SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Histone deacetylases (HDACs) regulate cardiac plasticity; however, their molecular targets are unknown. As autophagy contributes to pathological cardiac remodeling, we hypothesized that HDAC inhibitors target autophagy. The prototypical HDAC inhibitor (HDACi), trichostatin A (TSA), attenuated both load-and agonistinduced hypertrophic growth and abolished the associated activation of autophagy. Phenylephrine (PE)-triggered hypertrophy and autophagy in cultured cardiomyocytes were each blocked by a panel of structurally distinct HDAC inhibitors. RNAi-mediated knockdown of either Atg5 or Beclin 1, two essential autophagy effectors, was similarly capable of suppressing ligand-induced autophagy and myocyte growth. RNAi experiments uncovered the class I isoforms HDAC1 and HDAC2 as required for the autophagic response. To test the functional requirement of autophagic activation, we studied mice that overexpress Beclin 1 in cardiomyocytes. In these animals with a fourfold amplified autophagic response to TAC, TSA abolished TAC-induced increases in autophagy and blunted load-induced hypertrophy. Finally, we subjected animals with preexisting hypertrophy to HDACi, finding that ventricular mass reverted to nearnormal levels and ventricular function normalized completely. Together, these data implicate autophagy as an obligatory element in pathological cardiac remodeling and point to HDAC1/2 as required effectors. Also, these data reveal autophagy as a previously unknown target of HDAC inhibitor therapy.

Background Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that SAHA, a histone deacetylase (HDAC) inhibitor FDA-approved for cancer treatment, will blunt reperfusion injury. Methods and Results Twenty-one rabbits were randomized into 3 groups: a) vehicle control, b) SAHA pretreatment (one day prior and at surgery), and c) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (I/R, 30min coronary ligation, 24h reperfusion). Additionally cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated I/R (sI/R) to probe mechanism. SAHA reduced infarct (those reduction inhibitor, SAHA, infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during I/R occur, at least in part, through induction of autophagic flux. assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to sI/R, SAHA pretreatment reduced cell death by 40%. This eduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA's cardioprotective effects. Conclusions The FDS-approved anti-cancer HDAC inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during I/R occur, at least in part, through induction of autophagic flux.

Background L-type calcium channel (LTCC) activity is critical to afterload-induced hypertrophic growth of the heart. However, mechanisms governing mechanical stress-induced activation of LTCC activity are obscure. Polycystin-1 (PC-1) is a G-protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results We subjected neonatal rat ventricular myocytes (NRVMs) to mechanical stretch by exposing them to hypo-osmotic (HS) medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on LTCC activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Over-expression of a C-terminal fragment of PC-1 was sufficient to trigger NRVM hypertrophy. Exposing NRVMs to HS medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 KO) and subjected them to mechanical stress in vivo (transverse aortic constriction, TAC). At baseline, PC-1 KO mice manifested decreased cardiac function relative to littermate controls, and α1C LTCC protein levels were significantly lower in PC-1 KO hearts. Whereas control mice manifested robust TAC-induced increases in cardiac mass, PC-1 KO mice showed no significant growth. Likewise, TAC-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusions PC-1 is a cardiomyocyte mechanosensor and is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein.

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs), a group of chromatin remodeling enzymes, has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that as part of mTOR complex I (mTORC1) drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. Here, we demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to TAC and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2), an mTOR inhibitor, was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes, loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity, and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease.

Purpose of review Despite maximum medical and mechanical support therapy, heart failure remains a relentlessly progressive disorder with substantial morbidity and mortality. Autophagy, an evolutionarily conserved process of cellular cannibalization, has been implicated in virtually all forms of cardiovascular disease. Indeed, its role is context dependent, antagonizing or promoting disease depending on the circumstance. Here, we review current understanding of the role of autophagy in the pathogenesis of heart failure and explore this pathway as a target of therapeutic intervention. Recent findings In preclinical models of heart disease, cardiomyocyte autophagic flux is activated; indeed, its role in disease pathogenesis is the subject of intense investigation to define mechanism. Similarly, in failing human heart of a variety of etiologies, cardiomyocyte autophagic activity is upregulated, and therapy, such as with mechanical support systems, elicits declines in autophagy activity. However, when suppression of autophagy is complete, rapid and catastrophic cell death occurs, consistent with a model in which basal autophagic flux is required for proteostasis. Thus, a narrow zone of ‘optimal’ autophagy seems to exist. The challenge moving forward is to tune the stress-triggered autophagic response within that ‘sweet spot’ range for therapeutic benefit. Summary Whereas we have known for some years of the participation of lysosomal mechanisms in heart disease, it is only recently that upstream mechanisms (autophagy) are being explored. The challenge for the future is to dissect the underlying circuitry and titrate the response into an optimal, proteostasis-promoting range in hopes of mitigating the ever-expanding epidemic of heart failure.

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CβII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

Significance: Autophagy is an evolutionarily ancient process of intracellular protein and organelle recycling required to maintain cellular homeostasis in the face of a wide variety of stresses. Dysregulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage. Both autophagy and ROS/RNS serve pathological or adaptive roles within cardiomyocytes, depending on the context. Recent Advances: ROS/RNS and autophagy communicate with each other via both transcriptional and post-translational events. This cross talk, in turn, regulates the structural integrity of cardiomyocytes, promotes proteostasis, and reduces inflammation, events critical to disease pathogenesis. Critical Issues: Dysregulation of either autophagy or redox state has been implicated in many cardiovascular diseases. Cardiomyocytes are rich in mitochondria, which make them particularly sensitive to oxidative damage. Maintenance of mitochondrial homeostasis and elimination of defective mitochondria are each critical to the maintenance of redox homeostasis. Future Directions: The complex interplay between autophagy and oxidative stress underlies a wide range of physiological and pathological events and its elucidation holds promise of potential clinical applicability. Antioxid. Redox Signal. 20, 507-518.