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 2

Tat–Beclin 1–treated CMs show decreased levels of cellular membranes.

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Tat–Beclin 1–treated CMs show decreased levels of cellular membranes. (A...
(A–D) NRCMs were treated with Scrambled or Tat–Beclin 1 (5 and 10 μM) for 3 hours and subjected to membrane fractionation assays. The heavy membrane and cytosolic fractions were analyzed by Western blotting using anti-calnexin (ER), anti-PDH (mitochondria), anti-PMCA (plasma membrane), and anti–α-tubulin antibodies (A). Expression ratios of calnexin (B), PDH (C), and PMCA to α-tubulin (D) were quantified; mean ± SD, n = 5 (B), n = 4 (C and D); *P < 0.05, **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (E and F) NRCMs were transfected with siControl (siCtrl) or siVAPA and siVAPB. After 60 hours, cells were treated with Scrambled or Tat–Beclin 1 at the indicated doses for 3 hours and subjected to membrane fractionation assays. (E) Whole cell lysates (WCL) and heavy membrane (mem.) fractions were used for immunoblot analyses with anti-calnexin, anti-VAPA, anti-LC3, and anti–α-tubulin antibodies. (F) Cell death induced by 10 μM Tat–Beclin 1 was quantified with CellTiter-Blue assays; mean ± SD, n = 4 values were measured from more than 4 different wells per experiment; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. See also Supplemental Figure 2.