Figure 1.
Generation of the RyR2 Ex3-del mutant mouse model.
(A) The mouse ryr2 locus (top line), the construction of the knock-in (KI) vector containing the deletion of the exon-3 and flanking intron sequences in the mouse ryr2 gene, the selectable markers neo (neomycin resistant gene) and DTA (diphtheria toxin A), the FRT sites (middle line), the generation of the exon-3 deletion mutant allele, and the removal of the neo gene via homologous recombination (bottom line) are depicted. (B) Intron and exon sequences that were deleted in the RyR2 Ex3-del mutant mice. (C) Southern blot analysis of recombinant embryonic stem cells (WT, wild type; Het, heterozygous). (D) PCR genotyping using tail samples from WT and Ex3-del heterozygous (Het) mice.
Figure 2.
Deletion of exon-3 in the RyR2 mRNA from heterozygous RyR2 Ex3-del mice.
A fragment of the mouse RyR2 mRNA covering exon-3 was converted to cDNA and amplified using RT-PCR from total RNAs isolated from wild type (WT) and heterozygous RyR2 Ex3-del (Het) mutant mice (A). The RT-PCR products were isolated and sequenced. The sequence of the RyR2 Ex3-del cDNA was shown (B). Note that the exon-2 sequence is directly followed by the exon-4 sequence, i.e. the exon-3 sequence has been deleted.
Figure 3.
Heterozygous RyR2 Ex3-del mutant mice are not susceptible to CPVT.
Representative ECG recordings of wild type (WT) (A) and RyR2 Ex3-del+/− mutant (B) mice (1.5–3 months of age) before (top) and after (bottom) the injection of epinephrine (1.6 mg/kg) and caffeine (120 mg/kg). Note that no VTs were detected in either WT (n = 16) or mutant (n = 14) mice during the 30-min period of ECG recording after the injection of the triggers.
Figure 4.
Depolarization-induced Ca2+ transients in WT and heterozygous RyR2 Ex3-del mutant cardiomyocytes.
Ventricular myocytes isolated from RyR2 WT and Ex3-del+/− mutant hearts were loaded with Rhod-2-AM and perfused with 2 mM extracellular Ca2+ in KRH solution and paced at 3Hz. Ca2+ transients were monitored by line-scan confocal Ca2+ imaging. Representative images/traces of WT (A) and Ex3-del+/− mutant (B) cardiomyocytes, and average data of the amplitude (C), time to peak (D), and time to 50% decay (E) of Ca2+ transients in WT and Ex3-del+/− mutant cells are shown. Data shown are mean ± SEM from 35 WT and 58 mutant cells (**P<0.001; *P<0.05).
Figure 5.
Reduced RyR2 protein expression in heterozygous RyR2 Ex3-del mutant hearts.
(A) Whole heart homogenates were prepared from wild type (WT) (n = 4) and RyR2 Ex3-del−/− mutant (n = 4) mice (2–3 months) and used for immunoblotting analysis using antibodies against RyR2 or β-actin. (B) The expression of RyR2 in the Ex3-del hearts was significantly reduced (58±3%) as compared to that in WT hearts (*P<0.001).
Figure 6.
Cardiac-specific, conditional knockout of the WT RyR2 allele in heterozygous RyR2 Ex3-del mutant mice results in early death.
iRyR2flox/flox (n = 13, black circles) and iRyR2flox/Ex3-del (n = 11, white circles) mice were injected with tamoxifen (75 mg/kg/day) for 3 consecutive days. The percentage of live mice (survival rate) on day 6–14 post tamoxifen treatment is shown.
Figure 7.
Heterozygous RyR2 Ex3-del mutant mice with cardiac specific, conditional KO of the WT RyR2 allele exhibit bradycardia, but no CPVT.
iRyR2flox/flox (n = 11) and iRyR2flox/Ex3-del (n = 8) mice were treated with tamoxifen. ECG recording was performed 12 days post tamoxifen treatment to determine their basal heart rates (before epinephrine/caffeine challenge) (A) and their susceptibility to CPVT (B, C). Representative ECG recordings of the tamoxifen-treated iRyR2flox/flox mice (B) and the tamoxifen-treated iRyR2flox/Ex3-del mice (C) before (top panel) and after (bottom panel) the injection of epinephrine (1.6 mg/kg) and caffeine (120 mg/kg). Note that no VTs were detected in either the tamoxifen-treated iRyR2flox/flox mice (B) or the tamoxifen-treated iRyR2flox/Ex3-del mice during the 30-min period of ECG recording after the injection of the triggers (*P<0.05).
Figure 8.
Cardiac specific, conditional knockout of the WT RyR2 allele results in markedly reduced RyR2 expression.
Whole heart homogenates were prepared from iRyR2flox/flox (A) and iRyR2flox/Ex3-del (B) mice before and after tamoxifen treatment, and used for immunoblotting analysis using antibodies against RyR2 or β-actin. Note that the expression of RyR2 in iRyR2flox/flox hearts (n = 6) or iRyR2flox/Ex3-del hearts (n = 6) was significantly reduced 12 days post tamoxifen treatment (*P<0.001).