MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotidegated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between ؊60 and ؊95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a  subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.MinK-related protein (MiRP1 or KCNE2) is purported to be a  subunit for several voltage-gated potassium channels, including the rapid delayed rectifier (1), slow delayed rectifier (2), and transient outward current (3). In addition, we have reported that it can act as a  subunit for the hyperpolarizationactivated, cyclic nucleotide-gated (HCN) 1 family of pacemaker channels (4). We found that co-expression of MiRP1 with HCN1 or HCN2 in Xenopus oocytes resulted in larger and more rapidly activating currents than when either HCN isoform was expressed alone but that MiRP1 did not shift the midpoint of activation of either isoform. Further, co-immunoprecipitation experiments involving HA-tagged MiRP1 and HCN1 demonstrated that the two proteins interact when co-expressed in oocytes.A more recent study demonstrated MiRP1 interaction with the HCN4 isoform. Here, co-expression with MiRP1, either in oocytes or Chinese hamster ovary cells, also resulted in increased current amplitude, but this was associated with slowing of kinetics and a negative shift of the midpoint of activation (5). However, another laboratory did not detect any effect of MiRP1 when co-expressed in HEK293 cells with either HCN4 or an HCN1-HCN4 tandem construct (6). Finally, a study of MiRP1 and HCN2 co-expression in Chinese hamster ovary cells reported a reduced time-dependent current component and increased instantaneous ...
Background We sought to assess the impact and predictors of Coronavirus Disease 2019 (COVID‐19) infection and severity in a cohort of congenital heart disease (CHD) patients at a large CHD center in New York City. Methods and Results We performed a retrospective review of all individuals with CHD followed at Columbia University Irving Medical Center who were diagnosed with COVID‐19 between 3/1/2020 and 7/1/2020. The primary endpoint was moderate/severe response to COVID‐19 infection defined as a) death during COVID‐19 infection; or 2) need for hospitalization and/or respiratory support secondary to COVID‐19 infection. Among 53 COVID‐19 positive patients with CHD, 10 (19%) were <18 years old (median age 34 years). 31 (58%) had complex congenital anatomy including 10 (19%) with a Fontan repair. Eight (15%) had a genetic syndrome, six (11%) had pulmonary hypertension (PH), and nine (17%) were obese. Among adults, 18 (41%) were physiologic class C or D. For the entire cohort, nine (17%) had a moderate/severe infection, including three deaths (6%). After correcting for multiple comparisons, the presence of a genetic syndrome (OR=35.82: p=0.0002), and in adults, physiological Stage C or D (OR=19.38: p=0.002) were significantly associated with moderate/severe infection. Conclusions At our CHD center, the number of symptomatic COVID‐19 patients was relatively low. CHD patients with a genetic syndrome and adults at advanced physiological stage were at highest risk for moderate/severe infection.
Objective A recent STS database study showed that low weight (<2.5 kg) at surgery was associated with a high operative mortality (16%). We thought to assess outcomes after cardiac repair in patients <2.5 kg as compared to 2.5-4.5kg in an institution with a dedicated neonatal cardiac program; and to determine the potential role played by prematurity, STAT risk categories, uni/biventricular pathway, and timing of surgery. Methods We analyzed outcomes (hospital mortality, early reintervention, postoperative length of stay (postopLOS), mortality (at last follow-up) in patients <2.5kg at time of surgery (n=146; group1) and 2.5-4.5kg (n=622; group2), who underwent open or closed cardiac repairs from January 2006 to December 2012 at our institution. The statistical analysis was stratified by prematurity, STAT risk categories, uni/biventricular pathway and “usual”/“delayed” timing of surgery. A uni/multivariate risk analysis was performed. Mean follow-up was 21.6±25.6 months. Results Hospital mortality in group 1 was 10.9% (n=16) vs. 4.8% (n=30) in group 2 (p=0.007). PostopLOS and early un-planned reintervention rates were similar between the two groups. Late mortality in group 1 was 0.7% (n=1). In Group 1, early outcomes were independent of STAT risk categories, uni/biventricular pathway or timing of surgery, as opposed to group 2. Lower gestational age at birth was an independent risk factor for early mortality in group 1. Conclusions A dedicated multidisciplinary neonatal cardiac program yields good outcomes for neonates and infants <2.5kg independently of STAT risk categories and uni/biventricular pathway. Lower gestational age at birth was an independent risk factor for hospital mortality.
We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160⌬CT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160⌬CT was released by cells in the form of membrane-bound vesicles. gp160⌬CT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virusneutralizing activity by vaccination may be realized.
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