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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.

SUBMITTER: Nah J

PROVIDER: S-EPMC7260042 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Upregulation of Rubicon promotes autosis during myocardial ischemia/reperfusion injury.

Nah Jihoon J Zhai Peiyong P Huang Chun-Yang CY Fernández Álvaro F ÁF Mareedu Satvik S Levine Beth B Sadoshima Junichi J

The Journal of clinical investigation 20200601 6

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 ...[more]

PMID: 32364533

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Project description:ObjectiveThe incidence of acute myocardial infarction (AMI) is increasing yearly. With the use of thrombolysis or percutaneous coronary intervention (PCI), the mortality rate of acute myocardial infarction has been significantly reduced. However, reperfusion can cause additional myocardial injury. There is still a lack of effective drugs to treat I/R injury, and it is urgent to find new therapeutic drugs.MethodsIn this study, network pharmacology was used to predict potential targets and biological processes involved in Muscone-mediated treatment of acute myocardial infarction. To model ischemia‒reperfusion injury, a hypoxia-reoxygenation model and in vivo ischemia‒reperfusion injury C57BL/6 mice model was constructed. Mice were treated with Muscone i.p. for 4 weeks. We detected the cardiac function on day 28.The expression levels of the apoptotic proteins Caspase-3 and Bax and the anti-apoptotic protein Bcl-2 were detected by immunoblotting after Muscone treatment of AC16 cells and in vivo. Additionally, the gene expression levels of the PUMA and p53 were analyzed by qRT‒PCR. Molecular docking was used to evaluate the binding energy between Muscone and NLRP3-related proteins. Immunoblotting and qRT‒PCR were used to assess the expression levels of NLRP3 signaling pathway-related proteins (NLRP3, ASC, and Caspase-1) and the NLRP3 gene, respectively. Moreover, the extracellular acidification rate of AC16 cells was measured using the Seahorse system to evaluate glycolysis levels after Muscone treatment. The expression of the key glycolytic enzyme PKM2 was analyzed by immunoblotting and qRT‒PCR. Finally, ChIP‒qPCR was performed to determine the levels of histone modifications (H3K4me3, H3K27me3, and H2AK119Ub) in the PKM2 promoter region.ResultsGO functional enrichment analysis revealed that muscone was involved in regulating the biological processes (BP) of AMI, which mainly included negative regulation of the apoptosis signaling pathway, the response to lipopolysaccharide, and blood pressure regulation. The cellular components (CC) involved in muscone-mediated regulation of AMI mainly included lipid rafts, membrane microdomains, and membrane regions. The molecular functions (MF) involved in muscone-mediated regulation of AMI mainly included oxidoreductase activity, nuclear receptor activity, and transcription factor activity. In vitro results indicated that muscone treatment could inhibit the expression levels of Bax and Caspase-3 in AC16 cells after ischemia‒reperfusion while increasing the expression level of the antiapoptotic protein Bcl-2. Muscone significantly suppressed the transcription levels of p53 and PUMA in AC16 cells. Molecular docking suggested that muscone could bind well with the Cryo-EM structure of NEK7(PDB ID:6NPY). Further investigation of inflammatory pathways revealed that muscone could inhibit the expression level of NLRP3 in AC16 cells and reduce the expression levels of Caspase-1 and Caspase recruitment domain. Fluorescent quantitative PCR experiments showed that muscone significantly inhibited the transcription of NLRP3. Moreover, we found that muscone could enhance the glycolytic efficiency of AC16 cells, which may be related to the increased protein expression of PKM2 in AC16 cells. Fluorescent quantitative PCR showed that muscone could increase the transcription level of PKM2. Chromatin immunoprecipitation assays showed that muscone treatment increased the expression level of H3K4me3 in the PKM2 promoter region and inhibited the levels of H3K27me3 and H2AK119Ub in the PKM2 promoter region.ConclusionMuscone promoted myocardial glycolysis and inhibited NLRP3 pathway activation to improve myocardial ischemia‒reperfusion injury.

Project description:Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1?±?9.3 vs. 14.3?±?9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3?±8.4% and 42.2?±?11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

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Upregulation of Rubicon promotes autosis during myocardial ischemia/reperfusion injury.

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Upregulation of Rubicon promotes autosis during myocardial ischemia/reperfusion injury.

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