Endoplasmic reticulum–mitochondria crosstalk in NIX-mediated murine cell death
Abhinav Diwan, Scot J. Matkovich, Qunying Yuan, Wen Zhao, Atsuko Yatani, Joan Heller Brown, Jeffery D. Molkentin, Evangelia G. Kranias, Gerald W. Dorn II
Abhinav Diwan, Scot J. Matkovich, Qunying Yuan, Wen Zhao, Atsuko Yatani, Joan Heller Brown, Jeffery D. Molkentin, Evangelia G. Kranias, Gerald W. Dorn II
View: Text | PDF
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

×

Figure 5

ER- and mitochondria-targeted Nix are equally effective in killing cultured cardiac myocytes but utilize different mediators.

Options: View larger image (or click on image) Download as PowerPoint
ER- and mitochondria-targeted Nix are equally effective in killing cultu...
(A) Cultured neonatal rat cardiac myocytes were infected with adenoviruses encoding WT Nix or 1 of the 3 Nix mutants and subjected to fluorescence microscopy for subcellular localization. Shown are overlay images with FLAG-Nix (green) and MitoFluor Red 589 (red). Original magnification, ×1,000. Scale bar: 10 μm (shown for comparison). (B) Cultured neonatal rat cardiac myocytes were infected with adenoviruses encoding WT Nix or 1 of the 3 Nix mutants, and Nix expression was analyzed by immunoblotting for FLAG epitope and GAPDH (loading control; 25 μg/lane). Arrows indicate Nix mutants with varying molecular weights. (C) Quantitative analysis of cardiomyocyte death (left y axis, white bars) and TUNEL positivity in the absence (right y axis, black bars) and presence (right y axis, gray bars) of 25 μM BAPTA-AM induced by subcellular targeting of Nix. Means ± SEM of 4 independent experiments for death and 8 (–BAPTA) and 5 for TUNEL (+BAPTA) are shown. (D) Confocal microscopy of TMRE (red) and fluorescent caspase substrate (rhodamine 100 bis-l-aspartic acid amide; green) in cultured neonatal rat cardiac myocytes infected with adenoviruses encoding WT Nix or 1 of the 3 Nix mutants. Original magnification, ×400. Nuclei are blue (Hoechst 33342). Scale bar: 20 μm (shown for comparison).