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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.

The heterogeneity of endothelium across different organs was recently explored using single-cell RNA-sequencing analysis. Compared to other organs, the lung exhibits a distinct structure composed of a thin layer of capillary for efficient gas exchange. In this study, we demonstrate that Tmem100 is a lung-specific endothelium gene.

RationaleRare genetic variants and genetic variation at loci in an enhancer in SRY-Box Transcription Factor 17 (SOX17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutation in SOX17 in PAH pathogenesis has not been reported.ObjectivesTo investigate the role of SOX17 deficiency in pulmonary hypertension (PH) development.MethodsHuman lung tissue and endothelial cells (ECs) from IPAH patients were used to determine the expression of SOX17. Tie2Cre-mediated and EC-specific deletion of Sox17 mice were assessed for PH development. Single-cell RNA sequencing analysis, human lung ECs, and smooth muscle cell culture were performed to determine the role and mechanisms of SOX17 deficiency. A pharmacological approach was used in Sox17 deficiency mice for therapeutic implication.Measurement and Main ResultsSOX17 expression was downregulated in the lungs and pulmonary ECs of IPAH patients. Mice with Tie2Cre mediated Sox17 knockdown and EC-specific Sox17 deletion developed spontaneously mild PH. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced PH in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and anti-apoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP signaling. E2F Transcription Factor 1 (E2F1) signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction and PH development.ConclusionsOur study demonstrated that endothelial SOX17 deficiency induces PH through E2F1 and targeting E2F1 signaling represents a promising approach in PAH patients.

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.

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 (H(2)O(2)). 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.

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.

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 (ER) stress is a pathological hallmark of numerous ischemic diseases, including stroke and myocardial infarction (MI). In these diseases, ER stress leads to activation of the unfolded protein response (UPR) and subsequent adaptation of cellular physiology in ways that dictate cellular fate following ischemia. Recent evidence highlights a protective role for the activating transcription factor 6 (ATF6) arm of the UPR in mitigating adverse outcomes associated with ischemia/reperfusion (I/R) injury in multiple disease models. This suggests ATF6 as a potential therapeutic target for intervening in diverse ischemia-related disorders. Here, we discuss the evidence demonstrating the importance of ATF6 signaling in protecting different tissues against ischemic damage and discuss preclinical results focused on defining the potential for pharmacologically targeting ATF6 to intervene in such diseases.

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo. Here we show, using a mouse model of myocardial ischemia/reperfusion, that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 is also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis., Imbalanced proteostasis is associated with diverse diseases, including ischemia/reperfusion injury in the heart. Here the authors show that the ATF6 arm of the unfolded protein response can be pharmacologically activated with a small molecule in vivo, providing protection from ischemia/reperfusion injury in the heart, the brain, and the kidney.

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.

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 Ca(2+) 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 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.

Activating transcription factor-6 &alpha, (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 &beta, (TGF&beta, ), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGF&beta, &ndash, Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of &alpha, smooth muscle actin (&alpha, SMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGF&beta, Smad axis of cardiac fibroblast activation.

The integrity of the proteome in cardiac myocytes is critical for robust heart function. Proteome integrity in all cells is managed by protein homeostasis or proteostasis, which encompasses processes that maintain the balance of protein synthesis, folding, and degradation in ways that allow cells to adapt to conditions that present a potential challenge to viability (1). While there are processes in various cellular locations in cardiac myocytes that contribute to proteostasis, those in the cytosol, mitochondria and endoplasmic reticulum (ER) have dominant roles in maintaining cardiac contractile function. Cytosolic proteostasis has been reviewed elsewhere (2, 3); accordingly, this review focuses on proteostasis in the ER and mitochondria, and how they might influence each other and, thus, impact heart function in the settings of cardiac physiology and disease.

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.

Peptidergic signaling is an ancient manner of intertissue communication in multicellular organisms. Even the early eukaryote Trichoplax , with its limited 6-tissue repertoire, uses peptides to communicate between its tissues (1). Humans use peptidergic communication not only to transfer signals between tissues, but also to employ peptide signals in brain and peripheral nerve tracts to efficiently transfer information regarding hunger, anxiety, and many other types of physiologic states (reviewed in ref. 2). In PNAS, a study by Powers et al. (3), “Identifying roles for peptidergic signaling in mice,” describes an approach to the study of peptidergic function that depends on a unique aspect of how signaling peptides are synthesized. The manner in which signaling peptides are synthesized has remained remarkably constant over millions of years of eukaryotic evolution. Within the regulated secretory pathway (present in neurons and neuroendocrine cells), small peptides are typically excised from larger precursors by “eukaryotic subtilases” at sites marked by pairs of basic amino acids, typically Lys-Arg, followed by a series of enzymatic reactions. These reactions serve to trim, modify, and/or protect the termini of the excised peptides and are catalyzed by a variety of enzymes in addition to the subtilases. These include a specific carboxypeptidase, carboxypeptidase E, which removes terminal basic residues, and an amidating enzyme, which protects the carboxyl terminus of the trimmed peptide from degradation and often confers receptor-specific information. This latter enzyme, peptidylglycine α-amidating monooxygenase (PAM) (Fig. 1 A ), represents a particularly fascinating molecular entity. Formed from 2 entirely different catalytic species, a monooxygenase and a lyase, in neuroendocrine cells such as neurons in the brain, this complex enzyme catalyzes a 2-step reaction that transforms a terminal glycine residue into an amide (Fig. 1 B ) (4). The importance of PAM is underscored by the fact that more than half of all known peptides are … [↵][1]1To whom correspondence may be addressed. Email: ILindberg{at}som.umaryland.edu. [1]: #xref-corresp-1-1

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.

Background Junctophilin-2 (JPH2) is the primary structural protein for the coupling of transverse (T)-tubule associated cardiac L-type Ca channels and type-2 ryanodine receptors on the sarcoplasmic reticulum within junctional membrane complexes (JMCs) in cardiomyocytes. Effective signaling between these channels ensures adequate Ca-induced Ca release required for normal cardiac contractility. Disruption of JMC subcellular domains, a common feature of failing hearts, has been attributed to JPH2 downregulation. Here, we tested the hypothesis that adeno-associated virus type 9 (AAV9) mediated overexpression of JPH2 could halt the development of heart failure in a mouse model of transverse aortic constriction (TAC). Methods and results Following TAC, a progressive decrease in ejection fraction was paralleled by a progressive decrease of cardiac JPH2 levels. AAV9-mediated expression of JPH2 rescued cardiac contractility in mice subjected to TAC. AAV9-JPH2 also preserved T-tubule structure. Moreover, the Ca 2+ spark frequency was reduced and the Ca 2+ transient amplitude was increased in AAV9-JPH2 mice following TAC, consistent with JPH2-mediated normalization of SR Ca 2+ handling. Conclusions This study demonstrates that AAV9-mediated JPH2 gene therapy maintained cardiac function in mice with early stage heart failure. Moreover, restoration of JPH2 levels prevented loss of T-tubules and suppressed abnormal SR Ca 2+ leak associated with contractile failure following TAC. These findings suggest that targeting JPH2 might be an attractive therapeutic approach for treating pathological cardiac remodeling during heart failure.

Fibroblasts in the heart respond to myocardial injury by infiltrating the affected area and differentiating into new cell types called myofibroblasts. These cells are characterized both by the induction of contractile proteins and the secretion of extracellular matrix proteins which form fibrotic scar tissue. Investigating the factors governing fibroblast activation is key to understanding how these cells function in the heart and may be key to future therapeutic strategies. Activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum unfolded protein response, plays critical roles in development, as well as in the differentiation of certain cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective in vivo during heart disease. However, ATF6 has not been studied in cardiac fibroblasts and its effect on fibrosis in the heart is unknown. We hypothesized that ATF6 in fibroblasts is an important regulator of their function. Fibroblast activation markers including αSMA were increased in infarcted hearts with global ATF6 deletion. Additionally, hearts with pressure overload showed increased fibrosis staining in global ATF6-null mice relative to WT hearts. In isolated adult murine ventricular fibroblasts (AMVF), loss of ATF6 induced myofibroblast markers with and without the activation stimulus TGFβ. ATF6 loss of function also enhanced the effect of TGFβ on fibroblast contraction. These effects were associated with an increase in Smad phosphorylation, a crucial step in the TGFβ pathway. Interestingly, the effect of ATF6 loss of function in AMVF was erased when treated with a TGFβ receptor inhibitor. Additionally, when ATF6 was overexpressed or when endogenous ATF6 was chemically activated, myofibroblast markers were reduced and activation by TGFβ was blunted. ATF6 activation was associated with induction of several TGFβ/Smad pathway negative regulators including SMURF1, SMURF2, and PMEPA1, though none of these are known to be ATF6 target genes. These data suggest that ATF6 plays an important role in moderating fibroblast activation and this may contribute to previously reported roles for ATF6 in preserving cardiac function post-injury.

[Image: see text] 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.

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.

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.

Recent studies have suggested that multipotent stem cells residing in the adult heart, called cardiac stem cells (CSCs), mitigate damage in the infarcted or failing heart. Investigating the factors governing CSC proliferation and differentiation is key to understanding what role these cells play in the heart and in future therapeutic strategies. Additionally, activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum (ER) unfolded protein response (UPR), plays critical roles in development, as well as in the differentiation of certain stem cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective in vivo during ischemia/reperfusion partly by virtue of its ability to induce an antioxidant gene program that reduces damaging reactive oxygen species (ROS). However, ATF6, and its involvement in antioxidant gene induction, have not been studied in CSCs. Therefore, here we hypothesized that activation of the ATF6 branch of the UPR in CSCs is important for their proliferation and differentiation, given that ROS is known to be essential for these processes. To address this hypothesis, we subjected cultured mouse CSCs to simulated ischemia and observed increased ATF6 target gene mRNA levels. This demonstrates that, despite their undifferentiated status, CSCs have a functional UPR, which can be activated in response to ischemic stress. ATF6 loss of function (LOF) in CSCs, via RNAi or chemical inhibitor, yielded a basal decrease in cell viability and an increase in several differentiation markers, similar to the effect of dexamethasone differentiation stimulus. Increased ROS was also observed in an ATF6 LOF model. Strikingly, cotreatment with a chemical ROS inhibitor significantly rescued cell viability and reduced markers of differentiation in CSCs with reduced ATF6 function. These results suggest that CSCs require a basal level of ATF6 activity to maintain their proliferation and pluripotentcy in vitro and that this is mediated by the role of ATF6 in the mitigation of ROS. This is an important finding given that stem cell expansion in vitro is a critical step in the characterization of stem cells and their use in many therapeutic treatment strategies.

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo . Here, using a mouse model of myocardial ischemia/reperfusion, we showed that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 was also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a first-in-class proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.

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: Hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (Hrd1) is an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase that has been studied in yeast, where it contributes to ER protein quality control by ER-associated degradation (ERAD) of misfolded proteins that accumulate during ER stress. Neither Hrd1 nor ERAD has been studied in the heart, or in cardiac myocytes, where protein quality control is critical for proper heart function. Objective: The objective of this study were to elucidate roles for Hrd1 in ER stress, ERAD, and viability in cultured cardiac myocytes and in the mouse heart, in vivo. Methods and Results: The effects of small interfering RNA–mediated Hrd1 knockdown were examined in cultured neonatal rat ventricular myocytes. The effects of adeno-associated virus–mediated Hrd1 knockdown and overexpression were examined in the hearts of mice subjected to pressure overload–induced pathological cardiac hypertrophy, which challenges protein-folding capacity. In cardiac myocytes, the ER stressors, thapsigargin and tunicamycin increased ERAD, as well as adaptive ER stress proteins, and minimally affected cell death. However, when Hrd1 was knocked down, thapsigargin and tunicamycin dramatically decreased ERAD, while increasing maladaptive ER stress proteins and cell death. In vivo, Hrd1 knockdown exacerbated cardiac dysfunction and increased apoptosis and cardiac hypertrophy, whereas Hrd1 overexpression preserved cardiac function and decreased apoptosis and attenuated cardiac hypertrophy in the hearts of mice subjected to pressure overload. Conclusions: Hrd1 and ERAD are essential components of the adaptive ER stress response in cardiac myocytes. Hrd1 contributes to preserving heart structure and function in a mouse model of pathological cardiac hypertrophy.

Background Aberrant Ca 2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca 2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl‐prolyl cis ‐ trans isomerase peptidyl‐prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca 2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. Methods and Results Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca 2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATP ase ( SERCA 2a) and Na 2+ /Ca 2+ exchanger 1 protein levels. However, SERCA 2a ATP ase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1‐deficient hearts, suggesting that Pin1 influences SERCA 2a function. SERCA 2a and Na 2+ /Ca 2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca 2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA 2a and Na 2+ /Ca 2+ exchanger 1 proteins. Conclusions Pin1 serves as a modulator of SERCA 2a and Na 2+ /Ca 2+ exchanger 1 Ca 2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Rationale: ATF6α and ATF6β are endoplasmic reticulum (ER) transmembrane proteins that sense the accumulation of toxic misfolded proteins in the ER of cardiomyocytes, which can be brought about by ER stresses as ischemia. Upon ER stress, ATF6α is proteolytically cleaved into a transcription factor that binds to ER stress response elements (ERSEs) and increases expression of cardioprotective genes that restore ER protein folding. If ER proteostasis is not restored, maladaptive signaling is initiated. ATF6β is also proteolytically cleaved during ER stress, binds to the same ERSEs as ATF6α, but does not induce transcription. Hence it is clear from the above studies done in cancer cells that there are some marked similarities and differences between ATF6α and ATF6β. However, the relative roles of ATF6α and ATF6β have not been studied in the heart, where they might work in concert to mediate the dynamic switch from adaptive to maladaptive gene programing during myocardial pathology. Methods: We used neonatal rat ventricular myocytes (NRVMs) to explore the effects of ATF6α or ATF6β loss-of-function in cells treated with the ER stressor, thapsigargin (TG), which mimics ischemic heart disease. Results: In NRVM treated with TG, knockdown of ATF6β resulted in much more pronounced cell death in isolated myocytes than knockdown of ATF6α. Consistent with this finding, transcriptome analyses showed that compared to knocking down ATF6α, knockdown of ATF6β upregulated much more maladaptive, cell death-inducing genes and downregulated more cardioprotective genes. Surprisingly, knockdown of either ATF6α or ATF6β downregulated some common adaptive ER stress response genes, such as GRP78 and Derlin while also upregulating common maladaptive ER stress response genes, such as CHOP, Bcl2, Bax. Conclusion: These data indicate that both ATF6α and ATF6 β are needed for optimal viability of NRVM subjected to ER stress. There is a common, as well as differential gene regulation program controlled by these two isoforms of ATF6. Importantly, this study demonstrates a novel mechanism by which these two isoforms of ATF6 interact to govern the progression from adaptive to maladaptive ER stress signaling during chronic misfolding of ER proteins that occurs in ischemic heart disease.

Rationale: Reactive oxygen species generated during myocardial ischemia/reperfusion (I/R) potentiate myocyte death and cardiac dysfunction. Recently, our lab published a newly described role for the adaptive ER stress sensor and transcription factor, ATF6, as a novel inducer of an adaptive antioxidant gene family. These results highlight the need for the development of small molecule drug candidates that preferentially activate endogenous ATF6 to promote the adaptive effects of ER stress and ameliorate myocardial I/R damage. To this end we used of a cell-based high throughput-screen to identify a novel small molecular activator of endogenous ATF6, herein called compound 147, and tested its efficacy in cardiac myocytes and in the heart. Objective/Methods: The ability of compound 147 to activate endogenous ATF6, as measured by nuclear localization of ATF6 and ATF6-specific target gene induction was examined in cultured neonatal rat ventricular myocytes (NRVM). The effects of compound 147 on the viability of NRVM treated with H2O2 to generate ROS, or simulated I/R were assessed. Finally, the effects of compound 147 in the mouse heart were examined in vivo by administering the compound to mice and, 24h later, determining the effects of simulated I/R on cardiac myocytes isolated from the mice, or determining the effects of ex vivo I/R on hearts isolated from compound 147-treated mice. Results: Compared to a control compound, treatment of NRVM with compound 147 specifically and acutely activated ATF6 and primed cells to mount an adaptive response when treated with H2O2 or subjected to simulated I/R. Treatment of both neonatal and adult ventricular myocytes with compound 147 increased survival in cells subjected to simulated I/R. Compared to control, the cardiac myocytes and hearts from mice treated with compound 147 exhibited increased viability and functional recovery in response to I/R, respectively. Conclusions: Compound 147 specifically activates ATF6 in cardiac myocytes and confers cardioprotection during I/R, in vitro and in vivo . Thus, compound 147 represents a potential first- in-class small molecule drug candidate that enhances myocardial recovery from I/R damage, specifically by activating the endogenous adaptive ATF6 gene program in the heart.

Rationale: The ER stress response is activated by the accumulation of misfolded, toxic proteins in the endoplasmic reticulum (ER), and upregulates proteins that restore ER protein-folding capacity. The ER-transmembrane protein, activating transcription factor 6 (ATF6) senses ER stress and responds by transcriptionally inducing many of these genes and is thus a key component of the adaptive ER stress response. We previously showed that in the heart, ischemia activates ATF6. Furthermore, transgenic mouse hearts expressing a conditionally activated form of ATF6, and subjected to ex vivo ischemia/reperfusion, exhibited preserved heart function and smaller infarcts. Our lab also showed that by serving as a novel inducer of a global anti-oxidant gene program, endogenous ATF6 limits cardiac damage caused by reactive oxygen species during reperfusion. However, the effect of endogenous ATF6 in the failing heart is not known. Given that acute ischemia caused by occlusion of the coronary arteries is the cause of myocardial infarction (MI), we hypothesized that endogenous ATF6 limits infarct size and preserves heart function during MI. Additionally, since deleterious cardiac remodeling and heart failure can be long-term consequences of MI, we hypothesized that ATF6 can mitigate these effects. Objective/Methods: To examine the role of endogenous ATF6 in heart failure, in vivo, we used a mouse model of MI-induced heart failure in mice with a global deletion of the ATF6 gene (ATF6 KO). Infarct size was measured by TTC staining and heart function was observed via longitudinal echocardiogram. Results: We found that following infarction, ATF6 KO mouse hearts had larger infarcts compared to control. Thus, ischemic cardiac tissue in the peri-infarct region requires ATF6 to limit cardiac myocyte death. Interestingly, ejection fraction following MI decreased more over 13 weeks in ATF6 KO mice relative to control. While control and ATF6 KO mouse hearts hypertrophied to a similar degree, KO mice showed greater cardiac dilation. Conclusions: Together these findings show for the first time that endogenous ATF6 acts to preserve heart structure and function in an MI model of heart failure, suggesting that ATF6 may be a viable therapeutic target for treatment of this disease.

Recent interest in mechanisms of stem cell–mediated repair in the heart have spawned the paracrine hypothesis, which posits that stem cells release beneficial substances that improve regeneration and function of the injured and diseased myocardium. In support of this hypothesis are studies showing that stem cells release small membranous vesicles called exosomes that deliver beneficial cargo to other cells in the heart. However, in addition to exosomes, which are released by the unconventional secretory pathway, are many other potentially beneficial factors released by the unconventional and the conventional secretory pathways. Therefore, a broader perspective of mechanisms of secretion, as well as an appreciation for the ways in which the secretion of a wide range of different types of molecules can be regulated, will spawn new avenues of thought necessary to move us beyond the exosome-centric view that drives much of the current thinking of those who study of stem cell–mediated repair in the heart. Secretion is what cells do; excretion is what the kidney does. Secretion is an active process by which cells release materials into the surrounding environment. Among the first secretion mechanisms studied were those involved in the regulated release of peptide hormones from endocrine cells. After release, substances can signal to cells afar via the blood stream (endocrine) and to neighboring cells (paracrine), as well as the cell of origin (autocrine). Additionally, some substances signal within the cell of origin (intracrine). Endocrine, paracrine, and autocrine signaling all involve the release of communicator substances directly into the interstitial spaces surrounding the cells of origin. Secretion into a duct, such as salivary or digestive enzyme secretion, is exocrine. Cells secrete many different substances, including proteins, lipids, steroids, nucleic acids, nucleotides, metabolites, and ions. Generally, these substances are secreted to facilitate communication with other cells and to affect the structure …

Deletion of Ca2+/calmodulin-dependent protein kinase II delta (CaMKII delta) has been shown to protect against in vivo ischemia/reperfusion (I/R) injury. It remains unclear which CaMKII delta isoforms and downstream mechanisms are responsible for the salutary effects of CaMKII delta gene deletion. In this study we sought to compare the roles of the CaMKII delta(B) and CaMKII delta(C) subtypes and the mechanisms by which they contribute to ex vivo I/R damage. WT, CaMKII delta KO, and mice expressing only CaMKII delta(B) or delta(C) were subjected to ex vivo global ischemia for 25 min followed by reperfusion. Infarct formation was assessed at 60 min reperfusion by triphenyl tetrazolium chloride (TTC) staining. Deletion of CaMKII delta conferred significant protection from ex vivo I/R. Re-expression of CaMKII delta(C) in the CaMKII delta KO background reversed this effect and exacerbated myocardial damage and dysfunction following I/R, while re-expression of CaMKII delta(B) was protective. Selective activation of CaMKII delta(C) 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-kappa B) signaling and tumor necrosis factor alpha (TNF-alpha) expression by CaMKII delta(B) and CaMKII8c. Selective activation of CaMKII delta(C) was also observed and associated with NF-kappa B activation in neonatal rat ventricular myocytes (NRVMs) subjected to oxidative stress. Pharmacological inhibition of NF-kappa B or TNF-alpha significantly ameliorated infarct formation in WT mice and those that re-express CaMKII8c, demonstrating distinct roles for CaMKII delta subtypes in I/R and implicating acute activation of CaMKII delta(C) and NF-kappa B in the pathogenesis of reperfusion injury. (C) 2017 Published by Elsevier Ltd.

Cardiac myocytes are the cells responsible for the robust ability of the heart to pump blood throughout the circulatory system. Cardiac myocytes grow in response to a variety of physiological and pathological conditions; this growth challenges endoplasmic reticulum-protein quality control (ER-PQC), a major feature of which includes the unfolded protein response (UPR). ER-PQC and the UPR in cardiac myocytes growing under physiological conditions, including normal development, exercise, and pregnancy, are sufficient to support hypertrophic growth of each cardiac myocyte. However, the ER-PQC and UPR are insufficient to respond to the challenge of cardiac myocyte growth under pathological conditions, including myocardial infarction and heart failure. In part, this insufficiency is due to a continual decline in the expression levels of important adaptive UPR components as a function of age and during myocardial pathology. This chapter will discuss the physiological and pathological conditions unique to the heart that involves ER-PQC, and whether the UPR is adaptive or maladaptive under these circumstances.

The hypertrophic growth of cardiac myocytes is a highly dynamic process that underlies physiological and pathological adaptation of the heart. Accordingly, a better understanding of the molecular underpinnings of cardiac myocyte hypertrophy is required in order to fully appreciate the causes and functional consequences of the changes in the size of the healthy and diseased heart. Hypertrophy is driven by increases in cardiac myocyte protein, which must be balanced by cellular ability to maintain protein quality in order to avoid maladaptive accumulation of toxic misfolded proteins. Recent studies have shown that the endoplasmic reticulum (ER), which, in cardiac myocytes, comprises the sarco/endoplasmic reticulum (SR/ER), is the site of most protein synthesis. Thus, the protein quality control machinery located at the SR/ER is likely to be an important determinant of whether the heart responds adaptively to hypertrophic growth stimuli. The SR/ER-transmembrane protein, ATF6, serves a critical protein quality control function as a first responder to the accumulation of potentially toxic, misfolded proteins. Misfolded proteins transform ATF6 into a transcription factor that regulates a gene program that is partly responsible for enhancing protein quality control. Two ATF6-inducible genes that have been studied in the heart and shown to be adaptive are RCAN1 and Derl3, which encode proteins that decrease protein-folding demand, and enhance degradation of misfolded proteins, respectively. Thus, the ATF6-regulated SR/ER protein quality control system is important for maintaining protein quality during growth, making ATF6, and other components of the system, potentially attractive targets for the therapeutic management pathological cardiac hypertrophy. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy".

Background— The mechanistic target of rapamycin (mTOR) comprises 2 structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown. Methods and Results— Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Conclusions— These results suggest that selectively increasing mTORC2 while concurrently inhibiting mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.