HuR/Cx40 downregulation causes coronary microvascular dysfunction in type 2 diabetes.

Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.

Ncor2/PPARα-Dependent Upregulation of MCUb in the Type 2 Diabetic Heart Impacts Cardiac Metabolic Flexibility and Function.

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. In this study, we show that the T2D heart is metabolically inflexible and almost exclusively dependent on mitochondrial fatty acid oxidation as a consequence of mitochondrial calcium uniporter complex (MCUC) inhibitory subunit MCUb overexpression. Using a recombinant endonuclease-deficient Cas9-based gene promoter pulldown approach coupled with mass spectrometry, we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor corepressor 2 repression, and chromatin immunoprecipitation assays identified peroxisome proliferator-activated receptor α as a mediator of MCUb gene expression in T2D cardiomyocytes. Upregulation of MCUb limits mitochondrial matrix Ca2+ uptake and impairs mitochondrial energy production via glucose oxidation by depressing pyruvate dehydrogenase complex activity. Gene therapy displacement of endogenous MCUb with a dominant-negative MCUb transgene (MCUbW246R/V251E) in vivo rescued T2D cardiomyocytes from metabolic inflexibility and stimulated cardiac contractile function and adrenergic responsiveness by enhancing phospholamban phosphorylation via protein kinase A. We conclude that MCUb represents one newly discovered molecular effector at the interface of metabolism and cardiac function, and its repression improves the outcome of the chronically stressed diabetic heart.

Overexpression of p53 due to excess protein O-GlcNAcylation is associated with coronary microvascular disease in type 2 diabetes.

AIMS: We previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility. METHODS AND RESULTS: We conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice. CONCLUSIONS: The data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.

Restoring mitochondrial calcium uniporter expression in diabetic mouse heart improves mitochondrial calcium handling and cardiac function.

Diabetes mellitus is a growing health care problem, resulting in significant cardiovascular morbidity and mortality. Diabetes also increases the risk for heart failure (HF) and decreased cardiac myocyte function, which are linked to changes in cardiac mitochondrial energy metabolism. The free mitochondrial calcium level ([Ca2+] m ) is fundamental in activating the mitochondrial respiratory chain complexes and ATP production and is also known to regulate pyruvate dehydrogenase complex (PDC) activity. The mitochondrial calcium uniporter (MCU) complex (MCUC) plays a major role in mediating mitochondrial Ca2+ import, and its expression and function therefore have a marked impact on cardiac myocyte metabolism and function. Here, we investigated MCU's role in mitochondrial Ca2+ handling, mitochondrial function, glucose oxidation, and cardiac function in the heart of diabetic mice. We found that diabetic mouse hearts exhibit altered expression of MCU and MCUC members and a resulting decrease in [Ca2+] m , mitochondrial Ca2+ uptake, mitochondrial energetic function, and cardiac function. Adeno-associated virus-based normalization of MCU levels in these hearts restored mitochondrial Ca2+ handling, reduced PDC phosphorylation levels, and increased PDC activity. These changes were associated with cardiac metabolic reprogramming toward normal physiological glucose oxidation. This reprogramming likely contributed to the restoration of both cardiac myocyte and heart function to nondiabetic levels without any observed detrimental effects. These findings support the hypothesis that abnormal mitochondrial Ca2+ handling and its negative consequences can be ameliorated in diabetes by restoring MCU levels via adeno-associated virus-based MCU transgene expression.

Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

Coronary microvascular rarefaction, due to endothelial cell (EC) dysfunction, is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial function. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca2+ concentration ([Ca2+]mito) compared with the control. Among several regulatory proteins for [Ca2+]mito, hexokinase 1 (HK1) and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from nondiabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in MCECs. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca2+]mito, but also reduced mitochondrial reactive oxygen species production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Mitochondrial connexin40 regulates mitochondrial calcium uptake in coronary endothelial cells.

Connexins (Cxs) are a group of integral membrane proteins that can form gap junctions between adjacent cells. Recently, it was reported that Cx43 is expressed not only in the plasma membrane but also in the inner mitochondrial membrane and that it regulates mitochondrial functions. Cx40 is predominantly expressed in vascular endothelial cells (ECs) and plays an important role in the electrical propagation between ECs and endothelial/smooth muscle cells. However, it is unknown whether Cx40 is expressed in the mitochondria and what the role of mitochondrial Cx40 is in endothelial functions. We observed in coronary ECs that Cx40 protein was expressed in the mitochondria, as determined by Western blot and immunofluorescence studies. We found that mouse coronary ECs (MCECs) isolated from Cx40 knockout (Cx40 KO) mice exhibited significantly lower resting mitochondrial calcium concentration ([Ca2+]mito) than MCECs from wild-type (WT) mice. After increase in cytosolic Ca2+ concentration ([Ca2+]cyto) with cyclopiazonic acid, calcium uptake into the mitochondria was significantly attenuated in MCECs from Cx40 KO mice compared with WT MCECs. There was no difference in resting [Ca2+]cyto and store-operated calcium entry in MCECs from WT and Cx40 KO mice. We also detected a significant decrease in the concentration of mitochondrial reactive oxygen species (ROS) in Cx40 KO MCECs. Cx40 overexpression in ECs significantly increased resting [Ca2+]mito level and calcium uptake by mitochondria in response to increased [Ca2+]cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.

O-GlcNAcylation of 8-Oxoguanine DNA Glycosylase (Ogg1) Impairs Oxidative Mitochondrial DNA Lesion Repair in Diabetic Hearts.

mtDNA damage in cardiac myocytes resulting from increased oxidative stress is emerging as an important factor in the pathogenesis of diabetic cardiomyopathy. A prevalent lesion that occurs in mtDNA damage is the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which can cause mutations when not repaired properly by 8-oxoguanine DNA glycosylase (Ogg1). Although the mtDNA repair machinery has been described in cardiac myocytes, the regulation of this repair has been incompletely investigated. Here we report that the hearts of type 1 diabetic mice, despite having increased Ogg1 protein levels, had significantly lower Ogg1 activity than the hearts of control, non-type 1 diabetic mice. In diabetic hearts, we further observed increased levels of 8-OHdG and an increased amount of mtDNA damage. Interestingly, Ogg1 was found to be highly O-GlcNAcylated in diabetic mice compared with controls. In vitro experiments demonstrated that O-GlcNAcylation inhibits Ogg1 activity, which could explain the mtDNA lesion accumulation observed in vivo Reducing Ogg1 O-GlcNAcylation in vivo by introducing a dominant negative O-GlcNAc transferase mutant (F460A) restored Ogg1 enzymatic activity and, consequently, reduced 8-OHdG and mtDNA damage despite the adverse hyperglycemic milieu. Taken together, our results implicate hyperglycemia-induced O-GlcNAcylation of Ogg1 in increased mtDNA damage and, therefore, provide a new plausible biochemical mechanism for diabetic cardiomyopathy.

Expression of the mitochondrial calcium uniporter in cardiac myocytes improves impaired mitochondrial calcium handling and metabolism in simulated hyperglycemia.

Diabetic cardiomyopathy is associated with metabolic changes, including decreased glucose oxidation (Gox) and increased fatty acid oxidation (FAox), which result in cardiac energetic deficiency. Diabetic hyperglycemia is a pathophysiological mechanism that triggers multiple maladaptive phenomena. The mitochondrial Ca2+ uniporter (MCU) is the channel responsible for Ca2+ uptake in mitochondria, and free mitochondrial Ca2+ concentration ([Ca2+]m) regulates mitochondrial metabolism. Experiments with cardiac myocytes (CM) exposed to simulated hyperglycemia revealed reduced [Ca2+]m and MCU protein levels. Therefore, we investigated whether returning [Ca2+]m to normal levels in CM by MCU expression could lead to normalization of Gox and FAox with no detrimental effects. Mouse neonatal CM were exposed for 72 h to normal glucose [5.5 mM glucose + 19.5 mM mannitol (NG)], high glucose [25 mM glucose (HG)], or HG + adenoviral MCU expression. Gox and FAox, [Ca2+]m, MCU levels, pyruvate dehydrogenase (PDH) activity, oxidative stress, mitochondrial membrane potential, and apoptosis were assessed. [Ca2+]m and MCU protein levels were reduced after 72 h of HG. Gox was decreased and FAox was increased in HG, PDH activity was decreased, phosphorylated PDH levels were increased, and mitochondrial membrane potential was reduced. MCU expression returned these parameters toward NG levels. Moreover, increased oxidative stress and apoptosis were reduced in HG by MCU expression. We also observed reduced MCU protein levels and [Ca2+]m in hearts from type 1 diabetic mice. Thus we conclude that HG-induced metabolic alterations can be reversed by restoration of MCU levels, resulting in return of [Ca2+]m to normal levels.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: ß-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.

Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor.

Thyroid hormone (TH) is essential for vertebrate development and the homeostasis of most adult tissues, including bone. TH stimulates target gene expression through the nuclear thyroid receptors TRα and TRß; however, TH also has rapid, transcription-independent (nongenomic) effects. We found a previously uncharacterized plasma membrane-bound receptor that was necessary and sufficient for nongenomic TH signaling in several cell types. We determined that this receptor is generated by translation initiation from an internal methionine of TRα, which produces a transcriptionally incompetent protein that is palmitoylated and associates with caveolin-containing plasma membrane domains. TH signaling through this receptor stimulated a pro-proliferative and pro-survival program by increasing the intracellular concentrations of calcium, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), which led to the sequential activation of protein kinase G II (PKGII), the tyrosine kinase Src, and extracellular signal-regulated kinase (ERK) and Akt signaling. Hypothyroid mice exhibited a cGMP-deficient state with impaired bone formation and increased apoptosis of osteocytes, which was rescued by a direct stimulator of guanylate cyclase. Our results link nongenomic TH signaling to a previously uncharacterized membrane-bound receptor, and identify NO synthase, guanylate cyclase, and PKGII as TH effectors that activate kinase cascades to regulate cell survival and proliferation.

In vivo selective expression of thyroid hormone receptor α1 in endothelial cells attenuates myocardial injury in experimental myocardial infarction in mice.

Ischemic heart disease (IHD) is the single most common cause of death. New approaches to enhance myocardial perfusion are needed to improve outcomes for patients with IHD. Thyroid hormones (TH) are known to increase blood flow; however, their usefulness for increasing perfusion in IHD is limited because TH accelerates heart rate, which can be detrimental. Therefore, selective activation of TH effects is desirable. We hypothesized that cell-type-specific TH receptor (TR) expression can increase TH action in the heart, while avoiding the negative consequences of TH treatment. We generated a binary transgenic (BTG) mouse that selectively expresses TRα1 in endothelial cells in a tetracycline-inducible fashion. In BTG mice, endothelial TRα1 protein expression was increased by twofold, which, in turn, increased coronary blood flow by 77%, coronary conductance by 60%, and coronary reserve by 47% compared with wild-type mice. Systemic blood pressure was decreased by 20% in BTG mice after TRα1 expression. No effects on heart rate were observed. Endothelial TRα1 expression activated AKT/endothelial nitric oxide synthase pathway and increased A2AR adenosine receptor. Furthermore, hearts from BTG mice overexpressing TRα1 that were submitted to 20 min ischemia and 20 min reperfusion showed a 20% decline in left ventricular pressure (LVP) compared with control mice where LVP was decreased by 42%. Studies using an infarction mouse model demonstrated that endothelial overexpression of TRα1 decreased infarct size by 45%. In conclusion, selective expression of TRα1 in endothelial cells protects the heart against injury after an ischemic insult and does not result in adverse cardiac or systemic effects.

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase γ was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage.

Sorcin modulates mitochondrial Ca(2+) handling and reduces apoptosis in neonatal rat cardiac myocytes.

Sorcin localizes in cellular membranes and has been demonstrated to modulate cytosolic Ca(2+) handling in cardiac myocytes. Sorcin also localizes in mitochondria; however, the effect of sorcin on mitochondrial Ca(2+) handling is unknown. Using mitochondrial pericam, we measured mitochondrial Ca(2+) concentration and fluxes in intact neonatal cardiac myocytes overexpressing sorcin. Our results showed that sorcin increases basal and caffeine-stimulated mitochondrial Ca(2+) concentration. This effect was associated with faster Ca(2+) uptake and release. The effect of sorcin was specific for mitochondria, since similar results were obtained with digitonin-permeabilized cells, where cytosolic Ca(2+) flux was disrupted. Furthermore, mitochondria of cardiac myocytes in which sorcin was overexpressed were more Ca(2+)-tolerant. Experiments analyzing apoptotic signaling demonstrated that sorcin prevented 2-deoxyglucose-induced cytochrome c release. Furthermore, sorcin prevented hyperglycemia-induced cytochrome c release and caspase activation. In contrast, antisense sorcin induced caspase-3 activation. Thus, sorcin antiapoptotic properties may be due to modulation of mitochondrial Ca(2+) handling in cardiac myocytes.

VDAC: old protein with new roles in diabetes.

A decrease in capillary density due to an increase in endothelial cell apoptosis in the heart is implicated in cardiac ischemia in diabetes. The voltage-dependent anion channel (VDAC) plays a crucial role in the regulation of mitochondrial metabolic function and mitochondria-mediated apoptosis. This study is designed to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in diabetic left ventricle of diabetic mice and mouse coronary ECs (MCECs) isolated from diabetic mice exhibited significantly higher mitochondrial Ca(2+) concentration and VDAC protein levels than control MCECs. The expression of VDAC-short hairpin RNA (shRNA) not only decreased the resting mitochondrial Ca(2+) concentration but also attenuated mitochondrial Ca(2+) uptake in diabetic MCECs. Furthermore, the downregulation of VDAC in diabetic MCECs significantly decreased mitochondrial superoxide anion (O(2)(-)) production and the activity of the mitochondrial permeability transition pore (mPTP) opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC level in diabetic MCECs is responsible for increased mitochondrial Ca(2+) concentration, mitochondrial O(2)(-) production, and mPTP opening activity. Normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.

Excess protein O-GlcNAcylation and the progression of diabetic cardiomyopathy.

We examined the role that enzymatic protein O-GlcNAcylation plays in the development of diabetic cardiomyopathy in a mouse model of Type 2 diabetes mellitus (DM2). Mice injected with low-dose streptozotocin and fed a high-fat diet developed mild hyperglycemia and obesity consistent with DM2. Studies were performed from 1 to 6 mo after initiating the DM2 protocol. After 1 mo, DM2 mice showed increased body weight, impaired fasting blood glucose, and hyperinsulinemia. Echocardiographic evaluation revealed left ventricular diastolic dysfunction by 2 mo and O-GlcNAcylation of several cardiac proteins and of nuclear transcription factor Sp1. By 4 mo, systolic dysfunction was observed and sarcoplasmic reticulum Ca(2+) ATPase expression decreased by 50%. Fibrosis was not observed at any timepoint in DM2 mice. Levels of the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine:fructose-6-phosphate amidotransferase (GFAT) were increased as early as 2 mo. Fatty acids, which are elevated in DM2 mice, can possibly be linked to excessive protein O-GlcNAcylation levels, as cultured cardiac myocytes in normal glucose treated with oleic acid showed increased O-GlcNAcylation and GFAT levels. These data indicate that the early onset of diastolic dysfunction followed by the loss of systolic function, in the absence of cardiac hypertrophy or fibrosis, is associated with increased cardiac protein O-GlcNAcylation and increased O-GlcNAcylation levels of key calcium-handling proteins. A link between excessive protein O-GlcNAcylation and cardiac dysfunction is further supported by results showing that reducing O-GlcNAcylation by O-GlcNAcase overexpression improved cardiac function in the diabetic mouse. In addition, fatty acids play a role in stimulating excess O-GlcNAcylation. The nature and time course of changes observed in cardiac function suggest that protein O-GlcNAcylation plays a mechanistic role in the triggering of diabetic cardiomyopathy in DM2.

Thyroid hormone receptor-α and vascular function.

Thyroid hormone (TH) treatment exerts beneficial effects on the cardiovascular system: it lowers cholesterol and LDL levels and enhances cardiac contractile function. However, little is known about the effect of TH on vascular function or the functional role of TH receptors (TRs) in the regulation of vascular tone. We have investigated the contribution of TRs to vascular contractility in the heart. Among different TR subtype-specific knockout (KO) mice, vascular contraction was significantly enhanced in coronary arteries isolated from TRα KO compared with wild-type mice, while chronic TH treatment significantly attenuated coronary vascular contraction. We found that TRα is the predominant TR in mouse coronary smooth muscle cells (SMCs). Coronary SMCs isolated from TRα KO mice exhibited a significant decrease in K(+) channel activity, whereas TH treatment increased K(+) channel activity in a dose-dependent manner. These data suggest that TRα in SMCs has prominent effects on regulation of vascular tone and TH treatment helps decrease coronary vascular tone by increasing K(+) channel activity through TRα in SMCs.

Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes.

Mitochondria are crucial organelles in cell life serving as a source of energy production and as regulators of Ca(2+) homeostasis, apoptosis, and development. Mitochondria frequently change their shape by fusion and fission, and recent research on these morphological dynamics of mitochondria has highlighted their role in normal cell physiology and disease. In this study, we investigated the effect of high glucose on mitochondrial dynamics in neonatal cardiac myocytes (NCMs). High-glucose treatment of NCMs significantly decreased the level of optical atrophy 1 (OPA1) (mitochondrial fusion-related protein) protein expression. NCMs exhibit two different kinds of mitochondrial structure: round shape around the nuclear area and elongated tubular structures in the pseudopod area. High-glucose-treated NCMs exhibited augmented mitochondrial fragmentation in the pseudopod area. This effect was significantly decreased by OPA1 overexpression. High-glucose exposure also led to increased O-GlcNAcylation of OPA1 in NCMs. GlcNAcase (GCA) overexpression in high-glucose-treated NCMs decreased OPA1 protein O-GlcNAcylation and significantly increased mitochondrial elongation. In addition to the morphological change caused by high glucose, we observed that high glucose decreased mitochondrial membrane potential and complex IV activity and that OPA1 overexpression increased both levels to the control level. These data suggest that decreased OPA1 protein level and increased O-GlcNAcylation of OPA1 protein by high glucose lead to mitochondrial dysfunction by increasing mitochondrial fragmentation, decreasing mitochondrial membrane potential, and attenuating the activity of mitochondrial complex IV, and that overexpression of OPA1 and GCA in cardiac myocytes may help improve the cardiac dysfunction in diabetes.

Thyroid hormone inhibits ERK phosphorylation in pressure overload-induced hypertrophied mouse hearts through a receptor-mediated mechanism.

Pressure overload-induced cardiac hypertrophy results in a pathological type of hypertrophy with activation of signaling cascades like the extracellular signal-regulated kinase (ERK) pathway, which promotes negative cardiac remodeling and decreased contractile function. In contrast, thyroid hormone mediates a physiological type of hypertrophy resulting in enhanced contractile function. In addition, thyroid hormone action is diminished in pressure overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone status modulates ERK activity and that administration of thyroid hormone could alter the activity of this kinase in cardiac hypertrophy induced by pressure overload. ERK is activated by phosphorylation; accordingly, we investigated phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice. In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied hearts from transverse aortic-constricted (TAC) mice was investigated. Results showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice presented a greater than fourfold increase of p-ERK compared with control mice. Interestingly, T3 administration dramatically canceled TAC-induced ERK phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3 treatment inhibited this effect of pressure overload and further decreased p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid hormone receptor-α in cultured cardiomyocytes potentiated the inhibitory effect of T3 on ERK phosphorylation. We concluded that thyroid hormone has an inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent mechanism.

Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose.

Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.