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.

Background Diabetic patients 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 HuR is a key regulator of mRNA stability and translation of many genes, and there is growing evidence showing the potential role of HuR in cardiovascular disease. In this study, we investigated the role of HuR and its target genes in the development of CMD in type 2 diabetic mice. Methods Type 2 diabetes was induced in male mice by a high-fat diet combined with a single injection of low-dose streptozotocin. We assessed coronary flow velocity reserve (CFVR, a determinant of coronary microvascular function) in vivo and isolated cardiac endothelial cells (CECs) from those mice for in vitro experiment. Coronary endothelial function was evaluated in the 3rd order of coronary arteries using a wire myograph. Human CECs from 4 control subjects and 4 diabetic patients were purchased from the company. Results Diabetic mice exhibited decreases in CFVR and capillary density in the left ventricle (LV). HuR protein levels in CECs were significantly lower in diabetic mice and diabetic patients than in 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 in comparison to control mice. Cx40 protein level and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice, and Cx40 -KO mice exhibited decreased CFVR, attenuated endothelium-dependent relaxation, and reduced capillary density in the LV. Furthermore, endothelium-specific Cx40 overexpression ameliorated endothelial functions by augmenting endothelium-dependent relaxation and increasing capillary density in the LV, and resulted in the improvement of CFVR in diabetic mice. Conclusions These data suggest that decreased HuR, a specific mRNA binding protein that downregulates gap junction protein Cx40 in CECs, plays an important role in the development of coronary microvascular disease in diabetes. Restoration of Cx40 expression and function is potentially a novel therapeutic strategy for diabetic cardiovascular complications.

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. Here, 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 (dCas9)-based gene promoter pull-down approach coupled with mass spectrometry we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor co-repressor 2 (Ncor2) repression, and ChIP assays identified PPARα 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 (PDC) 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 (PLN) phosphorylation via Protein Kinase A (PKA). 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.

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.

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.

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.

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.

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 ([Ca 2+ ] mito ) than MCECs from wild-type (WT) mice. After increase in cytosolic Ca 2+ concentration ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] mito level and calcium uptake by mitochondria in response to increased [Ca 2+ ] cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.

DNA damage in cardiac myocytes resulting from increased oxidative stress is an important factor in the pathogenesis of diabetic cardiomyopathy. Although DNA repair machinery has been described in the cardiac myocyte, the regulation of oxidative DNA damage repair has been incompletely investigated. Moreover, in hyperglycemia the O‐GlcNAc post‐translational protein modification has been reported to interfere with the regular intracellular activity of metabolically relevant proteins in either an adaptive or a maladaptive manner. The enzyme 8‐oxoguanosine DNA glycosylase (OGG1) is key in DNA repair. Thus, the objective of this work was to investigate the influence of O‐GlcNAcylation of OGG1 in excessive glucose concentrations, simulating the levels observed in diabetic animals or humans. Neonatal cardiac myocytes (NCM) were isolated and cultured for 72 h in either normal glucose (NG; 5.5 mM glucose, 19.5 mM mannitol) or high glucose (HG; 25 mM glucose). The relationship between OGG1 O‐GlcNAcylation levels, enzymatic functionality and subcellular localization was analyzed. OGG1 was found to be up‐regulated and highly O‐GlcNAcylated in HG cultured NCM and was associated with increased 8‐OHdG (8‐hydroxy‐deoxyguanosine) levels, a common oxidative stress biomarker targeted by OGG1. In addition, decreased DNA quality and mitochondrial function were observed. Interestingly, OGG1 enzymatic activity in cell lysates was lower in HG compared to NG. Furthermore, in vitro experiments with recombinant OGG1 and O‐GlcNac transferase (OGT) demonstrated that O‐GlcNAcylation of OGG1 diminishes enzyme activity. We performed rescue experiments in NCM in HG by either rectifying OGG1 levels or reducing OGG1 O‐GlcNAcylation. NCM were either transduced with adenoviral particles (Adv) carrying OGG1 (Adv‐OGG1) or co‐transduced with Adv‐OGG1 and a dominant negative OGT (Adv‐dnOGT). Co‐transduced NCM showed improved OGG1 enzymatic activity compared to controls cells in spite of HG. These results were associated with lower 8‐OHdG levels. Finally, a different sub‐cellular enzyme distribution and an improvement in mitochondrial function were also detected. In conclusion, these results suggest that O‐GlcNAcylation of OGG1 could be considered a maladaptive phenomenon caused by hyperglycemia, which directly interferes with DNA repair by altering OGG1 activity and translocation, and indirectly leads to mitochondrial dysfunction.Support or Funding InformationThis work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation.

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.

Cardiac aging results in impaired cardiac function along with mitochondrial (Mito) dysfunction. The mechanisms for diminished function of the aging heart are incompletely investigated. Mito metabolism is regulated by free calcium concentration ([Ca2+]m) in the mitochondrial matrix. The Mito calcium uniporter complex (MCUC) is the main calcium transporter in the mitochondria. We hypothesized that Mito dysfunction could be the result of aging‐induced maladaptive changes in protein levels of the MCUC that results in deficient Mito Ca2+ handling that, in turn, impairs Mito metabolism. Therefore, the objective of this work was to determine the protein levels of members of the MCUC and analyze Mito Ca2+ handling as well as glucose oxidation (Gox). We measured protein levels by Western blot of MCU, MCUb, EMRE and MICU1/2 in hearts of young (2–3 months) and old (19–22 months) mice. In addition, we analyzed Mito Ca2+ uptake, release, and [Ca2+]m using Mito pericam. Gox was measured in an isolated “working heart” preparation. Our results showed that MCU and EMRE, which stimulate Ca2+ import, are decreased by 22% and 83% in hearts from old mice, respectively. In addition, MCUb which inhibits Ca2+ import is increased by 65% with no changes in MICU1/2. Furthermore, Mito Ca2+ uptake and [Ca2+]m were decreased by 35% and 37%, respectively. These effects of aging on Mito Ca2+ handling were consistent with reduced Gox by 45% that we measured in hearts from old mice.In conclusion, aging is associated with abnormal Mito Ca2+ handling produced by decrease in proteins of the MCUC that stimulate Mito Ca2+ uptake and increase in expression of MCUb, which inhibits Mito Ca2+ uptake. These maladaptive changes in aging result in impaired Mito metabolism and reduced Mito function in the heart.Support or Funding InformationThis work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation. Diaz‐Juarez J. received support from UCMEXUS‐CONACYT.

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.

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: β-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.

Cardiac ischemia is a leading cause of morbidity and mortality in diabetes, and endothelial cell (EC) dysfunction is implicated in coronary arterial disease. O‐linked N‐acetyglucosamine (O‐GlcNAcylation) is a post‐translational modification that is tightly controlled by two enzymes: O‐GlcNAc transferase (OGT, catalyzes the addition of an O‐GlcNAc to proteins) and O‐GlcNAcase (GCA, removes GlcNAc from proteins). In this study, we examine the role of GCA in coronary endothelial dysfunction in type 1 diabetic mice.We generated EC‐specific, inducible GCA transgenic mice and induced diabetes in these mice with streptozotocin. In diabetic mouse coronary endothelial cells (MCECs), GCA expression was significantly decreased and protein O‐GlcNAcylation was increased. GCA overexpression in diabetic mice decreased O‐GlcNAcylation in MCECs. Capillary density in the left ventricle was decreased in diabetes, while GCA overexpression significantly increased the density to the control level. Endothelium‐dependent relaxation was attenuated in diabetic coronary arteries (CAs) and restored by GCA overexpression. These data suggest that elevated protein O‐GlcNAcylation due to decreased GCA protein expression in MCECs contributes to coronary endothelial dysfunction in diabetes, whereas overexpression of GCA in ECs improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.Grant Funding Source: supported by NIH/NHLBI(HL115578)

Increased nuclear protein O‐linked β‐N‐acetylglucosamine glycosylation (O‐GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. Mitochondrial proteins in cardiac myocytes are also submitted to O‐GlcNAcylation, however, whether excessive O‐GlcNAcylation alters mitochondrial function is incompletely studied. We have previously demonstrated decreased mitochondrial calcium concentration in cardiac myocytes exposed to high glucose. We hypothesize that excessive O‐GlcNAcylation may be responsible of this maladaptive phenomenon and that reducing excessive O‐GlcNAcylation may return mitochondrial calcium levels to normal. Our objective was to test this hypothesis, therefore, we incubated cultured neonatal mouse myocytes in high glucose (HG, 25mM) for 72 hours and inhibited O‐GlcNAcylation by adenoviral expression of a dominant negative mutant of the O‐GlcNAc transferase (dnOGT). As expected, mitochondrial calcium levels were reduced by 20% after HG exposure but were returned to control values after dnOGT expression. In addition, mitochondrial calcium uptake decreased by 52% in HG and was improved by expression of dnOGT, however, did not reach control values. We examined the mitochondrial calcium uniporter (MCU) and found that it is susceptible to be O‐GlcNAcylated and HG further increased O‐GlcNAcylation. In conclusion, our results suggest that excessive O‐GlcNAcylation in hyperglycemia alters mitochondrial calcium homeostasis by increasing O‐GlcNAcylated MCU.Grant Funding Source: Supported by UC MEXUS and VA‐5 I01BX001121‐02‐

Signaling from a plasma membrane–associated receptor contributes to the effects of thyroid hormones on bones.

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.

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.