ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.

BACKGROUND: ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance. METHODS: Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied. RESULTS: Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses. CONCLUSIONS: ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.

An antisense oligonucleotide efficiently suppresses splicing of an alternative exon in vascular smooth muscle in vivo.

Targeting alternative exons for therapeutic gain has been achieved in a few instances and potentially could be applied more broadly. The myosin phosphatase (MP) enzyme is a critical hub upon which signals converge to regulate vessel tone. Alternative exon 24 of myosin phosphatase regulatory subunit (Mypt1 E24) is an ideal target as toggling between the two isoforms sets smooth muscle sensitivity to vasodilators such as nitric oxide (NO). This study aimed to develop a gene-based therapy to suppress splicing of Mypt1 E24 thereby switching MP enzyme to the NO-responsive isoform. CRISPR/Cas9 constructs were effective at editing of Mypt1 E24 in vitro; however, targeting of vascular smooth muscle in vivo with AAV9 was inefficient. In contrast, an octo-guanidine conjugated antisense oligonucleotide targeting the 5' splice site of Mypt1 E24 was highly efficient in vivo. It reduced the percent splicing inclusion of Mypt1 E24 from 80% to 10% in mesenteric arteries. The maximal and half-maximal effects occurred at 12.5 and 6.25 mg/kg, respectively. The effect persisted for at least 1 mo without toxicity. This highly effective splice-blocking antisense oligonucleotide could be developed as a novel therapy to reverse vascular dysfunction common to diseases such as hypertension and heart failure.NEW & NOTEWORTHY Alternative exon usage is a major driver of phenotypic diversity in all cell types including smooth muscle. However, the functional significance of most of the hundreds of thousands of alternative exons has not been defined, nor in most cases even tested. If their importance to vascular function were known these alternative exons could represent novel therapeutic targets. Here, we present injection of Vivo-morpholino splice-blocking antisense oligonucleotides as a simple, efficient, and cost-effective method for suppression of alternative exon usage in vascular smooth muscle in vivo.

Depressed Proximal Glycolysis in Myocardium Of Human Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) accounts for >50% of all heart failure world-wide and remains a major unmet medical need. The most effective recently approved treatments were first developed for diabetes, suggesting metabolic defects are paramount. Myocardial metabolomics in human HFpEF has identified reduced fatty acid and branched chain amino acid catabolism, but the status of glycolysis is unknown. Here we performed targeted metabolomics and protein analysis of glycolytic pathway enzymes in myocardial biopsies of patients with HFpEF versus HF with reduced ejection fraction (HFrEF0 or non-failing controls. Glucose was increased in HFpEF myocardium, but immediate downstream glycolytic metabolites (glucose-6 phosphate, fructose 1,6 diphosphate), were more reduced in HFpEF than the other groups, as were their associated synthetic enzymes hexokinase and phosphofructokinase. Pyruvate was also reduced in HFpEF versus controls. These changes were either not present or substantially less so in HFrEF. Suppression of proximal glycolysis was also coupled to lower metabolites and proteins in the pentose phosphate pathway but was independent of diabetes or obesity. These findings support marked metabolic inflexibility in HFpEF and identifies very proximal blockade in glucose metabolism. Efforts to improve metabolic use of carbohydrates in HFpEF will likely need to target these proximal glycolytic enzymes.

Dominant vector changes during early wavebreak/spiral wave (Wiggers stage 1) in ventricular fibrillation: insights from the analysis of 100 electrophysiology studies.

PURPOSE: To describe electrocardiographic vector patterns during early VF transition (Wiggers stage 1). METHODS: In 100 electrophysiology studies with VF induction, the first 3 beats of VF were analyzed in lead I for left/right axis (LA/RA), V1 for left/right bundle (LB/RB), and aVF for superior/inferior axis (SA/IA). Correlation with demographic/clinical factors was performed using regression analyses and mixed effect modeling. RESULTS: VF initiated more likely with LA than RA (P < 0.001) and LB than RB (P = 0.04) suggesting original wavebreak in the right ventricle. The 3-dimensional morphology changed in 69% of VF during the first 3 beats, with predominant increase in RB, suggesting a transition of QRS-originating vector to septum/left ventricle. Conservation of morphology (31%) was favored by initial RB (P = 0.002) and LA morphology (P = 0.01). Initiation of VF with LA vs RA was more likely in African-Americans (P = 0.016) and increasing age (P = 0.032). Ischemic cardiomyopathy favored VF initiation with RB 6.7-fold (P = 0.025), possibly linking LV myocardial scar to initial VF wavebreak location. Male gender and ischemic cardiomyopathy prolonged time-to-loss of predominant vector by 119% (P = 0.002) and 71% (P = 0.017), respectively, suggesting more preserved anatomic/functional reentry. CONCLUSION: The predominant QRS vectors during early Wiggers stage 1 VF are not random and suggest an initial wavebreak more commonly in the right ventricle, followed by a transitional shift to the septum/left ventricle. Ethnicity, male gender, age, and co-morbidities result in directional preservation of initiating VF vectors possibly due to myocardial mass/fibrosis. Findings may allow new treatment/ablation approaches.

Prospective Observational Study of Bisphosphonate-Related Osteonecrosis of the Jaw in Multiple Myeloma: Microbiota Profiling and Cytokine Expression.

PURPOSE: Define incidence and risk factors of osteonecrosis of the jaw (ONJ) and explore oral microbial signatures and host immune response as reflected by cytokine changes in saliva and serum in multiple myeloma (MM) patients on bisphosphate (BP) therapy. PATIENTS AND METHODS: A single center observational prospective study of MM patients (n = 110) on >2 years of BP, none had ONJ at enrollment. Patients were followed every 3 months for 18 months with clinical/dental examination and serial measurements of inflammatory cytokines, bone turnover markers, and angiogenic growth factors. Oral microbiota was characterized by sequencing of 16S rRNA gene from saliva. RESULTS: Over the study period 14 patients (13%) developed BRONJ, at a median of 5.7 years (95% CI: 1.9-12.0) from MM diagnosis. Chronic periodontal disease was the main clinically observed risk factor. Oral microbial profiling revealed lower bacterial richness/diversity in BRONJ. Streptococcus intermedius, S. mutans, and S. perioris were abundant in controls; S. sonstellatus and S anginosus were prevalent in BRONJ. In the saliva, at baseline patients who developed BRONJ had higher levels of MIP-1ß; TNF-α and IL-6 compared to those without BRONJ, cytokine profile consistent with M-1 macrophage activation. In the serum, patients with BRONJ have significantly lower levels of TGF beta and VEGF over the study period. CONCLUSION: Periodontal disease associated with low microbial diversity and predominance of invasive species with a proinflammatory cytokine profile leading to tissue damage and alteration of immunity seems to be the main culprit in pathogenesis of BRONJ.

The Heart as a Site of Metastasis of Benign Metastasizing Leiomyoma: Case Report and Review of the Literature.

Uterine leiomyomas are the most common gynecological tumors in premenopausal women. While the lung is the most common extrauterine organ afflicted, benign metastasizing leiomyomas (BML) of the heart are rarities. We report an incidental finding of a cardiac mass in a 36-year-old woman who presented to the Emergency Department after a motor vehicle accident. CT scan of the chest revealed 2 well-circumscribed pulmonary nodules and a filling defect in the right ventricle. Echocardiogram showed a 4 cm mass attached to the right ventricular (RV) septum. The cardiac tumor was resected and showed benign histologic features. Immunohistochemical staining was positive for smooth muscle α-actin and desmin, as well as estrogen and progesterone receptors, consistent with the diagnosis of uterine leiomyoma.

Neutralizing Antibody-Mediated Response and Risk of BK Virus-Associated Nephropathy.

BK virus-associated nephropathy (BKVAN) causes renal allograft dysfunction. The current management of BKVAN relies on pre-emptive adaptation of immunosuppression according to viral load monitoring. However, this empiric strategy is not always successful. Therefore, pretransplant predictive markers are needed. In a prospective longitudinal study, we enrolled 168 kidney transplant recipients and 69 matched donors. To assess the value of BKV genotype-specific neutralizing antibody (NAb) titers as a predictive marker for BKV replication, we measured BKV DNA load and NAb titers at transplant and followed patients for 24 months. After transplant, 52 (31%) patients displayed BKV replication: 24 (46%) patients were viruric and 28 (54%) patients were viremic, including 13 with biopsy-confirmed BKVAN. At any time, patients with high NAb titers against the replicating strain had a lower risk of developing BKV viremia (hazard ratio [HR], 0.44; 95% confidence interval [95% CI], 0.26 to 0.73; P=0.002). Each log10 increase in NAb titer decreased the risk of developing viremia by 56%. Replicating strains were consistent with donor transmission in 95% of cases of early BKV replication. Genotype mismatch between recipients' neutralization profiles before transplant and their subsequently replicating strain significantly increased the risk of developing viremia (HR, 2.27; 95% CI, 1.06 to 4.88; P=0.04). A NAb titer against the donor's strain <4 log10 before transplant significantly associated with BKV replication after transplant (HR, 1.88; 95% CI, 1.06 to 3.45; P=0.03). BKV genotype-specific NAb titers may be a meaningful predictive marker that allows patient stratification by BKV disease risk before and after transplant.

Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato.

In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether "species-related" inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved.

Comparison between a Broad-Range Real-Time and a Broad-Range End-Point PCR Assays for the Detection of Bacterial 16S rRNA in Clinical Samples.

Broad range PCR targeting the 16S rRNA gene is widely used to test clinical samples for the presence of bacterial DNA. End-point 16S PCR is both time-consuming and at high risk of cross-contamination. Prior to the replacement of the 16S end-point PCR assay routinely used in our clinical laboratory by a new 16S real-time PCR assay, we aimed to compare the performances of both techniques for the direct diagnosis of bacterial infections in clinical samples. In this prospective study, 129 clinical samples were included for direct comparison of both techniques. The sensitivity of 16S real-time PCR assay (76%) was significantly higher than that of end-point 16S PCR assay (41%) (p<0.01). Specificities of both PCR assays did not differ significantly (p=0.43). The 16S real-time PCR assay yielded an etiological diagnosis in 19% of culture-negative samples. It constitutes a reliable and complementary diagnostic tool to the bacterial culture.

Sequence Variation in Amplification Target Genes and Standards Influences Interlaboratory Comparison of BK Virus DNA Load Measurement.

International guidelines define a BK virus (BKV) load of ≥4 log10 copies/ml as presumptive of BKV-associated nephropathy (BKVN) and a cutoff for therapeutic intervention. To investigate whether BKV DNA loads (BKVL) are comparable between laboratories, 2 panels of 15 and 8 clinical specimens (urine, whole blood, and plasma) harboring different BKV genotypes were distributed to 20 and 27 French hospital centers in 2013 and 2014, respectively. Although 68% of the reported results fell within the acceptable range of the expected result ±0.5 log10, the interlaboratory variation ranged from 1.32 to 5.55 log10. Polymorphisms specific to BKV genotypes II and IV, namely, the number and position of mutations in amplification target genes and/or deletion in standards, arose as major sources of interlaboratory disagreements. The diversity of DNA purification methods also contributed to the interlaboratory variability, in particular for urine samples. Our data strongly suggest that (i) commercial external quality controls for BKVL assessment should include all major BKV genotypes to allow a correct evaluation of BKV assays, and (ii) the BKV sequence of commercial standards should be provided to users to verify the absence of mismatches with the primers and probes of their BKV assays. Finally, the optimization of primer and probe design and standardization of DNA extraction methods may substantially decrease interlaboratory variability and allow interinstitutional studies to define a universal cutoff for presumptive BKVN and, ultimately, ensure adequate patient care.

Toward standardization of BK virus monitoring: evaluation of the BK virus R-gene kit for quantification of BK viral load in urine, whole-blood, and plasma specimens.

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel (r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log10 copies/ml), plasma (1.17 ± 0.63 log10 copies/ml), and WB (1.28 ± 0.37 log10 copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient (r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.

[Comparison of routine use of two chromogenic media ChromID CPS (bioMérieux) and UriSelect4 (Bio-Rad) for the detection of Escherichia coli and major uropathogenics in urine]. / Comparaison de l'utilisation en routine de deux milieux chromogènes ChromID CPS (bioMérieux) et UriSelect4 (Bio-Rad) pour la détection d'Escherichia coli et des principaux uropathogènes dans les urines.

Escherichia coli is the most common bacterial cause of urinary tract infections. Its rapid and specific identification in urine samples represents a major challenge within the rendering results and optimizing the management of the patient. We aimed to compare the sensitivity and specificity of two commercially available chromogenic media for E. coli: ChromID CPS (Biomérieux) and UriSelect4 (Bio(-)Rad), without carrying out further tests. 99 consecutive and non-redundant urine samples considered to be infected were simultaneously plated onto blood agar and the two chromogenic media. Colony color and bacterial growth quantification were compared 18 and 48 hours after incubation. Bacteria were identified with mass spectrometry. A complementary analysis on 80 bacterial strains known to pose potential identification problems was performed. 43 urines samples grew E. coli, and 42 of them were pink-colored on the two chromogenic mediums, as expected (sensibility=97.7%). Growth quantification was significantly greater on blood agar than on chromogenic media (p<0.001).We noted specificity issues at the complementary analysis with the UriSelect4 medium: Citrobacter freundii and some strains of Citrobacter brakii, Enterobacter cloacae and Hafnia alvei were pink-colored, and could be misidentified as E. coli. ChromID CPS medium did not show such misidentification. In conclusion, the agar ChromID CPS proved to be greater than the UriSelect4 agar in our work in terms of specificity of direct identification of E. Coli, without the use of additional test.