Proteomic analysis identifies subgroups of patients with active systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a clinically and biologically heterogenous autoimmune disease. We aimed to investigate the plasma proteome of patients with active SLE to identify novel subgroups, or endotypes, of patients. METHOD: Plasma was collected from patients with active SLE who were enrolled in the British Isles Lupus Assessment Group Biologics Registry (BILAG-BR). The plasma proteome was analysed using a data-independent acquisition method, Sequential Window Acquisition of All theoretical mass spectra mass spectrometry (SWATH-MS). Unsupervised, data-driven clustering algorithms were used to delineate groups of patients with a shared proteomic profile. RESULTS: In 223 patients, six clusters were identified based on quantification of 581 proteins. Between the clusters, there were significant differences in age (p = 0.012) and ethnicity (p = 0.003). There was increased musculoskeletal disease activity in cluster 1 (C1), 19/27 (70.4%) (p = 0.002) and renal activity in cluster 6 (C6) 15/24 (62.5%) (p = 0.051). Anti-SSa/Ro was the only autoantibody that significantly differed between clusters (p = 0.017). C1 was associated with p21-activated kinases (PAK) and Phospholipase C (PLC) signalling. Within C1 there were two sub-clusters (C1A and C1B) defined by 49 proteins related to cytoskeletal protein binding. C2 and C6 demonstrated opposite Rho family GTPase and Rho GDI signalling. Three proteins (MZB1, SND1 and AGL) identified in C6 increased the classification of active renal disease although this did not reach statistical significance (p = 0.0617). CONCLUSIONS: Unsupervised proteomic analysis identifies clusters of patients with active SLE, that are associated with clinical and serological features, which may facilitate biomarker discovery. The observed proteomic heterogeneity further supports the need for a personalised approach to treatment in SLE.

Isoform a4 of the vacuolar ATPase a subunit promotes 4T1-12B breast cancer cell-dependent tumor growth and metastasis in vivo.

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that governs the pH of various intracellular compartments and also functions at the plasma membrane in certain cell types, including cancer cells. Membrane targeting of the V-ATPase is controlled by isoforms of subunit a, and we have previously shown that isoforms a3 and a4 are important for the migration and invasion of several breast cancer cell lines in vitro. Using CRISPR-mediated genome editing to selectively disrupt each of the four a subunit isoforms, we also recently showed that a4 is critical to plasma membrane V-ATPase localization, as well as in vitro migration and invasion of 4T1-12B murine breast cancer cells. We now report that a4 is important for the growth of 4T1-12B tumors in vivo. We found that BALB/c mice bearing a4-/- 4T1-12B allografts had significantly smaller tumors than mice in the control group. In addition, we determined that a4-/- allografts showed dramatically reduced metastases to the lung and reduced luminescence intensity of metastases to bone relative to the control group. Taken together, these results suggest that the a4 isoform of the V-ATPase represents a novel potential therapeutic target to limit breast cancer growth and metastasis.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Mechanisms underlying neonate-specific metabolic effects of volatile anesthetics.

Volatile anesthetics (VAs) are widely used in medicine, but the mechanisms underlying their effects remain ill-defined. Though routine anesthesia is safe in healthy individuals, instances of sensitivity are well documented, and there has been significant concern regarding the impact of VAs on neonatal brain development. Evidence indicates that VAs have multiple targets, with anesthetic and non-anesthetic effects mediated by neuroreceptors, ion channels, and the mitochondrial electron transport chain. Here, we characterize an unexpected metabolic effect of VAs in neonatal mice. Neonatal blood ß-hydroxybutarate (ß-HB) is rapidly depleted by VAs at concentrations well below those necessary for anesthesia. ß-HB in adults, including animals in dietary ketosis, is unaffected. Depletion of ß-HB is mediated by citrate accumulation, malonyl-CoA production by acetyl-CoA carboxylase, and inhibition of fatty acid oxidation. Adults show similar significant changes to citrate and malonyl-CoA, but are insensitive to malonyl-CoA, displaying reduced metabolic flexibility compared to younger animals.

Vasodilators for primary Raynaud's phenomenon.

BACKGROUND: Numerous agents have been suggested for the symptomatic treatment of primary Raynaud's phenomenon. Apart from calcium channel blockers, which are considered to be the drugs of choice, evidence of the effects of alternative pharmacological treatments is limited. This is an update of a review first published in 2008. OBJECTIVES: To assess the effects of drugs with vasodilator effects on primary Raynaud's phenomenon as determined by frequency, severity, and duration of vasospastic attacks; quality of life; adverse events; and Raynauds Condition Score. SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Cochrane Vascular Specialised Register, CENTRAL, MEDLINE, Embase, and CINAHL databases, and the World Health Organization International Clinical Trials Registry Platform and the ClinicalTrials.gov trial register to November 16, 2020. SELECTION CRITERIA: We included randomized controlled trials evaluating effects of oral, intravenous, and topical formulations of any drug with vasodilator effects on subjective symptoms, severity scores, and radiological outcomes in primary Raynaud's phenomenon. Treatment with calcium channel blockers was not assessed in this review, nor were these agents compared. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies for inclusion, assessed studies using the Cochrane "Risk of bias" tool, and extracted study data. Outcomes of interest included frequency, severity, and duration of attacks; quality of life (QoL); adverse events (AEs); and the Raynaud Condition Score (RCS). We assessed the certainty of the evidence using GRADE. MAIN RESULTS: We identified seven new studies for this update. In total, we included 15 studies involving 635 participants. These studies compared different vasodilators to placebo. Individual studies used different methods and measures to report different outcomes. Angiotensin-converting enzyme (ACE) inhibitors Combining data from three studies revealed a possible small increase in the frequency of attacks per week after treatment (captopril or enalapril) compared to placebo (mean difference [MD] 0.79, 95% confidence interval [CI] 0.43 to 1.17; low-certainty evidence). There was no evidence of a difference between groups in severity of attacks (MD -0.17, 95% CI -4.66 to 4.31; 34 participants, 2 studies; low-certainty evidence); duration of attacks (MD 0.54, 95% CI -2.42 to 1.34; 14 participants, 1 study; low-certainty evidence); or AEs (risk ratio [RR] 1.35, 95% CI 0.67 to 2.73; 46 participants, 3 studies; low-certainty evidence). QoL and RCS were not reported. Alpha blockers Two studies used alpha blockers (buflomedil or moxisylyte). We were unable to combine data due to the way results were presented. Buflomedil probably reduced the frequency of attacks compared to placebo (MD -8.82, 95% CI -11.04 to -6.60; 31 participants, 1 study; moderate-certainty evidence) and may improve severity scores (MD -0.41, 95% CI -0.62 to -0.30; moderate-certainty evidence). With moxisylyte, investigators reported fewer attacks (P < 0.02), less severe symptoms (P < 0.01), and shorter duration of attacks, but the clinical relevance of these results is unclear. No evidence of a difference in AEs between buflomedil and placebo groups was noted (RR 1.41, 95% CI 0.27 to 7.28; 31 participants, 1 study; moderate-certainty evidence). More AEs were observed in participants in the moxisylyte group than in the placebo group. Prostaglandin/prostacyclin analogues One study compared beraprost versus placebo. There was no evidence of benefit for frequency (MD 2.00, 95% CI -0.35 to 4.35; 118 participants, low-certainty evidence) or severity (MD -0.06, 95% CI -0.34 to 0.22; 118 participants, low-certainty evidence) of attacks. Overall, more AEs were noted in the beraprost group (RR 1.59, 95% CI 1.05 to 2.42; 125 participants; low-certainty evidence). This study did not report on duration of attacks, QoL, or RCS. Thromboxane synthase inhibitors One study compared a thromboxane synthase inhibitor (dazoxiben) versus placebo. There was no evidence of benefit for frequency of attacks (MD 0.8, 95% CI -1.81 to 3.41; 6 participants; very low-certainty evidence). Adverse events were not reported in subgroup analyses of participants with primary Raynaud's phenomenon, and the study did not report on duration of attacks, severity of symptoms, QoL, or RCS. Selective serotonin reuptake inhibitors One study compared ketanserin with placebo. There may be a slight reduction in the number of attacks per week with ketanserin compared to placebo (MD -14.0, 95% CI -27.72 to -0.28; 41 participants; very low-certainty evidence) and reduced severity score (MD -133.00, 95% CI -162.40 to -103.60; 41 participants; very low-certainty evidence). There was no evidence that ketanserin reduced the duration of attacks (MD -4.00, 95% CI -14.82 to 6.82; 41 participants; very low-certainty evidence), or that AEs were increased in either group (RR 1.54, 95% CI 0.89 to 2.65; 41 participants; very low-certainty evidence). This study did not report on QoL or RCS. Nitrate/nitrate derivatives Four studies compared topical treatments of nitroglycerin or glyceryl trinitrate versus placebo, each reporting on limited outcomes. Meta-analysis demonstrated no evidence of effect on frequency of attacks per week (MD -1.57, 95% CI -4.31 to 1.17; 86 participants, 2 studies; very low-certainty evidence). We were unable to pool any data for the remaining outcomes. Phosphodiesterase inhibitors Three studies compared phosphodiesterase inhibitors (vardenafil, cilostazol or PF-00489791) to an equivalent placebo. Results showed no evidence of a difference in frequency of attacks (standardized MD [SMD] -0.05, 95% CI -6.71 to 6.61; 111 participants, 2 studies; low-certainty evidence), severity of attacks (MD -0.03, 95% CI -1.04 to 0.97; 111 participants, 2 studies; very low-certainty evidence), duration of attacks (MD -1.60, 95% CI -7.51 to 4.31; 73 participants, 1 study; low-certainty evidence), or RCS (SMD -0.8, 95% CI -1.74 to 0.13; 79 participants, 2 studies; low-certainty evidence). Study authors reported that 35% of participants on cilostazol complained of headaches, which were not reported in the placebo group. PF-00489791 caused 34 of 54 participants to experience AEs versus 43 of 102 participants receiving placebo (RR 1.49). Headache was most common, affecting 14 participants (PF-00489791) versus nine participants (placebo). AUTHORS' CONCLUSIONS: The included studies investigated several different vasodilators (topical and oral) for treatment of primary Raynaud's phenomenon. Small sample sizes, limited data, and variability in outcome reporting yielded evidence of very low to moderate certainty. Evidence is insufficient to support the use of vasodilators and suggests that vasodilator use may even worsen disease.

Low-Level Mouse DNA in Conditioned Medium Generates False Positive Cross-Species Contamination Results in Human Organoid Cultures.

Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.

Development of a Personalized mHealth Program for Cancer Symptom and Complication Management.

Based on care need assessment of colorectal and bladder cancer (CBC) patients with newly created ostomies (stomas) and caregivers, and with the input of the Wound, Ostomy, and Continence Nurses (WOCNs), we have developed an mHealth complication and symptom monitoring and self-management program, titled PRISMS.

Cardiomyocyte d-dopachrome tautomerase protects against heart failure.

The mechanisms contributing to heart failure remain incompletely understood. d-dopachrome tautomerase (DDT) is a member of the macrophage migration inhibitory factor family of cytokines and is highly expressed in cardiomyocytes. This study examined the role of cardiomyocyte DDT in the setting of heart failure. Patients with advanced heart failure undergoing transplantation demonstrated decreased cardiac DDT expression. To understand the effect of loss of cardiac DDT in experimental heart failure, cardiomyocyte-specific DDT-KO (DDT-cKO) and littermate control mice underwent surgical transverse aortic constriction (TAC) to induce cardiac pressure overload. DDT-cKO mice developed more rapid cardiac contractile dysfunction, greater cardiac dilatation, and pulmonary edema after TAC. Cardiomyocytes from DDT-cKO mice after TAC had impaired contractility, calcium transients, and reduced expression of the sarcoplasmic reticulum calcium ATPase. The DDT-cKO hearts also exhibited diminished angiogenesis with reduced capillary density and lower VEGF-A expression after TAC. In pharmacological studies, recombinant DDT (rDDT) activated endothelial cell ERK1/2 and Akt signaling and had proangiogenic effects in vitro. The DDT-cKO hearts also demonstrated more interstitial fibrosis with enhanced collagen and connective tissue growth factor expression after TAC. In cardiac fibroblasts, rDDT had an antifibrotic action by inhibiting TGF-ß-induced Smad-2 activation. Thus, endogenous cardiomyocyte DDT has pleiotropic actions that are protective against heart failure.

Macrophage migration inhibitory factor mediates metabolic dysfunction induced by atypical antipsychotic therapy.

Atypical antipsychotics are highly effective antischizophrenic medications but their clinical utility is limited by adverse metabolic sequelae. We investigated whether upregulation of macrophage migration inhibitory factor (MIF) underlies the insulin resistance that develops during treatment with the most commonly prescribed atypical antipsychotic, olanzapine. Olanzapine monotherapy increased BMI and circulating insulin, triglyceride, and MIF concentrations in drug-naive schizophrenic patients with normal MIF expression, but not in genotypic low MIF expressers. Olanzapine administration to mice increased their food intake and hypothalamic MIF expression, which led to activation of the appetite-related AMP-activated protein kinase and Agouti-related protein pathway. Olanzapine also upregulated MIF expression in adipose tissue, which reduced lipolysis and increased lipogenic pathways. Increased plasma lipid concentrations were associated with abnormal fat deposition in liver and skeletal muscle, which are important determinants of insulin resistance. Global MIF-gene deletion protected mice from olanzapine-induced insulin resistance, as did intracerebroventricular injection of neutralizing anti-MIF antibody, supporting the role of increased hypothalamic MIF expression in metabolic dysfunction. These findings uphold the potential pharmacogenomic value of MIF genotype determination and suggest that MIF may be a tractable target for reducing the metabolic side effects of atypical antipsychotic therapy.

Differential regulation of tefluthrin and telmisartan on the gating charges of INa activation and inactivation as well as on resurgent and persistent INa in a pituitary cell line (GH3).

Voltage-gated Na+ currents (INa), known to contain many components (e.g., transient, resurgent and persistent INa) with unique gating properties, are involved in the generation and propagation of action potentials in excitable cells. In this study, how tefluthrin (Tef), a synthetic pyrethoid, and telmisartan (TEL), blocker of angiotensin II receptors can perturb those components of INa was investigated. The presence of either Tef or TEL increased the values of the gating charges of INa involved in the activation (za) and inactivation (zi). Tef also increased the amplitude of resurgent INa (INa(R)) or persistent INa (INa(P)) in a pituitary cell line (GH3), while TEL produced minimal effects on them. Subsequent addition of either ranolazine (a blocker of late INa) or d-limonene (a monoterpene), could reverse the changes by TEL or Tef on za or zi. In SCN5A-expressing HEK293T cells, addition of Tef or TEL also increased the peak amplitude and the inactivation time constant of INa which was accompanied by the increased za value of INa. Taken together, the results indicated that Tef- or TEL-mediated changes in the gating kinetics of INa are linked to their actions on functional activity of neurons, neuroendocrine or endocrine cells.

Racial and Ethnic Disparities in Oncotype DX Test Receipt in a Statewide Population-Based Study.

Background: Racial disparities have been reported in breast cancer care, yet little is known about disparities in access to gene expression profiling (GEP) tests. Given the impact of GEP test results, such as those of Oncotype DX (ODx), on treatment decision-making for hormone receptor-positive (HR+) breast cancer, it is particularly important to assess disparities in its use. Methods: We conducted a retrospective population-based study of 8,784 patients diagnosed with breast cancer in Connecticut during 2011 through 2013. We assessed the association between race, ethnicity, and ODx receipt among women with HR+ breast cancer for whom NCCN does and does not recommend ODx testing, using bivariate and multivariate logistic analyses. Results: We identified 5,294 women who met study inclusion criteria: 83.8% were white, 6.3% black, and 7.4% Hispanic. Overall, 50.9% (n=4,131) of women in the guideline-recommended group received ODx testing compared with 18.5% (n=1,163) in the nonrecommended group. More white women received the ODx test compared with black and Hispanic women in the recommended and nonrecommended groups (51.4% vs 44.6% and 47.7%; and 21.2% vs 9.0% and 9.7%, respectively). After adjusting for tumor and clinical characteristics, we observed significantly lower ODx use among black (odds ratio [OR], 0.64; 95% CI, 0.47-0.88) and Hispanic women (OR, 0.59; 95% CI, 0.45-0.77) compared with white women in the recommended group and in the guideline-discordant group (blacks: OR, 0.39; 95% CI, 0.20-0.78, and Hispanics: OR, 0.44; 95% CI, 0.23-0.85). Conclusions: In this population-based study, we identified racial disparities in ODx testing. Disparities in access to innovative cancer care technologies may further exacerbate existing disparities in breast cancer outcomes.

Spontaneous hepatic artery dissection-a rare presentation of fibromuscular dysplasia.

Fibromuscular dysplasia (FMD) is a rare condition that causes structural compromise of the blood vessel presenting either as an incidental radiological finding, dissection or stenosis usually of the renal or craniocervical arteries. Seldom, patients present with spontaneous dissection in visceral arteries and there are few reports of hepatic involvement. This report outlines the case of a 43-year-old female who presented with severe right upper quadrant pain with a subsequent diagnosis of FMD manifesting as spontaneous hepatic artery dissection. The patient was treated with conservative antiplatelet therapy and regular radiographic follow-up, decided by the treating team as no clear guidelines exist for management of this particular presentation of FMD. Surgical management is not currently recommended to this patient due to the risk of further dissection, but may be considered if there is severe haemodynamic compromise or refractory pain.

Surveillance Practice Patterns after Curative Intent Therapy for Stage I Non-Small-Cell Lung Cancer in the Medicare Population.

OBJECTIVES: Recurrence after treatment for non-small cell lung cancer (NSCLC) is common, and routine imaging surveillance is recommended by evidence-based guidelines. Little is known about surveillance patterns after curative intent therapy for early stage NSCLC. We sought to understand recent practice patterns for surveillance of stage I NSCLC in the first two years after curative intent therapy in the Medicare population. MATERIALS AND METHODS: Using the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database we selected patients diagnosed with stage I NSCLC between 1998 and 2008. We studied adherence to surveillance guidelines based on specialty society recommendations for chest radiography and computed tomography (CT) scanning. We also tracked the use of Positron Emission Tomography (PET) scans, which are not recommended for surveillance. We calculated the percent of patients who received guideline-adherent surveillance imaging and used logistic regression to determine associations between patient and provider factors and guideline adherence. RESULTS: Overall, 61.4% of patients received guideline-adherent surveillance during the initial 2 years after treatment. Use of CT scans in the first year after treatment increased from 47.4% in 1998-78.5% in 2008, and PET use increased from 5.8% to 28.9%. Adherence with surveillance imaging was associated with younger age, higher income, more comorbidities, access to primary care, and receipt of SBRT as the primary treatment. CONCLUSIONS: Adherence to specialty society guidelines for surveillance after treatment for stage I NSCLC was poor in this population of Medicare beneficiaries, with less than two-thirds of patients receiving recommended imaging, and almost 30% receiving non-recommended PET scans.

AMPK is critical for mitochondrial function during reperfusion after myocardial ischemia.

AMP-activated kinase (AMPK) is a stress responsive kinase that regulates cellular metabolism and protects against cardiomyocyte injury during ischemia-reperfusion (IR). Mitochondria play an important role in cell survival, but the specific actions of activated AMPK in maintaining mitochondrial integrity and function during reperfusion are unknown. Thus, we assessed the consequences of AMPK inactivation on heart mitochondrial function during reperfusion. Mouse hearts expressing wild type (WT) or kinase-dead (KD) AMPK were studied. Mitochondria isolated from KD hearts during reperfusion had intact membrane integrity, but demonstrated reduced oxidative capacity, increased hydrogen peroxide production and decreased resistance to mitochondrial permeability transition pore opening compared to WT. KD hearts showed increased activation of the mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) and greater necrosis during reperfusion after coronary occlusion. Transgenic expression of mitochondrial catalase (MCAT) prevented the excessive cardiac JNK activation and attenuated the increased myocardial necrosis observed during reperfusion in KD mice. Inhibition of JNK increased the resistance of KD hearts to mPTP opening, contractile dysfunction and necrosis during IR. Thus, intrinsic activation of AMPK is critical to prevent excess mitochondrial reactive oxygen production and consequent JNK signaling during reperfusion, thereby protecting against mPTP opening, irreversible mitochondrial damage and myocardial injury.

Association of 21-gene recurrence score assay and adjuvant chemotherapy use in the medicare population, 2008-2011.

OBJECTIVES: The 21-gene recurrence score (RS) assay helps guide adjuvant chemotherapy use for patients with breast cancer, and is predicted to reduce overall chemotherapy use. Little is known about recent patterns of testing in the Medicare program and the impact of testing on chemotherapy use as a function of patient age. MATERIALS AND METHODS: We conducted a national claims-based study of Medicare beneficiaries age ≥ 66 years. We assessed trends in assay use (using multivariable regression), adjuvant chemotherapy use, and associated expenditures, for all patients and for two age strata: age 66-74 years and 75-94 years. Geographic variations in assay adoption and regional-level correlation between assay and chemotherapy use were measured. RESULTS: We identified 132,222 women who underwent breast surgery from 2008-2011. Assay use increased from 9.0% to 17.2% from 2008-2011 (p<.001), but chemotherapy use remained stable at 12.5% (p=.49). In younger patients, assay use increased from 14.3% to 23.7% (p<.001), while chemotherapy use decreased from 18.2% to 16.2% (p<.001). In older patients, assay use increased from 4.1% to 9.9% (p<.001), while chemotherapy use remained stable at 6.8% (p=.67). Mean per-beneficiary expenditures for testing and chemotherapy increased from $2030 to $2430 (p<.001). Regions with increased assay adoption were not more likely to reduce chemotherapy. CONCLUSION: Despite increased RS testing for both younger and older Medicare patients, there has only been a modest decrease in chemotherapy use for younger patients and no change for older patients, resulting in an overall increase in costs associated with gene expression profiling.

LKB1 deletion causes early changes in atrial channel expression and electrophysiology prior to atrial fibrillation.

AIMS: Liver kinase B1 (LKB1) is a protein kinase that activates the metabolic regulator AMP-activated protein kinase (AMPK) and other related kinases. Deletion of LKB1 in mice leads to cardiomyopathy and atrial fibrillation (AF). However, the specific role of the LKB1 pathway in early atrial biology remains unknown. Thus, we investigated whether LKB1 deletion altered atrial channel expression and electrophysiological function in a cardiomyocyte-specific knockout mouse model. METHODS AND RESULTS: We performed a systematic comparison of αMHC-Cre LKB1(fl/fl) and littermate LKB1(fl/fl) male mice. This included analysis of gene expression, histology, and echocardiography, as well as cellular and tissue-level electrophysiology using patch-clamp recordings in vitro, optical mapping ex vivo, and ECG recordings in vivo. At postnatal day 1, atrial depolarization was prolonged, and Nav1.5 and Cx40 expression were markedly down-regulated in MHC-Cre LKB1(fl/fl) mice. Inward sodium current density was significantly decreased in MHC-Cre LKB1(fl/fl) neonatal atrial myocytes. Subsequently, additional alterations in atrial channel expression, atrial fibrosis, and spontaneous onset of AF developed by 2 weeks of age. In adult mice, abnormalities of interatrial conduction and bi-atrial electrical coupling were observed, likely promoting the perpetuation of AF. Mice with AMPK-inactivated hearts demonstrated modest overlap in channel expression with MHC-Cre LKB1(fl/fl) hearts, but retained normal structure, electrophysiological function and contractility. CONCLUSIONS: Deletion of LKB1 causes early defects in atrial channel expression, action potential generation and conduction, which precede widespread atrial remodelling, fibrosis and AF. LKB1 is critical for normal atrial growth and electrophysiological function.

Analysis of autophagosome formation using lentiviral biosensors for live fluorescent cellular imaging.

Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process. EMD Millipore has developed technologies where prepackaged, ready-to-use, high-titer lentiviral particles, "lentiviral biosensors," encoding GFP- or RFP-tagged proteins provide a convenient and robust solution for fluorescent imaging of cells undergoing autophagy. Compared to nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are nondisruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes.

Can three-dimensional echocardiography accurately predict complexity of mitral valve repair?

OBJECTIVE: Feasibility of mitral repair is a key factor in the decision to operate for mitral regurgitation. Repair feasibility is highly dependent on surgical experience and repair complexity. We sought an objective means of predicting complexity of repair using three-dimensional (3D) transoesophageal echocardiography. METHODS: In a cohort of 786 patients who underwent mitral valve surgery between 2007 and 2010, 3D transoesophageal echocardiography was performed in 66 patients with mitral regurgitation prior to the institution of cardiopulmonary bypass. The surgeon reviewed the 2D echocardiographic images for all patients pre-operatively, but did not view the 3D echocardiographic quantitative data or volumetric analysis until after surgery. Repairs involving no or a single-segment leaflet resection, sliding-plasty, cleft closure, chordal or commissural repair techniques were classed as standard repairs. Complex repairs were defined as those involving bileaflet repair techniques, requiring multiple resections or patch augmentation. Disease aetiology included Barlow's disease (n = 18), fibroelastic deficiency (n = 22), ischaemic (n = 5), endocarditis (n = 5), rheumatic (n = 2) and dilated cardiomyopathy (n = 2). RESULTS: No patient required mitral replacement or had more than mild mitral regurgitation on pre-discharge echocardiography. Anterior and posterior leaflet areas, annular circumference, anterior and posterior leaflet angles, prolapse and tenting heights and volumes were most strongly predictive of repair complexity. As 21 of the 22 patients with bileaflet pathology and multisegment prolapse were complex repairs, we sought to develop a model predicting repair complexity in the remaining patients. The most predictive model with a c-statistic of 0.91 included three predictors: multisegment pathology, prolapsing height and posterior leaflet angle. After bootstrap validation, the revised c-statistic was 0.88. CONCLUSIONS: 3D transoesophageal echocardiography provides an objective means of predicting mitral repair complexity in mitral regurgitation due to a range of aetiology.

FLICE-like inhibitory protein blocks transforming growth factor beta 1-induced caspase activation and apoptosis in prostate epithelial cells.

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.