Expression of tropomyosin in relation to myofibrillogenesis in axolotl hearts
© Zajdel et al.; licensee BioMed Central Ltd. 2013
Received: 9 August 2013
Accepted: 8 October 2013
Published: 4 December 2013
The anatomy, function and embryonic development of the heart have been of interest to clinicians and researchers alike for centuries. A beating heart is one of the key criteria in defining life or death in humans. An understanding of the multitude of genetic and functional elements that interplay to form such a complex organ is slowly evolving with new genetic, molecular and experimental techniques. Despite the need for ever more complex molecular techniques some of our biggest leaps in knowledge come from nature itself through observations of mutations that create natural defects in function. Such a natural mutation is found in the Mexican axolotl, Ambystoma mexicanum. It is a facultative neotenous salamander well studied for its ability to regenerate severed limbs and tail. Interestingly it also well suited to studying segmental heart development and differential sarcomere protein expression due to a naturally occurring mendelian recessive mutation in cardiac mutant gene “c”. The resultant mutants are identified by their failure to beat and can be studied for extended periods before they finally die due to lack of circulation. Studies have shown a differential expression of tropomyosin between the conus and the ventricle indicating two different cardiac segments. Tropomyosin protein, but not its transcript have been found to be deficient in mutant ventricles and sarcomere formation can be rescued by the addition of TM protein or cDNA. Although once thought to be due to endoderm induction our findings indicate a translational regulatory mechanism that may ultimately control the level of tropomyosin protein in axolotl hearts.
The Mexican axolotl, a facultative neotenous salamander, provides a valuable model to study heart development due to a cardiac lethal mutation (gene c) that affects only heart muscle [1, 2]. It has also been used extensively for organ regeneration research, particularly of its limbs and tails  but have included initial studies into the regeneration of the heart . The axolotl cardiac gene c mutation is a Mendelian, autosomal recessive lethal mutation with significant effects on tropomyosin protein levels in the cardiac tissue. Morphological studies of the abnormal cardiomyogenesis in mutants have shown they lack organized myofibrils, have large collections of amorphous material, but still retain normal electrophysiological properties [5–7]. The embryos can survive for up to a fortnight post-hatching which is ideal for studying this process before the lack of circulation, secondary to abnormal sarcomere formation is lethal. The mutant axolotl heart provides a unique opportunity for studying the intricate process of cardiac development and for examining the specific functional role of each tropomyosin (TM) isoform in this process. Ultimately the protein level of TM is profoundly diminished in the ventricle of c/c mutant hearts, resulting in an absence of organized myofibrils and subsequently the inability to beat [5–8]. It is important to note that the conus is not deficient in tropomyosin protein, retains organized myofibrils and is capable of beating independently, unlike the atria and ventricle in mutant hearts . Notably, the mutant hearts can be rescued in situ by supplying exogenous TM protein or TM cDNA in an expression construct under the control of an appropriate promoter(s) [9, 10]. Mutant hearts can also be rescued in situ by a specific non-coding RNA that is unrelated to TM [11–13]. However, the exact mechanism by which this RNA modulates the expression of tropomyosin is yet to be elucidated. To better understand the mechanism(s) effecting tropomyosin expression in mutant hearts, we undertook an extensive molecular characterization of the various isoforms of tropomyosin in the Mexican axolotl.
Isoform diversity of tropomyosin in vertebrates
The thin filaments of striated muscle in vertebrate consist of actin, tropomyosin, the troponin (Tn) complex (Tn-I, Tn-C and Tn-T), tropomodulin, and a few other proteins . Actin filaments interaction with Ca +2 governs muscle contraction and relaxation. Tropomyosin is a coiled coil actin-binding protein found along the length of seven actin monomers. A set of four known genes (TPM1, TPM2, TPM3 and TPM4) that encode tropomyosin in vertebrates [15–19] give rise to various tropomyosin protein isoforms that play important roles in striated, smooth and non-muscle cells. There remains even further diversity in other models such as zebrafish where six tropomyosin genes have been identified . The creation of different tropomyosin isoforms occurs through various mechanisms, including the use of different promoters, alternative mRNA splicing, different 3’ end mRNA processing and tissue specific translation control .
Nomenclature of various isoforms of TM referred to in this article
TPM Gene encoding the Isoforms: New Nomenclature (Old Nomenclature)
Various isoforms of TM currently known as
Nomenclature used in previous publications on axolotl
Accounting for the other tropomyosin genes other than TPM1, TPM2α (sarcomeric isoform of the TPM2 gene) is also expressed in mammalian hearts in addition to the previously described TPM1α and TPM1κ The sarcomeric isoform of the TPM3 gene, TPM3α, is only expressed in slow-twitch skeletal muscle. No sarcomeric isoform of the TPM4 gene is expressed in mammalian striated muscles because the TPM4 gene is truncated in mammals [15–17]. On the contrary, TPM4α is a major TM isoform in amphibian cardiac tissues [23, 31] and is the only isoform for sarcomeric TM in adult avian hearts [27, 30, 32].
Sarcomeric tm protein in cardiac mutant axolotl hearts
Although mutant axolotl hearts are deficient in sarcomere specific TM proteins, mRNA levels of each of three striated muscle isoforms (TPM1α, TPM1κ, and TPM4α) are comparable in normal and mutant hearts [12, 23]. Hence, the tropomyosin deficiency in mutant heart is not due to an insufficiency in transcription or post-transcriptional splicing . We cloned and sequenced cDNAs of three isoforms from mutant hearts; no mutation(s) was detected in any of these cDNAs that may cause truncated non-functional TM isoform(s). Additionally, we have cloned and sequenced the promoter region of the TPM4 gene from the DNA isolated from normal and mutant axolotl hearts and again, no differences were observed . Hence, the possibility of insufficient transcription of the cardiac specific TPM4α isoform is highly unlikely. The most plausible explanation based on available evidence of TM deficiency in mutant hearts is a translational insufficiency of the tropomyosin transcripts in mutant hearts .
Molecular analysis and manipulation of tropomyosin isoforms in normal and mutant axolotl hearts
As stated earlier, there are at least three striated muscle isoforms of tropomyosin present in the axolotl. Two isoforms of tropomyosin cDNA have been identified which apparently are derived from the single alpha-tropomyosin gene (TPM1) through alternative splicing [21, 22]. Spinner et al.  cloned another tropomyosin cDNA, which is the product of a TM4 type tropomyosin gene from axolotl heart. An expression construct with each of these isoforms upon transfection into mutant hearts canaugment tropomyosin proteinlevels and promotes myofibrillogenesis. The important question is whether or not any one of these isoforms alone and/or in various combination(s) is necessary for myofibrllogenesis in axolotl hearts in vivo. In order to address this issue we developed procedures for disruption of myofibrils in normal axolotl hearts mimicking the mutant hearts by lipofecting antibodies against sarcomeric TM into normal hearts in situ. Myofibrils in lipofected normal hearts indeed became greatly disorganized [10, 35]. As CH1 antibodies react with all three sarcomeric tropomyosins, it is not possible to evaluate the requirement of a particular isoform that is involved in cardiac myofibrillogenesis. Later we developed antibody against TPM1κ in rabbits using a 15-mer peptide sequence (LDELHKSEESLLTAD) derived from axolotl exon 2a . Recall, TPM1κ is unique as a sarcomeric TM in that it contains exon 2a instead of exon 2b which is found in TPM1α. The affinity purified anti-TPM1κ antibody upon transfection could disarray the organized myofibrils in axolotl hearts . The results strongly suggest that TPM1κ plays a critical role in myofibril formation in axolotl hearts.
Differential expression of tropomyosin in conus and the ventricle
Promotion of myofibrillogenesis in mutant hearts in situ by a non-coding rna
A noncoding RNA, Myofibril-Inducing RNA (MIR) is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotl hearts in situ . Zhang et al.  demonstrated that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development at the level of translation or post-translation. qRT-PCR using isoform-specific primer-pairs showed that mRNA expression of three sarcomeric tropomyosin isoforms (TPM1α, TPM1κ, and TPM4α) in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, sarcomeric tropomyosin isoforms detected with CH1 monoclonal antibodies, are significantly reduced in mutant and dsMIR treated normal hearts. However, this study neither showed the mechanism by which MIR may induce sarcomeric TM synthesis in axolotl hearts nor addressed the role of specific tropomyosin isoforms in cardiac myofibrillogenesis.
Recently, Kochegarovr et al.  randomly cloned RNAs from fetal human heart. RNA from one of the clones (clone #291) was found to promote myofibril formation in mutant axolotl in situ. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. The nucleotide sequences of the cloned RNA matches in partial with the human microRNA-499a and b, although it differs in length. qRT-PCR data suggest this RNA may induce the TPM4α (ATmC-3) isoform in mutant heart, producing more sarcomeric tropomyosin protein and subsequently promote myofibril formation. The mechanism by which this non-coding RNA induces tropomyosin in mutant hearts may well be different from that of MIR, which acts at the post transcriptional level .
Review and conclusions
Although our complete understanding of the mechanism of tropomyosin expression in mutant axolotl hearts as well as the nature and function of gene “c” is far from over, we would like to end this review with a positive note. Our finding of sarcomeric TM isoform TPM1κ in axolotl led to the discovery of this isoform in human hearts . Unlike in axolotl, it is not expressed in human skeletal muscle. Most importantly, an upregulation of TPM1κ protein has been reported in hearts from human dilated cardiomyopathy patients . However, it is not yet known whether the upregulation of TPM1κ is the cause or a consequence of cardiomyopathy in this patient. Our anti-sense experiments suggest strongly the functional significance of TPM1κ in axolotl hearts. In addition, the lower expression level of TPM1κ protein in axolotl heart and skeletal muscle  and also in human hearts  points towards translational repression of TPM1κ. Further, upon injection intraperitoneally into juvenile axolotl, Shz-1, a cardiogenic small molecule, augmented the expression levels of transcripts of TPM1α, TPM1κ, and TPM4α in hearts. But the increased transcript level did not resulted into increased sarcomeric TM protein expression . Finally, although the transcript levels of all TPM isoforms in normal and mutant axolotl heart ventricles are comparable, the proteins of all three isoforms are diminished significantly. This observation also point towards the translational repression of TM in cardiac tissues . The evidence for translational control of sarcomeric TM in mammalian hearts was originally came from the works from the laboratory of Dr. David Wieczorek, University of Cincinnati, Cincinnati, OH [29, 43]. Rethinesamy et al.  and Blanchard et al.  independently ablated one of the two alleles of the TPM1 gene in mice that resulted in half of the TPM1α transcripts in ablated mice hearts compared to wild-type. However, TPM1α protein level was unchanged in ablated mice hearts suggesting a higher translational efficiency of TPM1α transcripts in ablated mice hearts. Again, Rajan et al.  reported that although the level of transcripts of TPM1α and TPM1κ human hearts is parallel, TPM1κ protein is only ~5% of the total sarcomeric TM whereas TPM1α protein constitutes about 90-95% of the total sarcomeric TM. The results strongly suggest the translational repression of TPM1κ transcripts in human hearts. The immediate future goal of our laboratory is to explore further the translational repression of tropomyosin expression in vertebrate hearts as well as to find out the functional role of TPM1κ.
The work in this laboratory was supported by grants from American Heart Association (both National & New York State affiliate), CNY Children’s Miracle Network, Syracuse, NY, grants from Golisano Children’s Hospital, Syracuse, NY to DKD, an AHA (NY Affiliate) grant to RW.
- Humphrey RR: Genetic and experimental studies on a mutant gene (c) determining absence of heart action in embryos of the Mexican axolotl (Ambystoma mexicanum). Dev Biol 1972, 27: 365–375. 10.1016/0012-1606(72)90175-3PubMedView ArticleGoogle Scholar
- Lemanski LF: Morphology of developing heart in cardiac lethal mutant Mexican axolotls, ambystoma mexicanum. Devel Biol 1973, 33: 312–333. 10.1016/0012-1606(73)90140-1View ArticleGoogle Scholar
- Voss GJ, Kump DK, Walker JA, Voss SR: Variation of Salamender tail regeneration is associated with genetic factors that determine tail morphology. PLos ONE 2013,8(7):e67274. doi:10,1371/journal.pone.0067274 10.1371/journal.pone.0067274PubMed CentralPubMedView ArticleGoogle Scholar
- Cano-Martinez A, Vargas-Gonzaleez A, Guarner-Lans V, Prado-Zayago E, Leon-Oleda M, Nieto-Lima B: Functional and structural regeneration in the axolotl heart (Ambystom mexicanum) after partial ventricular amputation. Arch Cariol Mexi 2010, 80: 79–86.Google Scholar
- Lemanski LF: Role of tropomyosin in actin filament formation in embryonic salamander heart cells. J Cell Biol 1979, 82: 227–238. 10.1083/jcb.82.1.227PubMedView ArticleGoogle Scholar
- LaFrance SM, Lemanski LF: Imunofluorescent confocal analysis of tropomyosin in developing hearts of normal and cardiac mutant axolotls. Int J Devel Biol 1994, 38: 695–700.Google Scholar
- Zajdel RW, Dube DK, Lemanski LF: The cardiac mutant axolotl is a unique animal modelfor evaluation of cardiac myofibrillogenesis. Exp Cell Res 1999, 248: 557–566. 10.1006/excr.1999.4419PubMedView ArticleGoogle Scholar
- Zajdel RW, MeLean MD, Denz CR, Dube S, Thurston H, Poiesz BJ, Dube DK: Differential expression of tropomyosin during segmental heart development in Mexican axolotl. J Cell Biochem 2006, 99: 952–965. 10.1002/jcb.20954PubMedView ArticleGoogle Scholar
- Zajdel RW, McLean MD, Lemanski SL, Muthuchamy M, Wieczorek DF, Lemanski LF, Dube DK: Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts. Dev Dynamics 1998, 213: 412–420. 10.1002/(SICI)1097-0177(199812)213:4<412::AID-AJA6>3.0.CO;2-CView ArticleGoogle Scholar
- Zajdel RW, McLean MD, Lemanski LF, Dube DK: Alteration of cardiac myofibrillogenesis by lipofectin-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts. Anat Embryol 2000, 210: 217–228.View ArticleGoogle Scholar
- Lemanski LF, Nakatsugawa M, Bhatia R, Erginel-Unaltuna N, Spinner BJ, Dube DK: A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl. Biochem Biophys Res Com 1996, 229: 974–981. 10.1006/bbrc.1996.1910PubMedView ArticleGoogle Scholar
- Zhang C, Jia P, Huang X, Sferrazza GF, Athauda G, Achary MP, Wang J, Lemanski SL, Dube DK, Lemanski LF: Myofibril-inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts. J Biomed Sci 2009, 16: 81. 10.1186/1423-0127-16-81PubMed CentralPubMedView ArticleGoogle Scholar
- Zhang C, Dube DK, Huang X, Zajdel RW, Bhatia R, Foster D, Lemanski SL, Lemanski LF: A point mutation in bioactive RNA results in the failure of mutant heart correction in Mexican axolotls. Anat Embryol 2003, 206: 495–506.PubMedGoogle Scholar
- Perry SV: Vertebrate tropomyosin, properties and function. J Muscle Res Cell Motil 2001, 22: 5–49. 10.1023/A:1010303732441PubMedView ArticleGoogle Scholar
- Schevzov G, Whittaker SP, Fath T, Lin JJ, Gunning PW: Tropomyosin isoforms and reagents. Bioarchitecture 2011, 1: 135–164. 10.4161/bioa.1.4.17897PubMed CentralPubMedView ArticleGoogle Scholar
- Gunning P, O’Neill G, Hardmen E: Tropomyosin-based regulation of actin cytoskeleton in time and space. Physiol Rev 2008, 88: 1–35. 10.1152/physrev.00001.2007PubMedView ArticleGoogle Scholar
- Lees-Miller JP, Helfman DM: The molecular basis for tropomyosin isoform diversity. Bioessays 1991, 13: 429–437. 10.1002/bies.950130902PubMedView ArticleGoogle Scholar
- Wieczorek DF: Regulation of alternatively spliced alpha-tropomyosin gene expression by nerve extract. J Bol Chem 1988, 263: 10456–10463.Google Scholar
- Pittenger MF, Kazzaz JA, Helfman DM: Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol 1994, 1: 96–104.View ArticleGoogle Scholar
- Piples K, Wieczorek DF: Tropomyosin 3 increases striated muscle isoform diversity. Biochemistry 2000, 39: 8291–8297. 10.1021/bi000047xView ArticleGoogle Scholar
- Luque EA, Lemanski LF, Dube DK: Molecular cloning, sequencing and expression of atropomyosin form cardiac muscle of the Mexican axolotl, ambystoma mexicanum. Biochem Biophys Res Comm 1994, 203: 319–325. 10.1006/bbrc.1994.2184PubMedView ArticleGoogle Scholar
- Luque EA, Spinner BJ, Dube S, Dube DK, Lemanski LF: Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum ). Gene 1997, 185: 175–180. 10.1016/S0378-1119(96)00606-3PubMedView ArticleGoogle Scholar
- Spinner BJ, Lemanski LF, Dobbins N, Dube DK: Transient expression of the cardiac specific TM4-type tropomyosin suggest a unique role in heart development in the Mexican axolotl. J Cell Biochem 2002, 85: 747–761. 10.1002/jcb.10178PubMedView ArticleGoogle Scholar
- Thomas A, Rajan S, Thurston H, Masineni L, Sreeharsha N, Dube P, Bose A, Muthu V, Dube T, Wieczorek DF, Dube DK: Expression of a novel tropomyosin isoform in axoltl heart and skeltel muscle. J Cell Biochem 2010, 110: 875–881. 10.1002/jcb.22599PubMed CentralPubMedView ArticleGoogle Scholar
- Rajan S, Jagatheesan G, Karam CN, Alves ML, Bodi I, Schwartz A, Buclago CF, D’Souza KM, Akhter SA, Boivin GP, Dube DK, Petrasheveskaya N, Herr AB, Hullin R, Liggett SB, Beata MW, Solaro JR, Wieczorek DF: Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform. Circulation 2010, 121: 410–418. 10.1161/CIRCULATIONAHA.109.889725PubMed CentralPubMedView ArticleGoogle Scholar
- Denz CR, Narshi A, Zajdel RW, Dube DK: Expression of a novel cardiac-specific tropomyosin isoform in humnas. Biochem Biophys Res Comm 2004, 320: 1291–1297. 10.1016/j.bbrc.2004.06.084PubMedView ArticleGoogle Scholar
- Zajdel RW, Denz CR, Lee S, Dube S, Ehler E, Perriard E, Periard J-C, Dube DK: Identification, characterization, and expression of a novel alpha tropomyosin isoform in cardiac tissue in developing chicken. J Cell Biochem 2003, 89: 427–439. 10.1002/jcb.10504PubMedView ArticleGoogle Scholar
- Ward SM, Dube DK, Fransen ME, Lemanski LF: Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls ( Ambystoma mexicanum ). Devel Dynamics 1996, 205: 93–103. 10.1002/(SICI)1097-0177(199602)205:2<93::AID-AJA1>3.0.CO;2-ZView ArticleGoogle Scholar
- Rethinasamy P, Muthuchamy M, Hewett T, Boivin G, Wolska BM, Evans C, Solaro RJ, Wieczorek DF: Molecular and physiological effects of alpha-tropomyosin ablation in the mouse. Circ Res 1998, 82: 116–123. 10.1161/01.RES.82.1.116PubMedView ArticleGoogle Scholar
- Fleenor DE, Hickman KH, Lindquester GJ, Devlin RB: Avian cardiac tropomyosin gene produces tissue-specific isoforms through alternative TNA splicing. J Muscle Res Cell Motel 1992, 13: 55–63. 10.1007/BF01738428View ArticleGoogle Scholar
- Hardy S, Theze N, Lepetit D, Allo MR, Thiebaud P: The Xenopus laevis TM-4 gene encodes non-muscle and cardiac tropomyosin isoforms through alternative splicing. Gene 1995, 156: 265–270. 10.1016/0378-1119(94)00929-MPubMedView ArticleGoogle Scholar
- Forry-Schaudies S, Gruber CE, Hughes SH: Chicken tropomyosin and a low molecular weight non-muscle tropomyosin are related by alternative splicing. Cell Growth Diff 1990, 1: 473–481.PubMedGoogle Scholar
- McLean MD, Zajdel RW, Dube S, Thurston H, Dube DK: Tropomodulin expression in developing hearts of normal and cardiac mutant Mexican axolotl. Cardiovasc Toxicol 2006, 6: 85–98. 10.1385/CT:6:2:85PubMedView ArticleGoogle Scholar
- Denz CR, Zhang C, Jia P, Du J, Huang X, Dube S, Thomas A, Poiesz BJ, Dube DK: Absence of mutation at the 5’-upstream promoter region of the TPM4 gene from cardiac mutant axolotl. (Ambystoma mexicanum) Cardiovasc Toxicol 2011, 11: 235–243. 10.1007/s12012-011-9117-zPubMedView ArticleGoogle Scholar
- Zajdel RW, Denz CR, Narshi A, Dube S, Dube DK: Anti-sense-mediated inhibition of expression of the novel striated tropomyosin isoform TPM1κ disrupts myofibril organization in embryonic axolotl hearts. J Cell Biochem 2005, 95: 840–848. 10.1002/jcb.20456PubMedView ArticleGoogle Scholar
- Spinner BJ: Molecular analysis of isoform diversity of cardiac tropomyosin in the Mexican axolotl. Upstate Medical University, Department of Anatomy and Cell Biology: Ph.D. thesis; 1998.Google Scholar
- Zajdel RW, McLean MD, Denz CR, Dube S, Lemanski LF, Dube DK: Manipulation of myofibrillogenesis in whole heart. In Myofibrillogenesis. Edited by: Dube DK. Boston: Birkhauser; 2002:87–100.View ArticleGoogle Scholar
- Satin J, Fujii S, DeHaan RI: Devekopment of cardiac beat rate in early chick embryos is regulated by regional cues. Dev Biol 1988, 129: 103–113. 10.1016/0012-1606(88)90165-0PubMedView ArticleGoogle Scholar
- Zhang R, Xu X: Transient and transgenic analysis of the zebrafish ventricular myosin hearvy chain (vmhc ) promoter: an ingibitory mechanism of ventricle-specific gene expression. Dev Dyn 2009, 238: 1564–1573. 10.1002/dvdy.21929PubMed CentralPubMedView ArticleGoogle Scholar
- Kochegarov A, Moses A, Lian W, Meyer J, Michael C, Hanna MC, Lemanski LF: A new unique form of microRNA from human heart, microRNA-499c, promotes myofibril formation and rescues cardiac development in mutant axolotl embryos. J Biomed Sci 2013, 20: 20. 10.1186/1423-0127-20-20PubMed CentralPubMedView ArticleGoogle Scholar
- Pinnamaneni S, Dube S, Welch C, Shrestha R, Benz PM, Lynn Abbott L, Poiesz BJ, Dube DK: Effect of Shz-1, a cardiogenic small molecule, on expression of tropomyosin in axolotl heart. American Based Res J 2013, 2: 24–40.Google Scholar
- Dube DK, Benz PM, Dube S, Poiesz BJ: Do we know the complete story of TPM1κ expression in vertebrate hearts? J Cytol Histol 2012, 3: 3.View ArticleGoogle Scholar
- Schevzov G, Fath T, Vrhovski B, Vlahovich N, Rajan S, Hook J, Joya JE, Lemckert F, Puttur F, Lin JJ, Hardeman EC, Wieczorek DF, O’Neill GM, Gunning PW: Divergent regulation of the sarcomere and the cytoskeleton. J Biol Chem 2008, 283: 275–283. 10.1074/jbc.M704392200PubMedView ArticleGoogle Scholar
- Blanchard EM, Iizuka K, Christe M, Conner DA, Geisterfer-Lowrance A, Schoen FJ, Maughan DW, Seidman CE, Seidman JG: Targeted ablation of the murine alpha-tropomyosin gene. Circ Res 1997, 81: 1005–1010. 10.1161/01.RES.81.6.1005PubMedView ArticleGoogle Scholar
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