Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

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.

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator.

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

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.

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.

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.

IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism.

BACKGROUND: Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS: HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS: An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE: Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE: Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.

Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.