Attempts to Create Transgenic Mice Carrying the Q3924E Mutation in RyR2 Ca2+ Binding Site

Figures

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  Figure 1. Calcium signaling from WT and Q3925E hiPSC-CMs. (A) The diagram shows that RyR2 Ca2+-binding residues mutation cause defective CICR. (B) Application of 5 mM 4-CmC and 5 mM caffeine triggered ER Ca2+ release and cytosolic Ca2+ signal measured by R-CEPIA1er and Fluo-4 simultaneously. (C) Quantification of 4-CmC and caffeine-triggered Ca2+ signals measured by Fluo-4 and R-CEPIA1er simultaneously. n = 12 for WT and n = 10 for Q3925E cells. ** p < 0.01 vs. WT by unpaired t-test. (D) Spontaneous cytosolic Ca2+ transients and SR Ca2+ release measured by Fluo-4 and R-CEPIA1er in WT and Q3925E cells. (E) SR Ca2+ leak measured with Fura-2 and ER-GCamP6 probe in WT and Q3925E mutant cells. (F) Quantification of SR Ca2+ leak measured by ER-GCamMP6 and Fura-2 in WT cells and SR Ca2+ leak measured by ER-GCamMP6 in Q3925E cells. ** p < 0.01 vs. WT by unpaired t-test.

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  Figure 2. Heatmap of RNA sequencing results from WT and Ca2+ binding site mutant Q3925E and E3848A hiPSC-CMs. (A) GO: 0055074. Calcium homeostasis genes. (B) GO:0006816. Calcium ion transport genes. (C) GO: 0045823. Positive regulation of heart contraction genes. Lower panel: quantification of ADRB1 transcription levels in WT and the two mutant hiPSC-CMs by quantitative RT-PCR. * p < 0.05 vs. WT by one-way ANOVA. (D) GO: 0080135. Regulation of cellular response to stress genes. The lines on the left of the heatmaps are a simple dendrogram, which attempts to find trends within the data and cluster them together.

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  Figure 3. Generation of mutant mice harboring RyR2-Q3924E. (A) CRISPR/Cas9 design and genome sequences of heterozygous founder F3. CRISPR/Cas9 introduced 4 mutations, 5′-GAATATATTGAG-3′, in mouse Ryr2 gene. One results in Q3924E mutation (CAG to GAG), and the other 3 are silent mutations (no amino acid changes). Two different peaks were observed at four nucleotide positions in Sanger sequencing, because the heterozygous mouse carry both wild-type and mutant sequences. (B) Genome sequencing results of founders A9, B1 and E1. For founders A9 and B1, 3 mutations, 5′-GAATATATCGAG-3′, were introduced in mouse Ryr2 gene. One results in Q3924E mutation (CAG to GAG), and the other two are silent mutations. Three different peaks are observed in A9 because the chimera mouse carried 25% of the Q3924E mutation. Founders B1 and E1 are both heterozygous.

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  Figure 4. Histological analysis of heart sections from RyR2-WT and RyR2-Q3924E+/− mutant mice. (A) H&E-stained longitudinal direction heart sections of WT and mutant hearts (founder A9). Images were captured from Keyence microscope. (B) Cross-section of WT and mutant heart (founder F3). (C) Comparison of cardiomyocytes from the same area of left ventricle of WT and mutant heart sections from panel (B). Images were captured from TIRF microscope with 20× objective lens.