Sharon L. Lemanski

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse α‐tropomyosin cDNA under the control of a cardiac‐specific α‐myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well‐organized sarcomeric myofibrils. Transfection of a β‐tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a β‐galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of α‐tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412–420.

Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart.

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an Important role in maintaining myofibril alignment in cardiac cells.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol‐located and mitochondria‐enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications. J. Cell. Biochem. 111: 94–103, 2010.

A point mutation in bioactive RNA results in the failure of mutant heart correction in mexican axolotls

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. The defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or endoderm/mesoderm-conditioned medium. By screening a cDNA library made of total conditioned medium RNA from normal axolotl embryonic endoderm, we isolated a single clone (MIR), the synthetic RNA from which corrects the mutant heart defect by promoting myofibrillogenesis and thus was named MIR (myofibrillogenesis inducing RNA). In the present study, we have examined MIR gene expression in mutant axolotl hearts at early pre-heart-beat developmental stages and found its quantitative expression, as detected by RT-PCR, to be the same as in normal hearts. However, careful analysis of sequence data revealed a G➙U point mutation in the mutant MIR RNA. Further computational analyses, using GENEBEE software to compare normal and mutant MIR RNAs show a significant alteration in RNA secondary structure of the point-mutated MIR RNA. The results from bioassay and confocal microscopy immunofluorescent studies demonstrate that, unlike MIR RNA derived from normal embryos, the mutated MIR RNA does not promote myofibrillogenesis in mutant embryonic hearts and fails to rescue/correct the mutant heart defect.

Expression of axolotl RNA-binding protein during development of the Mexican axolotl

Abstract Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10–12 (gastrula), reaches a maxima around stage 15–20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full‐length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development. J. Cell. Biochem. 100: 1–15, 2007.

Anoxia, acidosis, and intergenic interactions selectively regulate methionine sulfoxide reductase transcriptions in mouse embryonic stem cells.

Methionine sulfoxide reductases (Msr) belong to a gene family that contains one MsrA and three MsrBs (MsrB1, MsrB2, and MsrB3). We have identified all four of the genes that are expressed in mouse embryonic stem cell cultures. The vital cellular functions of the Msr family of genes are to protect cells from oxidative damage by enzymatically reducing the oxidized sulfide groups of methionine residues in proteins from the sulfoxide form (SO) back to sulfide thus restoring normal protein functions as well as reducing intracellular reactive oxygen species (ROS). We have performed studies on the Msr family genes to examine the regulation of gene expression. Our studies using real‐time RT‐PCR and Western blotting have shown that expression levels of the four Msr family genes are under differential regulation by anoxia/reoxygenation treatment, acidic culture conditions and interactions between MsrA and MsrB. Results from these in vitro experiments suggest that although these genes function as a whole in oxidative stress protection, each one of the Msr genes could be responsive to environmental stimulants differently at the tissue level. J. Cell. Biochem. 112: 98–106, 2011.

Creation of chimeric mutant axolotls: a model to study early embryonic heart development in Mexican axolotls

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development since it carries a cardiac lethal mutation in gene c that results in failure of contraction of mutant embryonic myocardium. In cardiac mutant axolotls (c/c) the hearts do not beat, apparently because of an absence of organized myofibrils. To date, there has been no way to analyze the genotypes of embryos from heterozygous spawnings (+/c×+/c) until stage 35 when the normal (+/c or +/+) embryos first begin to have beating hearts; mutant (c/c) embryos fail to develop normal heartbeats. In the present study, we created chimeric axolotls by using microsurgical techniques. The general approach was to transect tailbud embryos and join the anterior and posterior halves of two different individuals. The chimeric axolotl is composed of a normal head and heart region (+/+), permitting survival and a mutant body containing mutant gonads (c/c) that permits the production of c/c mutant offspring: 100% c/c offspring were obtained by mating c/c chimeras (c/c×c/c). The mutant phenotypes were confirmed by the absence of beating hearts and death at stage 41 in 100% of the embryos. Examination of the mutant hearts with electron microscopy and comfocal microscopy after immunofluorescent staining for tropomyosin showed identical images to those described previously in naturally-occurring c/c mutant axolotls (i.e., lacking organized sarcomeric myofibrils). These ”c/c chimeric” axolotls provide a useful and unique way to investigate early embryonic heart development in cardiac mutant Mexican axolotls.

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development.

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.

Cellular, Molecular, and Developmental Studies on Heart Development in Normal and Cardiac Mutant Axolotls, Ambystoma mexicanum

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development because it carries a simple recessive cardiac lethal mutation that results in a failure of the mutant embryonic myocardium to contract. In cardiac mutant axolotls, the hearts do not beat, apparently due to an absence of organized myofibrils. The mutant hearts can be rescued by coculturing them with normal anterior endoderm/mesoderm tissue, by a medium conditioned with normal anterior endoderm/mesoderm, or by an RNA isolated from the conditioned medium. We have previously isolated a single cDNA clone from a library prepared with total RNA from conditioned medium; this 166-nt-long in vitro synthesized RNA, directed by the unique cDNA clone (Clone #4), has the ability to correct the heart defect and promote myofibrillogenesis in mutant hearts. The criteria for rescue include contraction of mutant hearts throughout their lengths, an increase in sarcomeric tropomyosin arrays as shown by immunofluorescent confocal microscopy, and the ultrastructural appearance of organized sarcomeric myofibrils. More recently, we carried out RT-PCR with total RNAs extracted from normal or mutant axolotl embryos. A point mutation (G-T) was found in Clone #4 RNA derived from the mutant embryos at stages 20 and 30 as compared to normal axolotls at the same stages. Furthermore, we searched for the full-length Clone #4 gene in an axolotl genomic library. Primer extension yielded a product of —500 by at the 5’ end of the Clone #4 gene. The nucleotide sequence of the extended Clone #4 (Ext-Clone #4) was determined and was found to be unique because there was no significant homology with other known sequences available from the gene databases. Interestingly, T7 sense RNA from the Ext-Clone #4 (-500-nt RNA) showed a higher efficiency in rescuing mutant hearts than the T7 RNA from original Clone #4 (166-nt RNA). Our working hypothesis is that the bioactive RNA is a regulatory RNA that may directly or indirectly upregulate tropomyosin production in the axolotl heart.