Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1µ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and µ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1µ non-muscle isoform protein expression before and during cardiac differentiation.

Delayed seroconversion to STLV-1 infection is associated with mutations in the pol and rex genes.

BACKGROUND: Simian T-cell lymphoma/leukemia virus-1 (STLV-1) infection of non-human primates can serve as a model for human T-cell lymphoma/leukemia virus infection. METHODS: Two tantalus and 2 patas monkeys were transfused with intraspecies whole blood infected with STLV-1. Infection was determined by ELISA, western blot and DNA PCR analyses. The entire genome of the STLV-1 Tan 90 strain and some of the STVL-1 Pat74 strain were amplified using over-lapping primer-pairs and subsequently sequenced. RESULTS: Followup studies conducted over 2 years indicated that all 4 monkeys remained healthy despite being infected with STLV-1, as determined by PCR, cloning and sequencing analyses. ELISA and Western blot analyses indicated that both patas monkeys seroconverted within 2 months of transfusion, while one tantalus monkey required one year to seroconvert and the other never fully seroconverted. The tantalus monkey which never fully seroconverted, failed to react to HTLV-1 p24 Gag antigen. Sequence analyses indicated that, while unique, the deduced p24 Gag amino acid sequence of the STLV-1 Tan 90 strain used for infection was still highly homologous to the HTLV-1 p24 Gag amino acids present in the ELISA and WB assays. However, a mutation in the pol sequence of STLV-1 Tan 90 encoded a putative stop codon, while a common deletion in the pol/rex regulatory gene causes significant changes in the Pol, and p27 Rex proteins. These same mutations were also observed in the viral DNA of both recipient infected tantalus monkeys and were not present in the STLV-1 Pat 74 strain. CONCLUSION: Our data suggest that seroconversion to STLV-1 infection may be prolonged due to the above mutations, and that compensatory molecular events must have occurred to allow for virus transmission.

Tropomyosin Isoform Diversity in the Cynomolgus Monkey Heart and Skeletal Muscles Compared to Human Tissues.

Old world monkeys separated from the great apes, including the ancestor of humans, about 25 million years ago, but most of the genes in humans and various nonhuman primates are quite similar even though their anatomical appearances are quite different. Like other mammals, primates have four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing. Only TPM1 produces two sarcomeric isoforms (TPM1α and TPM1κ), and TPM2, TPM3, and TPM4 each generate one sarcomeric isoform. We have cloned and sequenced TPM1α, TPM1κ, TPM2α, TPM3α, and TPM4α with RNA from cynomolgus (Cyn) monkey hearts and skeletal muscle. We believe this is the first report of directly cloning and sequencing of these monkey transcripts. In the Cyn monkey heart, the rank order of TPM isoform expression is TPM1α > TPM2α > TPM1κ > TPM3α > TPM4α. In the Cyn monkey skeletal muscle, the rank order of expression is TPM1α > TPM2α > TPM3α > TPM1κ > TPM4α. The major differences in the human heart are the increased expression of TPM1κ, although TPM1α is still the dominant transcript. In the Cyn monkey heart, the only sarcomeric TPM isoform at the protein level is TPM1α. This is in contrast to human hearts where TPM1α is the major sarcomeric isoform but a lower quantity of TPM1κ, TPM2α, and TPM3α is also detected at the protein level. These differences of tropomyosin and/or other cardiac protein expression in human and Cyn monkey hearts may reflect the differences in physiological activities in daily life.

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Inhibitors of the ubiquitin proteasome system block myofibril assembly in cardiomyocytes derived from chick embryos and human pluripotent stem cells.

Details of sarcomeric protein assembly during de novo myofibril formation closely resemble myofibrillogenesis in skeletal and cardiac myocytes in birds, rodents, and zebrafish. The arrangement of proteins during myofibrillogenesis follows a three-step process: beginning with premyofibrils, followed by nascent myofibrils, and concluding with mature myofibrils (reviewed in Sanger et al., 2017). Assembly and maintenance of myofibrils in living muscle cells. In: Handbook of experimental pharmacology, 2017 [pp. 39-75]. Our aim is to determine if the same pathway is followed in human cardiomyocytes derived from human inducible pluripotent stem cells. We found that the human cardiomyocytes developed patterns of protein organization identical to the three-step series seen in the model organisms cited above. Further experiments showed that myofibril assembly can be blocked at the nascent myofibril by five different inhibitors of the Ubiquitin Proteasome System (UPS) stage in both avian and human cardiomyocytes. With the exception of Carfilzomib, removal of the UPS inhibitors allows nascent myofibrils to proceed to mature myofibrils. Some proteasomal inhibitors, such as Bortezomib and Carfilzomib, used to treat multiple myeloma patients, have off-target effects of damage to hearts in three to 6 % of these patients. These cardiovascular adverse events may result from prevention of mature myofibril formation in the cardiomyocytes. In summary, our results support a common three-step model for the formation of myofibrils ranging from avian to human cardiomyocytes. The Ubiquitin Proteasome System is required for progression from nascent myofibrils to mature myofibrils. Our experiments suggest a possible explanation for the cardiac and skeletal muscle off-target effects reported in multiple myeloma patients treated with proteasome inhibitors.

Identification of a novel TPM4 isoform transcript and comparison to the expression of other tropomyosin isoforms in bovine cardiac and skeletal muscles.

In mammals, there are four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) each of which generate a multitude of alternatively spliced mRNAs. TPM isoform diversity in bovine unlike in humans are not well characterized. The purpose of this investigation is to perform an extensive analysis of the expression of both transcripts and corresponding protein of sarcomeric TPMs in bovine strated muscles. We have cloned and sequenced the transcripts of the sarcomeric isoform of the TPM4 gene designated as TPM4α as well as a new splice variant TPM4ε from bovine striated muscles. Additionally, we have determined the expression of various sarcomeric TPM isoforms and TPM4ε in bovine heart and skeletal muscles. Relative expression as well as absolute copy number determination by qRT-PCR suggests that TPM1α expression is significantly higher in bovine cardiac muscle, whereas TPM2α is higher in skeletal muscle. The relative expression of TPM3α in bovine heart and skeletal muscle is very similar. The relative expression of TPM4α and TPM4ε is higher in bovine heart and skeletal muscle, respectively. We have evaluated the protein expression levels of various TPM isoforms by 2D western blot analyses in commercially available protein extracts of heart and skeletal muscles with the CH1 monoclonal antibody against TPM. Protein from each CH1-positive spot was extracted for LC-MS/MS analyses, which show that bovine heart extract contains 91.66% TPM1 and 8.33% TPM2, whereas skeletal muscle extract contains 57% TPM1 and 42.87% TPM2. We have failed to detect the presence of unique peptide(s) for TPM3α, TPM4α, and TPM4ε.

Effect of MG-132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes.

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle.

TPM1kappa is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1kappa expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1kappa protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1kappa mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1kappa specific antibody, we demonstrated the expression and incorporation of TPM1kappa protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1kappa in myofibrillogenesis and sarcomeric function.

Sarcomeric TPM3 expression in human heart and skeletal muscle.

In mammals, four tropomyosin genes TPM1, TPM2, TPM3, and TPM4 are known. One isoform of the TPM3 gene, encoding 285 amino acid residues designated as TPM3α, has been reported. TPM3α protein expression in human hearts is not definitively established. We have cloned from human heart and skeletal muscle transcripts of TPM3α and three novel TPM3 isoforms, TPM3ν, TPM3ξ, and TPM3ο. TPM3ν and TPM3ο are alternatively spliced RNAs with different 3'-UTRs encoding an identical novel protein with 285 amino acid differing from TPM3α and TPM3ξ in exon 6 only. TPM3α and TPM3ξ, which have different 3'UTRs, also encode an identical protein. qRT-PCR data show that the transcripts of TPM3α, TPM3ν, TPM3ξ, and TPM3ο are expressed in both heart and skeletal muscle. We have evaluated the expression of various TPM proteins in fetal and adult human hearts, and also in skeletal muscle samples. Western blots using CG3 antibody show a stronger signal of TPM3 protein in fetal heart and adult skeletal muscle compared to adult heart. LC-MS/MS studies with the protein spots separated and identified by CH1 antibody after 2D Western blot analyses, confirm the expression of TPM3α/TPM3ξ in heart, but some peptides detected could be either TPM3α or TPM3ν. In heart samples, TPM1 protein was the dominant with varying amount of TPM2 and TPM3, while TPM4 expression was not observed. In skeletal muscles, TPM2 was the majority TPM protein expressed. The biological consequences of these varying expression of individual tropomyosin proteins are yet to be established.

Myofibril assembly and the roles of the ubiquitin proteasome system.

De novo assembly of myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in premyofibrils, followed by insertions of additional proteins and increased organization in nascent myofibrils, ending with mature contractile myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent myofibrils from transitioning to mature myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature myofibrils.

Myofibril-inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts.

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms 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, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.

The complete genomic sequence of an in vivo low replicating BLV strain.

DNA was extracted from lamb lymphocytes that were infected in vivo with a BLV strain after inoculation with the peripheral blood mononuclear cells from a persistently sero-indeterminate, low viral load, BLV-infected Holstein cow (No. 41) from Argentina. The DNA was PCR amplified with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 41 DNA was cloned, sequenced, and compared phylogenetically to other BLV sequences including an in vivo high replicating strain (BLV ARG 38) from the same herd in Argentina. Characterization of BLV ARG 41's deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Tropomyosin expression and dynamics in developing avian embryonic muscles.

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.

Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes.

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.

Qualitative and quantitative evaluation of TPM transcripts and proteins in developing striated chicken muscles indicate TPM4α is the major sarcomeric cardiac tropomyosin from early embryonic life to adulthood.

The chicken has been used since the 1980s as an animal model for developmental studies regarding tropomyosin isoform diversity in striated muscles, however, the pattern of expression of transcripts as well as the corresponding TPM proteins of various tropomyosin isoforms in avian hearts are not well documented. In this study, using conventional and qRT-PCR, we report the expression of transcripts for various sarcomeric TPM isoforms in striated muscles through development. Transcripts of both TPM1α and TPM1κ, the two sarcomeric isoforms of the TPM1 gene, are expressed in embryonic chicken hearts but disappear in post hatch stages. TPM1α transcripts are expressed in embryonic and adult skeletal muscle. The sarcomeric isoform of the TPM2 gene is expressed mostly in embryonic skeletal muscles. As reported earlier, TPM3α is expressed in embryonic heart and skeletal muscle but significantly lower in adult striated muscle. TPM4α transcripts are expressed from embryonic to adult chicken hearts but not in skeletal muscle. Our 2D Western blot analyses using CH1 monoclonal antibody followed by mass spectra evaluations found TPM4α protein is the major sarcomeric tropomysin expressed in embryonic chicken hearts. However, in 7-day-old embryonic hearts, a minute quantity of TPM1α or TPM1κ is also expressed. This finding suggests that sarcomeric TPM1 protein may play some important role in cardiac contractility and/or cardiac morphogenesis during embryogenesis. Since only the transcripts of TPM4α are expressed in adult chicken hearts, it is logical to presume that TPM4α is the only sarcomeric TPM protein produced in adult cardiac tissues.

Sarcomeric TPM3α in developing chicken.

Cloning and sequencing of various tropomyosin isoforms expressed in chickens have been described since the early 1980s. However, to the best of our knowledge, this is the first report on the molecular characterization and the expression of the sarcomeric isoform of the TPM3 gene in cardiac and skeletal muscles from developing as well as adult chickens. Expression of TPM3α was performed by conventional RT-PCR as well as qRT-PCR using relative expression (by ΔCT as well as ΔΔCT methods) and by determining absolute copy number. The results employing all these methods show that the expression level of TPM3α is maximum in embryonic (10-day/15-day old) skeletal muscle and can barely be detected in both cardiac and skeletal muscles from the adult chicken. Our various RT-PCR analyses suggest that the expression of high molecular weight TPM3 isoforms are regulated at the transcription level from the proximal promoter at the 5'-end of the TPM3 gene.

A reduction of tropomyosin limits development of sarcomeric structures in cardiac mutant hearts of the Mexican axolotl.

The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin, a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a later stage. The formation of functional sarcomeres coincided with contractions in normal hearts at stage 35. A-bands and I-bands were formed at stage 35 and did not change at stage 39. The widening of Z-bodies into z-lines was the main developmental difference between stage 35 and 39 normal hearts. Relative to normal hearts, a reduction of sarcomeric protein levels in mutant hearts at stage 35 was found, and a greater reduction occurred at later stages. The lower level of tropomyosin limited the areas where organized myofibrils formed in the mutant. The areas that had tropomyosin staining also had staining for alpha-actinin and myosin. Early myofibrils formed in these areas but the A-bands and I-bands were shorter than normal. At a later stage in the mutant, A-bands and I-bands remained shorter and importantly the Z-bodies also did not form wider z-lines.

Delayed Seroconversion to HTLV-II Is Associated with a Stop-Codon Mutation in the pol Gene.

A known HIV-1-positive intravenous drug user was found to be human T cell lymphoma/leukemia virus-II (HTLV-II) DNA positive by polymerase chain reaction but seronegative in a screening ELISA. He was consistently DNA positive but took 2 years to fully seroconvert. Sequencing of the HTLV-II strain in his cultured T lymphocytes indicated that it is a prototypical type A strain with no major differences in the long terminal repeat DNA sequence, nor major amino acid differences in the Gag, Env, Tax, and Rex proteins. However, a mutation in its pol gene created a stop codon at amino acid 543 of the Pol protein, a region that encodes for the RNase function. This mutation may account for the subject's slow seroconversion.

Expression of various sarcomeric tropomyosin isoforms in equine striated muscles.

In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.