Treatment with SP600125 prevents abnormal elongation of cardiomyocyte nuclei in Lmna^H222P/H222P mice. (A) Histological analysis of cross-sections of hearts from Lmna^H222P/H222P mice treated with SP600125 or placebo (DMSO). Heart from Lmna^+/+ mice was used for comparison. Sections are stained with hematoxylin and eosin. Yellow lines with arrowheads demonstrate the measurement of nuclear length. Scale bar: 10 μm. (B) Quantification of nuclear elongation in cardiomyocytes from mice. Cardiomyocyte nuclei are measured along the yellow lines with arrowheads. Bars indicate the length of cardiomyocyte nuclei in the indicated hearts. Values are means ± standard errors for n=230 cardiomyocytes. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; ***P<0.0005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P mice was performed using Student unpaired t-test; ^‡‡‡P<0.0005.

Treatment with SP600125 prevents cardiac fibrosis in Lmna^H222P/H222P mice. (A) Representative heart tissue sections from Lmna^H222P/H222P mice treated with SP600125 or DMSO stained with Gomori’s Trichrome are shown. Heart tissue section from Lmna^+/+ mice is shown for comparison. Scale bar: 50 μm. Bar graph represents the quantification of fibrotic area in hearts from mice. Micrographs (n=3) for each heart were processed (JMicroVision software) and blue-staining fibrotic tissue was quantified (ImageJ64 software). Bars indicate the percentage of fibrosis in heart from Lmna^+/+ and Lmna^H222P/H222P mice treated with SP600125 or DMSO. Values are means ± standard errors for n=3 mice per group. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; ***P<0.0005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P was performed using Student unpaired t-test ^‡P<0.05. (B) Effect of SP600125 inhibitor on cardiac expression of collagens genes in Lmna^H222P/H222P mice. Bars indicate the expression of Col1a1 and Col1a2 in heart from Lmna^+/+ and Lmna^H222P/H222P mice treated with SP600125 or placebo (DMSO). Values are means ± standard errors for n=3 hearts. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; *P<0.05, **P<0.005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P mice was performed using Student unpaired t-test; ^‡P<0.05, ^‡‡‡P<0.0005.

Treatment of male Lmna^H222P/H222P mice with SP600125 inhibits expression of natriuretic peptides and myosin chains. Quantitative real-time RT-PCR showing the expression of (A) mRNAs from NppA and NppB genes encoding natriuretic peptide precursors A and B, respectively and (B) mRNAs from Myh7 encoding myosing heavy chain and Myl4 and Myl7 encoding myosin light chain. Results from hearts from Lmna^+/+ mice are shown for comparison. Bars indicate the fold over-expression of the indicated mRNA calculated by the ΔΔC_T method. Values are means ± standard errors for n=5 samples from different animals per group. Reactions were performed in triplicate for each different RNA sample. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; *P<0.05, **P<0.005, ***P<0.0005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P was performed using Student unpaired t-test; ^‡P<0.05, ^‡‡P<0.005.

Treatment of male Lmna^H222P/H222P mice with SP600125 inhibits phosphorylation of JNK signaling. (A) Representative immunoblots using antibodies against phophorylated JNK (p-JNK), total JNK, phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 to probe proteins extracted from hearts from Lmna^H222P/H222P mice treated with SP600125 or DMSO. Blots of proteins extracted from hearts of Lmna^+/+ mice are shown for comparison. Data in bar graphs show quantification of phosphorylated JNK compared with total JNK measured by scanning immunoblots and using ImageJ64 software. Values are means ± standard errors for n=5 samples from different animals per group. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; ***P<0.0005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P was performed using Student unpaired t-test; ^‡‡P<0.005. (B) Representative immunoblots using antibody against phosphorylated cJun (p-cJun) to probe proteins extracted from hearts from Lmna^H222P/H222P mice treated with SP600125 or DMSO. Blot of proteins extracted from hearts of Lmna^+/+ mice is shown for comparison. Antibody against GAPDH is used as a loading control. (C) Quantitative real-time RT-PCR showing the expression of mRNA from JunD. Results from hearts from Lmna^+/+ mice are shown for comparison. Bars indicate the fold over-expression of the indicated mRNA calculated by the ΔΔC_T method. Values are means ± standard errors for n=5 samples from different animals per group. Reactions were performed in triplicate for each different RNA sample. Comparison between DMSO-treated Lmna^H222P/H222P and Lmna^+/+ mice was performed using Student unpaired t-test; ***P<0.0005. Comparison between SP600125-treated and DMSO-treated Lmna^H222P/H222P was performed using Student unpaired t-test; ^‡‡P<0.005.