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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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 1

Tat–Beclin 1 induces autosis in CMs.

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Tat–Beclin 1 induces autosis in CMs. (A) The autophagic flux probe GFP-L...
(A) The autophagic flux probe GFP-LC3-RFP. (B) NRCMs were transduced with Ad-GFP-LC3-RFP and treated with Tat–Beclin 1 (TB) for 3 hours. GFP/RFP ratio is shown. n = 5 each; scale bar: 20 μm. (C) NRCMs were treated with Tat–Beclin 1 for 2 hours and analyzed by Western blotting. See complete unedited blots in the supplemental material. (D) Ratios of LC3-II to GAPDH and p62 to GAPDH. Mean ± SD, n = 3; *P < 0.05, **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. (E) NRCMs were treated with Tat–Beclin 1 for 3 hours and analyzed with CellTiter-Blue assays. Mean ± SD, n = 4 values were measured from more than 8 different wells per experiment; **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (F) NRCMs were pretreated with Z-VAD, Nec1, or 3MA for 1 hour; treated with Tat–Beclin 1 for 3 hours; and analyzed with CellTiter-Blue assays. Mean ± SD, n = 11 (Scrambled, 5 μM Tat–Beclin 1), n = 8 (10 μM Tat-Beclin 1); *P < 0.05, **P < 0.01 vs. Scrambled, ##P < 0.01 vs. 10 μM Tat–Beclin 1 with vehicle (Veh.), 2-way ANOVA. (G–I) NRCMs were treated with Tat–Beclin 1 for 3 hours and subjected to EM analyses. (G) Nuclei (Nu), ballooning of the PNS, empty vacuoles (EVs), mitochondria (Mito), sarcomeres, and autophagic vacuoles (AVs) (scale bars: 2 μm). Number of AVs per cell (H) and percentage of cells with PNS (I) were evaluated from more than 10 different areas (H) and more than 20 CM nuclei (I) in each experiment. Mean ± SEM, n = 6 (Tat-Scrambled [TS], 5 μM Tat–Beclin 1), n = 3 (1 μM Tat–Beclin 1); **P < 0.01 vs. Tat-Scrambled; ##P < 0.01, 1-way ANOVA with Tukey’s post hoc test. (J) NRCMs were transduced with Ad–shNa+,K+-ATPase α1 and analyzed by Western blotting. (K) NRCMs were transduced with Ad-shNa+,K+-ATPase α1, then treated with Tat–Beclin 1 for 3 hours, and analyzed with CellTiter-Blue assays (mean ± SD, n = 4, values were measured from more than 16 wells per experiment; *P < 0.05, **P < 0.01 vs. Scrambled, ##P < 0.01, 2-way ANOVA). See also Supplemental Figure 1.