Regulation of Kv11.1 Isoform Expression by Polyadenylate Binding Protein Nuclear 1.

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression.

Effects of consecutive monoculture of sweet potato on soil bacterial community as determined by pyrosequencing.

Soil bacteria play key roles in determining soil health and plant growth. In this study, four sweet potato fields that had been consecutively monocultured for 1, 2, 3, and 4 years were used to investigate the effect of monoculture on soil physicochemical properties and soil bacterial communities. The results revealed that continuous cropping led to a significant decline in soil pH, soil organic carbon, and soil bacterial abundance. Miseq pyrosequencing analysis of 16S rRNA genes revealed that Proteobacteria and Bacteroidetes were the main phyla in the sweet potato monoculture soils, comprising up to 66.24% of the total sequences. The relative abundances of beneficial bacteria, including Actinobacteria, Gemmatimonadetes, Firmicutes, Xanthomonadaceae, Rhodospirillaceae, and Syntrophobacteraceae, as well as their subgroups at the genus and operational taxonomic unit (OTU) levels, decreased considerably as the number of continuous cropping years increased. In contrast, the number of potentially pathogenic bacteria, such as Acidobacteria, Sphingomonadaceae, and Pedobacter accumulated with increasing years. The results also showed the alterations to the bacterial community in the sweet potato monoculture soils were mainly driven by soil pH and soil organic matter. Overall, the decline in soil quality after successive sweet potato monoculture can be attributed to the imbalance in soil properties and soil microbes, including the decrease in soil pH and soil organic carbon, and the enrichment of pathogenic bacteria at the expense of plant-beneficial bacteria.

Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD.

The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 isoform expression. We show that HuR and HuD inhibit activity at the intron 9 polyadenylation site. When co-expressed with the KCNH2 gene, HuR and HuD increased levels of the Kv11.1a isoform and decreased the Kv11.1a-USO isoform in the RNase protection assays and immunoblot analyses. In patch clamp experiments, HuR and HuD significantly increased the Kv11.1 current. siRNA-mediated knockdown of HuR protein decreased levels of the Kv11.1a isoform and increased those of the Kv11.1a-USO isoform. Our findings suggest that the relative expression levels of Kv11.1 C-terminal isoforms are regulated by the RNA-binding HuR and HuD proteins.

Upregulation of functional Kv11.1a isoform expression by modified U1 small nuclear RNA.

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a due to the weak 5' splice site of intron 9. We previously showed that the weak 5' splice site is caused by sequence deviation from the consensus, and that mutations toward the consensus sequence increased the efficiency of intron 9 splicing. It is well established that 5' splice sites are recognized by complementary base-paring with U1 small nuclear RNA (U1 snRNA). In this study, we modified the sequence of U1 snRNA to increase its complementarity to the 5' splice site of KCNH2 intron 9 and observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis showed that modified U1 snRNA increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform. In patch-clamp experiments, modified U1 snRNA significantly increased Kv11.1 current. Our findings suggest that relative expression of Kv11.1 C-terminal isoforms can be regulated by modified U1 snRNA.

Nonsense-Mediated mRNA Decay of hERG Mutations in Long QT Syndrome.

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-à-go-go related gene (hERG), which encodes the Kv11.1 potassium channel in the heart. Over 30% of identified LQT2 mutations are nonsense or frameshift mutations that introduce premature termination codons (PTCs). Contrary to intuition, the predominant consequence of LQT2 nonsense and frameshift mutations is not the production of truncated proteins, but rather the degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD), an RNA surveillance mechanism that selectively eliminates the mRNA transcripts that contain PTCs. In this chapter, we describe methods to study NMD of hERG nonsense and frameshift mutations in long QT syndrome.

Regulation of Isoform Expression by Blocking Polyadenylation Signal Sequences with Morpholinos.

Alternative polyadenylation is increasingly being recognized as an important layer of gene regulation. Antisense-mediated modulation of alternative polyadenylation represents an attractive strategy for the regulation of gene expression as well as potential therapeutic applications. In this chapter, we describe methods to upregulate the functional Kv11.1 isoform expression by blocking intronic polyadenylation signal sequences with antisense morpholinos.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Brief Report: Oxidative Stress Mediates Cardiomyocyte Apoptosis in a Human Model of Danon Disease and Heart Failure.

Danon disease is a familial cardiomyopathy associated with impaired autophagy due to mutations in the gene encoding lysosomal-associated membrane protein type 2 (LAMP-2). Emerging evidence has highlighted the importance of autophagy in regulating cardiomyocyte bioenergetics, function, and survival. However, the mechanisms responsible for cellular dysfunction and death in cardiomyocytes with impaired autophagic flux remain unclear. To investigate the molecular mechanisms responsible for Danon disease, we created induced pluripotent stem cells (iPSCs) from two patients with different LAMP-2 mutations. Danon iPSC-derived cardiomyocytes (iPSC-CMs) exhibited impaired autophagic flux and key features of heart failure such as increased cell size, increased expression of natriuretic peptides, and abnormal calcium handling compared to control iPSC-CMs. Additionally, Danon iPSC-CMs demonstrated excessive amounts of mitochondrial oxidative stress and apoptosis. Using the sulfhydryl antioxidant N-acetylcysteine to scavenge free radicals resulted in a significant reduction in apoptotic cell death in Danon iPSC-CMs. In summary, we have modeled Danon disease using human iPSC-CMs from patients with mutations in LAMP-2, allowing us to gain mechanistic insight into the pathogenesis of this disease. We demonstrate that LAMP-2 deficiency leads to an impairment in autophagic flux, which results in excessive oxidative stress, and subsequent cardiomyocyte apoptosis. Scavenging excessive free radicals with antioxidants may be beneficial for patients with Danon disease. In vivo studies will be necessary to validate this new treatment strategy.

Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides.

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study, we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of Kv11.1a mRNA, protein and channel current. More importantly, blocking these elements by antisense morpholino oligonucleotides shifted the alternative processing of KCNH2 intron 9 from the polyadenylation to the splicing pathway, leading to the predominant production of Kv11.1a and a significant increase in Kv11.1 current. Our findings indicate that the expression of the Kv11.1a isoform can be upregulated by an antisense approach. Antisense inhibition of KCNH2 intronic polyadenylation represents a novel approach to increase Kv11.1 channel function.

Identification of Kv11.1 isoform switch as a novel pathogenic mechanism of long-QT syndrome.

BACKGROUND: The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. The relative expression of the full-length Kv11.1a isoform and the C-terminally truncated Kv11.1a-USO isoform plays an important role in regulation of channel function. The formation of C-terminal isoforms is determined by competition between the splicing and alternative polyadenylation of KCNH2 intron 9. It is not known whether changes in the relative expression of Kv11.1a and Kv11.1a-USO can cause long-QT syndrome. METHODS AND RESULTS: We identified a novel KCNH2 splice site mutation in a large family. The mutation, IVS9-2delA, is a deletion of the A in the AG dinucleotide of the 3' acceptor site of intron 9. We designed an intron-containing full-length KCNH2 gene construct to study the effects of the mutation on the relative expression of Kv11.1a and Kv11.1a-USO at the mRNA, protein, and functional levels. We found that this mutation disrupted normal splicing and resulted in exclusive polyadenylation of intron 9, leading to a switch from the functional Kv11.1a to the nonfunctional Kv11.1a-USO isoform in HEK293 cells and HL-1 cardiomyocytes. We also showed that IVS9-2delA caused isoform switch in the mutant allele of mRNA isolated from patient lymphocytes. CONCLUSIONS: Our findings indicate that the IVS9-2delA mutation causes a switch in the expression of the functional Kv11.1a isoform to the nonfunctional Kv11.1a-USO isoform. Kv11.1 isoform switch represents a novel mechanism in the pathogenesis of long-QT syndrome.

Position of premature termination codons determines susceptibility of hERG mutations to nonsense-mediated mRNA decay in long QT syndrome.

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs 25 and D1037Rfs 82. R1032Gfs 25 introduces a PTC in exon 14, whereas D1037Rfs 82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs 25, but not D1037Rfs 82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs 25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned >54-60 nt upstream of the 3'-most exon-exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD.

LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation.

The human ether-a-go-go-related gene (hERG) encodes a voltage-activated K(+) channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met(124), resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met(60). Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

Early LQT2 nonsense mutation generates N-terminally truncated hERG channels with altered gating properties by the reinitiation of translation.

Mutations in the human ether-a-go-go-related gene (hERG) result in long QT syndrome type 2 (LQT2). The hERG gene encodes a K(+) channel that contributes to the repolarization of the cardiac action potential. We have previously shown that hERG mRNA transcripts that contain premature termination codon mutations are rapidly degraded by nonsense-mediated mRNA decay (NMD). In this study, we identified a LQT2 nonsense mutation, Q81X, which escapes degradation by the reinitiation of translation and generates N-terminally truncated channels. RNA analysis of hERG minigenes revealed equivalent levels of wild-type and Q81X mRNA while the mRNA expressed from minigenes containing the LQT2 frameshift mutation, P141fs+2X, was significantly reduced by NMD. Western blot analysis revealed that Q81X minigenes expressed truncated channels. Q81X channels exhibited decreased tail current levels and increased deactivation kinetics compared to wild-type channels. These results are consistent with the disruption of the N-terminus, which is known to regulate hERG deactivation. Site-specific mutagenesis studies showed that translation of the Q81X transcript is reinitiated at Met124 following premature termination. Q81X co-assembled with hERG to form heteromeric channels that exhibited increased deactivation rates compared to wild-type channels. Mutant channels also generated less outward current and transferred less charge at late phases of repolarization during ventricular action potential clamp. These results provide new mechanistic insight into the prolongation of the QT interval in LQT2 patients. Our findings indicate that the reinitiation of translation may be an important pathogenic mechanism in patients with nonsense and frameshift LQT2 mutations near the 5' end of the hERG gene.

Isoform-specific dominant-negative effects associated with hERG1 G628S mutation in long QT syndrome.

BACKGROUND: Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K(+) channel with properties similar to the rapidly activating delayed rectifying K(+) current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a(USO) isoforms. METHODS: A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation. CONCLUSIONS: The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a(USO) isoform. hERG1a(USO)-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.

Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome.

BACKGROUND: Nonsense and frameshift mutations are common in congenital long QT syndrome type 2 (LQT2). We previously demonstrated that hERG nonsense mutations cause degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD) and are associated with mild clinical phenotypes. The impact of NMD on the expression of hERG frameshift mutations and their phenotypic severity is not clear. OBJECTIVE: The purpose of this study was to examine the role of NMD in the pathogenesis of a hERG frameshift mutation, P926AfsX14, identified in a large LQT2 kindred and characterize genotype-phenotype correlations. METHODS: Genetic screening was performed among family members. Phenotyping was performed by assessment of ECGs and LQTS-related cardiac events. The functional effect of P926AfsX14 was studied using hERG cDNA and minigene constructs expressed in HEK293 cells. RESULTS: Significant cardiac events occurred in carriers of the P926AfsX14 mutation. When expressed from cDNA, the P926AfsX14 mutant channel was only mildly defective. However, when expressed from a minigene, the P926AfsX14 mutation caused a significant reduction in mutant mRNA, protein, and hERG current. Inhibition of NMD by RNA interference knockdown of up-frameshift protein 1 partially restored expression of mutant mRNA and protein and led to a significant increase in hERG current in the mutant cells. These results suggest that NMD is involved in the pathogenic mechanism of the P926AfsX14 mutation. CONCLUSION: Our findings suggest that the hERG frameshift mutation P926AfsX14 primarily results in degradation of mutant mRNA by the NMD pathway rather than production of truncated proteins. When combined with environmental triggers and genetic modifiers, LQT2 frameshift mutations associated with NMD can manifest with a severe clinical phenotype.

Inhibition of nonsense-mediated mRNA decay by antisense morpholino oligonucleotides restores functional expression of hERG nonsense and frameshift mutations in long-QT syndrome.

Mutations in the human ether-a-go-go-related gene (hERG) cause long-QT syndrome type 2 (LQT2). We previously described a homozygous LQT2 nonsense mutation Q1070X in which the mutant mRNA is degraded by nonsense-mediated mRNA decay (NMD) leading to a severe clinical phenotype. The degradation of the Q1070X transcript precludes the expression of truncated but functional mutant channels. In the present study, we tested the hypothesis that inhibition of NMD can restore functional expression of LQT2 mutations that are targeted by NMD. We showed that inhibition of NMD by RNA interference-mediated knockdown of UPF1 increased Q1070X mutant channel protein expression and hERG current amplitude. More importantly, we found that specific inhibition of downstream intron splicing by antisense morpholino oligonucleotides prevented NMD of the Q1070X mutant mRNA and restored the expression of functional Q1070X mutant channels. The restoration of functional expression by antisense morpholino oligonucleotides was also observed in LQT2 frameshift mutations. Our findings suggest that inhibition of NMD by antisense morpholino oligonucleotides may be a potential therapeutic approach for some LQT2 patients carrying nonsense and frameshift mutations.

Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome.

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5' splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

A major role for HERG in determining frequency of reentry in neonatal rat ventricular myocyte monolayer.

RATIONALE: the rapid delayed rectifier potassium current, I(Kr), which flows through the human ether-a-go-go-related (hERG) channel, is a major determinant of the shape and duration of the human cardiac action potential (APD). However, it is unknown whether the time dependency of I(Kr) enables it to control APD, conduction velocity (CV), and wavelength (WL) at the exceedingly high activation frequencies that are relevant to cardiac reentry and fibrillation. OBJECTIVE: to test the hypothesis that upregulation of hERG increases functional reentry frequency and contributes to its stability. METHODS AND RESULTS: using optical mapping, we investigated the effects of I(Kr) upregulation on reentry frequency, APD, CV, and WL in neonatal rat ventricular myocyte (NRVM) monolayers infected with GFP (control), hERG (I(Kr)), or dominant negative mutant hERG G628S. Reentry frequency was higher in the I(Kr)-infected monolayers (21.12 ± 0.8 Hz; n=43 versus 9.21 ± 0.58 Hz; n=16; P<0.001) but slightly reduced in G628S-infected monolayers. APD(80) in the I(Kr)-infected monolayers was shorter (>50%) than control during pacing at 1 to 5 Hz. CV was similar in both groups at low frequency pacing. In contrast, during high-frequency reentry, the CV measured at varying distances from the center of rotation was significantly faster in I(Kr)-infected monolayers than controls. Simulations using a modified NRVM model predicted that rotor acceleration was attributable, in part, to a transient hyperpolarization immediately following the AP. The transient hyperpolarization was confirmed experimentally. CONCLUSIONS: hERG overexpression dramatically accelerates reentry frequency in NRVM monolayers. Both APD and WL shortening, together with transient hyperpolarization, underlies the increased rotor frequency and stability.

Alternative splicing and polyadenylation contribute to the generation of hERG1 C-terminal isoforms.

The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Several hERG1 isoforms with different N- and C-terminal ends have been identified. The hERG1a, hERG1b, and hERG1-3.1 isoforms contain the full-length C terminus, whereas the hERG1(USO) isoforms, hERG1a(USO) and hERG1b(USO), lack most of the C-terminal domain and contain a unique C-terminal end. The mechanisms underlying the generation of hERG1(USO) isoforms are not understood. We show that hERG1 isoforms with different C-terminal ends are generated by alternative splicing and polyadenylation of hERG1 pre-mRNA. We identified an intrinsically weak, noncanonical poly(A) signal, AGUAAA, within intron 9 of hERG1 that modulates the expression of hERG1a and hERG1a(USO). Replacing AGUAAA with the strong, canonical poly(A) signal AAUAAA resulted in the predominant production of hERG1a(USO) and a marked decrease in hERG1 current. In contrast, eliminating the intron 9 poly(A) signal or increasing the strength of 5' splice site led to the predominant production of hERG1a and a significant increase in hERG1 current. We found significant variation in the relative abundance of hERG1 C-terminal isoforms in different human tissues. Taken together, these findings suggest that post-transcriptional regulation of hERG1 pre-mRNA may represent a novel mechanism to modulate the expression and function of hERG1 channels.

Molecular cloning and expression analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in grass carp.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a novel antimicrobial peptide (AMP) recently found in vertebrates, and exhibits distinct amino acid sequence, secondary structure and expression pattern from other peptides. In this study, the LEAP-2 gene and its full-length cDNA were cloned from grass carp. Grass carp LEAP-2 gene consists of two introns and three exons. The translated product contains 92 amino acids, including a 26 amino acids signal peptide and a mature peptide of 41 amino acids. Grass carp LEAP-2 gene was expressed in a wide range of tissues except blood, with the highest level of transcripts found in liver. Upon induction by Aeromonas hydrophila, its expression was significantly up-regulated in liver, gill, skin, muscle, spleen, blood, head kidney, heart and intestine, but down-regulated in trunk kidney and brain. The transcript level was high in embryos at the 16-cell stage but declined gradually afterwards, suggesting that LEAP-2 transcripts in early embryos might be maternal. Mature peptides obtained by in vitro expression displayed selective antimicrobial activities. These results together further our understanding of the physiological function of LEAP-2 in vertebrates.