Expression studies of neogenin and its ligand hemojuvelin in mouse tissues.

Juvenile hemochromatosis is a severe hereditary iron overload disease caused by mutations in the HJV (hemojuvelin) and HAMP (hepcidin) genes. Hepcidin is an important iron regulatory hormone, and hemojuvelin may regulate hepcidin synthesis via the multifunctional membrane receptor neogenin. We explored the expression of murine hemojuvelin and neogenin mRNAs and protein. Real-time RT-PCR analysis of 18 tissues from male and female mice was performed to examine the mRNA expression profiles. To further study protein expression and localization we used immunohistochemistry on several tissues from three mouse strains. Mouse Neo1 mRNA was detectable in the 18 tissues tested, the highest signals being evident in the ovary, uterus, and testis. Neogenin protein was observed in the brain, skeletal muscle, heart, liver, stomach, duodenum, ileum, colon, renal cortex, lung, testis, ovary, oviduct, and uterus. The spleen, thymus, and pancreas were negative for neogenin. The highest signals for Hjv mRNA were detectable in the skeletal muscle, heart, esophagus, and liver. The results indicate that Neo1 mRNA is widely expressed in both male and female mouse tissues with the highest signals detected in the reproductive system. Moreover, Hjv and Neo1 mRNAs are simultaneously expressed in skeletal muscle, heart, esophagus, and liver.     

Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system

LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development, in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in developing outflow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development. Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease.     

Troponin I switching in the developing heart

Monoclonal antibodies identify two distinct isoforms of troponin I in rat cardiac muscle, one predominant in the embryonic and fetal heart and one predominant in the adult heart. The two isoforms can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 27,000 and 31,500, respectively. The adult isoform is specifically recognized by a monoclonal antibody that is unreactive with the embryonic variant, while two other monoclonal antibodies recognize both isoforms. A monoclonal antibody to cardiac troponin T was used to isolate by affinity chromatography the troponin complex from adult and neonatal rat heart. Affinity purified troponin from neonatal heart was found to contain both the embryonic and adult isoforms of troponin I. Comparative immunoblotting analysis with different muscle tissues shows that embryonic troponin I is identical with respect to electrophoretic mobility and pattern of immunoreactivity to the major troponin I isoform found in adult slow skeletal muscle. Troponin I switching may be implicated in developmental changes involving Ca2+ and pH sensitivity of the contractile system and response to beta-adrenergic stimulation.     

Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.