- Open Access
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.
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