PEDF protects cardiomyocytes by promoting FUNDC1‑mediated mitophagy via PEDF-R under hypoxic condition

Abstract

Pigment epithelial-derived factor (PEDF) is known to exert diverse physiological activities. Previous studies suggest that hypoxia could induce mitophagy. Astoundingly, under hypoxic condition, we found that PEDF decreased the mitochondrial density of cardiomyocytes. In this study, we evaluated whether PEDF could decrease the mitochondrial density and play a protective role in hypoxic cardiomyocytes via promoting mitophagy. Immunostaining and western blotting were used to analyze mitochondrial density and mitophagy of hypoxic cardiomyocytes. Gas chromatography‑mass spectrometry and ELISA were used to analyze levels of palmitic acid and diacylglycerol. Transmission Electron Microscopy was used to detect mitophagy and the mitochondrial density in adult male Sprague-Dawley rat model of acute myocardial infarction. Compared to the control group, we observed that PEDF decreased mitochondrial density through promoting hypoxic cardiomyocyte mitophagy. PEDF increased the levels of palmitic acid and diacylglycerol, and then upregulated the levels of protein kinase Cα (PKC-α) and its activation. Furthermore, inhibition of PKC-α by Go6976 could effectively suppress PEDF-induced mitophagy. Besides, we found that PEDF promoted FUNDC1-mediated cardiomyocyte mitophagy via ULK1, which depended on the activation of PKC-α. Finally, we discovered that mitophagy was increased and mitochondrial density was reduced in adult male Sprague-Dawley rat model of acute myocardial infarction. We concluded that PEDF promotes mitophagy to protect hypoxic cardiomyocytes, through PEDF/PEDF-R/PA/DAG/PKC-α/ULK1/FUNDC1 pathway.

Figure 1

Pigment epithelial-derived factor (PEDF) decreased the mitochondrial density of primary cardiomyocytes under hypoxic condition. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 0, 2, 4, 8 and 12 h with or without PEDF (10 nM). (A) Cell viability was assayed by the cell counting kit-8 (CCK-8). Approximately 1×10⁴ cells were grown in each well of 96-well plates with 100 µl medium and the absorbance at 450 nm was directly proportional to the number of viable cells (n=4, ^*p<0.05). (B) Samples were collected for western blotting to analyze the level of mitochondrial protein COX IV by ImageJ software (n=4, ^*p<0.05). (C) Samples were stained with MitoTracker Red. For each indicated times, total mitochondrial number per cultured cardiomyocytes was quantified from stacks of images through the entire thickness of cardiomyocytes by ImageJ software (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). (D) Five randomly picked regions of each sample were captured by confocal z-axis scanning and the total volume of mitochondria was calculated and quantified (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Data are expressed in fold induction, relative to control. Values are means ± SD.

Figure 2

Pigment epithelial-derived factor (PEDF) played a protective role in primary cardiomyocytes through promoting mitophagy, and has no effect on mitochondrial fusion. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 0, 2, 4, 8 and 12 h with or without PEDF (10 nM). Lysosome inhibitor BAF1 (50 nM) was added. (A) LC3-II, Opa1, Mfn1 and Mfn2 proteins in the mitochondrial fractions were tested by western blotting (n=4, ^*p<0.05). (B) Samples were stained with MitoTracker Red and anti-LC3 (green) antibody. Quantitative analysis of cardiomyocytes that contain co-localization of LC3 dots with mitochondria per cardiomyocytes. (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Lysosome inhibitor BAF1 (50 nM) was added. (C and D) Cell counting kit-8 (CCK-8) and LDH released assays were employed to assessed cell viability and the rate of cell death (n=4, ^*p<0.05). (E) Samples were stained with MitoTracker Red. Five randomly picked regions of each sample were captured by confocal z-axis scanning and the total volume of mitochondria was calculated and quantified (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Data were expressed in fold induction, relative to control. Values are means ± SD.

Figure 2

Pigment epithelial-derived factor (PEDF) played a protective role in primary cardiomyocytes through promoting mitophagy, and has no effect on mitochondrial fusion. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 0, 2, 4, 8 and 12 h with or without PEDF (10 nM). Lysosome inhibitor BAF1 (50 nM) was added. (A) LC3-II, Opa1, Mfn1 and Mfn2 proteins in the mitochondrial fractions were tested by western blotting (n=4, ^*p<0.05). (B) Samples were stained with MitoTracker Red and anti-LC3 (green) antibody. Quantitative analysis of cardiomyocytes that contain co-localization of LC3 dots with mitochondria per cardiomyocytes. (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Lysosome inhibitor BAF1 (50 nM) was added. (C and D) Cell counting kit-8 (CCK-8) and LDH released assays were employed to assessed cell viability and the rate of cell death (n=4, ^*p<0.05). (E) Samples were stained with MitoTracker Red. Five randomly picked regions of each sample were captured by confocal z-axis scanning and the total volume of mitochondria was calculated and quantified (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Data were expressed in fold induction, relative to control. Values are means ± SD.

Figure 3

Pigment epithelial-derived factor (PEDF) increases the level of PA and diacylglycerol (DAG) via PEDF-R, leading to protein kinase Cα (PKC-α) activation, which induced mitophagy. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 4 h with or without PEDF (10 nM). RNA interference assays were used to silence PEDF-R. (A) The PA level was quantified using GC-MS (n=4, ^*p<0.05). (B) The DAG level was quantified by enzyme-linked immunosorbent assay (ELISA) (n=4, ^*p<0.05). (C) p-PKC-α and PKC-α proteins were analyzed by western blotting (n=4, ^*p<0.05). (D) PKC-α inhibitor Go6976 (1 µM) was added. Samples were stained with MitoTracker Red and anti-LC3 (green) antibody. Quantitative analysis of cardiomyocytes that contain co-localization of LC3 dots with mitochondria per cardiomyocytes (^*p<0.05, n=90 cardiomyocytes from three independent experiments; scale bar, 20 µm). Data are expressed in fold induction, relative to control. Values are means ± SD.

Figure 4

Pigment epithelial-derived factor (PEDF)-activated protein kinase Cα (PKC-α) promotes FUNDC1-mediated primary cardiomyocyte mitophagy under hypoxic condition. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 4 h with or without PEDF (10 nM). RNA interference assays were used to silence PEDF-R. PKC-α inhibitor Go6976 (1 µM) was added. (A-C) Nix, Parkin, FUNDC1 and p-FUNDC1 proteins in the mitochondrial fractions were tested by western blotting (n=4, ^*p<0.05). Data were expressed in fold induction, relative to control. Values are means ± SD.

Figure 5

Pigment epithelial-derived factor (PEDF)-induced hypoxic primary cardiomyocyte mitophagy through FUNDC1 is dependent on ULK1 signaling pathway. Primary cardiomyocytes were maintained in normoxic or hypoxic conditions for 4 h with or without PEDF (10 nM). RNA interference assays were used to silence PEDF-R. PKC-α inhibitor Go6976 (1 µM) was added. ULK1, FUNDC1, p-FUNDC1 and LC3-II levels were tested by western blotting (n=4, ^*p<0.05). Data were expressed in fold induction, relative to control. Values are means ± SD.

Figure 6

Pigment epithelial-derived factor (PEDF) decreases the mitochondrial density by promoting mitophagy in vivo. Among the 55 rats entered into our experiment, 5 died after 4 h surgery. So 50 rats were used for analysis of transmission electron microscopy and randomized into the four group (n=5). (A) Electron micrographs show cardiomyocyte mitophagy from border zone after 1, 2, 3 and 4 h surgery. Typical mitophagy (arrows) was observed. Scale bar, 500 nm. (B) Electron micrographs show cardiomyocyte mitochondrial density and mitochondrial volume from border zone after 1, 2, 3 and 4 h surgery. Scale bar, 2 µm.