Rationale: ER stress dysregulates ER proteostasis, which activates the transcription factor, ATF6, an inducer of genes that enhance protein folding and restore proteostasis. Due to increased protein synthesis, it is possible that protein folding and, thus, ER proteostasis are challenged during cardiac myocyte growth. However, it is not known whether ATF6 is activated, and if so, what its function is during hypertrophic growth of cardiac myocytes. Objective: To examine the activity and function of ATF6 during cardiac hypertrophy. Methods and Results: We found that ATF6 was activated and ATF6-target genes were induced in mice subjected to an acute model of trans-aortic constriction (TAC), or to free-wheel exercise, which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO) blunted TAC- and exercise-induced cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript profiling and chromatin immunoprecipitation identified RHEB as an ATF6-target gene in the heart. RHEB is an activator of mTORC1, a major inducer of protein synthesis and subsequent cell growth. Both TAC and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in WT mouse hearts, but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine (PE)-, and insulin-like growth factor 1 (IGF1)-mediated Rheb induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, AAV9-RHEB restored cardiac growth to ATF6 cKO mice subjected to TAC. Finally, ATF6 induced RHEB in response to growth factors, but not in response to other activators of ATF6 that do not induce growth, indicating that ATF6 target gene induction is stress-specific. Conclusions: Compensatory cardiac hypertrophy activates ATF6, which induces Rheb and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.
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.
Rationale: In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during myocardial ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. In response to ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF is induced by ATF6, is ER-localized, and possesses a conserved pattern of cysteines found in all known species of MANF, we hypothesized that MANF is a redox-regulated chaperone that optimizes cardiomyocyte viability during ischemia. Methods: The ability of MANF to bind misfolded proteins during reductive ER stress or ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF (rMANF) to suppress aggregation of misfolded proteins was examined in an in vitro chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo , were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. Results: In NRVM subjected to reductive ER stress or simulated ischemia, MANF formed disulfide-linked complexes with misfolded proteins. Under reducing conditions, rMANF suppressed aggregation of model misfolded proteins in vitro , and mutant rMANF in which the cysteine residues were mutated to alanine did not suppress misfolded protein aggregation. MANF knockdown in the heart, in vivo , increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency, in vivo . Conclusions: MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.
Rationale: In cardiomyocytes, secreted and membrane proteins critical for heart function are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that myocardial ischemia decreases oxygen required for disulfide bond formation in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins, which contributes to cardiomyocyte death. In response to ER stress, the transcription factor ATF6 induces various ER-resident proteins that restore SR/ER protein folding, including ER chaperones. We found that ATF6 induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function could not be inferred by analogy to other proteins. Since we found that MANF is an ATF6-inducible ER-resident protein we hypothesized that it functions as a chaperone, and since MANF has 8 cysteine residues that are conserved in a wide range of species, that its chaperone function is redox-regulated and protective in the ischemic heart. Methods: The ability of recombinant MANF (rMANF) to suppress misfolded protein aggregation was examined in an in vitro chaperone assay. The effect of MANF knockdown on cell viability during simulated ischemia (sI) was determined in neonatal rat ventricular myocytes (NRVM). The effect of MANF loss-of-function in the ischemic heart, in vivo , was determined in a novel mouse model in which MANF is knocked down in cardiomyocytes. Results: rMANF formed disulfide-dependent complexes with and suppressed aggregation of model misfolded proteins in vitro , and these effects were lost when the cysteines in rMANF were mutated to alanine. In NRVM, MANF knockdown decreased viability during simulated ischemia; this viability deficit was restored upon ectopic expression of wild type, but not mutant MANF. MANF knockdown in the heart, in vivo , increased ischemia/reperfusion damage, and this damage was mitigated using an AAV9-based gene therapy approach to restore MANF expression. Conclusions: MANF is a novel redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and mitigates ischemia/reperfusion damage in the heart.
Rationale: In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during myocardial ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. In response to ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF is induced by ATF6, is ER-localized, and contains a conserved redox-sensitive motif found in PDIs, we hypothesized that MANF is a redox-sensitive chaperone that optimizes cardiomyocyte viability during ischemia. Methods: The redox status of MANF during reductive ER stress and the ability of MANF to bind misfolded proteins during ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to suppress aggregation of misfolded proteins was examined in an in vitro chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo , were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. Results: In NRVM subjected to ER stress MANF was as sensitive to changes in ER redox status as the sentinel PDI, PDIA1. Moreover, MANF formed disulfide-linked complexes with misfolded proteins during ischemia-mediated ER stress. Under reducing conditions, recombinant MANF suppressed aggregation of model misfolded proteins, in vitro . MANF knockdown in the heart, in vivo , increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency, in vivo. Conclusions: MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.
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