High-Resolution Respirometry in Diagnostics of Mitochondrial Diseases Caused by Mitochondrial Complex I Deficiency

Mitochondrial complex I deficiency (CID) is one of the most common defects in the OXPHOS system; it represents more than 30% cases of mitochondrial diseases. The group is characterized by clinical and genetic heterogeneity and comprises several nosological forms. The most prevalent phenotypes of CID are Leber hereditary optic neuropathy (LHON) and Leigh syndrome. In this study we have analyzed skin fibroblasts from 11 patients with mutations in mtDNA, which cause LHON or Leigh-like phenotypes (m.11778 G>A (n = 3), m.3460 A>G (n = 2), m.3635 G>A (n = 1), m.3308 T>G (n = 2), m.3472 T>C (n = 1)), and 2 patients with earlier unknown substitutions m.3945 C>A and m.14441 T>C. High-resolution respirometry (HRR) was performed for complex analysis of the mitochondrial respiratory function in intact and permeabilized fibroblasts of patients and healthy controls. Flux control ratios in intact cells R/E, (R-L)/E were raised compared to the control. Rates of R, E, L normalized on the citrate synthase (CS) activity were statistically different (p < 0.05) between patients and controls. In permeabilized fibroblasts we have found statistically significant differences (p < 0.05) in ratios СII/E, Rot/E, R/CII, CI/CII between groups. These data highlight dysfunctions of the OXPHOS system, particularly CI. Increased CS activity and decreased CI/CII ratio suggest a compensatory metabolic response to the respiratory chain dysfunction. Our results show applicability of HRR in revealing biochemical abnormalities of mitochondrial complex I in fibroblasts of patients with LHON and Leigh-like syndrome. We also suggest HRR to be a useful method for inspection of new mutations causing mitochondrial complex I deficiency.     

Capacity of oxidative phosphorylation in human skeletal muscle: New perspectives of mitochondrial physiology

Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I + II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1–3 mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate + malate + succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate + malate or glutamate + malate yield submaximal oxygen fluxes at 0.50–0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 °C (pmol O₂ s⁻¹ mg⁻¹ wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100–150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60–120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.     

VEGF and IL-4 gene variability and its association with the risk of coronary heart disease in north Indian population

Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that has been shown to play a significant role in neovascularization during inflammation in atherosclerotic plaques, formation of collateral vessels to an area of ischemic myocardium and neovascularization at the edges of a myocardial infarction during its repair. Interleukin-4 (IL-4) has important role in immune cell chemotaxis, formation of endothelial cell adhesion molecules and has numerous anti-inflammatory effects which prevent the complications of atherosclerosis, the primary cause of coronary heart disease (CHD). In this study, we have analyzed the effect of 1154 A/G polymorphism of VEGF and 70 bp VNTR polymorphism of intron 3 in IL-4 genes in coronary heart disease (CHD) patients (n = 300) and their age matched controls (n = 300). To analyze polymorphic alleles, ARMS-PCR and RFLP techniques were used. Multiple logistic regression analysis was carried out with statistical software. GG genotype was associated with a decreased risk of development of CHD (OR 0.22, 95% CI 0.12–0.38, P < 0.001). However, A allele showed an increased risk whereas G allele decreased the risk of CHD with diabetes mellitus, hypertension, chronic mental stress and positive familial history of myocardial infarction (MI)/CHD. GG genotype was found to have protective effect with alcohol intake (OR 0.34, 95% CI 0.14–0.82, P < 0.01) and central obesity (OR 0.15, 95% CI 0.04–0.56, P < 0.001). GG genotype of VEGF has also shown significant association with IL-4 (P2P2 and P1P2) genotypes.     

Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle

Background

Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.

Methodology/Principal Findings

We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.Oxidative phosphorylation (OXPHOS) capacity (pmol O₂•s⁻¹•mg⁻¹ wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness.

Conclusions/Significance

The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.     

Effects of acute cigarette smoke concentrate exposure on mitochondrial energy transfer in fast- and slow-twitch skeletal muscle

The mechanisms underlying cigarette smoke-induced mitochondrial dysfunction in skeletal muscle are still poorly understood. Accordingly, this study aimed to examine the effects of cigarette smoke on mitochondrial energy transfer in permeabilized muscle fibers from skeletal muscles with differing metabolic characteristics. The electron transport chain (ETC) capacity, ADP transport, and respiratory control by ADP were assessed in fast- and slow-twitch muscle fibers from C57BL/6 mice (n = 11) acutely exposed to cigarette smoke concentrate (CSC) using high-resolution respirometry. CSC decreased complex I-driven respiration in the white gastrocnemius (CONTROL:45.4 ± 11.2 pmolO₂.s⁻¹.mg⁻¹ and CSC:27.5 ± 12.0 pmolO₂.s⁻¹.mg⁻¹; p = 0.01) and soleus (CONTROL:63.0 ± 23.8 pmolO₂.s⁻¹.mg⁻¹ and CSC:44.6 ± 11.1 pmolO₂.s⁻¹.mg⁻¹; p = 0.04). In contrast, the effect of CSC on Complex II-linked respiration increased its relative contribution to muscle respiratory capacity in the white gastrocnemius muscle. The maximal respiratory activity of the ETC was significantly inhibited by CSC in both muscles. Furthermore, the respiration rate dependent on the ADP/ATP transport across the mitochondrial membrane was significantly impaired by CSC in the white gastrocnemius (CONTROL:-70 ± 18 %; CSC:-28 ± 10 %; p < 0.001), but not the soleus (CONTROL:47 ± 16 %; CSC:31 ± 7 %; p = 0.08). CSC also significantly impaired mitochondrial thermodynamic coupling in both muscles. Our findings underscore that acute CSC exposure directly inhibits oxidative phosphorylation in permeabilized muscle fibers. This effect was mediated by significant perturbations of the electron transfer in the respiratory complexes, especially at complex I, in both fast and slow twitch muscles. In contrast, CSC-induced inhibition of the exchange of ADP/ATP across the mitochondrial membrane was fiber-type specific, with a large effect on fast-twitch muscles.     

A novel haplotype within C-reactive protein gene influences CRP levels and coronary heart disease risk in Northwest Indians

According to several epidemiological and clinical studies, the concentration of C-reactive protein (CRP) in blood is associated with the risk of coronary heart disease (CHD). However, these studies are limited in high incidence and prevalence area of North-West India. The present case control study investigated the contribution of three relevant CRP single nucleotide polymorphisms: ?717A>G located in the promoter region (rs2794521), +1059G>C on exon2 (rs1800947) and +1444C>T in the 3′ UTR (rs1130864) in 180 angiographically verified CHD cases and 175 control subjects. Minor allele frequencies (G, C and T) of rs2794521, rs1800947 and rs1130864 are observed to be 21.1, 11.7, 29.4 and 11.4, 10.0, 19.7 % in CHD cases and controls respectively. AA genotype of ?717A>G and TT genotype of +1444C>T were significantly associated (P = 0.02 & 0.03 respectively) with the risk of CHD whereas, +1059G and +1444T were found to be strongly related (P = 0.023 & P = 0.008 respectively) with multivariable adjusted CRP levels. AGT Haplotype was significantly associated with the adjusted CRP levels (P < 0.05). Disease association analysis revealed that haplotype AGT influences CHD risk (OR 2.4, 95 % CI 1.23–4.84, P = 0.006) which exacerbates after correcting the confounding effects of risk variables (OR 2.5, 95 % CI 1.27–4.99, P = 0.004). With the global index of Akaike information criterion, it has been observed that the carrying each single unit of this susceptibility haplotype increases CHD risk by a value of 2.41 ± 0.439 (β ± SE) in the recessive mode.     

Association of ACE and FACTOR VII gene variability with the risk of coronary heart disease in north Indian population

The angiotensin converting enzyme (ACE) is a key factor in the production of angiotensin II and in the degradation of bradykinin. Chronic exposure to high levels of circulating and tissue ACE predispose to vascular wall thickening and atherosclerosis. Factor VII (FACTOR VII) is the first enzyme in the extrinsic pathway of the blood coagulation system and plays a key role in hemostasis; it also contributes to the occurrence of thrombotic events. In this study, we have examined the association of ACE and FACTOR VII gene in coronary heart disease patients (n = 300) and their age-matched controls (n = 300). Genotyping was done by PCR-RFLP method. No significant difference was observed in the distribution of I/D genotypes of ACE between cases and controls. In case of FACTOR VII R353Q polymorphism, there was not much difference in the distribution of alleles. AA genotype had protective effect for CHD (OR 0.56, 95% CI 0.37–0.83, P = 0.001). In case of FACTOR VII VNTR, there was difference in the distribution of alleles, H6 (73.5) and H7 (25.5) in cases, and H6 (70.5) and H7 (30.5) in controls. H6H7 and H7H7 genotypes had a protective effect for CHD with OR 0.27, 95% CI 0.18–0.41, P < 0.001, and OR 0.18, 95% CI 0.09–0.36, P < 0.001. Our study showed D allele of ACE to be associated with marginal risk of CHD, AA genotype of FACTOR VII R353Q and H6H7 and H7H7 genotypes of FACTOR VII VNTR showed protective effect for CHD.     

Human coronary heart disease: importance of blood cellular miR-2909 RNomics

The characterization of atherosclerosis as a chronic inflammatory disease has triggered extensive research worldwide to dissect the pro- and anti-inflammatory, cellular as well as molecular mechanisms governing the pathogenesis of this dreadful disease. Though several microRNAs have been shown to play crucial role in regulating lipid metabolism and inflammation, we are far from resolving the role of epigenomic signals in etiology of coronary heart disease (CHD). The present study was addressed to understand the role of a novel microRNA, miR-2909, in the regulation of genes involved in the initiation and progression of human coronary occlusion. Peripheral blood mononuclear cells were isolated from human CHD subjects at various stages of coronary occlusion (n = 80) and their corresponding normal healthy counterparts (n = 20). Various experimental strategies involving gene expression and silencing, reporter plasmid assays, and flow cytometric analysis were blend together to address the current problem. The present study shows for the first time that the blood cellular miR-2909 expression increases with the severity of coronary occlusion, exhibiting a strong positive correlation (r = 0.943 at p < 0.01). Further, miR-2909 was shown to regulate genes involved in inflammation, immunity, and oxLDL uptake, thereby contributing significantly to the initiation and progression of CHD patho-physiological process. Based upon these results, we propose that miR-2909 RNomics may be a step forward in understanding human CHD at the epigenomic level and can be exploited for designing new therapeutic strategies as well as diagnostic and prognostic markers for this disease in future.     

The association between CD14 gene C-260T polymorphism and coronary heart disease risk: a meta-analysis

Monocyte differentiation antigen CD14 is considered an important cell-activating mediator of inflammatory responses that may result in atherosclerosis, coronary heart disease (CHD), thrombus formation, and myocardial infarction (MI). A common C-260T polymorphism in the promoter of the CD14 gene, the trans-membrane receptor of lipopolysaccharides, has been inconsistently associated with CHD. To investigate this inconsistency, we performed a meta-analysis of 28 studies involving a total of 13,335 CHD cases and 7,979 controls for C-260T of the CD14 gene to evaluate the effect of CD14 on genetic susceptibility for CHD. An overall random effects odds ratio of 1.24 (95 % CI: 1.12–1.36, P < 10^?5) was found for T allele. Significant results were also observed using dominant (OR = 1.34, 95 % CI: 1.17–1.54, P < 10^?4) or recessive genetic model (OR = 1.25, 95 % CI: 1.10–1.41, P = 0.0004). There was strong evidence of heterogeneity (P < 10^?5), which largely disappeared after stratification by ethnicity. After stratified by ethnicity, significant results were found in East Asians; whereas no significant associations were found among Caucasians and other ethnic populations in all genetic models. In the stratified analysis according to sample size, CHD endpoints, and HWE status, significantly increased risks for the polymorphism were found in all genetic models. In conclusion, our results indicate that the CD14 C-260T polymorphism is a risk factor of CHD, especially in East Asians. However, additional very large-scale studies are warranted to confirm our results.     

Prevalence and risk of viral infection in patients with acute exacerbation of chronic obstructive pulmonary disease: a meta-analysis

Exacerbations of chronic obstructive pulmonary disease (COPD) lead to substantial morbidity and mortality. Viral infections could be an important cause of acute exacerbations of COPD (AECOPD) and only a few studies report the prevalence of respiratory viruses on this disease. We aimed to update the review on the prevalence of respiratory viral infection in patients with AECOPD with a meta-analysis. We reviewed the prevalence of respiratory viruses on this disease by searching PubMed systematically to identify primary studies published from Jan 1990 to March 2012. Studies met with seven criteria were extracted for meta-analysis. A total of 17 studies were eligible for the meta-analysis. Weighted overall prevalence of respiratory viruses in patients with AECOPD was 39.3 % (95 % CI 36.9–41.6) with a high degree of a heterogeneity (I ² > 75 %). In contrast, the rate in stable COPD patients from four studies was 13.6 % (95 % CI 9.0–18.2) without any apparent heterogeneity. Pooled risk ratio for respiratory viral infection was 4.1 (95 % CI 2.0–8.5) for AECOPD as compared with stable COPD. Rhinovirus was the most common virus and with a weighted prevalence of 14.8 % (95 % CI 13.3–16.5). Respiratory viruses probably are important etiological agents in patients with AECOPD as compared with the stable COPD patients. This result would help to provide better strategies for management of AECOPD and health-care planning.     

Correlations of SELE genetic polymorphisms with risk of coronary heart disease and myocardial infarction: a meta-analysis

This meta-analysis of case–control studies was conducted to determine whether SELE genetic polymorphisms contribute to the pathogenesis of coronary heart disease (CHD) and myocardial infarction (MI). The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched for relevant articles published before November 1st, 2013 without any language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Twenty case–control studies met the inclusion criteria, with a total of 2,292 CHD patients, 901 MI patients and 3,233 healthy controls. Six common polymorphisms in the SELE gene were evaluated, including 554L/F, 98G/T, 128S/R, 2692G/A, 1901C/T, and 1856A/G. The results of our meta-analysis suggest that SELE genetic polymorphisms might be strongly correlated with an increased risk of CHD (allele model: OR 2.08, 95 % CI 1.67–2.58, P < 0.001; dominant model: OR 2.12, 95 % CI 1.68–2.68, P < 0.001; respectively), especially the SELE 554L/F, 98G/T and 128S/R polymorphisms. Furthermore, our findings indicated that SELE genetic polymorphisms were closely linked to the risk of CHD in Asians but not Caucasians. However, our findings reveal no positive correlations between SELE genetic polymorphisms and MI risk (allele model: OR 1.39, 95 % CI 1.00–1.94, P = 0.054; dominant model: OR 1.40, 95 % CI 0.96–2.04, P = 0.081; respectively). The current meta-analysis suggests that SELE genetic polymorphisms may contribute to an increased risk of CHD, especially the SELE 554L/F, 98G/T and 128S/R polymorphisms in Asians. However, SELE genetic polymorphisms may not be important determinants of susceptibility to MI.     

Additive Antiatherogenic Effects of CETP rs708272 on Serum LDL Subfraction Levels in Patients with CHD Under Statin Therapy

Recently, subfraction analysis of serum low density lipoprotein (LDL) is considered to be a better predictor of the risk of coronary heart disease (CHD) compared to the other lipid parameters. The aim of this study was to examine the effects of the HDL-associated Taq1B (rs708272) SNP of cholesterol ester transfer protein (CETP) gene on serum LDL subfractions in patients with CHD. Serum lipid levels were measured enzymatically and LDL subfraction analysis was carried out by the Lipoprint System (Quantimetrix, CA, USA). The CETP rs708272 SNP was studied in 66 healthy controls and 79 patients with CHD receiving statin therapy by the PCR–RFLP technique. The CHD patients had elevated antiatherogenic LDL-1 subfraction (p = 0.042), decreased atherogenic IDL-C subfraction (p = 0.023), and total IDL (p = 0.030) levels compared to the healthy controls. The CETP rs708272 Taq1B minor B2 allele was associated with increased levels of antiatherogenic LDL-1 (B2: 0.40 ± 0.20 vs. B1B1: 0.25 ± 0.08, p = 0.004) and large-LDL (LDL 1–2) subfractions in the CHD group (B2 allele: 0.68 ± 0.41 vs. B1B1: 0.42 ± 0.20; p < 0.05), while it was associated with reduced levels of the large-LDL subfraction in healthy subjects (B2 allele: 0.29 ± 0.14 vs. B1B1: 0.54 ± 0.24; p = 0.017). However, there was no statistically significant association between the CETP rs708272 SNP and small dense LDL subfraction (LDL 3–7) and lipoprotein levels (p > 0.05). Our findings have indicated that the CETP rs708272 SNP together with statin therapy may show a favorable effect on antiatherogenic LDL-1 and large-LDL subfractions in CHD patients with an atherogenic effect on large-LDL subfraction in healthy subjects. Based on these results, it can be concluded that the effects of the CETP variation on LDL subfraction could change in cardiometabolic events such as CHD and statin therapy.     

Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Association of two variants in the interleukin-6 receptor gene and premature coronary heart disease in a Chinese Han population

Two novel single nucleotide polymorphisms (SNPs; rs7529229 and rs2228145) in the interleukin-6 receptor (IL6R) gene have recently been associated with coronary heart disease (CHD) in a European population. We sought to replicate this finding and to investigate associations of these two SNPs with the severity and clinical phenotypes of premature CHD in a Chinese Han population. A total of 418 patients were studied, including 187 cases with coronary stenosis ≥50 % or acute myocardial infarction (males < 55 years and females < 65 years) and 231 controls without documented CHD. A ligase detection reaction was performed to detect rs7529229 and rs2228145. There were no differences between the controls and premature CHD groups in the frequencies for the three genotypes and alleles of rs7529229 and rs2228145 (all P > 0.05), nor did they differ between the two groups when grouped by gender (all P > 0.05). There were also no associations between these two SNPs and the severity of coronary lesions or clinical phenotypes of premature CHD (all P > 0.05). Our results do not support an association between rs7529229 or rs2228145 with premature CHD in the Chinese Han population. Further studies are warranted to elucidate the role of these two SNPs in the development of atherosclerosis and CHD.     

Changes in cardiac nucleotide metabolism in Huntington's disease

ABSTRACT

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.     

Reliability and Determinants of Self-Evaluation of Breathing Questionnaire (SEBQ) Score: A Symptoms-Based Measure of Dysfunctional Breathing

Dysfunctional breathing is characterised by an abnormal breathing pattern leading to respiratory symptoms. The 25-item Self Evaluation of Breathing Questionnaire (SEBQ) has been developed to measure breathing-related symptoms and their severity but lacks thorough evaluation. To determine reproducibility, internal consistency and predictors of SEBQ score, 180 participants completed an online SEBQ with additional demographic and lifestyle questions. Two weeks later, 155 of those repeated SEBQ. Test–retest correlation of the SEBQ was high [intraclass correlation coefficient (3, 1) = 0.89; 95 % CI 0.85–0.92]. There was no difference in SEBQ score between test and retest (15.1 (11.6) [mean (SD)] versus 14.7 (12.4); P = 0.4) and the score showed a typical error (standard error of measurement) of 4.0. Internal consistency was high (Cronbach’s α = 0.93), and a single factor structure for items was shown. Smoking status, reported respiratory disease, recent respiratory illness and female gender were positively-associated predictors of SEBQ score, and together explained 25.6 % of score variance (P ≤ 0.001). The SEBQ has high test–retest reproducibility and its score may be predicted by current smoking, chronic respiratory disease, recent respiratory illness and female gender, thus may be a useful clinical screening tool for dysfunctional breathing.