Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Abstract

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca(2+) have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca(2+) chelator BAPT A-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy independent of Ca(2+), ROS generation, and mPTP opening.

Figure 1

Bnip3 is a potent inducer of autophagy in adult cardiac myocytes. (A) Adult cardiac myocytes were infected with adenoviruses encoding β-gal or Bnip3 plus GFP-LC3 for 24 hours and then stained with LysoTracker Red. Live cells were examined by fluorescence microscopy. Merged image shows co-localization between autophagosomes and lysosomes (yellow). (B) Quantitation of autophagy (n = 3). (C) Western blot analysis of LC3-I and LC3-II levels in myocytes overexpressing Bnip3. (D) Bnip3 increased autophagy in a dose-dependent manner when overexpressed at increasing multiples of infection (MOI) (n = 3, *p < 0.05 compared to 0 MOI of Ad-Bnip3). Overexpression of Bnip3 was verified by western blotting.

Figure 2

Bnip3 localizes to the mitochondria where it induces mitochondrial autophagy. (A) A cardiac myocyte infected with Ad-Bnip3 was stained with antibodies against Bnip3 and Tom20. Fluorescence microscopy shows co-localization between Bnip3 (red) and Tom20 (green). (B) Western blotting of cytosolic and mitochondrial fractions confirms mitochondrial localization of Bnip3 when overexpressed in myocytes. Tom20 served as a mitochondrial marker. (C) Cells were stained with anti-COX IV to label mitochondria in µ-gal and Bnip3 overexpressing cells at 24 and 48 hrs post-infection. Co-localization between GFP -LC3 and COX IV at 24 hrs demonstrated extensive co-localization between autophagosomes and mitochondria (marked by arrows). After 48 hrs, there was substantial loss of mitochondria with accumulation of autophagosomes around the nucleus. (D) Electron micrographs revealed autophagosomes containing mitochondria (white arrows) in myocytes overexpressing Bnip3 (scale bar = 500 nm).

Figure 3

Bnip3 induces autophagy independent of Ca²⁺, ROS and mPTP opening. (A) Starvation-induced autophagy is inhibited in the presence of 0.2 µM BAPTA-AM, 1 µM CsA or 1 mM NAC in adult myocytes (n = 3, *p < 0.05 compared with non-starved cells). (B) Bnip3-mediated autophagy is not inhibited in the presence of 0.2 µM BAPTA-AM, 1 mM NAC or 1 µM CsA. (C) Quantification of Bnip3-mediated autophagy in the presence of DMSO or inhibitors (*, **, *** and ****p ≤ 0.05 compared with control, n = 3). (D) The presence of CsA failed to inhibit Bnip3-mediated mitochondrial autophagy. Representative image of an adult myocyte infected with Bnip3 for 24 h in the presence of 1 µm CsA. Maximum intensity projection of a cardiac myocyte observed along the XY (center), XZ (top) and YZ (right) planes. Colocalization scatter plot of mitochondria (quadrant 1) and LC3-GFP (quadrant 2). Quadrant 3 represents colocalization of mitochondria and LC3GFP. (E) Mouse embryonic fibroblasts isolated from WT and CypD^-/- mice were infected with adenoviruses encoding µ-gal or Bnip3 plus GFP-LC3 for 24 hours and then examined by fluorescence microscopy. Shown are representative images. (F) Quantitation of autophagy (*p ≤ 0.05 compared with control, n = 3).