Cardioprotection by the mitochondrial unfolded protein response requires ATF5

Abstract

The mitochondrial unfolded protein response (UPR^mt) is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. UPR^mt activation upregulates chaperones, proteases, antioxidants, and glycolysis at the gene level to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5) is a proposed mediator of the mammalian UPR^mt. Herein, we hypothesized pharmacological UPR^mt activation may protect against cardiac ischemia-reperfusion (I/R) injury in an ATF5-dependent manner. Accordingly, in vivo administration of the UPR^mt inducers oligomycin or doxycycline 6 h before ex vivo I/R injury (perfused heart) was cardioprotective in wild-type but not global Atf5^-/- mice. Acute ex vivo UPR^mt activation was not cardioprotective, and loss of ATF5 did not impact baseline I/R injury without UPR^mt induction. In vivo UPR^mt induction significantly upregulated many known UPR^mt-linked genes (cardiac quantitative PCR and Western blot analysis), and RNA-Seq revealed an UPR^mt-induced ATF5-dependent gene set, which may contribute to cardioprotection. This is the first in vivo proof of a role for ATF5 in the mammalian UPR^mt and the first demonstration that UPR^mt is a cardioprotective drug target.NEW & NOTEWORTHY Cardioprotection can be induced by drugs that activate the mitochondrial unfolded protein response (UPR^mt). UPR^mt protection is dependent on activating transcription factor 5 (ATF5). This is the first in vivo evidence for a role of ATF5 in the mammalian UPR^mt.

Keywords: cardioprotection; chaperone; ischemia; metabolism; mitochondria; unfolded protein response.

Fig. 1.

Loss of activating transcription factor 5 (ATF5), baseline cardiac function, and ischemic-reperfusion injury. A: example PCR genotyping of Atf5, yielding amplicons at 1,006 base pair (bp) for wild type (WT), 439 bp for knockout, and both for heterozygotes. B: neonatal mortality (<21 days) for 93 litters (n = 769 mice) by genotype. Mortality is marked by white portion of bars, with fractional values above. C: baseline cardiac function [rate × pressure product (RPP)] of perfused hearts, by genotype and sex. bpm, beats/min; F, female; M, male. D: post-reperfusion functional recovery (RPP normalized to baseline) by genotype. Means ± SE, both sexes, n = 12 per genotype (WT: black, Atf5^+/−: green, Atf5^−/−: blue). E: infarct sizes of the hearts in D. Representative slices (E, top) and digitally parsed pseudocolor images (E, middle) show red healthy tissue and white infarct. Means ± SE shown to right of individual data. F: post-reperfusion functional recovery (data from D stratified by sex). Means ± SE, all genotypes sexes, n = 21 female (black), n = 15 male (gray). G: infarct data for hearts in F. No significant differences between any groups by 2-way ANOVA.

Fig. 2.

Mitochondrial unfolded protein response induction by oligomycin is cardioprotective and requires activating transcription factor 5 (ATF5). A: post-reperfusion functional recovery [rate × pressure product (RPP), normalized to baseline] of hearts from wild-type (WT) mice given oligomycin (orange) or vehicle (black); n = 5. *P < 0.05 vs. matching time point. B: infarct sizes of hearts in A. Representative and pseudocolor images as per Fig. 1E. *P < 0.05 vs. vehicle. C: post-reperfusion functional recovery of hearts from Atf5^−/− mice given oligomycin (pink) or vehicle (blue); n = 5 per group. *P < 0.05 vs. matching time point. D: infarct sizes of hearts in C. E: baseline cardiac functional data for hearts in this cohort. V, vehicle; O, oligomycin; bpm, beats/min. All data are means ± SE.

Fig. 3.

Mitochondrial unfolded protein response induction by doxycycline is cardioprotective and requires activating transcription factor 5 (ATF5). A: post-reperfusion functional recovery [rate × pressure product (RPP), normalized to baseline] of hearts from WT mice given doxycycline (red) or vehicle (black); n = 5 per group. *P < 0.05 vs. matching time point. B: infarct sizes of hearts in A. Representative and pseudocolor images as per Fig. 1E. *P < 0.05 vs. vehicle. C: post-reperfusion functional recovery of hearts from Atf5^−/− mice given doxycycline (purple) or vehicle (blue); n = 5 per group. D: infarct sizes of hearts in C. E: baseline cardiac functional data for hearts in this cohort. V, vehicle; D, doxycycline; bpm, beats/min. F: post-reperfusion functional recovery of WT hearts given acute doxycycline (red) or vehicle (black) ex vivo; n = 3. G: infarct sizes of the hearts in F. All data are means ± SE.

Fig. 4.

Mitochondrial unfolded protein response (UPR^mt) gene expression. A: relative wild-type (WT) cardiac expression of selected genes 6 h after oligomycin treatment by RT-qPCR (−ΔΔCq, normalized to Hprt1 reference gene and compared with vehicle treatment). Hspa5 (BiP) is an endoplasmic reticulum UPR chaperone, Hspd1 (HSP60) is a UPR^mt chaperone, and Clpp and Lonp are UPR^mt proteases. Horizontal lines represent means. *P < 0.05 vs. vehicle, n = 5. B: primers used for quantitative PCR. D: Western blot normalized to protein loading (Ponceau-stained membrane) was used to quantify relative levels of indicated proteins in hearts 6 h after vehicle or oligomycin. Values for each protein are expressed relative to abundance in vehicle-treated hearts. Representative blots are shown, with one biological replicate (animal) per lane. Note that in lane 2 of the oligomycin group, the sample was lost during preparation so the lane was left blank. *P < 0.05 compared with vehicle-treatment, n = 5/4 per group. Data are means ± SE. C: RNA-Seq heat map showing relative expression of 69 genes identified as upregulated by oligomycin/doxycycline treatment in hearts from WT mice with a blunted response in hearts from Atf5^−/−; 48 of the 69 genes (left) are color-coded for annotation by functional category. V, vehicle; D, doxycycline.