Fig. 5. Homozygous female knock-in animals exhibit enlarged hearts and extensive fibrosis and calcification 8 weeks after TAC surgery.

Two-month-old wild-type and knock-in female mice were subjected to either sham or TAC surgery and evaluated 8 weeks later. (A) Representative immunoblots of protein lysates prepared from left ventricles of sham- or TAC-subjected wild-type and knock-in animals using antibodies to the NH₂ and COOH termini of obscurins. Relative quantification revealed increased levels of obscurins in the knock-in–TAC hearts due to up-regulation of obscurin B (asterisk). HSP90α/β served as loading control; n = 3 animals per group, two to three replica blots per heart; t test, *P < 0.05, ^#P < 0.005; error bars represent SEM. (B to B‴) Immunofluorescence combined with confocal optics and antibodies to the COOH terminus of obscurins demonstrated that their localization is unaltered in knock-in left ventricles subjected to sham or TAC surgery; obscurins occupying M-bands are shown in red, and α-actinin, which is a Z-disk marker, is shown in green; scale bar, 5 μm. (C to C‴) Gross evaluation of wild-type and knock-in hearts 8 weeks after sham or TAC surgery indicated that the KI-TAC hearts are notably enlarged; scale bar, 2 mm. (D to D‴) H&E staining of cardiac sections from left ventricles revealed no obvious structural abnormalities in any group; scale bar, 50 μm. (E and E′) Masson’s trichrome staining of sections from left ventricles of wild-type–TAC and knock-in–TAC hearts revealed the presence of excessive interstitial fibrosis in the latter; scale bar, 50 μm. (F) Left ventricles from knock-in–TAC animals contained significantly increased amounts of hydroxyproline compared to left ventricles from wild-type–TAC animals; t test, *P < 0.01; error bars represent SEM; n = 3 animals per group, three repeats per heart. (G to G″) von Kossa staining of left ventricles demonstrated the presence of more prominent peripheral (G′) and interstitial (G″) dystrophic calcification in knock-in–TAC versus wild-type–TAC (G) hearts; scale bar, 25 μm; 40× objective.