Fig 2: Cathepsin D (CTSD) was released by human stem cell-derived cardiomyocytes in concert with troponin T. Extracellular levels of CTSD were elevated after stretch (A), while intracellular levels were only reduced after tumour necrosis factor alpha (TNFa) stimulation or hypoxia (B). Damage marker release of troponin T was also increased by mechanical stretch (C). Graphs show results from three independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. unstimulated control human stem cell-derived cardiomyocytes unless otherwise indicated. Statistical analysis was done with with a Kruskal–Wallis test followed by Dunn's post-hoc test. HIF1a, hypoxia-inducible factor 1 alpha.

Fig 3: Activities of (a) CTSD and (b) BH in corneocytes from subjects of Groups III and IV. Group III included subjects with a CUR value lower than 0.22, whereas Group IV comprised subjects with a CUR value higher than 0.22. Significance between each Group was determined by the Mann-Whitney U test (*p < 0.05, n = 20 per group). CTSD, cathepsin D; BH, bleomycin hydrolase; CUR, corneocyte unevenness ratio; RFU, relative fluorescence unit

Fig 4: Inhibition of lysosomal exocytosis by vacuolin-1 improved autophagic response by retention of intracellular cathepsin D (CTSD). Extracellular levels of CTSD after stretch were reduced compared to stretched vehicle control stem cell-derived cardiomyocytes (hPSC-CM) (A). Incubation with vacuolin-1 increased intracellular levels of CTSD (B). Inhibition of CTSD secretion marginally affected apoptosis (C) and prevented induction of necrosis (D). Autophagy was restored by vacuolin-1 in CTSD knockdown hPSC-CM after stretch (E), whereas metabolism was unaffected (F). Graphs show results from three independent experiments; *P < 0.05; **P < 0.01 vs. static scrambled control hPSC-CM (shSCR) unless otherwise indicated. Statistical analysis was done with with a Kruskal–Wallis test followed by Dunn's post-hoc test.

Fig 5: Cathepsin D (CTSD) knockdown resulted in increased cell death following mechanical stretch. Intracellular levels of CTSD were greatly reduced by shRNA-mediated CTSD gene silencing (A); extracellular levels were found to show a similar pattern (B). Troponin T levels were found to be increased following mechanical stretch with CTSD knockdown (C). CTSD knockdown resulted in deteriorated sarcomeric structure (D). Mechanical stretch induced apoptosis regardless of CTSD knockdown (E), whereas necrosis is increased after stretch in human stem cell-derived cardiomyocytes (hPSC-CM) with CTSD knockdown (F). Stretching induced autophagy in scrambled control hPSC-CM, which was abrogated in CTSD-deficient cells (G). Stretching resulted in increased metabolism in hPSC-CM with CTSD silencing exclusively (GH). Graphs show results from three independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. static scrambled control hPSC-CM (shSCR) unless otherwise indicated. Statistical analysis was done with with a Kruskal–Wallis test followed by Dunn's post-hoc test.