Name: functional characterization of endogenously expressed human ryr1 variants
Uploaded: 2021-06-09T13:45:00
Description: Watch this Scientific Journal Video about Functional Characterization of Endogenously Expressed Human RYR1 Variants at JoVE.com

The work described in this manuscript was supported by grants from the Swiss National Science Foundation (SNF) and the Swiss Muscle Foundation.

The protocols described below comply with the ethics guidelines of the Ethikkommission Nordwest- und Zentralschweiz EKNZ.
1. Preparation of Epstein Barr immortalized B-lymphocyte cell lines11
<ol>
	<li>After informed consent, collect 30 mL of whole blood in EDTA-treated sterile tubes from the proband carrying a RYR1 mutation and from healthy family members with no mutation. 
	NOTE: Keep all solutions sterile and work in a tissue culture hood.</li>
	<li>Isolate mo...

<ol>
	<li>Dlamini, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+RYR1+are+a+common+cause+of+exertional+myalgia+and+rhabdomyolysis.">Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.</a> Neuromuscular Disorders. 23 (7), 540-548 (2013).</li><li>Klein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+genetic+findings+in+a+large+cohort+of+patients+with+ryanodine+receptor+1+gene-associated+myopathies.">Clinical and genetic findings in a large cohort of patients with ryanodine receptor 1 gene-associated myopathies.</a> Human Mutation. 33, 981-988 (2012).</li><li>Robinson, R., Carpenter, D., Shaw, M. A., Halsall, J., Hopkins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+RYR1+in+malignant+hyperthermia+and+central+core+disease.">Mutations in RYR1 in malignant hyperthermia and central core disease.</a> Human Mutation. 27 (20), 977-989 (2006).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++mediated+activation+of+the+skeletal+muscle+ryanodine+receptor+ion+channel.">Ca2+ mediated activation of the skeletal muscle ryanodine receptor ion channel.</a> Journal of Biological Chemistry. 293 (50), 19501-19509 (2018).</li><li>Treves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+intracellular+Ca2++transients+in+COS-7+cells+transfected+with+the+cDNA+encoding+skeletal-muscle+ryanodine+receptor+carrying+a+mutation+associated+with+malignant+hyperthermia.">Alteration of intracellular Ca2+ transients in COS-7 cells transfected with the cDNA encoding skeletal-muscle ryanodine receptor carrying a mutation associated with malignant hyperthermia.</a> Biochemical Journal. 301 (3), 661-665 (1994).</li><li>Nakai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dihydropyridine+receptor+channel+activity+in+the+presence+of+ryanodine+receptor.">Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor.</a> Nature. 389 (6569), 72-75 (1996).</li><li>Durham, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RyR1+S-nitrosylation+underlies+environmental+heat+stroke+and+sudden+death+in+Y522S+RyR1+knockin+mice.">RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice.</a> Cell. 133 (81), 53-65 (2008).</li><li>Zvaritch, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++dysregulation+in+Ryr1(I4895T/wt)+mice+causes+congenital+myopathy+with+progressive+formation+of+minicores,+cores,+and+nemaline+rods.">Ca2+ dysregulation in Ryr1(I4895T/wt) mice causes congenital myopathy with progressive formation of minicores, cores, and nemaline rods.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (51), 21813-21818 (2009).</li><li>Elbaz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-allelic+expression+of+the+RyR1+p.A4329D+mutation+decreases+muscle+strength+in+slow-twitch+muscles+in+mice.">Bi-allelic expression of the RyR1 p.A4329D mutation decreases muscle strength in slow-twitch muscles in mice.</a> Journal of Biological Chemistry. 295, 10331-10339 (2020).</li><li>Censier, K., Urwyler, A., Zorzato, F., Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+homeostasis+in+human+primary+muscle+cells+from+malignant+hyperthermia+susceptible+and+normal+individuals.">Intracellular calcium homeostasis in human primary muscle cells from malignant hyperthermia susceptible and normal individuals.</a> Journal of Clinical Investigations. 101 (6), 1233-1242 (1998).</li><li>Girard, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-lymphocytes+from+Malignant+Hyperthermia-Susceptible+patients+have+an+increased+sensitivity+to+skeletal+muscle+ryanodine+receptor+activators.">B-lymphocytes from Malignant Hyperthermia-Susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators.</a> Journal of Biological Chemistry. 276 (51), 48077 (2001).</li><li>Ducreux, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ryanodine+receptor+mutations+on+interleukin-6+release+and+intracellular+calcium+homeostasis+in+human+myotubes+from+Malignant+Hyperthermia-+Susceptible+individuals+and+patients+affected+by+Central+Core+Disease.">Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from Malignant Hyperthermia- Susceptible individuals and patients affected by Central Core Disease.</a> Journal of Biological Chemistry. 279 (42), 43838-43846 (2004).</li><li>Ducreux, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+ryanodine+receptors+carrying+three+amino+acid+substitutions+identified+in+patients+affected+by+multi-minicore+disease+and+central+core+disease,+expressed+in+immortalized+lymphocytes.">Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes.</a> Biochemical Journal. 395, 259-266 (2006).</li><li>Tilgen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+four+novel+mutations+in+the+C-terminal+membrane+spanning+domain+of+the+ryanodine+receptor+1:+association+with+central+core+disease+and+alteration+of+calcium+homeostasis.">Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis.</a> Human Molecular Genetics. 10 (25), 2879-2887 (2001).</li><li>Treves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitation-coupled+Ca2++entry+induces+nuclear+translocation+of+NFAT+and+contributes+to+IL-6+release+from+myotubes+from+patients+with+central+core+disease.">Enhanced excitation-coupled Ca2+ entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease.</a> Human Molecular Genetics. 20 (3), 589-600 (2011).</li><li>Yasuda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JP-45/JSRP1+variants+affect+skeletal+muscle+excitation+contraction+coupling+by+decreasing+the+sensitivity+of+the+dihydropyridine+receptor.">JP-45/JSRP1 variants affect skeletal muscle excitation contraction coupling by decreasing the sensitivity of the dihydropyridine receptor.</a> Human Mutation. 34, 184-190 (2013).</li><li>Sei, Y., Gallagher, K. L., Basile, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+ryanodine+receptor+is+involved+in+calcium+signaling+in+human+B+lymphocytes.">Skeletal muscle ryanodine receptor is involved in calcium signaling in human B lymphocytes.</a> Journal of Biological Chemistry. 274 (9), 5995-6062 (1999).</li><li>Tegazzin, V., Scutari, E., Treves, S., Zorzato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorocresol,+an+additive+to+commercial+succinylcholine,+induces+contracture+of+human+malignant+Hyperthermia+Susceptible+muscles+via+activation+of+the+ryanodine+receptor+Ca2++channel.">Chlorocresol, an additive to commercial succinylcholine, induces contracture of human malignant Hyperthermia Susceptible muscles via activation of the ryanodine receptor Ca2+ channel.</a> Anesthesiology. 84, 1275-1279 (1996).</li><li>Kushnir, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+receptor+calcium+leak+in+circulating+B-lymphocytes+as+a+biomarker+for+heart+failure.">Ryanodine receptor calcium leak in circulating B-lymphocytes as a biomarker for heart failure.</a> Circulation. 138 (11), 1144-1154 (2018).</li><li>Zullo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+ryanodine+receptor+sequence+variants+using+a+metabolic+assay+in+immortalized+B-lymphocytes.">Functional characterization of ryanodine receptor sequence variants using a metabolic assay in immortalized B-lymphocytes.</a> Human Mutation. 30 (4), 575-590 (2009).</li><li>Hoppe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypermetabolism+in+B-lymphocytes+from+malignant+hyperthermia+susceptible+individuals.">Hypermetabolism in B-lymphocytes from malignant hyperthermia susceptible individuals.</a> Scientific Reports. 6, 33372 (2016).</li><li>Monnier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homozygous+splicing+mutation+causing+a+depletion+of+skeletal+muscle+RYR1+is+associated+with+multi-minicore+disease+congenital+myopathy+with+ophthalmoplegia.">A homozygous splicing mutation causing a depletion of skeletal muscle RYR1 is associated with multi-minicore disease congenital myopathy with ophthalmoplegia.</a> Human Molecular Genetics. 12, 1171-1178 (2003).</li><li>Schartner, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydropyridine+receptor+(DHPR,+CACNA1S)+congenital+myopathy.">Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.</a> Acta Neuropathologica. 133, 517-533 (2017).</li><li>Ullrich, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+excitation-contraction+coupling+and+excitation+coupled+Ca2++entry+in+human+myotubes+carrying+CAV3+mutations+linked+to+rippling+muscle+disease.">Alterations of excitation-contraction coupling and excitation coupled Ca2+ entry in human myotubes carrying CAV3 mutations linked to rippling muscle disease.</a> Human Mutation. 32, 1-9 (2010).</li><li>Rokach, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+skeletal+muscle-+derived+cell+line:+biochemical,+cellular+and+electrophysiological+characterization.">Characterization of a human skeletal muscle- derived cell line: biochemical, cellular and electrophysiological characterization.</a> Biochemical Journal. 455, 169-177 (2013).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RYR1+mutations+in+core+myopathies.">Characterization of RYR1 mutations in core myopathies.</a> Human Molecular Genetics. 15, 2791-2803 (2006).</li><li>Klinger, W., Baur, C., Georgieff, M., Lehmann-Horn, F., Melzer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proton+release+from+cultured+human+myotubes+to+identify+malignant+hyperthermia+susceptibility.">Detection of proton release from cultured human myotubes to identify malignant hyperthermia susceptibility.</a> Anesthesiology. 97, 1043-1056 (2002).</li></ol>

The protocols described in this paper have been successfully utilized by several laboratories to study the impact of RYR1 mutations on calcium homeostasis. The critical steps of the approaches outlined in this paper deal with sterility, cell culturing skills and techniques and availability of biological material. In principle, the use of EBV-immortalized B lymphocytes is simpler and allows one to generate cell lines containing mutant RyR1 channels. The cells can be frozen and stored in liquid nitrogen for many years and ...

To date more than 700 RYR1 variants have been identified in the human population and linked to various neuromuscular disorders including malignant hyperthermia susceptibility (MHS), exercise induced rhabdomyolysis, central core disease (CCD), multi-minicore disease (MmD), centronuclear myopathy (CNM)1,2,3; nevertheless, studies to characterize their functional effects are lagging and only approximately 10% of mutations have been tested functionally. Different experimental approaches can be used to assess the impact of a given RyR1 variant, including transfection of heterologous cells such as HEK293 and COS-7 cells with plasmid encoding for the WT and mutant RYR1 cDNA4,5, transduction of dyspedic mouse fibroblasts with plasmids and vectors encoding for the WT and mutant RYR1 cDNA, followed by transduction with myo-D and differentiation into myotubes6, generation of transgenic animal models carrying mutant RyR1s7,8,9, characterization of cells from patients expressing the RYR1 variant endogenously10,11,12. Such methods have helped established how different mutations functionally impact the RyR1 Ca2+ channel.
Here, methods developed to assess the functional effects of RYR1 mutations are described. Various parameters of intracellular calcium homeostasis are investigated in human cells endogenously expressing the RyR1 calcium channel, including myotubes and Epstein Barr Virus (EBV) immortalized B-lymphocytes. Cells are obtained from patients, expanded in culture and loaded with ratiometric fluorescent calcium indictors such as Fura-2 or indo-1. Parameters which have been reported to be altered because of pathogenic RYR1 mutations including the resting [Ca2+], the sensitivity to different pharmacological agonists and the size of the intracellular Ca2+ stores are measured either at the single cell level, using fluorescence microscopy, or in cell populations using a fluorimeter. Results obtained in cells from mutation carriers are then compared to those obtained from healthy control family members. This approach has demonstrated that: (i) many mutations linked to MHS lead to an increase in the resting [Ca2+] and a shift to the left in the dose response curve to either KCl-induced depolarization or pharmacological RyR1 activation with 4-chloro-m-cresol10,11,12,13; (ii) mutations linked to CCD lead to a decrease in the peak [Ca2+] released by pharmacological activation of the RyR1 and decreased size if the intracellular Ca2+ stores12,13,14,15; (iii) some variants do not impact Ca2+ homeostasis13. Advantages of this experimental approach are: the RyR1 protein is not over-expressed and physiological levels are present, cells can be immortalized (both muscle cells and B-lymphocytes) providing cell lines containing mutations. Some disadvantages relate to the fact that patients may carry mutations in more than one gene encoding proteins involved in calcium homeostasis and/or excitation contraction coupling (ECC) and this may complicate experimental conclusions. For example, two JP-45 variants were identified in the MHS and control population and their presence were shown to impact the sensitivity of the dihydropyridine receptor (DHPR) to activation16. Patients need to be available, biological material needs to be freshly collected and ethical permits need to be obtained from the local ethical boards.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-chloro-m-cresol</td>
			<td>Fluka</td>
			<td>24940</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>Sarstedt</td>
			<td>172202</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>caffeine</td>
			<td>Merk</td>
			<td>102584</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 125+ CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 128+ CCD</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine</td>
			<td>Sigma-Aldrich</td>
			<td>C-3630</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>41639</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Fluka</td>
			<td>3778</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque</td>
			<td>Cytiva</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal calf serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2/AM</td>
			<td>Invitrogen Life Sciences</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630049</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>ThermoFisher Scientific</td>
			<td>A11382II</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Lanthanum</td>
			<td>Fluka</td>
			<td>61490</td>
			<td></td>
		</tr>
		<tr>
			<td>Microperfusion system</td>
			<td>ALA-Scientific</td>
			<td>DAD VM 12 valve manifold</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin Software</td>
			<td>OriginLab Corp</td>
			<td>Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin/Streptomycin</td>
			<td>Gibco Life Sciences</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion chamber POC-R</td>
			<td>Pecon</td>
			<td>000000-1116-079</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>ThermoFisher Scientific</td>
			<td>21875091</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrofluorimeter</td>
			<td>Perkin Elmer</td>
			<td>LS50</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Calbiochem</td>
			<td>586005</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask</td>
			<td>Falcon</td>
			<td>353107</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture inserts</td>
			<td>Falcon</td>
			<td>353090</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA solution</td>
			<td>ThermoFisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Visiview</td>
			<td>Visitron Systems GmbH</td>
			<td>Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axiovert S100 TV microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss glass coverslips</td>
			<td>Carl Zeiss AG</td>
			<td>0727-016</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of endogenously expressed human ryr1 variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

[Ca2+]i measurements in populations of EBV-immortalized B lymphocytes 
Primary B-lymphocytes express the RyR1 isoform that functions as a Ca2+ release channel during B cell antigen receptor stimulated signaling processes17. Immortalization of B-cells with EBV, a procedure routinely used by geneticists to obtain cell lines containing genomic information of patients, provides th...

Watch this Scientific Journal Video about Functional Characterization of Endogenously Expressed Human RYR1 Variants at JoVE.com

Functional Characterization of Endogenously Expressed Human RYR1 Variants

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

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Medicine

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...