Schematic diagram illustrating the LMNA mutations involved in this study. A, Genetic disorders caused by nonsense and frame‐shift mutations in LMNA. R225X yielded a premature stop codon, TGA, due to a C‐to‐T substitution; Q354X also yielded a premature stop codon, TAG, due to a C‐to‐T substitution; and the frameshift mutation T518fs resulted from a C deletion. B, Splice variant of LMNA gene yielding lamin A/C protein: the mutation yielded 3 different lengths of truncated lamin A/C protein. Key: NLS indicates nuclear localization signal.
Effects of PTC124 on the expression of lamin A/C proteins in dermal fibroblasts and hiPSC‐derived cardiomyocytes (hiPSC‐CMC) derived from wild‐type (LMNAWT1/WT1 and LMNAWT2/WT2) and LMNA mutants (LMNAR225X/WT, LMNAQ354X/WT, and LMNAT518fs/WT). A, Representative immunoblot and (B) quantitative data of protein‐normalized lamin A/C protein expression. The band intensities of lamin A/C and β‐actin (Actb) were measured from the high‐resolution (300×300 dots per inch) images using Image J. At least 3 to 6 independent samples were prepared for Western blot analysis. The significant difference of quantitative data was tested by 2‐way ANOVA with post hoc Turkey multiple comparison test (**P<0.01 as indicated by arrows; #P<0.05, Control vs PTC). Original images of immunoblots in individual experiments are shown in Figure S2. hiPSC indicates human induced pluripotent stem cells.
Nuclear blebbing in the hiPSC‐derived cardiomyocytes. The 2 wild‐type lines (LMNAWT1/WT1 and LMNAWT2/WT2), R225X (LMNAR225X/WT), Q354X (LMNAQ354/WT), and T518fs (LMNAT518fs/WT), mutant hiPSC lines were differentiated into cardiomyocytes and treated with PTC124. The occurrence of cardiac nuclear blebbing was revealed with coimmunostaining of cardiac troponin T (red) and lamin A/C (green). A, A single representative hiPSC CMC, and (B through D) representative images of lower magnification were used to quantify the portion of nuclear blebbing in a single field. At least 3 countings were performed. E, Quantitative data of nuclear blebbing count. The significant difference of quantitative data was tested by 2‐way ANOVA with post hoc Turkey multiple comparison test (****P<0.0001 as indicated by arrows; ####P<0.0001 control vs treatment group). cTnT indicates cardiac troponin‐T; DAPI, 4,6‐diamidino‐2‐phenylindole dihydrochloride; hiPSC, human induced pluripotent stem cells; LMNA, lamin.
Evaluation of TUNEL‐positive apoptotic cell in electrically stressed and PTC124‐treated cardiomyocytes derived from wild‐type (LMNAWT1/WT1 and LMNAWT2/WT2) and LMNA mutants (LMNAR225X/WT, LMNAQ354X/WT, and LMNAT518fs/WT) by (A through E) immunostaining; (F through G) and also by Apo‐BrdU TUNEL‐FACS analysis to quantify apoptotic cells (Significant difference was analyzed by 2‐way ANOVA with Tukey multiple comparison post‐hoc test *P<0.05; n=3 to 5 (as indicted by arrows) and (control vs electrically stressed) #P<0.05; n=3 to 7). FACS indicates fluorescence‐activated cell sorting; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Normalized gene expression level of LMNA in PTC124‐treated hiPSC‐derived cardiomyocytes. The endogenous control gene was TUBB, and TNNT2 was used to normalize the number of cardiac cells within the differentiated population. Significant difference was analyzed by 1‐way ANOVA with Tukey multiple comparison post hoc test compared with wild‐type control group, *P<0.05 and **P<0.01; n=5. hiPSC indicates human induced pluripotent stem cells.
Excitation‐contraction (EC) coupling was improved by PTC124 in the cardiomyocytes derived from the R255X mutant. A, Representative tracing of calcium transient and cell‐shortening in LMNAWT/WT and LMNAR225X/WT cells treated with PTC124. Rate dependence of calcium transients and cell contractility pacing frequency range of 0.5, 1, and 1.5 to 2 Hz are shown in terms of (B) diastolic calcium, (C) peak calcium amplitude, (D) fractional shortening (%) (*P<0.05; n=5 to 7; WT:PTC vs R225X:PTC ####P<0.0001; R225X: 0.5‐Hz control vs R225X: 0.5‐Hz or 1‐Hz PTC: φ φ φ φ P<0.0001; R225X: 0.5 Hz PTC vs R225X: 1.5 or 2 Hz PTC: φ φ P<0.001); (E and F) maximal upstroke velocity (Vmax upstroke) of calcium transient and rate of cell shortening (ie, contraction); (G and H) maximal decay velocity (Vmax decay) and rate of cell relengthening (ie relaxation). Improved calcium decay kinetics and relaxation performance are shown (G and H). Significant difference was analyzed by comparing pacing rate of 0.5 Hz with the other rates of the same treatment group in 1 cell line, or otherwise indicated by arrows using 2‐way ANOVA with Turkey test as post hoc (*P<0.05).
AMP-activated protein kinase (AMPK) α2 deletion enhances Western diet–induced features of atherosclerotic plaque instability in the brachiocephalic arteries (BA). A, Representative images from BA lesions of Apoe−/− and Apoe−/−AMPKα2−/− mice with hematoxylin-eosin (H&E) staining for intraplaque hemorrhage (black arrow) and buried fibrous cap (black arrowhead). B–D, Incidence for intraplaque hemorrhage (B), presence of buried fibrous cap (C), and presence of fibrous cap discontinuity (D) in the BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. E and F, Representative images and quantification of necrotic core area in the BA based on H&E staining (black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. G and H, Representative images and quantification for the area of fibrous cap staining (black arrow) in BA based on Sirius Red staining (red staining) of Apoe−/− and Apoe−/−AMPKα2−/− mice. I and J, Representative images and quantification of plaque collagen content in BA based on Masson trichrome staining (blue staining, black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. K and L, Representative images and quantification of plaque macrophage content in BA based on CD68 immunohistochemistry (IHC) staining (brown staining, black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=20 to 21 in each group. Values represent the mean±SEM. *P<0.05 vs Apoe−/− mice. Scale bar, 100 µm.
AMP-activated protein kinase (AMPK) α2 deletion induces vascular smooth muscle cell phenotypic switching in advanced atherosclerotic plaque in the brachiocephalic arteries (BA). A, Representative images of immunohistochemistry staining of smooth muscle actin (SMA)-α (dark pink) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. Arrow represents representative staining of SMA-α. Scale bar, 100 µm. B, Quantification of plaque SMA-α coverage on the plaque cap in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. C, Quantification of total plaque SMA-α content in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. D, Representative images of immunofluorescence staining of vimentin (red) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. DAPI=blue staining of nucleus. Scale bar, 100 µm. E, Quantification of plaque vimentin coverage on the plaque cap in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. F, Quantification of total plaque vimentin content in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=10 in each group. Values represent the mean±SEM. *, P<0.05 vs Apoe−/− mice.
AMP-activated protein kinase (AMPK) α2 deletion upregulates Kruppel-like factor 4 (KLF4) expression in advanced atherosclerotic plaque in the brachiocephalic arteries (BA). A, Immunofluorescence staining of KLF4 (red) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. DAPI=blue staining of nucleus. Scale bar, 100 µm. B, Quantification of KLF4 expression in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=10 in each group. Values represent the mean±SEM. *P<0.05 vs Apoe−/− mice.
Pravastatin treatment alleviates Western diet–induced plaque instability in Apoe−/−AMPKα2sm+/+ mice, but not in Apoe−/−AMPKα2sm−/− mice.A, Representative images from hematoxylin-eosin staining of the brachiocephalic arteries (BA) in Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. Scale bar, 100 µm. B, Quantification of plaque size and (C) necrotic core size in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. D and E, Representative images and quantification of plaque collagen content in the BA based on Masson trichrome staining of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. Scale bar, 100 µm. F, Quantification of fibrous cap area in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. n=10 in each group. Values represent the means±SEM. *P<0.05 vs Apoe−/−AMPKα2sm+/+ mice without pravastatin treatment. #P<0.05 vs Apoe−/−AMPKα2sm+/+ mice with pravastatin treatment. G, Western blot analysis of pAMPKα (Thr172) in human aortic smooth muscle cell (HASMC) treated with 0.01 to 50 µmol/L pravastatin for 24 h. H, Western blot analysis of pAMPKα (Thr172) in aorta from Apoe−/−AMPKα2sm+/+ mice fed with Western diet for 10 wk and treated with or without pravastatin for 4 wk.
