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Mesencephalic astrocyte–derived neurotrophic factor is an ER-resident chaperone that protects against reductive stress in the heart

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We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6α (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca2+ depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress.

endoplasmic reticulum stress (ER stress)
protein folding
cardiomyocyte
ischemia
chaperone
cardiomyocyte-
heart function
ischemia/reperfusion
mesencephalic astrocyte-derived neurotrophic factor (MANF)
reductive stress
unfolded protein response

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Author contributions—A. A. and C. C. G. conceptualization; A. A., E. A. B., W. T. S., M. S. D., A. S. B., D. J. T., A. N. P., C. A., A. V. S., S. D., and C. C. G. data curation; A. A., E. A. B., W. T. S., A. S. B., D. J. T., A. N. P., A. V. S., S. D., and C. C. G. formal analysis; A. A., E. A. B., and C. C. G. funding acquisition; A. A., E. A. B., W. T. S., A. S. B., D. J. T., and C. C. G. validation; A. A., E. A. B., S. D., and C. C. G. investigation; A. A. and C. C. G. visualization; A. A., E. A. B., S. D., and C. C. G. methodology; A. A. and C. C. G. writing-original draft; A. A., E. A. B., W. T. S., D. J. T., S. D., and C. C. G. writing-review and editing; D. J. T., S. D., and C. C. G. supervision; C. C. G. project administration.

Funding and additional information—This work was supported by American Heart Association Grant 17PRE33670796; National Institutes of Health Grant 1F31HL140850 (to E. A. B.) and Grants R01 HL135893, R01 HL141463, and R01 HL149931 (to C. C. G.); the San Diego State University (SDSU) Heart Institute (to W. T. S., A. A., E. A. B., and C. C. G.); the Inamori Foundation (to E. A. B.); and the ARCS Foundation, Inc., San Diego Chapter (to W. T. S. and E. A. B). Additionally, W. T. S., A. A., and E. A. B are Rees-Stealy Research Foundation Phillips Gausewitz, M.D., Scholars of the SDSU Heart Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    ER

    endoplasmic reticulum

    AAV9

    adeno-associated virus serotype 9

    ANOVA

    analysis of variance

    ATF6

    activating transcription factor 6α

    GRP78 and -94

    78- and 94-kDa glucose-regulated protein, respectively

    HMGB1

    high-mobility group box 1 protein

    I/R

    ischemia/reperfusion

    LVDP

    left ventricular developed pressure

    MANF

    mesencephalic astrocyte derived neurotrophic factor

    MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

    NEM

    N-ethylmaleimide

    sI

    simulated ischemia

    sI/R

    simulated ischemia/reperfusion

    TG

    thapsigargin

    TTC

    2,3,5-triphenyl tetrazolium chloride

    UPR

    unfolded protein response

    aa

    amino acids

    KD

    knockdown

    AMVM

    adult mouse ventricular myocyte

    NRVM

    neonatal rat ventricular myocyte

    PI

    propidium iodide

    ROS

    reactive oxygen species

    α1AT

    α1-antitrypsin

    IP

    immunoprecipitation

    IB

    immunoblotting

    rMANF

    recombinant MANF

    TM

    tunicamycin

    DMEM

    Dulbecco's modified Eagle's medium

    FBS

    fetal bovine serum

    AdV

    adenovirus

    qPCR

    quantitative PCR

    DPBS

    Dulbecco's PBS

    HA

    hemagglutinin

    rLA

    reduced α-lactalbumin

    GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

    LDH

    lactate dehydrogenase

    MEM

    minimal essential medium.