Heart disease, in its many different forms, is the leading cause of death worldwide for which there is no cure. The lethality of heart failure stems both from its limited capacity to withstand injury, its inherent inability to regenerate upon injury. In order to develop effective therapies that mitigate the extent of injury, novel disease mechanisms must be discerned that will pave the way for development of new therapies. 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 6 (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 adeno-associated virus serotype 9 (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.

Numerous stresses on the heart, both physiological and pathological, present challenges to ER proteostasis in the heart. Simultaneously, various cardiac cell lineages respond to injury in ways requiring increased protein flux through the ER. While cardiac myocytes react to these stresses largely through hypertrophy or cell death mechanisms, other non-myocyte cells in the heart respond in more dynamic ways, including proliferation and differentiation. Additionally, virtually all cardiac cells secrete signaling cytokines or hormones both at baseline and during injury. Thus, precisely when ER proteostasis is most critical, cells are challenged with conditions which impair ER protein folding. ER unfolded protein response signaling attempts to respond to these challenges through the three main ER stress sensors, PERK, IRE1, and ATF6α. All sense protein misfolding and induce downstream signaling which, to varying degrees, includes global translational attenuation with concurrent upregulation of chaperones, protein disulfide isomerases, and ER-associated degradation machinery. Of these pathways, ATF6α is the most extensively characterized as adaptive in the heart and other tissues. Additionally, recent studies have identified multiple noncanonical ATF6α gene programs which are only indirectly related to ER proteostasis but are nevertheless critical to the ability of ATF6α to preserve tissue function. Research on the effect of ATF6α in the heart had previously been restricted to ventricular myocytes. Though these cells are the most direct effectors of cardiac function, this ignores critical roles played by other cardiac non-myocyte cells. This work examines the effects of ATF6α gain- and loss-of-function in three model systems designed to uncover previously unknown roles for ATF6α signaling in cardiac non-myocytes. ATF6α global knockout mice were found to progress to heart failure more quickly than wild-type counterparts following permanent-occlusion myocardial infarction, an unexplored disease model in the context of ATF6α signaling. We subsequently explored the role of ATF6α in two murine cardiac non-myocyte cell types. The first, c-Kit+ cardiac stem cells, required ATF6α both for survival and stemness while ATF6α loss-of-function induced multiple lineage markers. Second, we identified roles for ATF6α in limiting fibrosis in a pressure overload injury model and found that ATF6α blunted activation of isolated adult murine ventricular fibroblasts.

The heart exhibits incredible plasticity in response to both environmental and genetic alterations that affect workload. Over the course of development, or in response to physiological or pathological stimuli, the heart responds to fluctuations in workload by hypertrophic growth primarily by individual cardiac myocytes growing in size. Cardiac hypertrophy is associated with an increase in protein synthesis, which must coordinate with protein folding and degradation to allow for homeostatic growth without affecting the functional integrity of cardiac myocytes (i.e., proteostasis). This increase in the protein folding demand in the growing cardiac myocyte activates the transcription factor, ATF6 (activating transcription factor 6α, an inducer of genes that restore proteostasis. Previously, ATF6 has been shown to induce ER-targeted proteins functioning primarily to enhance ER protein folding and degradation. More recent studies, however, have illuminated adaptive roles for ATF6 functioning outside of the ER by inducing non-canonical targets in a stimulus-specific manner. This unique ability of ATF6 to act as an initial adaptive responder has bolstered an enthusiasm for identifying small molecule activators of ATF6 and similar proteostasis-based therapeutics.

ATF6α is an endoplasmic reticulum (ER) transmembrane protein that senses the accumulation of misfolded proteins in the ER of cells during ER stress. Upon ER stress, ATF6α is proteolytically cleaved. The resulting N-terminal fragment becomes an active transcription factor, i.e. N-ATF6α, which induces genes that adaptively reprogram ER protein folding. N-ATF6α exerts potent but transient transcriptional activation of its target genes; the transient nature of its activity is due in part to its rapid degradation upon engagement in transcription. The transcriptional activation domain (TAD) and degradation domain (degron) of N-ATF6α map to the same N-terminal region of N-ATF6α, implying that transcriptional activity and degradation are linked. Moreover, mutations introduced into the TAD of N-ATF6α that decrease its activity also decrease its degradation, indicating that N-ATF6α degradation is coupled with its transcriptional activity. To elucidate how and why N-ATF6α degradation and activation are coupled, we examined the mechanism of N-ATF6α degradation, positing that ubiquitylation may target it for proteasome-mediated degradation. Thus, this study focused primarily on delineating whether N-ATF6α and ATF6α are ubiquitylated, and if so, what domain(s) of N-ATF6α and ATF6α are ubiquitylated, as well as investigating the functional consequences of such ubiquitylation.

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Background

Methods and results

Conclusion