Cardiac remodeling pathways do not accelerate disease onset and severity in a mouse model of PLN-R14del cardiomyopathy

Figures

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  Fig. 1. TAC induced cardiac hypertrophy and cardiac dysfunction to a similar extend in WT and R14del mice. (A) Ejection fraction (EF%) of individual mice in each group, (B) diastolic left ventricular anterior wall thickness (LVAWd), and (C) diastolic left ventricular posterior wall thickness (LVPWd). (D) Representative cardiac echo graphic images of mice in sham and TAC groups. (E) The ratio of ventricle/tibia and (F) atria/tibia. The data significance was analyzed using Kruskal-Wallis tests, followed by Dunn’s multiple comparisons test. **p < 0.01, ***p < 0.001.

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  Fig. 2. Cardiomyocytes hypertrophy and cardiac fibrosis are induced to a similar extend in both WT and PLN-R14del mice post TAC. (A) Representative images of extracellular Matrix (wheat germ agglutinin, WGA, green) and nuclei (4’,6-diamidino-2 phenylindole, DAPI, blue) staining on left ventricular (LV) sections and (B) cardiomyocytes size measurement on transversally cut cardiomyocytes (scale bar 20 μm). (C) Representative (average based) Masson trichrome-staining on LV sections (scale bar 20 μm), and (D) quantification of fibrosis area. Relative mRNA level of (E) cardiac stress (NPPA) and (F, G) fibrosis-related genes (col1a1 and Timp). The data significance was analyzed using Kruskal-Wallis tests, followed by Dunn’s multiple comparisons test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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  Fig. 3. PLN and SERCA level remain similar in both WT and PLN-R14del mice. (A, B) Representative Western blot images of total protein, SERCA protein, and PLN protein levels in LVs. (C, D) SERCA and PLN protein quantification corrected by total protein. (E) SERCA/PLN protein ratio. The data significance was analyzed using Kruskal-Wallis tests, followed by Dunn’s multiple comparisons test. PVDF (polyvinylidene fluoride) membranes were sectioned according to the molecular weights of the target proteins and incubated with the corresponding antibodies. Full-length, uncropped Western blot images are included in the supplemental material for reference.

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  Fig. 4. TAC did not induce PLN-R14del S/ER clustering. Representative images immunofluorescence staining of PLN (red), together with extracellular matrix (green) and nuclei (blue) of the LV sections of WT Sham, WT TAC, R14Δ/+ Sham and R14Δ/+ TAC (scale bar 20 μm).

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  Fig. 5. RNA sequencing identified common biological processes activated in WT and R14del mice in response to TAC. (A) Principal component (PC) analysis plot of RNA sequencing results on mice LV tissue (n = 5 per group). Genes contributing to the variance in PC1, represent 66.09% of the total variance, and genes contributing to the PC2 is 7.87% of the total variance. (B) Top 20 most positive enriched gene biological processes in WT mice and R14Δ/+ TAC mice. Top 15 most negative enriched gene biological processes in WT and R14Δ/+ TAC mice.