ATF6 protects against protein misfolding during cardiac hypertrophy.

Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. METHODS: We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. RESULTS: ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. CONCLUSIONS: ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

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

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.

The peroxisomal enzyme, FAR1, is induced during ER stress in an ATF6-dependent manner in cardiac myocytes.

Although peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by pharmacological and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia-reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.NEW & NOTEWORTHY The peroxisomal enzyme, FAR1, was shown to be an ER stress- and ATF6-inducible protein that localizes to peroxisomes in cardiac myocytes. FAR1 decreases myocyte viability during oxidative stress.

ATF6 Regulates Cardiac Hypertrophy by Transcriptional Induction of the mTORC1 Activator, Rheb.

RATIONALE: Endoplasmic reticulum (ER) stress dysregulates ER proteostasis, which activates the transcription factor, ATF6 (activating transcription factor 6α), an inducer of genes that enhance protein folding and restore ER proteostasis. Because of increased protein synthesis, it is possible that protein folding and 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 ER stress and ATF6 were activated and ATF6 target genes were induced in mice subjected to an acute model of transverse aortic constriction, or to free-wheel exercise, both of which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO [conditional knockout]) blunted transverse aortic constriction 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 (Ras homologue enriched in brain) as an ATF6 target gene in the heart. RHEB is an activator of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a major inducer of protein synthesis and subsequent cell growth. Both transverse aortic constriction and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in wild type mouse hearts but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine- and IGF1 (insulin-like growth factor 1)-mediated RHEB induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, adeno-associated virus 9- RHEB restored cardiac growth to ATF6 cKO mice subjected to transverse aortic constriction. 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 ER stress and ATF6, which induces RHEB and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.

Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

Sledgehammer to Scalpel: Broad Challenges to the Heart and Other Tissues Yield Specific Cellular Responses via Transcriptional Regulation of the ER-Stress Master Regulator ATF6α.

There are more than 2000 transcription factors in eukaryotes, many of which are subject to complex mechanisms fine-tuning their activity and their transcriptional programs to meet the vast array of conditions under which cells must adapt to thrive and survive. For example, conditions that impair protein folding in the endoplasmic reticulum (ER), sometimes called ER stress, elicit the relocation of the ER-transmembrane protein, activating transcription factor 6α (ATF6α), to the Golgi, where it is proteolytically cleaved. This generates a fragment of ATF6α that translocates to the nucleus, where it regulates numerous genes that restore ER protein-folding capacity but is degraded soon after. Thus, upon ER stress, ATF6α is converted from a stable, transmembrane protein, to a rapidly degraded, nuclear protein that is a potent transcription factor. This review focuses on the molecular mechanisms governing ATF6α location, activity, and stability, as well as the transcriptional programs ATF6α regulates, whether canonical genes that restore ER protein-folding or unexpected, non-canonical genes affecting cellular functions beyond the ER. Moreover, we will review fascinating roles for an ATF6α isoform, ATF6ß, which has a similar mode of activation but, unlike ATF6α, is a long-lived, weak transcription factor that may moderate the genetic effects of ATF6α.

The ER Unfolded Protein Response Effector, ATF6, Reduces Cardiac Fibrosis and Decreases Activation of Cardiac Fibroblasts.

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor ß (TGFß), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFß-Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFß-Smad axis of cardiac fibroblast activation.

ATF6 Decreases Myocardial Ischemia/Reperfusion Damage and Links ER Stress and Oxidative Stress Signaling Pathways in the Heart.

RATIONALE: Endoplasmic reticulum (ER) stress causes the accumulation of misfolded proteins in the ER, activating the transcription factor, ATF6 (activating transcription factor 6 alpha), which induces ER stress response genes. Myocardial ischemia induces the ER stress response; however, neither the function of this response nor whether it is mediated by ATF6 is known. OBJECTIVE: Here, we examined the effects of blocking the ATF6-mediated ER stress response on ischemia/reperfusion (I/R) in cardiac myocytes and mouse hearts. METHODS AND RESULTS: Knockdown of ATF6 in cardiac myocytes subjected to I/R increased reactive oxygen species and necrotic cell death, both of which were mitigated by ATF6 overexpression. Under nonstressed conditions, wild-type and ATF6 knockout mouse hearts were similar. However, compared with wild-type, ATF6 knockout hearts showed increased damage and decreased function after I/R. Mechanistically, gene array analysis showed that ATF6, which is known to induce genes encoding ER proteins that augment ER protein folding, induced numerous oxidative stress response genes not previously known to be ATF6-inducible. Many of the proteins encoded by the ATF6-induced oxidative stress genes identified here reside outside the ER, including catalase, which is known to decrease damaging reactive oxygen species in the heart. Catalase was induced by the canonical ER stressor, tunicamycin, and by I/R in cardiac myocytes from wild-type but not in cardiac myocytes from ATF6 knockout mice. ER stress response elements were identified in the catalase gene and were shown to bind ATF6 in cardiac myocytes, which increased catalase promoter activity. Overexpression of catalase, in vivo, restored ATF6 knockout mouse heart function to wild-type levels in a mouse model of I/R, as did adeno-associated virus 9-mediated ATF6 overexpression. CONCLUSIONS: ATF6 serves an important role as a previously unappreciated link between the ER stress and oxidative stress gene programs, supporting a novel mechanism by which ATF6 decreases myocardial I/R damage.

CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α.

Deletion of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been shown to protect against in vivo ischemia/reperfusion (I/R) injury. It remains unclear which CaMKIIδ isoforms and downstream mechanisms are responsible for the salutary effects of CaMKIIδ gene deletion. In this study we sought to compare the roles of the CaMKIIδB and CaMKIIδC subtypes and the mechanisms by which they contribute to ex vivo I/R damage. WT, CaMKIIδKO, and mice expressing only CaMKIIδB or δC were subjected to ex vivo global ischemia for 25min followed by reperfusion. Infarct formation was assessed at 60min reperfusion by triphenyl tetrazolium chloride (TTC) staining. Deletion of CaMKIIδ conferred significant protection from ex vivo I/R. Re-expression of CaMKIIδC in the CaMKIIδKO background reversed this effect and exacerbated myocardial damage and dysfunction following I/R, while re-expression of CaMKIIδB was protective. Selective activation of CaMKIIδC in response to I/R was evident in a subcellular fraction enriched for cytosolic/membrane proteins. Further studies demonstrated differential regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and tumor necrosis factor alpha (TNF-α) expression by CaMKIIδB and CaMKIIδC. Selective activation of CaMKIIδC was also observed and associated with NF-κB activation in neonatal rat ventricular myocytes (NRVMs) subjected to oxidative stress. Pharmacological inhibition of NF-κB or TNF-α significantly ameliorated infarct formation in WT mice and those that re-express CaMKIIδC, demonstrating distinct roles for CaMKIIδ subtypes in I/R and implicating acute activation of CaMKIIδC and NF-κB in the pathogenesis of reperfusion injury.

Optimization of Large-Scale Adeno-Associated Virus (AAV) Production.

Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno-associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transduction in vivo. Importantly, our method differs from others in application of a streamlined, more cost-effective approach. From this method, as many as 2 × 1013 genome-containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011 vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AAV production Basic Protocol 2: AAV purification.

Dietary choline intake is necessary to prevent systems-wide organ pathology and reduce Alzheimer's disease hallmarks.

There is an urgent need to identify modifiable environmental risk factors that reduce the incidence of Alzheimer's disease (AD). The B-like vitamin choline plays key roles in body- and brain-related functions. Choline produced endogenously by the phosphatidylethanolamine N-methyltransferase protein in the liver is not sufficient for adequate physiological functions, necessitating daily dietary intake. ~90% of Americans do not reach the recommended daily intake of dietary choline. Thus, it's imperative to determine whether dietary choline deficiency increases disease outcomes. Here, we placed 3xTg-AD, a model of AD, and non-transgenic (NonTg) control mice on either a standard laboratory diet with sufficient choline (ChN; 2.0 g/kg choline bitartrate) or a choline-deficient diet (Ch-; 0.0 g/kg choline bitartrate) from 3 to 12 (early to late adulthood) months of age. A Ch- diet reduced blood plasma choline levels, increased weight, and impaired both motor function and glucose metabolism in NonTg mice, with 3xTg-AD mice showing greater deficits. Tissue analyses showed cardiac and liver pathology, elevated soluble and insoluble Amyloid-ß and Thioflavin S structures, and tau hyperphosphorylation at various pathological epitopes in the hippocampus and cortex of 3xTg-AD Ch- mice. To gain mechanistic insight, we performed unbiased proteomics of hippocampal and blood plasma samples. Dietary choline deficiency altered hippocampal networks associated with microtubule function and postsynaptic membrane regulation. In plasma, dietary choline deficiency altered protein networks associated with insulin metabolism, mitochondrial function, inflammation, and fructose metabolic processing. Our data highlight that dietary choline intake is necessary to prevent systems-wide organ pathology and reduce hallmark AD pathologies.

Hydrogen sulfide: the gas that fuels longevity.

The molecular determinants of lifespan can be examined in animal models with the long-term objective of applying what is learned to the development of strategies to enhance longevity in humans. Here, we comment on a recent publication examining the molecular mechanisms that determine lifespan in worms, Caenorhabditis elegans (C. elegans), where it was shown that inhibiting protein synthesis increased levels of the transcription factor, ATF4. Gene expression analyses showed that ATF4 increased the expression of genes responsible for the formation of the gas, hydrogen sulfide (H2S). Further examination showed that H2S increased longevity in C. elegans by modifying proteins in ways that stabilize their structures and enhance their functions. H2S has been shown to improve cardiovascular performance in mouse models of heart disease, and clinical trials are underway to test the effects of H2S on cardiovascular health in humans. These findings support the concept that nutrient deprivation, which slows protein synthesis and leads to ATF4-mediated H2S production, may extend lifespan by improving the function of the cardiovascular system and other systems that influence longevity in humans.

Design and Production of Heart Chamber-Specific AAV9 Vectors.

Adeno-associated virus serotype 9 (AAV9) is often used in heart research involving gene delivery due to its cardiotropism, high transduction efficiency, and little to no pathogenicity, making it highly applicable for gene manipulation, in vivo. However, current AAV9 technology is limited by the lack of strains that can selectively express and elucidate gene function in an atrial- and ventricular-specific manner. In fact, study of gene function in cardiac atria has been limited due to the lack of an appropriate tool to study atrial gene expression in vivo, hindering progress in the study of atrial-specific diseases such as atrial fibrillation, the most common cardiac arrhythmia in the USA.This chapter describes the method for the design and production of such chamber-specific AAV9 vectors, with the use of Nppa and Myl2 promoters to enhance atrial- and ventricular-specific expression. While several gene promoter candidates were considered and tested, Nppa and Myl2 were selected for use here because of their clearly defined regulatory elements that confer cardiac chamber-specific expression. Accordingly, Nppa (-425/+25) and Myl2 (-226/+36) promoter fragments are inserted into AAV9 vectors. The atrial- and ventricular-specific expression conferred by these new recombinant AAV9 was confirmed in a double-fluorescent Cre-dependent reporter mouse model. At only 450 and 262 base pairs of Nppa and Myl2 promoters, respectively, these AAV9 that drive chamber-specific AAV9 transgene expression address two major limitations of AAV9 technology, i.e., achieving chamber-specificity while maximizing space in the AAV genome for insertion of larger transgenes.

ATF6 as a Nodal Regulator of Proteostasis in the Heart.

Proteostasis encompasses a homeostatic cellular network in all cells that maintains the integrity of the proteome, which is critical for optimal cellular function. The components of the proteostasis network include protein synthesis, folding, trafficking, and degradation. Cardiac myocytes have a specialized endoplasmic reticulum (ER) called the sarcoplasmic reticulum that is well known for its role in contractile calcium handling. However, less studied is the proteostasis network associated with the ER, which is of particular importance in cardiac myocytes because it ensures the integrity of proteins that are critical for cardiac contraction, e.g., ion channels, as well as proteins necessary for maintaining myocyte viability and interaction with other cell types, e.g., secreted hormones and growth factors. A major aspect of the ER proteostasis network is the ER unfolded protein response (UPR), which is initiated when misfolded proteins in the ER activate a group of three ER transmembrane proteins, one of which is the transcription factor, ATF6. Prior to studies in the heart, ATF6 had been shown in model cell lines to be primarily adaptive, exerting protective effects by inducing genes that encode ER proteins that fortify protein-folding in this organelle, thus establishing the canonical role for ATF6. Subsequent studies in isolated cardiac myocytes and in the myocardium, in vivo, have expanded roles for ATF6 beyond the canonical functions to include the induction of genes that encode proteins outside of the ER that do not have known functions that are obviously related to ER protein-folding. The identification of such non-canonical roles for ATF6, as well as findings that the gene programs induced by ATF6 differ depending on the stimulus, have piqued interest in further research on ATF6 as an adaptive effector in cardiac myocytes, underscoring the therapeutic potential of activating ATF6 in the heart. Moreover, discoveries of small molecule activators of ATF6 that adaptively affect the heart, as well as other organs, in vivo, have expanded the potential for development of ATF6-based therapeutics. This review focuses on the ATF6 arm of the ER UPR and its effects on the proteostasis network in the myocardium.

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

Designing Novel Therapies to Mend Broken Hearts: ATF6 and Cardiac Proteostasis.

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.

Reactive Oxygen Species (ROS)-Activatable Prodrug for Selective Activation of ATF6 after Ischemia/Reperfusion Injury.

We describe here the design, synthesis, and biological evaluation of a reactive oxygen species (ROS)-activatable prodrug for the selective delivery of 147, a small molecule ATF6 activator, for ischemia/reperfusion injury. ROS-activatable prodrug 1 and a negative control unable to release free drug were synthesized and examined for peroxide-mediated activation. Prodrug 1 blocks activity of 147 by its inability to undergo metabolic oxidation by ER-resident cytochrome P450 enzymes such as Cyp1A2, probed directly here for the first time. Biological evaluation of ROS-activatable prodrug 1 in primary cardiomyocytes demonstrates protection against peroxide-mediated toxicity and enhances viability following simulated I/R injury. The ability to selectively target ATF6 activation under diseased conditions establishes the potential for localized stress-responsive signaling pathway activation as a therapeutic approach for I/R injury and related protein misfolding maladies.