Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jun 15;17(12):3309–3316. doi: 10.1093/emboj/17.12.3309

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

H Takeshima 1, S Komazaki 1, K Hirose 1, M Nishi 1, T Noda 1, M Iino 1
PMCID: PMC1170669  PMID: 9628868

Abstract

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

Full Text

The Full Text of this article is available as a PDF (712.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  2. Bertocchini F., Ovitt C. E., Conti A., Barone V., Schöler H. R., Bottinelli R., Reggiani C., Sorrentino V. Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles. EMBO J. 1997 Dec 1;16(23):6956–6963. doi: 10.1093/emboj/16.23.6956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhat M. B., Zhao J., Takeshima H., Ma J. Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor. Biophys J. 1997 Sep;73(3):1329–1336. doi: 10.1016/S0006-3495(97)78166-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cannell M. B., Cheng H., Lederer W. J. The control of calcium release in heart muscle. Science. 1995 May 19;268(5213):1045–1049. doi: 10.1126/science.7754384. [DOI] [PubMed] [Google Scholar]
  5. Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev. 1977 Jan;57(1):71–108. doi: 10.1152/physrev.1977.57.1.71. [DOI] [PubMed] [Google Scholar]
  6. Fabiato A., Fabiato F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat, and frog hearts and from fetal and new-born rat ventricles. Ann N Y Acad Sci. 1978 Apr 28;307:491–522. doi: 10.1111/j.1749-6632.1978.tb41979.x. [DOI] [PubMed] [Google Scholar]
  7. Fabiato A. Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol. 1985 Feb;85(2):291–320. doi: 10.1085/jgp.85.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fleischer S., Inui M. Biochemistry and biophysics of excitation-contraction coupling. Annu Rev Biophys Biophys Chem. 1989;18:333–364. doi: 10.1146/annurev.bb.18.060189.002001. [DOI] [PubMed] [Google Scholar]
  9. Flucher B. E. Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad. Dev Biol. 1992 Dec;154(2):245–260. doi: 10.1016/0012-1606(92)90065-o. [DOI] [PubMed] [Google Scholar]
  10. Franzini-Armstrong C., Jorgensen A. O. Structure and development of E-C coupling units in skeletal muscle. Annu Rev Physiol. 1994;56:509–534. doi: 10.1146/annurev.ph.56.030194.002453. [DOI] [PubMed] [Google Scholar]
  11. Giannini G., Conti A., Mammarella S., Scrobogna M., Sorrentino V. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J Cell Biol. 1995 Mar;128(5):893–904. doi: 10.1083/jcb.128.5.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ikemoto T., Komazaki S., Takeshima H., Nishi M., Noda T., Iino M., Endo M. Functional and morphological features of skeletal muscle from mutant mice lacking both type 1 and type 3 ryanodine receptors. J Physiol. 1997 Jun 1;501(Pt 2):305–312. doi: 10.1111/j.1469-7793.1997.305bn.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobson M. D., Weil M., Raff M. C. Programmed cell death in animal development. Cell. 1997 Feb 7;88(3):347–354. doi: 10.1016/s0092-8674(00)81873-5. [DOI] [PubMed] [Google Scholar]
  14. Klitzner T. S., Friedman W. F. A diminished role for the sarcoplasmic reticulum in newborn myocardial contraction: effects of ryanodine. Pediatr Res. 1989 Aug;26(2):98–101. doi: 10.1203/00006450-198908000-00005. [DOI] [PubMed] [Google Scholar]
  15. Komazaki S., Hiruma T. Calcium-containing vacuolated mitochondria during early heart development in chick embryos as demonstrated by cytochemistry and X-ray microanalysis. Anat Embryol (Berl) 1994 May;189(5):441–446. doi: 10.1007/BF00185439. [DOI] [PubMed] [Google Scholar]
  16. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  17. López-López J. R., Shacklock P. S., Balke C. W., Wier W. G. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science. 1995 May 19;268(5213):1042–1045. doi: 10.1126/science.7754383. [DOI] [PubMed] [Google Scholar]
  18. McGraw C. F., Somlyo A. V., Blaustein M. P. Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis. J Cell Biol. 1980 May;85(2):228–241. doi: 10.1083/jcb.85.2.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meissner G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu Rev Physiol. 1994;56:485–508. doi: 10.1146/annurev.ph.56.030194.002413. [DOI] [PubMed] [Google Scholar]
  20. Nakai J., Imagawa T., Hakamat Y., Shigekawa M., Takeshima H., Numa S. Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel. FEBS Lett. 1990 Oct 1;271(1-2):169–177. doi: 10.1016/0014-5793(90)80399-4. [DOI] [PubMed] [Google Scholar]
  21. Näbauer M., Callewaert G., Cleemann L., Morad M. Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science. 1989 May 19;244(4906):800–803. doi: 10.1126/science.2543067. [DOI] [PubMed] [Google Scholar]
  22. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shibata H., Toyama K., Shioya H., Ito M., Hirota M., Hasegawa S., Matsumoto H., Takano H., Akiyama T., Toyoshima K. Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science. 1997 Oct 3;278(5335):120–123. doi: 10.1126/science.278.5335.120. [DOI] [PubMed] [Google Scholar]
  24. Sitsapesan R., Williams A. J. Regulation of current flow through ryanodine receptors by luminal Ca2+. J Membr Biol. 1997 Oct 1;159(3):179–185. doi: 10.1007/s002329900281. [DOI] [PubMed] [Google Scholar]
  25. Sun X. H., Protasi F., Takahashi M., Takeshima H., Ferguson D. G., Franzini-Armstrong C. Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle. J Cell Biol. 1995 May;129(3):659–671. doi: 10.1083/jcb.129.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Takeshima H., Iino M., Takekura H., Nishi M., Kuno J., Minowa O., Takano H., Noda T. Excitation-contraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine-receptor gene. Nature. 1994 Jun 16;369(6481):556–559. doi: 10.1038/369556a0. [DOI] [PubMed] [Google Scholar]
  27. Takeshima H., Ikemoto T., Nishi M., Nishiyama N., Shimuta M., Sugitani Y., Kuno J., Saito I., Saito H., Endo M. Generation and characterization of mutant mice lacking ryanodine receptor type 3. J Biol Chem. 1996 Aug 16;271(33):19649–19652. doi: 10.1074/jbc.271.33.19649. [DOI] [PubMed] [Google Scholar]
  28. Takeshima H., Nishimura S., Matsumoto T., Ishida H., Kangawa K., Minamino N., Matsuo H., Ueda M., Hanaoka M., Hirose T. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 1989 Jun 8;339(6224):439–445. doi: 10.1038/339439a0. [DOI] [PubMed] [Google Scholar]
  29. Yamazawa T., Takeshima H., Sakurai T., Endo M., Iino M. Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling. EMBO J. 1996 Nov 15;15(22):6172–6177. [PMC free article] [PubMed] [Google Scholar]
  30. Yamazawa T., Takeshima H., Shimuta M., Iino M. A region of the ryanodine receptor critical for excitation-contraction coupling in skeletal muscle. J Biol Chem. 1997 Mar 28;272(13):8161–8164. doi: 10.1074/jbc.272.13.8161. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES