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The regulation of cardiomyocyte hypertrophy is a complex interplay among many known and unknown processes. One specific pathway involves the phosphatase calcineurin, which regulates nuclear translocation of the essential cardiac hypertrophy transcription factor, nuclear factor of activated T-cells (NFAT). Although metabolic dysregulation is frequently described during cardiac hypertrophy, limited insights exist regarding various accessory pathways. One metabolically derived signal, beta-O-linked N-acetylglucosamine (O-GlcNAc), has emerged as a highly dynamic posttranslational modification of serine and threonine residues regulating physiological and stress processes. Given the metabolic dysregulation during hypertrophy, we hypothesized that NFAT activation is dependent on O-GlcNAc signaling. Pressure overload-induced hypertrophy (via transverse aortic constriction) in mice or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced global O-GlcNAc signaling. NFAT-luciferase reporter activity revealed O-GlcNAc-dependent NFAT activation during hypertrophy. Reversal of enhanced OGlcNAc signaling blunted cardiomyocyte NFAT-induced changes during hypertrophy. Taken together, these results demonstrate a critical role of O-GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O-GlcNAc signaling is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in future in vivo studies of hypertrophy. [ABSTRACT FROM AUTHOR]

Molecular studies examining the impact of mitochondrial morphology on the mammalian heart have previously focused on dynamin related protein-1 (Drp-1) and mitofusin-2 (Mfn-2), while the role of the other mitofusin isoform, Mfn-1, has remained largely unexplored. In the present study, we report the generation and initial characterization of cardiomyocyte-specific Mfn-1 knockout (Mfn-1 KO) mice. Using electron microscopic analysis, we detect a greater prevalence of small, spherical mitochondria in Mfn-1 KO hearts, indicating that the absence of Mfn-1 causes a profound shift in the mitochondrial fusion/ fission balance. Nevertheless, Mfn-1 KO mice exhibit normal leftventricular function, and isolated Mfn-1 KO heart mitochondria display a normal respiratory repertoire. Mfn-1 KO myocytes are protected from mitochondrial depolarization and exhibit improved viability when challenged with reactive oxygen species (ROS) in the form of hydrogen peroxide (H2O2). Furthermore, in vitro studies detect a blunted response of KO mitochondria to undergo peroxideinduced mitochondrial permeability transition pore opening. These data suggest that Mfn-1 deletion confers protection against ROS-induced mitochondrial dysfunction. Collectively, we suggest that mitochondrial fragmentation in myocytes is not sufficient to induce heart dysfunction or trigger cardiomyocyte death. Additionally, our data suggest that endogenous levels of Mfn-1 can attenuate myocyte viability in the face of an imminent ROS overload, an effect that could be associated with the ability of Mfn-1 to remodel the outer mitochondrial membrane. [ABSTRACT FROM AUTHOR]

O-linked β- N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice ( n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs ( n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia-reoxygenation or HO, and changes in Ca levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly ( P > 0.05) alter baseline Ca or ROS levels. However, AdOGT significantly ( P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca overload and ROS generation. Additionally, OGA inhibition mitigated both HO-induced Ca overload and ROS generation. Although AdOGA exacerbated both hypoxia and HO-induced ROS generation, it had no effect on HO-induced Ca overload. We conclude that inhibition of Ca overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation. [ABSTRACT FROM AUTHOR]

We previously demonstrated that the O-linked β-N-acetylglucosarnine (O-GIcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GIcNAc signaling interfered with other mechanisms of cell death. Because ischemialhypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GIcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetainido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamatel to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GIcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death, Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiornyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stressinduced cell death. These results provide new insights into a possible interaction between O-GIcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection. [ABSTRACT FROM AUTHOR]

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outer mitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is a mediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance of mitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomorphic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrial dysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeability transition downstream of Ca2+ stimulation or due to local generation of reactive oxygen species (ROS). Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducing stimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude that in cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells to mitochondrial permeability transition and to trigger cell death. [ABSTRACT FROM AUTHOR]

The post-translational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), is a metabolic and nutrient sensor which exerts cardioprotective effects. However, a paucity of information exists regarding the direct regulation of O-GlcNAc levels in the heart. We evaluated the role of O-GlcNAc transferase (OGT, which adds OGlcNAc to proteins) on cardiac myocyte survival following hypoxiareoxygentaion. We infected isolated cardiac myocytes (n=4-5/group) with adenovirus carrying the OGT gene (AdOGT), or treated with an OGT inhibitor (TT04), and subjected them to hypoxia and reoxygenation. Whole cell lysates were immunoblotted for O-GlcNAc levels and myocyte damage following reoxygenation was spectrophotometrically assessed via LDH release. Timelapse fluorescence microscopy using tetramethylrhodamine methyl ester (TMRM) was used to assess the post-hypoxic loss of mitochondrial membrane potential. AdOGT significantly augmented O-GlcNAc levels (p<0.05 compared to control), produced a significant reduction in LDH release, and preserved mitochondrial membrane potential. OGT inhibition (TT04) significantly reduced (p<0.05) O-GlcNAc levels, exacerbated LDH release, and hastened the loss of mitochondrial membrane potential. We conclude that OGT is a previously unrecognized, pro-survival enzyme in cardiac myocytes that exerts its effects via augmentation of O-GlcNAc levels. [ABSTRACT FROM AUTHOR]