Gut lumen-leaked microbial DNA causes myocardial inflammation and impairs cardiac contractility in ageing mouse heart.

Emerging evidence indicates the critical roles of microbiota in mediating host cardiac functions in ageing, however, the mechanisms underlying the communications between microbiota and cardiac cells during the ageing process have not been fully elucidated. Bacterial DNA was enriched in the cardiomyocytes of both ageing humans and mice. Antibiotic treatment remarkably reduced bacterial DNA abundance in ageing mice. Gut microbial DNA containing extracellular vesicles (mEVs) were readily leaked into the bloodstream and infiltrated into cardiomyocytes in ageing mice, causing cardiac microbial DNA enrichment. Vsig4+ macrophages efficiently block the spread of gut mEVs whereas Vsig4+ cell population was greatly decreased in ageing mice. Gut mEV treatment resulted in cardiac inflammation and a reduction in cardiac contractility in young Vsig4-/- mice. Microbial DNA depletion attenuated the pathogenic effects of gut mEVs. cGAS/STING signaling is critical for the effects of microbial DNA. Restoring Vsig4+ macrophage population in ageing WT mice reduced cardiac microbial DNA abundance and inflammation and improved heart contractility.

Restoration of coronary microvascular function by OGA overexpression in a high-fat diet with low-dose streptozotocin-induced type 2 diabetic mice.

Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

[WITHDRAWN] Hexokinase-1 mitochondrial dissociation and protein O-GlcNAcylation drive heart failure with preserved ejection fraction.

The authors have requested that this preprint be removed from Research Square.

Microbial DNA Enrichment Promotes Adrenomedullary Inflammation, Catecholamine Secretion, and Hypertension in Obese Mice.

Background Obesity is an established risk factor for hypertension. Although obesity-induced gut barrier breach leads to the leakage of various microbiota-derived products into host circulation and distal organs, the roles of microbiota in mediating the development of obesity-associated adrenomedullary disorders and hypertension have not been elucidated. We seek to explore the impacts of microbial DNA enrichment on inducing obesity-related adrenomedullary abnormalities and hypertension. Methods and Results Obesity was accompanied by remarkable bacterial DNA accumulation and elevated inflammation in the adrenal glands. Gut microbial DNA containing extracellular vesicles (mEVs) were readily leaked into the bloodstream and infiltrated into the adrenal glands in obese mice, causing microbial DNA enrichment. In lean wild-type mice, adrenal macrophages expressed CRIg (complement receptor of the immunoglobulin superfamily) that efficiently blocks the infiltration of gut mEVs. In contrast, the adrenal CRIg+ cell population was greatly decreased in obese mice. In lean CRIg-/- or C3-/- (complement component 3) mice intravenously injected with gut mEVs, adrenal microbial DNA accumulation elevated adrenal inflammation and norepinephrine secretion, concomitant with hypertension. In addition, microbial DNA promoted inflammatory responses and norepinephrine production in rat pheochromocytoma PC12 cells treated with gut mEVs. Depletion of microbial DNA cargo markedly blunted the effects of gut mEVs. We also validated that activation of cGAS (cyclic GMP-AMP synthase)/STING (cyclic GMP-AMP receptor stimulator of interferon genes) signaling is required for the ability of microbial DNA to trigger adrenomedullary dysfunctions in both in vivo and in vitro experiments. Restoring CRIg+ cells in obese mice decreased microbial DNA abundance, inflammation, and hypertension. Conclusions The leakage of gut mEVs leads to adrenal enrichment of microbial DNA that are pathogenic to induce obesity-associated adrenomedullary abnormalities and hypertension. Recovering the CRIg+ macrophage population attenuates obesity-induced adrenomedullary disorders.

Constitutive protein kinase G activation exacerbates stress-induced cardiomyopathy.

BACKGROUND AND PURPOSE: Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long-term PKG activation in the heart are unknown. EXPERIMENTAL APPROACH: We characterized the cardiac phenotype of mice carrying a heterozygous knock-in mutation of PKG1 (Prkg1R177Q/+ ), which causes constitutive, cGMP-independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. KEY RESULTS: Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. CONCLUSION AND IMPLICATIONS: Long-term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Morphine induces physiological, structural, and molecular benefits in the diabetic myocardium.

The obesity epidemic has increased type II diabetes mellitus (T2DM) across developed countries. Cardiac T2DM risks include ischemic heart disease, heart failure with preserved ejection fraction, intolerance to ischemia-reperfusion (I-R) injury, and refractoriness to cardioprotection. While opioids are cardioprotective, T2DM causes opioid receptor signaling dysfunction. We tested the hypothesis that sustained opioid receptor stimulus may overcome diabetes mellitus-induced cardiac dysfunction via membrane/mitochondrial-dependent protection. In a murine T2DM model, we investigated effects of morphine on cardiac function, I-R tolerance, ultrastructure, subcellular cholesterol expression, mitochondrial protein abundance, and mitochondrial function. T2DM induced 25% weight gain, hyperglycemia, glucose intolerance, cardiac hypertrophy, moderate cardiac depression, exaggerated postischemic myocardial dysfunction, abnormalities in mitochondrial respiration, ultrastructure and Ca2+ -induced swelling, and cell death were all evident. Morphine administration for 5 days: (1) improved glucose homeostasis; (2) reversed cardiac depression; (3) enhanced I-R tolerance; (4) restored mitochondrial ultrastructure; (5) improved mitochondrial function; (6) upregulated Stat3 protein; and (7) preserved membrane cholesterol homeostasis. These data show that morphine treatment restores contractile function, ischemic tolerance, mitochondrial structure and function, and membrane dynamics in type II diabetic hearts. These findings suggest potential translational value for short-term, but high-dose morphine administration in diabetic patients undergoing or recovering from acute ischemic cardiovascular events.

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.

Mitochondrial calcium handling and heart disease in diabetes mellitus.

Diabetes mellitus-induced heart disease, including diabetic cardiomyopathy, is an important medical problem and is difficult to treat. Diabetes mellitus increases the risk for heart failure and decreases cardiac myocyte function, which are linked to changes in cardiac mitochondrial energy metabolism. The free mitochondrial calcium concentration ([Ca2+]m) is fundamental in activating the mitochondrial respiratory chain complexes and ATP production and is also known to regulate the activity of key mitochondrial dehydrogenases. The mitochondrial calcium uniporter complex (MCUC) plays a major role in mediating mitochondrial Ca2+ import, and its expression and function therefore may have a marked impact on cardiac myocyte metabolism and function. Here, we summarize the pathophysiological role of [Ca2+]m handling and MCUC in the diabetic heart. In addition, we evaluate potential therapeutic targets, directed to the machinery that regulates mitochondrial calcium handling, to alleviate diabetes-related cardiac disease.

Maintaining Myocardial Glucose Utilization in Diabetic Cardiomyopathy Accelerates Mitochondrial Dysfunction.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

Conditional Up-Regulation of SERCA2a Exacerbates RyR2-Dependent Ventricular and Atrial Arrhythmias.

Ryanodine receptor 2 (RyR2) and SERCA2a are two major players in myocyte calcium (Ca) cycling that are modulated physiologically, affected by disease and thus considered to be potential targets for cardiac disease therapy. However, how RyR2 and SERCA2a influence each others' activities, as well as the primary and secondary consequences of their combined manipulations remain controversial. In this study, we examined the effect of acute upregulation of SERCA2a on arrhythmogenesis by conditionally overexpressing SERCA2a in a mouse model featuring hyperactive RyR2s due to ablation of calsequestrin 2 (CASQ2). CASQ2 knock-out (KO) mice were crossbred with doxycycline (DOX)-inducible SERCA2a transgenic mice to generate KO-TG mice. In-vivo ECG studies have shown that induction of SERCA2a (DOX+) overexpression markedly exacerbated both ventricular and atrial arrhythmias in vivo, compared with uninduced KO-TG mice (DOX-). Consistent with that, confocal microscopy in both atrial and ventricular myocytes demonstrated that conditional upregulation of SERCA2a enhanced the rate of occurrence of diastolic Ca release events. Additionally, deep RNA sequencing identified 17 downregulated genes and 5 upregulated genes in DOX+ mice, among which Ppp1r13l, Clcn1, and Agt have previously been linked to arrhythmias. Our results suggest that conditional upregulation of SERCA2a exacerbates hyperactive RyR2-mediated arrhythmias by further elevating diastolic Ca release.

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.

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.

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.

Neuronal Na+ Channels Are Integral Components of Pro-arrhythmic Na+/Ca2+ Signaling Nanodomain That Promotes Cardiac Arrhythmias During ß-adrenergic Stimulation.

BACKGROUND: Cardiac arrhythmias are a leading cause of death in the US. Vast majority of these arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with increased levels of circulating catecholamines and involve abnormal impulse formation secondary to aberrant Ca2+ and Na+ handling. However, the mechanistic link between ß-AR stimulation and the subcellular/molecular arrhythmogenic trigger(s) remains elusive. METHODS AND RESULTS: We performed functional and structural studies to assess Ca2+ and Na+ signaling in ventricular myocyte as well as surface electrocardiograms in mouse models of cardiac calsequestrin (CASQ2)-associated CPVT. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) that colocalize with RyR2 and Na+/Ca2+ exchanger (NCX) are a part of the ß-AR-mediated arrhythmogenic process. Specifically, augmented Na+ entry via nNav in the settings of genetic defects within the RyR2 complex and enhanced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)-mediated SR Ca2+ refill is both an essential and a necessary factor for the arrhythmogenesis. Furthermore, we show that augmentation of Na+ entry involves ß-AR-mediated activation of CAMKII subsequently leading to nNav augmentation. Importantly, selective pharmacological inhibition as well as silencing of Nav1.6 inhibit myocyte arrhythmic potential and prevent arrhythmias in vivo. CONCLUSION: These data suggest that the arrhythmogenic alteration in Na+/Ca2+ handling evidenced ruing ß-AR stimulation results, at least in part, from enhanced Na+ influx through nNav. Therefore, selective inhibition of these channels and Nav1.6 in particular can serve as a potential antiarrhythmic therapy.

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.

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition.

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca(2+)ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca(2+)indicators to selectively measure mitochondrial and cytosolic Ca(2+)using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 - (small Ca(2+)intensity)/(large Ca(2+)intensity)]. Blocking of complex I and II, cytochrome-coxidase, F0F1synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P< 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P< 0.04).N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P< 0.001). CGP, an antagonist of the mitochondrial Na(+)-Ca(2+)exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P< 0.0001). The major findings of this study are that impairment of mitochondrial Ca(2+)cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca(2+)content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca(2+)signaling in myocytes from diseased hearts, leading to new therapeutic targets.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain Reverses Cardiac Dysfunction Caused by Sustained Beta-Adrenergic Receptor Stimulation.

OBJECTIVE: To test the hypothesis that cardiac-directed expression of the cytoplasmic domains of adenylyl cyclase-6 (AC6) would have beneficial effects on the heart. BACKGROUND: Eliminating the two transmembrane domains of AC6 yields a protein with an intact catalytic domain that is disengaged from membrane-associated ß-adrenergic receptor stimulation, but with enhanced propensity for intracellular interactions. METHODS: We constructed a peptide of the C1 and C2 segments of AC6 (C1C2), expressed C1C2 in an adenovirus vector and generated transgenic lines with cardiac-directed C1C2 expression, which underwent sustained isoproterenol (Iso) infusion. RESULTS: Gene transfer of C1C2 in cardiac myocytes showed reduced cAMP generation in response to Iso-stimulation. C1C2 transgenic mice had normal left ventricular (LV) structure and function. LV samples from C1C2 mice showed diminished Iso-stimulated cAMP generation but normal LV contractile responses, suggesting a compensatory mechanism. Cardiac myocytes from C1C2 mice showed increased Iso-stimulated Ca2+ release and reduced time to peak Ca2+ release. After 7 days Iso infusion, control mice tended to show reduced LV function, but C1C2 mice showed increases in both LV peak +dP/dt and peak -dP/dt indicating enhanced LV systolic and diastolic function. LV from C1C2 mice showed a 2.6-fold increase in SERCA2a protein, and cardiac myocytes showed increased Ca2+ release, reduced time to peak Ca2+ release and reduced Tau. CONCLUSIONS: In C1C2 mice, sustained isoproterenol infusion increases rather than decreases LV function. Reduced cAMP generation and resistance to catecholamine cardiomyopathy are attractive features of this novel AC-related protein.

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.