Steve W. Leung

Organic Solutes Rescue the Functional Defect in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of ΔF508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of ΔF508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested ΔF508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing ΔF508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-ΔF508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-ΔF508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the ΔF508 and wild type CFTR and inhibited ΔF508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of ΔF508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of ΔF508 CFTR.

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

MultiContrast Delayed Enhancement (MCODE) improves detection of subendocardial myocardial infarction by late gadolinium enhancement cardiovascular magnetic resonance: a clinical validation study.

BackgroundMyocardial infarction (MI) documented by late gadolinium enhancement (LGE) has clinical and prognostic importance, but its detection is sometimes compromised by poor contrast between blood and MI. MultiContrast Delayed Enhancement (MCODE) is a technique that helps discriminate subendocardial MI from blood pool by simultaneously providing a T2-weighted image with a PSIR (phase sensitive inversion recovery) LGE image. In this clinical validation study, our goal was to prospectively compare standard LGE imaging to MCODE in the detection of MI.MethodsImaging was performed on a 1.5 T scanner on patients referred for CMR including a LGE study. Prospective comparisons between MCODE and standard PSIR LGE imaging were done by targeted, repeat imaging of slice locations. Clinical data were used to determine MI status. Images at each of multiple time points were read on separate days and categorized as to whether or not MI was present and whether an infarction was transmural or subendocardial. The extent of infarction was scored on a sector-by-sector basis.ResultsSeventy-three patients were imaged with the specified protocol. The majority were referred for vasodilator perfusion exams and viability assessment (37 ischemia assessment, 12 acute MI, 10 chronic MI, 12 other diagnoses). Forty-six patients had a final diagnosis of MI (30 subendocardial and 16 transmural). MCODE had similar specificity compared to LGE at all time points but demonstrated better sensitivity compared to LGE performed early and immediately before and after the MCODE (p = 0.008 and 0.02 respectively). Conventional LGE only missed cases of subendocardial MI. Both LGE and MCODE identified all transmural MI. Based on clinical determination of MI, MCODE had three false positive MI’s; LGE had two false positive MI’s including two of the three MCODE false positives. On a per sector basis, MCODE identified more infarcted sectors compared to LGE performed immediately prior to MCODE (p < 0.001).ConclusionWhile both PSIR LGE and MCODE were good in identifying MI, MCODE demonstrated more subendocardial MI’s than LGE and identified a larger number of infarcted sectors. The simultaneous acquisition of T1 and T2-weighted images improved differentiation of blood pool from enhanced subendocardial MI.

Plasma levels of sphingosine 1-phosphate are strongly correlated with haematocrit, but variably restored by red blood cell transfusions

Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2=0.51, P<0.001) with no evidence for a plateau at Hct values as low as 19%. RBC transfusion in 23 anaemic patients with baseline mean Hct of 22.2±0.34% (value is the mean±S.D.) increased Hct to 28.3±0.6% at 72 h. Despite an Hct increase, RBC transfusion failed to elevate plasma S1P consistently. A trend towards an inverse correlation was observed between RBC storage duration and the post-transfusion increase in plasma S1P. After 30 days of storage, RBC S1P decreased to 19% of that observed in fresh (3–7-day-old) RBC segments. RBC membranes contain low levels of both S1P phosphatase and S1P lyase activities that may account for the decline in S1P levels with storage. Our results support a role for RBCs in buffering plasma S1P and identify a disturbance in the capacity after transfusion. Changes in S1P content may contribute to an RBC storage lesion. Further studies should investigate the clinical significance of alterations in circulating S1P levels and the potential value of enriching stored RBCs with S1P.

Expression of cyclic nucleotide-gated cation channels in airway epithelial cells.

Abstract. Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC50 of 154 μm, by 2′,4′-dichlorobenzamil with an IC50 of 50 μm and by amiloride with an IC50 of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC50 of 87 μm, by 2′4′-dichlorobenzamil with an IC50 of 38 μm and by amiloride with an IC50 of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC50 of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells.

Inside-out access: A new method of lead placement for patients with central venous occlusions

BACKGROUND Physicians will increasingly encounter patients who require rhythm management devices but have venous obstructions that prevent conventional access. Alternate access options, such as thoracotomy or transiliac approaches, exist but are associated with greater cost and morbidity. OBJECTIVE The purpose of this study is to describe a novel method of vascular access that allows prepectoral placement of conventional pacing and defibrillation leads in patients with complex central venous occlusions. METHODS Eight patients with central venous occlusions were referred for device implantation. Inside-out central venous access (IOCVA) was obtained via a percutaneous femoral approach. A catheter-dilator system was advanced via the right atrium to the most central point of venous occlusion. The occluded vein segment was punctured with a directionally guided needle, which was advanced along intravascular or extravascular tissue planes to the subclavian region. A solid wire needle was oriented toward the skin surface and advanced through the soft tissues until it exists from the body. The wire was used to pull rigid dilators through the occluded segment. Standard transvenous leads were implanted though the newly created channel. RESULTS All patients with total central venous occlusions (4 superior vena cava, 4 brachiocephalic and bilateral subclavian) had successful, prepectoral device implants (4 left-sided, 1 single-chamber, 4 dual-chamber, 3 biventricular). No procedure-related complications occurred. All patients had normal device function at follow-up of 485 ± 542 days. CONCLUSION IOCVA is an effective method of pacemaker and defibrillator implantation for patients with central venous occlusions. Further clinical evaluation of this novel method is needed.

Safety and tolerability of regadenoson CMR.

Aims Knowledge of adverse events associated with regadenoson perfusion cardiac magnetic resonance (CMR) and patient tolerability has implications for patient safety and staff training. We sought to assess the safety and tolerability of regadenoson stress CMR. Materials and methods A group of 728 consecutive patients (median age 58, 44% female) and 25 normal volunteers (median age 21, 24% female) were recruited from August 2009 to March 2012 using a prospective, cross-sectional study design. Subjects were stressed using fixed-dose regadenoson and imaged using a 1.5T MRI scanner. Symptoms and adverse events including death, myocardial infarction (MI), ventricular tachycardia (VT)/ventricular fibrillation (VF), hospitalization, arrhythmias, and haemodynamic stability were assessed. Results There were no occurrences of death, MI, VT/VF, high-grade atrioventricular block, or stress-induced atrial fibrillation. Notable adverse events included one case of bronchospasm and one case of heart failure exacerbation resulting in hospitalization. The most common symptoms in patients were dyspnoea (30%, n = 217), chest discomfort (27%, n = 200), and headache (15%, n = 111). There was minimal change between baseline and peak systolic and diastolic blood pressure in both patients and volunteers (P > 0.05). A blunted heart rate response to regadenoson was noted in patients with body mass index (BMI) ≥30 kg/m2 (P < 0.001), and diabetes (P = 0.001). Conclusions Regadenoson CMR is well tolerated and can be performed safely with few adverse events.

Mechanisms for overestimating acute myocardial infarct size with gadolinium-enhanced cardiovascular magnetic resonance imaging in humans: a quantitative and kinetic study †

Aims It remains controversial whether cardiovascular magnetic resonance imaging with gadolinium only enhances acutely infarcted or also salvaged myocardium. We hypothesized that enhancement of salvaged myocardium may be due to altered extracellular volume (ECV) and contrast kinetics compared with normal and infarcted myocardium. If so, these mechanisms could contribute to overestimation of acute myocardial infarction (AMI) size. Methods and results Imaging was performed at 1.5T ≤ 7 days after AMI with serial T1 mapping and volumetric early (5 min post-contrast) and late (20 min post-contrast) gadolinium enhancement imaging. Infarcts were classified as transmural (>75% transmural extent) or non-transmural. Patients with non-transmural infarctions (n = 15) had shorter duration of symptoms before reperfusion (P = 0.02), lower peak troponin (P = 0.008), and less microvascular obstruction (P < 0.001) than patients with transmural infarcts (n = 22). The size of enhancement at 5 min was greater than at 20 min (18.7 ± 12.7 vs. 12.1 ± 7.0%, P = 0.003) in non-transmural infarctions, but similar in transmural infarctions (23.0 ± 10.0 vs. 21.9 ± 9.9%, P = 0.21). ECV of salvaged myocardium was greater than normal (39.5 ± 5.8 vs. 24.1 ± 3.1%) but less than infarcted myocardium (50.5 ± 6.0%, both P < 0.001). In kinetic studies of non-transmural infarctions, salvaged and infarcted myocardium had similar T1 at 4 min but different T1 at 8–20 min post-contrast. Conclusion The extent of gadolinium enhancement in AMI is modulated by ECV and contrast kinetics. Image acquisition too early after contrast administration resulted in overestimation of infarct size in non-transmural infarctions due to enhancement of salvaged myocardium.

QRS duration predicts death and hospitalization among patients with atrial fibrillation irrespective of heart failure: evidence from the AFFIRM study

AIMS The association of QRS duration (QRSd) with morbidity and mortality is understudied in patients with atrial fibrillation (AF). We sought to assess any association of prolonged QRS with increased risk of death or hospitalization among patients with AF. METHODS AND RESULTS QRS duration was retrieved from the baseline electrocardiograms of patients enroled in the Atrial Fibrillation Follow-Up Investigation of Rhythm Management (AFFIRM) study and divided into three categories: <90, 90-119, ≥120 ms. Cox models were applied relating the hazards of mortality and hospitalizations to QRSd. Among 3804 patients with AF, 593 died and 2305 were hospitalized. Compared with those with QRS < 90 ms, patients with QRS ≥ 120 ms, had an increased mortality [hazard ratio (HR) 1.61, 95% confidence interval (CI): 1.29-2.03, P < 0.001] and hospitalizations (HR 1.14, 95% CI: 1.07-1.34, P = 0.043) over an average follow-up of 3.5 years. Importantly, for patients with QRS 90-119 ms, mortality and hospitalization were also increased (HR 1.31, P = 0.005 and 1.11, P = 0.026, respectively). In subgroup analysis based on heart failure (HF) status (previously documented or ejection fraction <40%), mortality was increased for QRS ≥ 120 ms patients with (HR 1.87, P < 0.001) and without HF (HR 1.63, P = 0.02). In the QRS 90-119 ms group, mortality was increased (HR 1.38, P = 0.03) for those with HF, but not significantly among those without HF (HR 1.23, P = 0.14). CONCLUSION Among patients with AF, QRSd ≥ 120 ms was associated with a substantially increased risk for mortality (all-cause, cardiovascular, and arrhythmic) and hospitalization. Interestingly, an increased mortality was also observed among those with QRS 90-119 ms and concomitant HF.

Smoking status and antithrombin therapy in patients with non-ST-segment elevation acute coronary syndrome.

BACKGROUND Smoking remains a major public health issue. We investigated the incidence of smoking and outcomes in high-risk patients with acute coronary syndromes. Differences in treatment effect of antithrombin therapies were also investigated. METHODS Using data from SYNERGY, patients were categorized by their self-reported smoking status. They were followed at 30 days and 6 months for death, nonfatal myocardial infarction (MI), revascularization procedures, stroke, and need for rehospitalization, and at 1 year for occurrences of death. RESULTS Overall, 9,971 patients were evaluated, of whom 2,404 (24%) were current smokers, 3,491 (35%) were former smokers, and 4076 (41%) had never smoked. Current smokers were younger (median age 61 years, interquartile range [IQR] 52-67) than former smokers (median age 69 years, IQR 63-75) and never smokers (median age 70 years, IQR 64-77) and had fewer additional coronary artery disease risk factors (hypertension, diabetes, hypercholesterolemia). The 30-day death/MI rate was similar for former versus never smokers (15% vs 13.6%, P = .079) and for current versus never smokers (14% vs 13.6%, P = .585). Adjusted odds ratios for 30-day death/MI in patients receiving enoxaparin compared with those receiving unfractionated heparin were 1.065 (95% CI 0.883-1.283, P = .51) in never smokers, 1.034 (95% CI 0.853-1.254, P = .733) in former smokers, and 0.742 (95% CI 0.582-0.948, P = .017) in current smokers. A significant interaction for treatment and smoking status was found at 30 days (P = .0215), but not at 6 months (P = .1381) or 1 year (P = .1054). One-year unadjusted mortality rates were higher for former versus never smokers (9.1% vs 6.7%, P = .0002) but were similar for current versus never smokers (6.5% vs 6.7%, P = .7226). On follow-up at 30 days, 62.3% (n =1397) of current smokers reported not smoking. CONCLUSIONS Smokers with non-ST-segment elevation acute coronary syndrome are generally younger and have fewer cardiac risk factors. A significant interaction of smoking and enoxaparin was seen at 30 days, but not sustained at 6 months and 1 year. More than 60% of smokers quit within 30 days of their cardiac event. There was little difference in outcomes from 30 days to 1 year for these smokers who quit versus those who did not.