Upregulation of Rubicon promotes autosis during myocardial ischemia/reperfusion injury
Jihoon Nah, … , Beth Levine, Junichi Sadoshima
Jihoon Nah, … , Beth Levine, Junichi Sadoshima
Published May 4, 2020
Citation Information: J Clin Invest. 2020;130(6):2978-2991. https://doi.org/10.1172/JCI132366.
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Research Article Cardiology Cell biology

Upregulation of Rubicon promotes autosis during myocardial ischemia/reperfusion injury

Abstract

Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by autophagy from death with autophagy, and whether autophagy contributes to death in cardiomyocytes (CMs) is still controversial. Excessive activation of autophagy induces a morphologically and biochemically defined form of cell death termed autosis. Whether autosis is involved in tissue injury induced under pathologically relevant conditions is poorly understood. In the present study, myocardial ischemia/reperfusion (I/R) induced autosis in CMs, as evidenced by cell death with numerous vacuoles and perinuclear spaces, and depleted intracellular membranes. Autosis was observed frequently after 6 hours of reperfusion, accompanied by upregulation of Rubicon, attenuation of autophagic flux, and marked accumulation of autophagosomes. Genetic downregulation of Rubicon inhibited autosis and reduced I/R injury, whereas stimulation of autosis during the late phase of I/R with Tat–Beclin 1 exacerbated injury. Suppression of autosis by ouabain, a cardiac glycoside, in humanized Na+,K+-ATPase–knockin mice reduced I/R injury. Taken together, these results demonstrate that autosis is significantly involved in I/R injury in the heart and triggered by dysregulated accumulation of autophagosomes due to upregulation of Rubicon.

Authors

Jihoon Nah, Peiyong Zhai, Chun-Yang Huang, Álvaro F. Fernández, Satvik Mareedu, Beth Levine, Junichi Sadoshima

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Figure 5

Inhibition of Na+,K+-ATPase α1 activity attenuates I/R injury in the mouse heart.

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Inhibition of Na+,K+-ATPase α1 activity attenuates I/R injury in the mou...
(A) Schematic diagram of the humanized sequence of Na+,K+-ATPase used to produce the Na+,K+-ATPase–knockin (NaK-KI) mice. (B) Schematic diagram of the experimental design for injection of ouabain during I/R. (C–E) Three-month-old WT and homozygous NaK-KI mice were subjected to 30 minutes of ischemia and 24 hours of reperfusion. They were injected with PBS or ouabain intraperitoneally as indicated in B. Hearts of PBS- or ouabain-injected WT and NaK-KI mice were subjected to TTC staining. (C) Representative images of LV myocardial sections after Alcian blue and TTC staining (scale bar: 1 mm). Ratios of AAR to total LV (E) and infarction area to AAR (D) were compared in WT and NaK-KI mice with PBS or ouabain injection (mean value ± SEM, n = 3 with PBS injected, n = 6 with ouabain injected; **P < 0.01, 2-way ANOVA). (F–J) Twenty-four hours after reperfusion, ouabain-injected WT and NaK-KI mice were subjected to EM analyses. (F) Representative images show AVs (arrowheads) and EVs (arrow) (scale bar: 2 μm). Cytoplasmic AVs and EVs (G) and electron-dense mitochondria (H) were counted. (I) Representative images of CM nuclei showing ballooning of the PNS (scale bar: 2 μm). (J) Percentage of cells with ballooning of the PNS was calculated. Mean ± SEM, n = 4; **P < 0.01 versus ouabain-injected WT; values were measured from more than 10 different areas (G and H) and more than 100 CM nuclei (J) per mouse; 2-way ANOVA (G) and unpaired Student’s t test (H and J). See also Supplemental Figure 5.