simultaneous-isolation-culture-atrial-myocytes-ventricular-myocytes

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

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

San Diego State University

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.

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

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

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

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule (2-hydroxy-5-methylphenyl)-3-phenylpropanamide () was previously identified (<xref ref-type="bibr" rid="bib35">Plate et al., 2016</xref>) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that -dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by  include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs -dependent induction of the ATF6-target gene, , implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with . Thus,  is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.

Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins.

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.

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

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.

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

Unfolding the Roles of Mitochondria as Therapeutic Targets for Heart Disease.

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.

Integrating ER and Mitochondrial Proteostasis in the Healthy and Diseased Heart.

There are more than 2000 transcription factors in eukaryotes, many of which are subject to complex mechanisms fine-tuning their activity and their transcriptional programs to meet the vast array of conditions under which cells must adapt to thrive and survive. For example, conditions that impair protein folding in the endoplasmic reticulum (ER), sometimes called ER stress, elicit the relocation of the ER-transmembrane protein, activating transcription factor 6α (ATF6α), to the Golgi, where it is proteolytically cleaved. This generates a fragment of ATF6α that translocates to the nucleus, where it regulates numerous genes that restore ER protein-folding capacity but is degraded soon after. Thus, upon ER stress, ATF6α is converted from a stable, transmembrane protein, to a rapidly degraded, nuclear protein that is a potent transcription factor. This review focuses on the molecular mechanisms governing ATF6α location, activity, and stability, as well as the transcriptional programs ATF6α regulates, whether canonical genes that restore ER protein-folding or unexpected, non-canonical genes affecting cellular functions beyond the ER. Moreover, we will review fascinating roles for an ATF6α isoform, ATF6β, which has a similar mode of activation but, unlike ATF6α, is a long-lived, weak transcription factor that may moderate the genetic effects of ATF6α.

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

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

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

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

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

We describe here the design, synthesis, and biological evaluation of a reactive oxygen species (ROS)-activatable prodrug for the selective delivery of , a small molecule ATF6 activator, for ischemia/reperfusion injury. ROS-activatable prodrug  and a negative control unable to release free drug were synthesized and examined for peroxide-mediated activation. Prodrug  blocks activity of  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  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.

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

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

ATF6 as a Nodal Regulator of Proteostasis 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 , 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 depletion or tunicamycin-mediated inhibition of ER protein glycosylation. , 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.

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

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.

Article	Year
ATF6 Decreases Myocardial Ischemia/Reperfusion Damage and Links ER Stress and Oxidative Stress Signaling Pathways in the Heart. Circulation research\| PubMed ID: 27932512	2017
CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α. Journal of molecular and cellular cardiology\| PubMed ID: 28077321	2017
Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins. eLife\| PubMed ID: 30084354	2018
ATF6 Regulates Cardiac Hypertrophy by Transcriptional Induction of the mTORC1 Activator, Rheb. Circulation research\| PubMed ID: 30582446	2019
Pharmacologic ATF6 activation confers global protection in widespread disease models by reprograming cellular proteostasis. Nature communications\| PubMed ID: 30643122	2019
Unfolding the Roles of Mitochondria as Therapeutic Targets for Heart Disease. Journal of the American College of Cardiology\| PubMed ID: 30975298	2019
Integrating ER and Mitochondrial Proteostasis in the Healthy and Diseased Heart. Frontiers in cardiovascular medicine\| PubMed ID: 32010709	2019
Sledgehammer to Scalpel: Broad Challenges to the Heart and Other Tissues Yield Specific Cellular Responses via Transcriptional Regulation of the ER-Stress Master Regulator ATF6α. International journal of molecular sciences\| PubMed ID: 32046286	2020
The ER Unfolded Protein Response Effector, ATF6, Reduces Cardiac Fibrosis and Decreases Activation of Cardiac Fibroblasts. International journal of molecular sciences\| PubMed ID: 32085622	2020
Designing Novel Therapies to Mend Broken Hearts: ATF6 and Cardiac Proteostasis. Cells\| PubMed ID: 32138230	2020
Reactive Oxygen Species (ROS)-Activatable Prodrug for Selective Activation of ATF6 after Ischemia/Reperfusion Injury. ACS medicinal chemistry letters\| PubMed ID: 32184959	2020
ATF6 as a Nodal Regulator of Proteostasis in the Heart. Frontiers in physiology\| PubMed ID: 32322217	2020
Mesencephalic astrocyte-derived neurotrophic factor is an ER-resident chaperone that protects against reductive stress in the heart. The Journal of biological chemistry\| PubMed ID: 32327487	2020
Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response. Journal of molecular and cellular cardiology\| PubMed ID: 32339566	2020

Erik A. Blackwood

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