Abstract 21206: Manf, a Structurally Unique Redox-Sensitive Chaperone, Restores ER-Protein Folding in the Ischemic Heart

Abstract

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.