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In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA

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Summary

A specific method for in situ-hybridization of slow myosin heavy chain MHCI (β-cardiac MHC) mRNA was established with the use of a nonradioactively labeled cRNA probe. The digoxigenin-labeled probe was the T7-RNA polymerase transcript from a 350 bp SacI fragment of a rabbit β-cardiac MHC cDNA. Northern blot analyses of RNA preparations from skeletal and cardiac muscles with homologous and complementary RNA proved the specificity of the hybridization. The in situ-hybridization was applied for studying the distribution of MHCI mRNA in normal fast- and slow-twitch muscles, as well as in muscles undergoing fast-to-slow transformation by chronic low-frequency stimulation. The majority of soleus muscle fibers was intensely stained, whereas fast-twitch muscles contained only a few positive fibers. The intracellular distribution of the hybridization product showed a clear relationship to the nuclei with intense staining of the perinuclear regions within the subsarcolemmal space. The more intensely stained fibers of transforming muscle displayed hybridization product also within the nuclei. As revealed by inspection of longitudinal sections at high magnification and polarized light, MHCI mRNA was also detectable in the myofibrils in a cross-striational pattern resulting from staining of the I-bands.

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References

  • Bandtlow CE, Heumann R, Schwab ME, Thoenen H (1987) Cellular localization of nerve growth factor synthesis by in situ hybridization. EMBO J 6:891–899

    Google Scholar 

  • Brahic M, Haase AT (1978) Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci USA 75:6125–6129

    Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Google Scholar 

  • Dix DJ, Eisenberg BR (1988a) Spatial distribution of myosin mRNA in cardiac tissue by in situ hybridization techniques. In: Clark WA, Decker RS, Borg TK (eds) Biology of isolated adult myocytes. Elsevier, Amsterdam New York, pp 147–160

    Google Scholar 

  • Dix DJ, Eisenberg BR (1988b) In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression. J Histochem Cytochem 36:1519–1526

    Google Scholar 

  • Fischman DA, Meltzer HY, Poppei RW (1973) The ultrastructure of human skeletal muscle: variations from archetypal morphology. In: Pearson CM, Mostofi FK (eds) The striated muscle. Williams & Wilkins, Baltimore, pp 58

    Google Scholar 

  • Hall ZW, Ralston E (1989) Nuclear domains in muscle cells. Cell 59:771–772

    Google Scholar 

  • Harris DA, Falls DL, Fischbach GD (1989) Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor. Nature 337:173–176

    Google Scholar 

  • Hood DA, Simoneau J-A (1989) Rapid isolation of total RNA from small mammal and human skeletal muscle. Am J Physiol 256:C1092-C1096

    Google Scholar 

  • Horne Z, Hesketh J (1990) Immunological localization of ribosomes in striated rat muscle. Evidence for myofibrillar association and ontological changes in the subsarcolemmal: myofibrillar distribution. Biochem J 268:231–236

    Google Scholar 

  • Huang WM, Gibson SJ, Facer P, Gu J, Polak JM (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers ofl-lysine as a slide coating. Histochemistry 77:275–279

    Google Scholar 

  • Jacoby J, Ko K, Weiss C, Rushbrook JI (1989) Systematic variation in myosin expression along extraocular muscle fibres of the adult rat. J Muscle Res Cell Motil 11:25–40

    Google Scholar 

  • Kirschbaum BJ, Heilig A, Härtner K-T, Pette D (1989) Electrostimulation-induced fast-to-slow transitions of myosin light and heavy chains in rabbit fast-twitch muscle at the mRNA level. FEBS Lett 243:123–126

    Google Scholar 

  • Leberer E, Pette D (1986) Immunochemical quantitation of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition. Eur J Biochem 156:489–496

    Google Scholar 

  • Lompré A-M, Nadal-Ginard B, Mahdavi V (1984) Expression of the cardiac ventricular α- and β-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem 259:6437–6446

    Google Scholar 

  • McNally EM, Kraft R, Bravo-Zehnder M, Taylor DA, Leinwand LA (1989) Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest molecular basis for functional differences. J Mol Biol 210:665–671

    Google Scholar 

  • Maier A, Gambke B, Pette D (1986) Degeneration-regeneration as a mechanism contributing to the fast to slow conversion of chronically stimulated fast-twitch rabbit muscle. Cell Tissue Res 244:635–643

    Google Scholar 

  • Maier A, Gorza L, Schiaffino S, Pette D (1988) A combined histochemical and immunohistochemical study on the dynamics of fast to slow fiber transformation in chronically stimulated rabbit muscle. Cell Tissue Res 254:59–68

    Google Scholar 

  • Pavlath GK, Rich K, Webster SG, Blau HM (1989) Localization of muscle gene products in nuclear domains. Nature 337:570–573

    Google Scholar 

  • Pette D, Smith ME, Staudte HW, Vrbová G (1973) Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. Pflügers Arch 338:257–272

    Google Scholar 

  • Sassoon DA, Garner I, Buckingham M (1988) Transcripts of α-cardiac and α-skeletal actins are early markers for myogenesis in the mouse embryo. Development 104:155–164

    Google Scholar 

  • Schiaffino S, Gorza L, Pitton G, Saggin L, Ausoni S, Sartore S, Lömo T (1988) Embryonic and neonatal myosin heavy chain in denervated and paralysed rat skeletal muscle. Dev Biol 127:1–11

    Google Scholar 

  • Schwarz G, Leisner E, Pette D (1983) Two telestimulation systems for chronic indirect muscle stimulation in caged rabbits and mice. Pflügers Arch 398:130–133

    Google Scholar 

  • Singer RH, Ward DC (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinylated nucleotide analog. Proc Natl Acad Sci USA 79:7331–7335

    Google Scholar 

  • Staron RS, Pette D (1987) Nonuniform myosin expression along single fibers of chronically stimulated and contralateral rabbit tibialis anterior muscles. Pflügers Arch 409:67–73

    Google Scholar 

  • Staron RS, Gohlsch B, Pette D (1987) Myosin polymorphism in single fibers of chronically stimulated rabbit fast-twitch muscle. Pflügers Arch 408:444–450

    Google Scholar 

  • Stockdale FE, Miller JB (1987) The cellular basis of myosin heavy chain isoform expression during development of avian skeletal muscles. Dev Biol 123:1–9

    Google Scholar 

  • Varndell IM, Polak JM, Sikri KL, Minth CD, Bloom SR, Dixon JE (1984) Visualisation of messenger RNA directing peptide synthesis by in situ hybridisation using a novel single-stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity. Histochemistry 81:597–601

    Google Scholar 

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Aigner, S., Pette, D. In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. Histochemistry 95, 11–18 (1990). https://doi.org/10.1007/BF00737222

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