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ISSN 2522-9028 (Print)
ISSN 2522-9036 (Online)
DOI: https://doi.org/10.15407/fz

Fiziologichnyi Zhurnal

is a scientific journal issued by the

Bogomoletz Institute of Physiology
National Academy of Sciences of Ukraine

Editor-in-chief: V.F. Sagach

The journal was founded in 1955 as
1955 – 1977 "Fiziolohichnyi zhurnal" (ISSN 0015 – 3311)
1978 – 1993 "Fiziologicheskii zhurnal" (ISSN 0201 – 8489)
1994 – 2016 "Fiziolohichnyi zhurnal" (ISSN 0201 – 8489)
2017 – "Fiziolohichnyi zhurnal" (ISSN 2522-9028)

Fiziol. Zh. 2018; 64(2): 26-33

OPTIMIZATION OF THE METHOD OF NUCLEI ISOLATION FOR ELECTROPHYSIOLOGICAL STUDIES OF ION CHANNELS IN THE NUCLEAR MEMBRANE OF THE RAT CARDIOMYOCYTES

O.A. Kotyk, A.B. Kotliarova, S.M. Marchenko

    O.O. Bogomoletz Institute of Physiology National Academy of Sciences of Ukraine, Kyiv, Ukraine
DOI: https://doi.org/10.15407/fz64.02.026


Abstract

The conditions for the nuclei isolation from cardiomyocytes for electrophysiological studies of ion channels in the native nuclear membrane were optimized. The structural and functional intactness of isolated nuclei is crucial for investigation of the biophysical properties of the transport systems in the native nuclear membrane. The quality of the obtained sample was evaluated by ion currents recording through the nuclear membrane in nucleus-attached or excised patch configuration at voltage-clamp mode of patch-clamp technique. In our study the solutions with different compositions were tested and the quality of the samples obtained using different methods of myocardium homogenization were compared. We found that the activity of ion channels in the nuclear membrane were most often registered when myocardium was homogenized in solution with the next composition (mmol/l): sucrose – 300, KCl – 60, HEPES – 10 (pH 7,2) with protease inhibitors cocktail, DL-dithiothreitol (1 mmol/l) and glycerol, using glass homogenizer (Dounce).

Keywords: nuclear envelope; cardiomyocytes; nuclear fraction isolation; native membrane; patch-clamp; ion channels

Full text: (PDF, in Ukrainian)

References

  1. Mazzanti M, Bustamante JO, Oberleithner H. Electrical dimension of the nuclear envelope. Physiol Rev. 2001; 81 (1): 1-19. doi.org/10.1152/physrev.2001.81.1.1. CrossRef  
  2. Baumann O, Walz B. Endoplasmic reticulum of animal cells and its organization into structural and functional domains. Int Rev Cytol. 2001; 205: 149-214. CrossRef  
  3. Arantes LAM, Aguiar CJ, Amaya MJ, Figueiro NCG, Andrade LM, Rocha-Resende C, et al. Nuclear inositol 1,4,5-trisphosphate is a necessary and conserved signal for the induction of both pathological and physiological cardiomyocyte hypertrophy. J Mol Cell Cardiol. 2012; 53 (4): 475-86. doi.org/10.1016/j.yjmcc.2012.06.017. CrossRef  
  4. Zima AV, Bare DJ, Mignery GA, Blatter LA. IP3- dependent nuclear Ca2+ signalling in the mammalian heart. J Physiol. 2007; 584 (2): 601-11. doi.org/10.1113/ jphysiol.2007.140731.
    5.  
  5. Bare DJ, Kettlun CS, Liang M, Bers DM, Mignery GA. Cardiac type 2 inositol 1,4,5-trisphosphate receptor Interaction and modulation by calcium/calmodulindependent protein kinase II. J Biol Chem. 2005; 280 (16): 15912-20. doi.org/10.1074/jbc.M414212200. CrossRef  
  6. Marchenko SM, Yarotskyy VV, Kovalenko TN, Kostyuk PG, Thomas RC. Spontaneously active and InsP3-activated ion channels in cell nuclei from rat cerebellar Purkinje and granule neurones. J Physiol. 2005; 565 (3): 897-910. doi. org/10.1113/jphysiol.2004.081299. CrossRef  
  7. Tabares L, Mazzanti M, Clapham DE. Chloride channels in the nuclear membrane. J Membr Biol. 1991; 123 (1): 49-54. CrossRef PubMed
    8.  
  8. Valenzuela SM, Martin DK, Por SB, Robbins JM, Warton K, Bootcov MR, et al. Molecular cloning and expression of a chloride ion channel of cell nuclei. J Biol Chem. 1997; 272 (19): 12575-82. CrossRef PubMed
    9.  
  9. Nabbi A, Riabowol K. Isolation of Nuclei. Cold Spring Harb Protoc. 2015; (8): 731-4. doi:10.1101/pdb. top074583.
    10.  
  10. Fedorenko OA, Marchenko SM. Ion channels of the nuclear membrane of hippocampal neurons. Hippocampus. 2014; 24 (7): 869-76. doi.org/10.1002/hipo.22276. CrossRef  
  11. Bergmann O, Jovinge S. Isolation of cardiomyocyte nuclei from post-mortem tissue. J Vis Exp. 2012 (65): 6. doi. org/10.3791/4205. CrossRef  
  12. Betzenhauser MJ, Wagner LE, II, Park HS, Yule DI. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs. J Biol Chem. 2009; 284 (24): 16156-63. doi. org/10.1074%2Fjbc.M109.006452.
    13.  
  13. Matzke AJM, Weiger TM, Matzke M. Ion channels at the nucleus: electrophysiology meets the genome. Mol Plant. 2010; 3 (4): 642-52. doi.org/10.1093/mp/ssq013. CrossRef  
  14. Marchenko SM, Thomas RC. Nuclear Ca2+ signalling in cerebellar Purkinje neurons. Cerebellum. 2006; 5 (1): 36-42. doi.org/10.1080/14734220600554438. CrossRef  
  15. Fedorenko OA, Marchenko SM. Spontaniously active ion cannels of the nuclear envelope membrane. Fiziol Zh. 2010; 56 (5): 95-105 [Ukrainian]. PubMed
    16.  
  16. Wilkie GS, Schirmer EC. Purification of nuclei and preparation of nuclear envelopes from skeletal muscle. Methods Mol Biol. 2008; 463: 23-41. doi.org/10.1007/978- 1-59745-406-3_2.
    17.  
  17. Brewer O. Isolation of myogenic nuclei from whole muscle tissue. Undergraduate Honors Theses. 2015; 954.
    18.  
  18. Ikeda Y, Nakamura T, Takano H, Kimura H, Obata JE, Takeda S, et al. Angiotensin II-induced cardiomyocyte hypertrophy and cardiac fibrosis in stroke-prone spontaneously hypertensive rats. J Lab Clin Med. 2000; 135 (4): 353-9. doi.org/10.1067/mlc.2000.105617. CrossRef  
  19. Mak D-OD, Vais H, Cheung K-H, Foskett JK. Isolating nuclei from cultured cells for patch-clamp electrophysiology of intracellular Ca2+ channels. Cold Spring Harb Protoc. 2013; (9): 880-4. doi.org/10.1101%2Fpdb.prot073056.
    20.  
  20. Tirziu D, Giordano FJ, Simons M. Cell communications in the heart. Circulation. 2010; 122 (9): 928-37. doi. org/10.1161/CIRCULATIONAHA.108.847731. CrossRef  
  21. Banerjee I, Fuseler JW, Price RL, Borg TK, Baudino TA. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse. Am J Physiol Heart Circ Physiol. 2007; 293 (3): H1883-H91. doi.org/10.1152/ajpheart.00514.2007. CrossRef  
  22. Zhou PZ, Pu WT. Recounting Cardiac Cellular Composition. Circulation Research. 2016;118(3):368-70. CrossRef PubMed PubMedCentral
    23.  

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