CTSD stained area was measured by ImageJ software and expressed as a percentage of the total cell area. diminished HG-induced cardiomyocyte death, while CTSD overexpression exaggerated HG-induced cell death. Together, these results suggested that HG increased CTSD expression, induced LMP and brought on CTSD release from the lysosomes, which collectively contributed to HG-induced cardiomyocyte injury. < 0.05 were considered statistically significant. 3.?Results 3.1. High glucose induced lysosomal membrane permeabilization (LMP) in cardiomyocytes Our previous results suggested that high glucose impaired the autophagy-lysosome system in cardiomyocytes . Lysosomal membrane permeabilization (LMP) is usually a prominent feature of lysosomal dysfunction which can lead to cellular damage under certain conditions [15, 17, 18]. LMP is usually characterized by the perturbation of membrane integrity and the consequent leakage of the lysosomal contents such as ions and proteins. We used the LysoSensor? Green and Galectin-3-RFP to assess the lysosomal acidity and membrane integrity of NRVCs. The LysoSensor? Green is usually a fluorescent pH indicator that exhibits a pH-dependent increase in fluorescence intensity upon acidification. Thus, LysoSensor labeled the functional acidic lysosomes as bright puncta around the fluorescent images of NRVC (Fig.1A). Compared with 5.5 mM glucose, high glucose (HG, 30 mM) markedly reduced the number and the RET-IN-1 fluorescent intensity of the puncta, suggesting that HG decreased the lysosomal acidification (Fig.1A). Galectin-3 is usually a cytosolic carbohydrate-binding lectin that is recruited to damaged lysosomes and has been used as a marker of lysosomal membrane injury . As shown in Fig. 1B, Galectin-3-RFP was diffusely distributed with very few puncta at 5.5 mM glucose. However, high glucose led to an RET-IN-1 increased accumulation of Galectin-3-RFP puncta, indicating increased lysosomal membrane injury. Galectin-3-RFP puncta colocalized with lysosomes stained by Lysotracker blue (Fig. 1C). These results suggested that high glucose treatment induced LMP and lysosomal damage. Open in a separate window Physique 1. High glucose (HG) induced LMP and brought on CTSD release in cardiomyocytes.Neonatal Rat Ventricle Cardiomyocytes (NRVC) were cultured in DMEM media containing 5.5 mM or 30 mM glucose for 72hrs. (A) LysoSensor?Green (2 uM) was added to the culture dish and the cells were imaged 30 min later under a fluorescent microscope. (B) NRVC were infected with AdtfGalectin3, cultured for 72 hrs and imaged with the Nikon C2 confocal microscope. (C) Galectin-3-RFP puncta colocalized with Lysotracker blue-stained lysosomes. NRVC were infected with AdtfGalectin3 and cultured for 72 hrs. The cells were incubated with Lysotracker? Blue dye at 100nM for 30 minutes and imaged using a fluorescent microscope. (D) NRVCs were cultured in DMEM for 72hrs, co-immunostained with antibodies against CTSD and LAMP1 and observed under the Nikon C2 confocal microscope. (E) NRVCs were cultured in DMEM for 72h and stained with CTSD antibodies. CTSD stained area was measured by ImageJ software and expressed as a percentage of the total cell area. (F) The CTSD enzymatic activity in culture media IGLC1 was decided. Both the LysoSensor?Green puncta and the Galectin3-RFP puncta were manually counted in at least 5 myocytes. All above experiments were repeated three times in triplicate culture dishes. All data were expressed as mean S.D, n3, *p<0.05. Scale bar represents 20um. 3.2. High glucose (HG) induced CTSD release from the lysosome LMP initially allows passage of small molecules such as ions, but eventually leads to the release of lysosomal proteases from the lysosomal lumen into the cytosolic compartments. We examined the subcellular localization of CTSD using co-immunostaining with lysosomal marker protein (Fig.1D). Most CTSD (red) was colocalized with lysosomal membrane protein LAMP1 (green) in NRVC cultured at 5.5 mM glucose, indicating that CTSD was normally restricted in the lysosome. However, HG treatment induced the leakage of CTSD into the cytosol as shown by its relocation from granular localization to a more diffused distribution in many areas which was no longer limited to the lysosomal lumen. As RET-IN-1 shown in Physique 1E, the anti-CTSD antibody-stained puncta in NRVC appeared mostly sharp-edged at 5.5 mM glucose, but the puncta looked swollen with diffused edges at HG. The total area stained by CTSD was markedly increased by HG as shown in the bar graph. Strikingly, HG was able to induce CTSD release from the lysosome not only into the cytosol but also into the culture media as shown by the increased CTSD enzymatic activity in the media (Fig. 1F). Together, these results suggest that HG induced lysosomal dysfunction RET-IN-1 in NRVC, which was characterized by altered pH, occurrence of LMP and CTSD leakage. These changes may contribute to HG-induced myocyte death as suggested before in other cell types. 3.3. CTSD expression is increased.