Journal: JCI Insight
Article Title: Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2
doi: 10.1172/jci.insight.126544
Figure Lengend Snippet: (A) Representative traces of the intra-ER FRET signal using the intra-ER Ca2+ sensor D1ER in cells expressing RyR2. Stable HEK293 cells with inducible expression of the mouse RyR2 were transfected with D1ER. RyR2 expression was induced with 1 μg/ml tetracycline 18–24 hours after transfection. Time-lapse imaging of D1ER FRET was performed after 18–24 hours of RyR2 induction. Images were collected for 3 minutes at 0 [Ca2+], 3 minutes at 1 mM [Ca2+], 5 minutes at 2 mM [Ca2+], 5 minutes in the presence of 1 mM tetracycline to inhibit RyR2 and measure maximum ER Ca2+ capacity (FMax), and about 3 minutes in the presence of 20 mM caffeine to empty the ER and measure the residual FRET signal (FMin). (B–D) Activation (B) and termination (C) thresholds and fraction of the ER Ca2+ content released (D) during spontaneous oscillations (*P < 0.05, **P < 0.01, rank sum test [B], 2-tailed t test [C–D]). (E) ER store capacity determined as (FMax–FMin) and expressed as percentage of WT (rank sum test). (F) Percentage of cells showing oscillations in the store [Ca2+] (z test). (G) Average number of [Ca2+] oscillations per cell (**P < 0.01, rank sum test) (n = 40 [B–D], 49 [E], 87 [F], and 39 [G] WT cells; 44 [B–D], 61 [E], 104 [F] and 43 [G] P1124L cells from 4 independent D1ER transfections and inductions of RyR2 expression).
Article Snippet: We thank Wayne Chen (Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada) for providing the RyR2-P1124L plasmid and stable cell lines expressing RyR2; Amy Palmer and Roger Tsien for providing the D1ER plasmid through Addgene (Watertown, Massachusetts, USA); and the Frankel Cardiovascular Center Animal Phenotyping Core and the Unit of Laboratory Animal Medicine In-Vivo Animal Core of the University of Michigan for their services.
Techniques: Expressing, Transfection, Imaging, Activation Assay
Addgene inc is a verified supplier 
![(A) Representative traces of the intra-ER FRET signal using the intra-ER Ca2+ sensor <t>D1ER</t> in cells expressing RyR2. Stable HEK293 cells with inducible expression of the mouse RyR2 were transfected with D1ER. RyR2 expression was induced with 1 μg/ml tetracycline 18–24 hours after transfection. Time-lapse imaging of D1ER FRET was performed after 18–24 hours of RyR2 induction. Images were collected for 3 minutes at 0 [Ca2+], 3 minutes at 1 mM [Ca2+], 5 minutes at 2 mM [Ca2+], 5 minutes in the presence of 1 mM tetracycline to inhibit RyR2 and measure maximum ER Ca2+ capacity (FMax), and about 3 minutes in the presence of 20 mM caffeine to empty the ER and measure the residual FRET signal (FMin). (B–D) Activation (B) and termination (C) thresholds and fraction of the ER Ca2+ content released (D) during spontaneous oscillations (*P < 0.05, **P < 0.01, rank sum test [B], 2-tailed t test [C–D]). (E) ER store capacity determined as (FMax–FMin) and expressed as percentage of WT (rank sum test). (F) Percentage of cells showing oscillations in the store [Ca2+] (z test). (G) Average number of [Ca2+] oscillations per cell (**P < 0.01, rank sum test) (n = 40 [B–D], 49 [E], 87 [F], and 39 [G] WT cells; 44 [B–D], 61 [E], 104 [F] and 43 [G] P1124L cells from 4 independent D1ER transfections and inductions of RyR2 expression).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3635/pmc06483635/pmc06483635__jciinsight-4-126544-g150.jpg)