Inhibition of the Anti-Apoptotic Bcl-2 Family by BH3 Mimetics Sensitize the Mitochondrial Permeability Transition Pore Through Bax and Bak.

Pooja Patel,Xander H T Wehrens,Meng C Wang,Arielys Mendoza,Dexter J Robichaux,Jason Karch

doi:10.3389/fcell.2021.765973

Abstract

Mitochondrial permeability transition pore (MPTP)-dependent necrosis contributes to numerous pathologies in the heart, brain, and skeletal muscle. The MPTP is a non-selective pore in the inner mitochondrial membrane that is triggered by high levels of matrix Ca2+, and sustained opening leads to mitochondrial dysfunction. Although the MPTP is defined by an increase in inner mitochondrial membrane permeability, the expression of pro-apoptotic Bcl-2 family members, Bax and Bak localization to the outer mitochondrial membrane is required for MPTP-dependent mitochondrial dysfunction and subsequent necrotic cell death. Contrary to the role of Bax and Bak in apoptosis, which is dependent on their oligomerization, MPTP-dependent necrosis does not require oligomerization as monomeric/inactive forms of Bax and Bak can facilitate mitochondrial dysfunction. However, the relationship between Bax and Bak activation/oligomerization and MPTP sensitization remains to be explored. Here, we use a combination of in vitro and ex vivo approaches to determine the role of the anti-apoptotic Bcl-2 family members, which regulate Bax/Bak activity, in necrotic cell death and MPTP sensitivity. To study the role of each predominantly expressed anti-apoptotic Bcl-2 family member (i.e., Mcl-1, Bcl-2, and Bcl-xL) in MPTP regulation, we utilize various BH3 mimetics that specifically bind to and inhibit each. We determined that the inhibition of each anti-apoptotic Bcl-2 family member lowers mitochondrial calcium retention capacity and sensitizes MPTP opening. Furthermore, the inhibition of each Bcl-2 family member exacerbates both apoptotic and necrotic cell death in vitro in a Bax/Bak-dependent manner. Our findings suggests that mitochondrial Ca2+ retention capacity and MPTP sensitivity is influenced by Bax/Bak activation/oligomerization on the outer mitochondrial membrane, providing further evidence of the crosstalk between the apoptotic and necrotic cell death pathways.

Highlights

The Bcl-2 family members are critical regulators of apoptotic cell death by regulating mitochondrial outer membrane permeabilization (MOMP) through the activation or inhibition of Bax and Bak oligomerization (Westphal et al, 2014; Kale et al, 2018)
To determine if the inhibition of various anti-apoptotic Bcl-2 family members through the treatment of BH3 mimetics can exacerbate necrotic cell death, we first determined the amount of each anti-apoptotic Bcl-2 family member expressed at baseline within the mitochondria in Wild type (WT) mouse embryonic fibroblasts (MEFs) by using tandem mass tagmass spectrometry (TMT-MS) (Figure 1A)
Each treatment led to a small increase in cell death, the BH3 mimetics exacerbated both apoptosis induced by staurosporine and necrosis induced by ionomycin in WT MEFs (Figure 1C)

Summary

Introduction

The Bcl-2 family members are critical regulators of apoptotic cell death by regulating mitochondrial outer membrane permeabilization (MOMP) through the activation or inhibition of Bax and Bak oligomerization (Westphal et al, 2014; Kale et al, 2018). During MOMP the outer mitochondrial membrane is permeable to proteins up to 100 kDa in size (Kalkavan and Green, 2018). This event leads to the release of cytochrome-c, which promotes apoptosome formation and subsequent caspase activation resulting in apoptotic cell death (Kalkavan and Green, 2018). The MPTP resides on the inner mitochondrial membrane and is permeable to solutes up to 1.5 kDa when open (Karch and Molkentin, 2015). Genetic deletion and pharmacological inhibition of either of these regulators leads to a desensitization of MPTP and increases mitochondrial Ca2+ retention capacity (Halestrap and Brenner, 2003; Kokoszka et al, 2004; Baines et al, 2005; Nakagawa et al, 2005)

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