Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-Induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypIs) represent such new drugs. We demonstrate that the nonimmunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we show that the HCV infection of humanized mice results in the progressive development of HCC. This is true for the four genotypes tested (1 to 4). Remarkably, we demonstrate that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks after infection when HCC is well established. Importantly, we show that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC, while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypI) represent such new drugs. We demonstrated that the non-immunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we showed that HCV infection of humanized mice results in the progressive development of HCC. This was true for four genotypes tested (1 to 4). Remarkably, we demonstrated that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks post-infection when HCC is well established. Importantly, we showed that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

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.

ATF6 as a Nodal Regulator of Proteostasis in the Heart.

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

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

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

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

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

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

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

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

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.

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.