Figure 7.

Transcription factor EB (TFEB) normalizes desmin localization via HSPB8 and promotes aggregate removal independent of HSPB8 expression. A, Representative confocal images demonstrating immunolocalization of desmin and αB‐crystallin in neonatal rat cardiac myocytes (NRCMs) adenovirally transduced with TFEB or control, with and without simultaneous knockdown of HSPB8 (ie, treated with adenoviral particles coding for R120G mutant of αB‐crystallin [multiplicity of infection {MOI}=10; for 48 hours] without or with HA‐tagged TFEB [MOI=10; for 24 hours] in the presence of adenoviral particles expressing short hairpin RNA targeting rat HSPB8 [short hairpin HSPB8 {shHSPB8}; MOI=100; for 72 hours]). Adenoviral particles coding for LacZ or short hairpin LacZ were added as controls simultaneously with Ad‐TFEB or Ad–αB‐crystallin overexpression and shHSPB8, respectively, to equalize the number of viral particles. Arrows point to desmin in Z‐discs, and arrowheads point to desmin colocalized with αB‐crystallin in aggregates. Representative of n=2 experiments. B, Representative confocal images demonstrating expression of desmin, ubiquitin, and p62 in NRCMs treated as in A. Arrows point to desmin in Z‐discs, and arrowheads point to desmin colocalized with p62 and ubiquitin in aggregates. Representative of n=2 experiments. C and D, Representative flow cytometric tracings demonstrating JC‐1 fluorescence (C) and quantitation of cells predominantly expressing JC‐1 monomers (lower right quadrants in C; D) for NRCMs treated as described for A. N=7/group. E, Mitochondrial DNA content indexed to nuclear DNA in cells treated as in A. N=4/group. F, Cell death in NRCMs treated as in A. N=7 to 8/group. *P<0.05 by post hoc test after 1‐way ANOVA.