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 ...

This protocol is significant because it uses patient material. If the patient has the disease, using their cells should more accurately reflect the patient's biological response. This technique is simple to perform, straightforward, and does not require sophisticated equipment.These methods can be used for diagnosis, for example, to prove that a RYR1 mutation functionally changes calcium homeostasis, as well as to test the potential therapeutic function of a compound. Depending on the knowledge and technical skills of the individual researcher, test experiments should be performed before actual patient samples are used. To monitor changes in the intracellular calcium concentration of EBV-transformed B lymphocytes, resuspend the immortalized B cells at a one times 10 to the seventh cells per milliliter concentration in Krebs Ringer's solution and Fura-2 AM to a final concentration of five micromolar for a 30-minute incubation at 37 degrees Celsius.At the end of the incubation, collect the cells by centrifugation, and resuspend the pellet in fresh Krebs Ringer's solution at a two times 10 to the sixth cells per milliliter concentration. Just before starting the experiment, centrifuge the cells again, and quickly resuspend the cells in 1.5 milliliters of Krebs Ringer's solution supplemented with 0.5-millimolar EGTA but no added calcium. Transfer the cells to a glass, three-milliliter spectrofluorometer cuvette, and record the fluorescence ratio on a spectrofluorometer equipped with a magnetic stirrer set at maximum velocity and 37 degrees Celsius for about 30 seconds.To assess the calcium release response in single cells, plate one times 106 Fura-2 AM loaded cells in one milliliter of Krebs Ringer's solution supplemented with one-millimolar calcium chloride onto individual poly-L-lysine coated glass cover slips, and incubate the cover slips at 37 degrees Celsius in a humidified cell culture incubator for 30 minutes. When the cells have attached, place a cover slip into a perfusion chamber, and begin perfusing the cells at a two milliliters of Krebs Ringer's solution supplemented with one-millimolar calcium per minute flow rate. Using an inverted fluorescent microscope equipped with a 40 times oil immersion objective and the appropriate filters, record online measurements with a software-controlled, charge-coupled device camera attachment at one-second intervals, at a fixed exposure time.To achieve cell stimulation, use a cell perfusion stimulator with 12 valves to add the different concentrations of agonist. To isolate myotubes from human muscle biopsies, first rinse the biopsy with sterile PBS to remove any excess blood before mincing the tissue into small, 0.5 to one millimeter fragments. Place two to three fragments into individual inserts in each well of a six-well tissue culture plate containing 1.5 milliliters of human muscle growth medium per well and 500 microliters of medium per insert, and place the plate into the cell culture incubator.To measure changes in calcium expression in human myotubes, when multinucleated myotubes can be observed, replace the myotube culture supernatants with fresh differentiation medium supplemented with 10 microliters of one-millimolar Fura-2 AM for a 30-minute incubation in the cell culture incubator. At the end of the incubation, transfer one glass cover slip culture to the perfusion chamber, and rinse the cells with Krebs Ringer's solution supplemented with two-millimolar calcium chloride. Then perform online calcium measurements as demonstrated using a 20 times water immersion objective.In this representative analysis, a rapid increase in calcium was observed in EBV-immortalized B lymphocytes after the addition of agonist that slowly declined back to resting levels over time. In a similar experiment, the addition of 400-nanomolar thapsigargin caused a large calcium transient that reached a peak fluorescence value of 2.4 arbitrary units. This fluorescence value was considered to be 100%when the corresponding dose response curve was constructed.In this analysis, immortalized B cells were stimulated with five-millimolar caffeine for 20 seconds, resulting in an immediate increase in the 340 and 380-nanometer fluorescence ratio. For each caffeine concentration tested, the caffeine-induced peak at the ratio of 340 by 380 nanometers of fluorescence was calculated and used to construct a dose response curve. Calcium release from differentiated myotubes can also be assessed in response to flushing with potassium chloride, different concentrations of agonist, and/or caffeine, and normal dose response curves can be generated.It is important to make sure that the cells are loaded properly with Fura-2 and that both the 340 and 380-nanometer wavelengths respond to the addition of the agonist. Intracellular calcium measurements with fluorescent calcium indicators can be investigated in most mammalian cells and can be applied to the study of intracellular calcium changes in response to different stimuli.

functional-characterization-endogenously-expressed-human-ryr1

Research

JoVE Journal

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 risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

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 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 membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

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 fluctuations over time. Temporal Lobe Epilepsy (TLE) is the most common type of adult epilepsy and involves multiple brain networks. The default mode network (DMN) is involved in conscious, resting state cognition and is thought to be affected in TLE where seizures cause impairment of consciousness. The DMN in epilepsy was examined using seed based fcMRI. The anterior and posterior hubs of the DMN were used as seeds in this analysis. The results show a disconnection between the anterior and posterior hubs of the DMN in TLE during the basal state. In addition, increased DMN connectivity to other brain regions in left TLE along with decreased connectivity in right TLE is revealed. The analysis demonstrates how seed-based fcMRI can be used to probe cerebral networks in brain disorders such as TLE.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

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

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

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 donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

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 step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

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

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

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

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

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 of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis 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 against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

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

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.