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 3

Autophagic flux is increased during early reperfusion but is inhibited during late reperfusion in the mouse heart.

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Autophagic flux is increased during early reperfusion but is inhibited d...
(A–C) Three-month-old C57BL/6J mice were subjected to 30 minutes of ischemia (Is.) followed by reperfusion for the indicated times, and the border region of infarction in the heart was analyzed by Western blotting using anti-p62, anti-LC3, and anti–α-tubulin antibodies (A). The ratios of LC3-II (B) and p62 to α-tubulin (C) were quantified (mean ± SEM, n = 6; *P < 0.05 vs. Sham, 1-way ANOVA with Dunnett’s post hoc test). (D–G) Three-month-old cardiac tissue–specific GFP-LC3-RFP-LC3ΔG transgenic mice were subjected to I/R for 4 and 24 hours. and hearts were analyzed by confocal microscopy to assess autophagic flux. Representative fluorescence ratio images of heart sections from cardiac tissue–specific GFP-LC3-RFP-LC3ΔG transgenic mice subjected to I/R for 4 (D) and 24 hours (E). Scale bars: 100 μm. The graph shows quantification of GFP/RFP fluorescence ratio intensities at 4 (F) and 24 hours (G) (mean ± SEM, n = 3; *P < 0.05, **P < 0.01 vs. remote region, 1-way ANOVA with Dunnett’s post hoc test). (H–K) Samples of hearts from mice subjected to I/R for various periods of time were subjected to EM analyses. Autolysosomes (ALs, arrow) and autophagosomes (APs, arrowhead) are indicated (H) and enlarged (J). Scale bars: 1 μm. Cytoplasmic ALs and APs (I) and enlarged APs with nondegraded materials (K) were counted. (I and K) Mean ± SEM, n = 7 for sham, n = 3 for ischemia, n = 5 for IR 2 hours, n = 4 for IR 6 hours, n = 7 for IR 24 hours; **P < 0.01 versus Sham, ##P < 0.01; values were measured from more than 10 different areas per mouse; 2-way ANOVA (I) and 1-way ANOVA with Dunnett’s post hoc test (K). See also Supplemental Figure 3.