Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model

Figures

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  Fig 1. sgRNAs and ssODN targeting Pln coding region. Targeting sequences of each sgRNA are capitalized and NGG PAM (protospacer-adjacent motif) sequences are underlined. Exons are indicated by closed boxes. The sgRNA targeting sites were designed to sandwich Pln coding region (filled with black). The ssODN is containing homologies of 60 bases on both sides flanking each of the sgRNA targeting sequences.

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  Fig 2. PCR analysis of mice obtained from 1st lot microinjection. (A) Genotyping of PLN KO mice by PCR. PLN wild type (WT) mice showed 1213 bp fragments and PLN KO mice showed 665 bp fragments. (B) Genotyping of CSQ-Tg mice by PCR. Mice which had canine Csq allele shows 407 bp fragments and Non-Tg mice does not show any fragments. M, DNA marker.

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  Fig 3. Survival curve in PLN wild type (WT)/CSQ-Tg mice and PLN homozygous KO/CSQ-Tg mice.

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  Fig 4. Hemodynamic parameters. Cardiac catheterization was performed in CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) dP/dtmax, (B) dP/dtmin, (C) Tau, (D) LVEDP, (E) MBP, (F) HR. Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, #P < 0.05 vs. PLN WT/CSQ-Tg mice by Student's t-test.

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  Fig 5. Immunoblots of PLN and SERCA2a, and actin in hearts from mice used for hemodynamic study. (A) CSQ non-Tg mice, (B) CSQ-Tg mice.

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  Fig 6. Heart and lung weight. Heart and lung weight were measured in CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) LV/BW (left ventricular weight/body weight), (B) RV/BW (right ventricular weight/body weight), (C) AW/BW (atria weight/body weight), (D) LW/BW (lung weight/body weight). Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, #P < 0.05, ##P < 0.01 vs. PLN WT/CSQ-Tg mice by Student's t-test.

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  Fig 7. mRNA levels in the left ventricle. Analysis of mRNA levels was performed in the left ventricle of CSQ non-Tg mice, PLN wild type (WT)/CSQ-Tg mice, and PLN homozygous KO/CSQ-Tg mice. (A) β-MHC, (B) ANF, (C) BNF. Values represent the mean ± SD, *P < 0.05, **P < 0.01 vs. CSQ non-Tg mice, #P < 0.05 vs. PLN WT/CSQ-Tg mice by Student's t-test.