Cardiomyocyte Contractility and Autophagy in a Premature Senescence Model of Cardiac Aging

Figure 5

Changes in autophagy for the culture of neonatal cardiomyocytes. To assess autophagy in the culture of murine, neonatal cardiomyocytes, upstream regulators and central constituents were determined using immunoblot and qPCR analyses ( mice). (a) AMPK activity was measured by detecting the subunit α and a comparison of the basal state to its phosphorylation at Thr¹⁷². For the determination of mTOR activity, (b) catalytic unit mTOR and (c) the target protein p70S6k in relation to its mTOR-dependent phosphorylation at Thr³⁸⁹ were quantified. Further analyzed were time-dependent changes in relative mRNA expression of (d) TFEB, (e) LC3, and (f) p62. To measure autophagic efficiency, protein levels of (g) LC3-I in relation to LC3-II, (h) p62, and (i) Lys⁶³-specific polyubiquitin were determined. Detected proteins were normalized to GAPDH as internal control, and representative immunoblots are shown. Data are presented as . Statistical significance was assessed by one-way ANOVA (); ^areference day 6; ^breference day 9; ^creference day 13; ^dreference day 17.

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