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
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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 4

Creation of mitochondria- and ER-specific Nix mutants.

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Creation of mitochondria- and ER-specific Nix mutants. (A) Calcium-induc...
(A) Calcium-induced swelling of purified WT (triangles) and Nix-knockout (diamonds) liver mitochondria. Calcium (250 μM) was added (arrow) and the decrease in absorbance of 540 nm light assessed over time. Each curve represents the mean of 2 separate experiments. (B) Schematic depiction of mutation strategy for organelle-specific Nix. TM, putative transmembrane domain. (C and D) FLAG epitope–tagged WT Nix (Nix-WT), Nix-ActA, Nix-cb5, or truncated Nix (Nix-trunc) were transiently expressed in HEK293 cells, fractionated into 10,000 g pellet, 100,000 g pellet, and 100,000 g supernatant, and immunoblotted with anti-FLAG, calnexin, or COX IV antibodies (25 μg protein/lane). (D) Transiently transfected WT Nix, Nix-ActA, Nix-cb5, or truncated Nix (all green) as in C were analyzed by fluorescence microscopy for colocalization with MitoFluor Red 589 or ER calnexin (both red). Nuclei are blue (DAPI). Overlays are shown at bottom. Original magnification, ×1,000. Scale bar: 10 μm (shown for comparison).