Expression of Multiple Subtypes of Muscarinic Receptors and Cellular Distribution in the Human Heart

  1. Huizhen Wang1,
  2. Hong Han1,
  3. Liming Zhang1,
  4. Hong Shi1,
  5. Gernot Schram1,
  6. Stanley Nattel1,2,3 and
  7. Zhiguo Wang1,2
  1. 1Research Center, Montreal Heart Institute, Montreal, Quebec, Canada (H.W., H.H., L.Z., H.S., G.S., S.N., Z.W.); 2Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada (S.N., Z.W.); and 3Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada (S.N.)

    Abstract

    Five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified by molecular cloning and designated m1–m5, of which four correspond to the functional subtypes M1, M2, M3, and M4 in primary tissues. The presence of M5receptors in tissues remains uncertain. The present study was designed to explore the diversity and cellular distribution of various mAChR subtypes in human hearts. Competition binding of [N-methyl-3H]-scopolamine methyl chloride with various mAChR antagonists yielded data consistent with the presence of multiple subtypes (M1/M2/M3/M5) of mAChRs in both human atrial (HA) and ventricular (HV) tissues. Expression of mRNAs encoding all five subtypes was readily detected by reverse transcription-polymerase chain reaction in both HA and HV samples. Immunoblotting with subtype-specific antibodies confirmed the presence of M1, M2, M3, and M5, but not M4, proteins in membrane preparations from both HA and HV. The protein levels of M1 and M2 were comparable between HA and HV. Although the density of M3appeared ∼10-fold higher in HV than HA, that of M5 was ∼5 times lower in HV than in HA. Positive immunostaining of single ventricular myocytes by M1, M2, M3, and M5 antibodies, respectively, was consistently detected. Under confocal microscopy, M5 showed characteristic localization to the intercalated discs, whereas other subtypes were more evenly distributed throughout the surface membrane. Our results provide the first molecular evidence for the presence of multiple subtypes of mAChR, including endogenous M5 receptors, in human hearts and suggest that different subtypes have different tissue distributions and cellular localization.

    Footnotes

    • Send reprint requests to: Zhiguo Wang, Ph.D, Research Center, Montreal Heart Institute, 5000 Bélanger St. East, Montreal, Quebec, Canada H1T 1C8. E-mail:wangz{at}icm.umontreal.ca

    • This work was supported in part by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, an Establishment Grant for young investigators from the Fonds de Recherche en Santé du Québec awarded to Dr. Wang, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Z.W. is a Research Scholar of the Heart and Stroke Foundation of Canada. H.W. and H.S. are Research Fellows of the Medical Research Council of Canada. H.H. is a Research Fellow of the Heart and Stroke Foundation of Canada.

    • Abbreviations:
      mAChR
      muscarinic acetylcholine receptor
      RT
      reverse transcription
      PCR
      polymerase chain reaction
      HA
      human atrium
      HV
      human ventricle
      PBS
      phosphate-buffered saline
      [3H]NMS
      [N-methyl-3H]scopolamine methyl chloride
      4-DAMP
      4-diphenylacetoxy-N-methylpiperidine methiodide
      bp
      base pair(s)
      HHSiD
      hexahydro-siladifenidol
      • Received August 22, 2000.
      • Accepted January 18, 2001.
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    1. Molecular Pharmacology May 1, 2001 vol. 59 no. 5 1029-1036
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