Evaluation of proteomic biomarkers associated with circulating microparticles as an effective means to stratify the risk of spontaneous preterm birth.

BACKGROUND: The analysis of circulating microparticles in pregnancy is of revolutionary potential because it represents an in vivo biopsy of active gestational tissues. OBJECTIVE: We hypothesized that circulating microparticle signaling will differ in pregnancies that experience spontaneous preterm birth from those delivering at term and that these differences will be evident many weeks in advance of clinical presentation. STUDY DESIGN: Utilizing plasma specimens obtained between 10 and 12 weeks' gestation as part of a prospectively collected birth cohort in which pregnancy outcomes are independently validated by 2 board-certified maternal-fetal medicine physicians, 25 singleton cases of spontaneous preterm birth ≤ 34 weeks were matched by maternal age, race, and gestational age of sampling (±2 weeks) with 50 uncomplicated term deliveries. Circulating microparticles from these first-trimester specimens were isolated and analyzed by multiple reaction monitoring mass spectrometry for potential protein biomarkers following previous studies. Markers with robust univariate performance in correlating spontaneous preterm birth were further evaluated for their biological relevance via a combined functional profiling/pathway analysis and for multivariate performance. RESULTS: Among the 132 proteins evaluated, 62 demonstrated robust power of detecting spontaneous preterm birth in a bootstrap receiver-operating characteristic curve analysis at a false discovery rate of < 20% estimated via label permutation. Differential dependency network analysis identified spontaneous preterm birth-associated coexpression patterns linked to biological processes of inflammation, wound healing, and the coagulation cascade. Linear modeling of spontaneous preterm birth using a multiplex of the candidate biomarkers with a fixed sensitivity of 80% exhibited a specificity of 83% with median area under the curve of 0.89. These results indicate a strong potential of multivariate model development for informative risk stratification. CONCLUSION: This project has identified functional proteomic factors with associated biological processes that are already unique in their expression profiles at 10-12 weeks among women who go on to deliver spontaneously ≤ 34 weeks. These changes, with further validation, will allow the stratification of patients at risk of spontaneous preterm birth before clinical presentation.

Circulating serum-derived microparticles provide novel proteomic biomarkers of spontaneous preterm birth.

OBJECTIVE: The purpose of this study was to determine whether the proteomic biosignature of circulating microparticles in maternal serum obtained in the second trimester could identify pregnancies that result in spontaneous preterm birth (SPTB). STUDY DESIGN: Microparticles were isolated from blinded biorepository-sourced serum samples from 48 pregnant women at 15 to 17 weeks of gestation. Microparticle proteins were extracted and analyzed using label-free liquid chromatography/mass spectrometry. Peptide features were analyzed to assess the association of specific protein patterns with subjects delivering at term (≥ 37 weeks gestation; n = 24) and those experiencing SPTB (≤ 34 weeks gestation; n = 24). RESULTS: We found 99 proteins that had statistically significant differences in signal intensity between term and SPTB women in both first (n = 26) and second (n = 22) singleton gestation pregnancy cohorts. Additional evaluation identified 18 biomarkers that met criteria for further priority evaluation (12 preterm, 6 term). Pathway analysis showed that differentiating SPTB biomarker proteins were predominantly associated with inflammation and cell injury, while differentiating term biomarkers were associated with cell growth and hematological parameters. CONCLUSION: This study shows for the first time that the proteomic content of serum microparticles isolated in the second trimester can identify with a high degree of accuracy pregnancies that result in SPTB.

The role of late I and antiarrhythmic drugs in EAD formation and termination in Purkinje fibers.

Multiple components of cardiac Na current play a role in determining electrical excitation in the heart. Recently, the role of nonequilibrium components in controlling cardiac action potential plateau duration, and their importance in regulating the occurrence of afterdepolarizations and arrhythmias have garnered more attention. In particular, late Na current (late I(Na)) has been shown to be important in LQT2 and LQT3 arrhythmias. Class III agents like dofetilide, clofilium, and sotalol, which can all cause a drug-induced form of LQT2, significantly lengthen action potential duration at 50% and 90% repolarization in isolated rabbit Purkinje fibers, and can initiate the formation of early afterdepolarizations, and extra beats. These actions can lead to the development of a serious ventricular tachycardia, torsades de pointes, in animal models and patients. However, pretreatment with agents that block late I(Na), like lidocaine, mexiletine, and RSD1235, a novel mixed ion channel blocker for the rapid pharmacologic conversion of atrial fibrillation, significantly attenuates the prolonging effects of Class III agents or those induced by ATX-II, a specific toxin that delays Na channel inactivation and amplifies late I(Na) greatly, mimicking LQT3. The Na channel block caused by lidocaine and RSD1235 can be through the open or inactivated states of the channel, but both equivalently inhibit a late component of Na current (I(Na)), recorded at 22 degrees C using whole-cell patch clamp of Nav 1.5 expressed in HEK cells. These protective actions of lidocaine, mexiletine, and RSD1235 may result, at least in part, from their ability to inhibit late I(Na) during action potential repolarization, and inhibition of the inward currents contributing to EAD and arrhythmia formation.

RSD1235 blocks late INa and suppresses early afterdepolarizations and torsades de pointes induced by class III agents.

OBJECTIVE: RSD1235 is a novel antiarrhythmic drug with atria-selective electrophysiological actions on Na(+) and K(+) currents. The mechanism for its protection of ventricular repolarization was assessed by its action on Purkinje fibers, and by block of late sodium current active during repolarization. Further, RSD1235's ability to reverse the pro-arrhythmic actions of the class III agents dofetilide and clofilium was assessed in isolated Purkinje fibers and an in vivo model of torsades de pointes (TdP). METHODS: Action potential and early after-depolarization (EAD) recordings were made from in situ and isolated rabbit Purkinje fibers at 37 degrees C using floating sharp microelectrodes; late I(Na) was recorded using a whole-cell patch clamp technique of Nav1.5 expressed in HEK cells at 22 degrees C; In vivo, anesthetized methoxamine-sensitized rabbits were used to test the ability of RSD1235 to suppress clofilium-induced TdP. RESULTS: RSD1235 (0.5-30 microM) had minor dose-dependent effects on action potential duration (APD) at 50% and 90% repolarization in Purkinje fibers, but pre-treatment significantly attenuated the APD-prolonging effects of dofetilide (300 nM). EADs induced by 300 nM dofetilide were terminated by 30 microM RSD1235 in all experiments (n=7). RSD1235 blocked a late component of Na current (I(Na)), which can produce inward currents contributing to EAD formation. RSD1235 pre-treatment (1 micromol/kg/min) or acute infusions prevented/terminated TdP induced by clofilium in 8 of 9 rabbits, and reduced the duration of TdP episodes from 71 +/- 23 s in control to 17 +/- 7 and 14 +/- 14 s at infusion rates of 0.3 and 1.0 micromol/kg/min, respectively (n = 9, p < 0.001). CONCLUSION: RSD1235 itself has minor actions on repolarization in Purkinje fibers, but can reverse the AP-prolonging actions of class III agents and terminate arrhythmias in a model of TdP. We suggest that these protective actions of RSD1235 may result, at least in part, from its ability to inhibit late I(Na) during action potential repolarization.

The mechanism of atrial antiarrhythmic action of RSD1235.

INTRODUCTION: RSD1235 is a novel drug recently shown to convert AF rapidly and safely in patients.(1) Its mechanism of action has been investigated in a rat model of ischemic arrhythmia, along with changes in action potential (AP) morphology in isolated rat ventricular myocytes and effects on cloned channels. METHODS AND RESULTS: Ischemic arrhythmias were inhibited with an ED50 of 1.5 micromol/kg/min, and repolarization times increased with non-significant effects on PR and QRS durations. AP prolongation was observed in rat myocytes at low doses, with plateau elevation and a reduction in the AP overshoot at higher doses. RSD1235 showed selectivity for voltage-gated K+ channels with IC50 values of 13 microM on hKv1.5 (1 Hz) versus 38 and 30 microM on Kv4.2 and Kv4.3, respectively, and 21 microM on hERG channels. RSD1235 did not block IK1 (IC50 > 1 mM) nor ICa,L (IC50= 220 microM) at 1 Hz in guinea pig ventricular myocytes (n = 4-5). The drug displayed mild (IC50= 43 microM at 1 Hz) open-channel blockade of Nav1.5 with rapid recovery kinetics after rate reduction (10-->1 Hz, 75% recovery with tau= 320 msec). Nav1.5 blocking potency increased with stimulus frequency from an IC50= 40 microM at 0.25 Hz, to an IC50= 9 microM at 20 Hz, and with depolarization increasing from 107 microM at -120 mV to 31 microM at -60 mV (1 Hz). CONCLUSIONS: These data suggest that RSD1235's clinical selectivity and AF conversion efficacy result from block of potassium channels combined with frequency- and voltage-dependent block of INa.

A randomized, controlled trial of RSD1235, a novel anti-arrhythmic agent, in the treatment of recent onset atrial fibrillation.

OBJECTIVES: The purpose of this study was to determine the efficacy and safety of intravenous RSD1235 in terminating recent onset atrial fibrillation (AF). BACKGROUND: Anti-arrhythmic drugs currently available to terminate AF have limited efficacy and safety. RSD1235 is a novel atrial selective anti-arrhythmic drug. METHODS: This was a phase II, multi-centered, randomized, double-blinded, step-dose, placebo-controlled, parallel group study. Fifty-six patients from 15 U.S. and Canadian sites with AF of 3 to 72 h duration were randomized to one of two RSD1235 dose groups or to placebo. The two RSD1235 groups were RSD-1 (0.5 mg/kg followed by 1 mg/kg) or RSD-2 (2 mg/kg followed by 3 mg/kg), by intravenous infusion over 10 min; a second dose was given only if AF was present. The primary end point was termination of AF during infusion or within 30-min after the last infusion. Secondary end points included the number of patients in sinus rhythm at 0.5, 1, and 24 h post-last infusion and time to conversion to sinus rhythm. RESULTS: The RSD-2 dose showed significant differences over placebo in: 1) termination of AF (61% vs. 5%, p < 0.0005); 2) patients in sinus rhythm at 30 min (56% vs. 5%, p < 0.001); 3) sinus rhythm at 1 h (53% vs. 5%, p = 0.0014); and 4) median time to conversion to SR (14 vs. 162 min, p = 0.016). There were no serious adverse events related to RSD1235. CONCLUSIONS: RSD1235, a new atrial-selective anti-arrhythmic agent, appears to be efficacious and safe for converting recent onset AF to sinus rhythm.