Endoplasmic reticulum–mitochondria crosstalk in NIX-mediated murine cell death
Abhinav Diwan, … , Evangelia G. Kranias, Gerald W. Dorn II
Abhinav Diwan, … , Evangelia G. Kranias, Gerald W. Dorn II
Published December 8, 2008
Citation Information: J Clin Invest. 2009;119(1):203-212. https://doi.org/10.1172/JCI36445.
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Research Article Cardiology

Endoplasmic reticulum–mitochondria crosstalk in NIX-mediated murine cell death

Abstract

Transcriptional upregulation of the proapoptotic BCL2 family protein NIX limits red blood cell formation and can cause heart failure by inducing cell death, but the requisite molecular events are poorly defined. Here, we show complementary mechanisms for NIX-mediated cell death involving direct and ER/sarcoplasmic reticulum–mediated (ER/SR-mediated) mitochondria disruption. Endogenous cardiac NIX and recombinant NIX localize both to the mitochondria and to the ER/SR. In genetic mouse models, cardiomyocyte ER/SR calcium stores are proportional to the level of expressed NIX. Whereas Nix ablation was protective in a mouse model of apoptotic cardiomyopathy, genetic correction of the decreased SR calcium content of Nix-null mice restored sensitivity to cell death and reestablished cardiomyopathy. Nix mutants specific to ER/SR or mitochondria activated caspases and were equally lethal, but only ER/SR-Nix caused loss of the mitochondrial membrane potential. These results establish a new function for NIX as an integrator of transcriptional and calcium-mediated signals for programmed cell death.

Authors

Abhinav Diwan, Scot J. Matkovich, Qunying Yuan, Wen Zhao, Atsuko Yatani, Joan Heller Brown, Jeffery D. Molkentin, Evangelia G. Kranias, Gerald W. Dorn II

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Figure 1

Nix localizes to mitochondria and ER and cofractionates with mitochondrial and ER/SR proteins.

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Nix localizes to mitochondria and ER and cofractionates with mitochondri...
(A) HEK293 cells were transfected with FLAG-Nix or β-gal control; fractionated into 10,000 g pellet (10p), 100,000 g pellet (100p), and 100,000 g supernatant (S); and immunoblotted with anti-FLAG, calnexin (Cal), or COX IV antibodies. (B) Neonatal rat ventricular myocytes were infected with adenoviruses encoding FLAG-Nix or β-gal control and processed as in A. E, empty lane. (C and D) Hearts from mice subjected to 1 week of pressure overload (TAC) and nonoperated controls (Non) were fractionated into a 10,000 g pellet and a 100,000 g pellet. The 100,000 g pellet was separated on a discontinuous sucrose gradient to yield the SR-rich fraction (SR, see Methods). 50 μg (C) and 20 μg (D) of the indicated fractions were separated by SDS-PAGE and immunoblotted with anti-Bnip3L (Nix), calnexin, or COX IV antibodies. Positive control (+C) (10 μg) was cellular extract from FLAG-Nix transfected HEK293 cells, showing multiple bands corresponding to Nix homodimers and heterodimers. (E) FLAG epitope–tagged Nix or β-gal control (both green) were transiently expressed in HEK293 cells and analyzed by fluorescence microscopy for colocalization with mitochondrial MitoFluor Red 589 (Mito) or ER calnexin (both red). Nuclei are blue (DAPI). Overlay for Nix is at bottom. Original magnification, ×1,000. Scale bar: 10 μm (shown for comparison).