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.

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.

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.

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.

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.

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.

Pharmacologic ATF6 activation confers global protection in widespread disease models by reprograming cellular proteostasis.

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.

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.

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.

Junctophilin-2 gene therapy rescues heart failure by normalizing RyR2-mediated Ca2+ release.

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 Ca2+ spark frequency was reduced and the Ca2+ transient amplitude was increased in AAV9-JPH2 mice following TAC, consistent with JPH2-mediated normalization of SR Ca2+ 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 Ca2+ 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.

S100A4 protects the myocardium against ischemic stress.

BACKGROUND: Myocardial infarction is followed by cardiac dysfunction, cellular death, and ventricular remodeling, including tissue fibrosis. S100A4 protein plays multiple roles in cellular survival, and tissue fibrosis, but the relative role of the S100A4 in the myocardium after myocardial infarction is unknown. This study aims to investigate the role of S100A4 in myocardial remodeling and cardiac function following infarct damage. METHODS AND RESULTS: S100A4 expression is low in the adult myocardium, but significantly increased following myocardial infarction. Deletion of S100A4 increased cardiac damage after myocardial infarction, whereas cardiac myocyte-specific overexpression of S100A4 protected the infarcted myocardium. Decreased cardiac function in S100A4 Knockout mice was accompanied with increased cardiac remodeling, fibrosis, and diminished capillary density in the remote myocardium. Loss of S100A4 caused increased apoptotic cell death both in vitro and in vivo in part mediated by decreased VEGF expression. Conversely, S100A4 overexpression protected cells against apoptosis in vitro and in vivo. Increased pro-survival AKT-signaling explained reduced apoptosis in S100A4 overexpressing cells. CONCLUSION: S100A4 expression protects cardiac myocytes against myocardial ischemia and is required for stabilization of cardiac function after MI.

Hrd1 and ER-Associated Protein Degradation, ERAD, are Critical Elements of the Adaptive ER Stress Response in Cardiac Myocytes.

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.

PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity.

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.

Mechanistic target of rapamycin complex 2 protects the heart from ischemic damage.

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.

Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1), necessary for cellular growth, is regulated by intracellular signaling mediating inhibition of mTORC1 activation. Among mTORC1 regulatory binding partners, the role of Proline Rich AKT Substrate of 40 kDa (PRAS40) in controlling mTORC1 activity and cellular growth in response to pathological and physiological stress in the heart has never been addressed. This report shows PRAS40 is regulated by AKT in cardiomyocytes and that AKT-driven phosphorylation relieves the inhibitory function of PRAS40. PRAS40 overexpression in vitro blocks mTORC1 in cardiomyocytes and decreases pathological growth. Cardiomyocyte-specific overexpression in vivo blunts pathological remodeling after pressure overload and preserves cardiac function. Inhibition of mTORC1 by PRAS40 preferentially promotes protective mTORC2 signaling in chronic diseased myocardium. In contrast, strong PRAS40 phosphorylation by AKT allows for physiological hypertrophy both in vitro and in vivo, whereas cardiomyocyte-specific overexpression of a PRAS40 mutant lacking capacity for AKT-phosphorylation inhibits physiological growth in vivo, demonstrating that AKT-mediated PRAS40 phosphorylation is necessary for induction of physiological hypertrophy. Therefore, PRAS40 phosphorylation acts as a molecular switch allowing mTORC1 activation during physiological growth, opening up unique possibilities for therapeutic regulation of the mTORC1 complex to mitigate pathologic myocardial hypertrophy by PRAS40.

Regulation of cardiac hypertrophic signaling by prolyl isomerase Pin1.

RATIONALE: Cardiac hypertrophy results from the complex interplay of differentially regulated cascades based on the phosphorylation status of involved signaling molecules. Although numerous critical regulatory kinases and phosphatases have been identified in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the nonmyocyte context, control of folding, activity, and stability of proteins is mediated by the prolyl isomerase Pin1, but the role of Pin1 in the heart is unknown. OBJECTIVE: To establish the role of Pin1 in the heart. METHODS AND RESULTS: Here, we show that either genetic deletion or cardiac overexpression of Pin1 blunts hypertrophic responses induced by transaortic constriction and consequent cardiac failure in vivo. Mechanistically, we find that Pin1 directly binds to Akt, mitogen activated protein kinase (MEK), and Raf-1 in cultured cardiomyocytes after hypertrophic stimulation. Furthermore, loss of Pin1 leads to diminished hypertrophic signaling of Akt and MEK, whereas overexpression of Pin1 increases Raf-1 phosphorylation on the autoinhibitory site Ser259, leading to reduced MEK activation. CONCLUSIONS: Collectively, these data support a role for Pin1 as a central modulator of the intensity and duration of 2 major hypertrophic signaling pathways, thereby providing a novel target for regulation and control of cardiac hypertrophy.

Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion.

The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.

Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia/reperfusion-mediated cell death.

Proper folding of secreted and transmembrane proteins made in the rough endoplasmic reticulum (ER) requires oxygen for disulfide bond formation. Accordingly, ischemia can impair ER protein folding and initiate the ER stress response, which we previously showed is activated in the ischemic heart and in culture cardiac myocytes subjected to simulated ischemia. ER stress and ischemia activate the transcription factor, activating transcription factor 6 (ATF6), which induces numerous genes, many of which have not been identified, or examined in the heart. Using an ATF6 transgenic mouse model, we previously showed that ATF6 protected the heart from ischemic damage; however, the mechanism of this protection remains to be determined. In this study, we showed that, in the mouse heart, and in cultured cardiac myocytes, ATF6 induced the protein disulfide isomerase associated 6 (PDIA6) gene, which encodes an ER enzyme that catalyzes protein disulfide bond formation. Moreover, in cultured cardiac myocytes, ER stress-mediated PDIA6 promoter activation was ATF6-dependent, and required an ER stress response element (ERSE) and a nearby CCAAT box element. Electromobility shift assays and chromatin immunoprecipitation showed that ATF6 bound to the ERSE in the PDIA6 promoter, in vitro, and in the mouse heart, in vivo. Gain- and loss-of-function studies showed that PDIA6 protected cardiac myocytes against simulated ischemia/reperfusion-induced death in a manner that was dependent on the catalytic activity of PDIA6. Thus, by facilitating disulfide bond formation, and enhanced ER protein folding, PDIA6 may contribute to the protective effects of ATF6 in the ischemic mouse heart.

Regulation of microRNA expression in the heart by the ATF6 branch of the ER stress response.

A nodal regulator of endoplasmic reticulum stress is the transcription factor, ATF6, which is activated by ischemia and protects the heart from ischemic damage, in vivo. To explore mechanisms of ATF6-mediated protection in the heart, a whole-genome microRNA (miRNA) array analysis of RNA from the hearts of ATF6 transgenic (TG) mice was performed. The array identified 13 ATF6-regulated miRNAs, eight of which were downregulated, suggesting that they could contribute to increasing levels of their mRNAs. The down-regulated miRNAs, including miR-455, were predicted to target 45 mRNAs that we had previously shown by microarray analysis to be up-regulated by ATF6 in the heart. One of the miR-455 targets was calreticulin (Calr), which is up-regulated in the pathologic heart, where it modulates hypertrophic growth, potentially reducing the impact of the pathology. To validate the effects of miR-455, we showed that Calr protein was increased by ATF6 in mouse hearts, in vivo. In cultured cardiac myocytes, treatment with the ER stressor, tunicamycin, or with adenovirus encoding activated ATF6 decreased miR-455 and increased Calr levels, consistent with the effects of ATF6 on miR-455 and Calr, in vivo. Moreover, transfection of cultured cardiac myocytes with a synthetic precursor, premiR-455, decreased Calr levels, while transfection with an antisense, antimiR-455, increased Calr levels. The results of this study suggest that ER stress can regulate gene expression via ATF6-mediated changes in micro-RNA levels. Moreover, these findings support the hypothesis that ATF6-mediated down-regulation of miR-455 augments Calr expression, which may contribute to the protective effects of ATF6 in the heart.