the relative amounts depending on the particular organism, developmental stage, and muscle-fiber type. In the murine heart, the /ß ratio changes during development, with the percentage of -TM increasing from 80% during the fetal stages to >95% to 98% in the mature heart. The unique functions of the two isoforms have been defined in transgenic overexpression studies.^{8 9}

Mutations in TM have been linked to cardiovascular disease. Beginning with the seminal study of Geisterfer-Lowrance et al,¹⁰ a genetic basis for the primary etiology of familial hypertrophic cardiomyopathy (FHC) began to be established. As a group of diseases, the familial hypertrophic cardiomyopathies are not particularly common, with independent studies yielding estimates that 1 in 5000 individuals are affected.^{11 12} However, FHC is a major cause of sudden death in otherwise healthy appearing young adults.¹³ Multiple mutations in the myosin heavy and light chains, troponin T and I subunits, myosin binding protein C, and -TM can cause hypertrophic cardiomyopathy, which is thus considered a "disease of the sarcomere."¹⁴ Mutations in -TM are quite rare and probably account for <5% of the total FHC cases. Only three -TM mutations, all of which result in single amino acid substitutions, have been identified to date, although this obviously doesn't preclude the possibility that others remain undiscovered at this time.

Existing data are consistent with the idea that FHC is caused by a "poison peptide." That is, the mutated genetic locus produces an aberrant polypeptide that is incorporated into the sarcomere and affects normal contractile function. The mutation that has been most completely characterized to date is carried on the myosin heavy chain at residue 403 (ArgGln), and multiple analyses show that the mutant protein is made and causes alterations in contractile function.^{15 16} A truncated cardiac troponin T, which causes FHC, can also be incorporated into the sarcomere in an overtly normal manner and affect function.^{17 18} However, an alternative hypothesis is that in some cases the disease can arise as the result of an effective change in gene dosage. This could occur if the mutated allele produced a nonfunctional polypeptide, leading to the decreased concentration of a normal protein essential for sarcomerogenesis and/or function. For example, in Drosophila, a null mutation in the myosin heavy chain gene can have dramatic effects on muscle structure and function, leading to sarcomere dysgenesis,¹⁹ and increases/decreases in gene dosage of the other contractile proteins can also compromise muscle function in these systems. Thus, in Drosophila, maintenance of normal sarcomere protein stoichiometry appears to be a direct function of the number of genes that encode the particular protein, and any changes in the absolute and/or relative numbers of the different sarcomeric genes can have severe consequences on muscle structure and function. Proof of principal for a similar effect in the mammalian contractile apparatus was obtained when gene targeting in the mouse was used to produce a null mutation in the cardiac -myosin heavy chain gene; sarcomere dysgenesis and altered cardiac function could be detected in a subset of the animals that were heterozygous for the null allele.²⁰

Several recent articles in Circulation Research address the poison peptide versus null allele hypothesis for -TM, both by creating mice containing null alleles of the gene^{2 4} and by examining the skeletal sarcomeric protein complement derived from muscles of patients carrying the -TM^Asp175Asn mutation, which is known to cause FHC.³ Blanchard et al⁴ ablated -TM in mice by inserting a neo-containing cassette into exon 9a, whereas Rethinasamy et al² carried out a different targeting event, replacing a 4.4-kbp -TM fragment containing the striated muscle–specific exons 12 to 13 with an HPRT minigene cassette. Each construct resulted in a null allele that did not produce a stable -TM RNA transcript. The phenotypes of both sets of animals were essentially identical. In the homozygous state, the null allele was embryonically lethal, confirming the essential nature of the protein for development. No compensatory upregulation of the other TM gene transcripts, which might have been able to effect a partial or complete rescue of embryonic lethality, was observed. This recapitulates the situation observed in the cardiac -myosin heavy chain knockout²⁰ but differs from the cardiac -actin knockout, in which upregulation of the vascular and skeletal genes occurred and "rescued" the developing fetus.²¹ The -TM mRNA levels in the heterozygotes were approximately half the levels found in the wild-type controls. Strikingly, however, -TM protein levels were unaffected. Again, no compensatory upregulation of the other TM genes could be detected. Functionally and structurally, the heterozygote animals were indistinguishable from the wild-type control animals, which is not surprising considering that -TM protein levels were maintained despite the decrease in -TM mRNA steady-state levels. No perturbations in morbidity, mortality, breeding behavior, or litter size were found. Gross morphology of the heterozygote hearts was normal, and electron microscopy of the sarcomeres also showed normal morphology. Functional analyses at the fiber and whole heart levels showed that all parameters were normal in terms of Ca²⁺ activation, cooperativity at the fiber level, and contractile and relaxation parameters as measured in the ex vivo working heart preparation.

That normal levels of -TM protein were maintained despite the presence of a single functional allele indicates that sarcomere protein stoichiometry and absolute mass are conserved even if only a single -TM allele produces protein, a result that is inconsistent with the null allele hypothesis. This was confirmed for one of the -TM FHC mutations by direct examination of the TM isoform content in muscles derived from patients carrying the -TM^Asp175Asn mutation.³ For ethical reasons, cardiac biopsies could not be obtained. However, the -TM isoform is expressed in both cardiac and skeletal muscles. When TM isoforms were isolated from the vastus lateralis, an aberrant protein was detected. This species comigrated with -TM^Asp175Asn that was produced in vitro. In the skeletal muscle samples, the mutant protein was expressed at high levels that were approximately equal to the wild-type protein levels.

There are no antibodies available that are able to distinguish between the wild-type and mutant TMs, so direct evidence that the -TM^Asp175Asn is incorporated into the sarcomere is lacking. However, this point was indirectly approached by analyzing muscle fibers that were "skinned," a treatment that results in the removal of the plasma membrane and the subsequent loss of soluble proteins or the removal of proteins that are not bound tightly to the sarcomere. The ratio of mutant to normal TM protein did not change when the total muscle biopsies and skinned fiber protein isolates were analyzed, a result consistent with the hypothesis that the mutant protein intercalates into the sarcomere and participates in fiber function.

The muscle fibers isolated from the patients were analyzed for Ca²⁺ sensitivity and cooperativity. Although the analyses were complicated by the heterogeneity of the different skeletal muscle fiber types, Ca²⁺ sensitivity (pCa²⁺₅₀) was higher in both type 1 and type 2A fibers derived from the FHC samples compared with normal fibers of the same isotype. No differences could be detected in cooperativity, the amount of force generated, or the V_max of shortening. The increased, rather than decreased, Ca²⁺ sensitivity raises the interesting possibility that muscle function at physiological Ca²⁺ levels might be enhanced rather than compromised in the affected fibers. However, this interpretation must be treated with caution when applied to cardiac function. For example, it has been reported that the cardiac troponin C^Ile79Asn FHC-causing mutation results in an increase in actin filament velocity in the in vitro motility assay²² ; enhanced velocity in the assay was also noted using the essential myosin light chain FHC mutation.²³ However, this assay measures unloaded motor speed, and when myotubes containing the human cardiac troponin C^Ile79Asn protein were made, a 25% decrease in maximum Ca²⁺-activated force production was noted.¹⁸

The -TM data, taken together with the data obtained for the myosin heavy chain and cardiac troponin T FHC, show that FHC occurs as a result of a poison peptide rather than haploinsufficiency. But it is premature to conclude that the paradigm is a general one. For example, as noted above, mutations in myosin binding protein C can also cause FHC, and a recent case study using endomyocardial biopsies from one of these patients reported that the transcript could be detected but the mutant protein could not.²⁴ However, a central tenet of the haploinsufficiency hypothesis, a reduction of protein, was not observed in the samples: the presence of a single wild-type allele did not appear to have a significant impact on the steady-state level of the normal protein. The authors hypothesized that this apparent contradiction, a dominant-negative effect in the absence of a polypeptide, might come about by the ability of an unstable mutant polypeptide to contribute, albeit transiently, to sarcomerogenesis.²⁴ A rigorous examination of the relevant animal model, produced either by partial transgenic replacement of the wild-type protein or by an "in-out" gene targeting event such as was used to produce the myosin heavy chain FHC model,¹⁶ should resolve these questions.

The -TM knockout data emphasize that posttranscriptional controls can have a major impact on maintaining normal protein stoichiometry in the contractile apparatus. Molecular genetic studies have defined the importance of transcriptional control for striated muscle development and differentiation. Transcriptional controls are generally recognized as playing the defining role in determining the specialized muscle cell protein complement. The posttranscriptional controls that might operate in these cells have, in comparison, received little attention. Perhaps it is time to reassess the potential of posttranscriptional control in maintaining normal cardiomyocyte protein levels in general and the stoichiometry of the sarcomere in particular. Both gene targeting studies (the -TM knockout and transgenic overexpression studies with the sarcomeric proteins)²⁵ show that despite the primacy of transcriptional controls in regulating the myofilament protein complement under normal circumstances, posttranscriptional controls can determine overall sarcomeric protein stoichiometry in the face of decreased (via gene ablations) or increased (via transgenic overexpression) message levels. In the -TM gene ablations, although message levels are decreased by half in the heterozygote nulls (implying that transcriptional controls do not result in upregulation of the remaining transcriptional activity of the allele), steady-state protein levels are unaffected. In a transgenic experiment, overexpression of a ventricular myosin light chain cDNA resulted in a 10-fold increase in steady-state mRNA levels, but the light chain protein complement was unaffected.²⁶

How can this occur? In the -TM knockouts, if message levels are decreased, is the remaining message more efficiently used by the translational apparatus, or is the resultant protein more efficiently recruited into the contractile apparatus and/or preferentially stabilized, effectively decreasing protein turnover times? Precedence for changes in the efficiencies of polysome loading and/or transit times in muscle exist.^{27 28} Rethinasamy et al² show some preliminary data comparing polysome loading of the -TM message in the wild-type and heterozygous nulls,² but the results are inconclusive, and further experiments bearing on mRNA utilization need to be performed. In the case of transgenic overexpression, is the "excess" RNA untranslated or translated less efficiently? Or, is excess protein made but, since it is not incorporated into the sarcomere, turned over so rapidly as to be undetectable? There are very few data bearing directly on these points, but clearly, these controls, since we now know that they are intrinsic to the protein complement of the diseased heart, warrant further detailed study. The different mouse models provide a suitable reagent set for studying these processes at a high level of resolution at both the translational and posttranslational levels and should prove generally useful in rigorously exploring these phenomena.

Footnotes

The opinions expressed in this article are not necessarily those of the editor or of the American Heart Association.

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