Placental Nkx2-5 and target gene expression in early-onset and severe preeclampsia.

OBJECTIVE: Preeclampsia (PE) affects 2-8% of pregnancies worldwide and is a significant source of maternal and neonatal morbidity and mortality. However, the mechanisms underlying PE are poorly understood and major questions regarding etiology and risk factors remain to be addressed. Our objective was to examine whether abnormal expression of the cardiovascular developmental transcription factor, Nkx2-5, was associated with early onset and severe preeclampsia (EOSPE). METHODS: Using qPCR and immunohistochemical assay, we examined expression of Nkx2-5 and target gene expression in EOSPE and control placental tissue. We tested resulting mechanistic hypotheses in cultured cells using shRNA knockdown, qPCR, and western blot. RESULTS: Nkx2-5 is highly expressed in racially disparate fashion (Caucasians > African Americans) in a subset of early EOSPE placentae. Nkx2-5 mRNA expression is highly correlated (Caucasians > African Americans) to mRNA expression of the preeclampsia marker sFlt-1, and of the Nkx2-5 target and RNA splicing factor, Sam68. Knockdown of Sam68 expression in cultured cells significantly impacts sFlt-1 mRNA isoform generation in vitro, supporting a mechanistic hypothesis that Nkx2-5 impacts EOSPE severity in a subset of patients via upregulation of Sam68 to increase sFlt-1 expression. Expression of additional Nkx2-5 targets potentially regulating metabolic stress response is also elevated in a racially disparate fashion in EOSPE. CONCLUSIONS: Expression of Nkx2-5 and its target genes may directly influence the genesis and racially disparate severity, and define a mechanistically distinct subclass of EOSPE.

Serum profiling by MALDI-TOF mass spectrometry as a diagnostic tool for domoic acid toxicosis in California sea lions.

BACKGROUND: There are currently no reliable markers of acute domoic acid toxicosis (DAT) for California sea lions. We investigated whether patterns of serum peptides could diagnose acute DAT. Serum peptides were analyzed by MALDI-TOF mass spectrometry from 107 sea lions (acute DAT n = 34; non-DAT n = 73). Artificial neural networks (ANN) were trained using MALDI-TOF data. Individual peaks and neural networks were qualified using an independent test set (n = 20). RESULTS: No single peak was a good classifier of acute DAT, and ANN models were the best predictors of acute DAT. Performance measures for a single median ANN were: sensitivity, 100%; specificity, 60%; positive predictive value, 71%; negative predictive value, 100%. When 101 ANNs were combined and allowed to vote for the outcome, the performance measures were: sensitivity, 30%; specificity, 100%; positive predictive value, 100%; negative predictive value, 59%. CONCLUSIONS: These results suggest that MALDI-TOF peptide profiling and neural networks can perform either as a highly sensitive (100% negative predictive value) or a highly specific (100% positive predictive value) diagnostic tool for acute DAT. This also suggests that machine learning directed by populations of predictive models offer the ability to modulate the predictive effort into a specific type of error.

Dystrophic heart failure blocked by membrane sealant poloxamer.

Dystrophin deficiency causes Duchenne muscular dystrophy (DMD) in humans, an inherited and progressive disease of striated muscle deterioration that frequently involves pronounced cardiomyopathy. Heart failure is the second leading cause of fatalities in DMD. Progress towards defining the molecular basis of disease in DMD has mostly come from studies on skeletal muscle, with comparatively little attention directed to cardiac muscle. The pathophysiological mechanisms involved in cardiac myocytes may differ significantly from skeletal myofibres; this is underscored by the presence of significant cardiac disease in patients with truncated or reduced levels of dystrophin but without skeletal muscle disease. Here we show that intact, isolated dystrophin-deficient cardiac myocytes have reduced compliance and increased susceptibility to stretch-mediated calcium overload, leading to cell contracture and death, and that application of the membrane sealant poloxamer 188 corrects these defects in vitro. In vivo administration of poloxamer 188 to dystrophic mice instantly improved ventricular geometry and blocked the development of acute cardiac failure during a dobutamine-mediated stress protocol. Once issues relating to optimal dosing and long-term effects of poloxamer 188 in humans have been resolved, chemical-based membrane sealants could represent a new therapeutic approach for preventing or reversing the progression of cardiomyopathy and heart failure in muscular dystrophy.

Parvalbumin corrects slowed relaxation in adult cardiac myocytes expressing hypertrophic cardiomyopathy-linked alpha-tropomyosin mutations.

Hypertrophic cardiomyopathy mutations A63V and E180G in alpha-tropomyosin (alpha-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the alpha-Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2+/-1.4 ms in A63V, 36.8+/-1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of alpha-Tm A63V and Parv caused a marked decrease in T50R (29.8+/-1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6+/-4.4 nm in A63+Parv, 137.8+/-5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from approximately 0.12 to approximately 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9+/-1.6 ms, sarcomere shortening amplitude=132.2+/-7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G alpha-Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the alpha-Tm mutation alone (E180G+/PV-) had slower sarcomere relengthening kinetics than the controls (T90R: 199+/-7 ms in E180G+/PV-, 130+/-4 ms in E180G-/PV-; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36+/-4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by alpha-Tm mutants can be corrected by modifying calcium handling with Parv.