Verapamil suppresses cardiac alternans and ventricular arrhythmias in acute myocardial ischemia via ryanodine receptor inhibition.

T-wave alternans (TWA) is a potent arrhythmia substrate under the conditions of acute myocardial ischemia. Abnormal intracellular calcium cycling contributes to the genesis of cardiac alternans. Ryanodine receptor (RyR) is a pivotal Ca2+ cycling protein central to Ca2+ signaling in the heart. Here, we investigated the potential role of RyR in cardiac alternans and ventricular arrhythmias in acute myocardial ischemia. Transmembrane action potentials were simultaneously recorded from epicardium and endocardium together with a transmural ECG and isometric contraction force in the arterially perfused left ventricular wedge preparations. Calcium alternans were induced by incremental frequency of field stimulation in rat ventricular myocytes. TWA, mechanical alternans and ventricular arrhythmias were reproducibly induced by rapid pacing in the acute ischemic wedge preparations. Compared with control group, calcium alternans ratio and spontaneous calcium release were increased in acute ischemic myocytes. Verapamil, a phenylalkylamine calcium channel blocker, can successfully abolish spontaneous calcium release, TWA, and ventricular arrhythmias. The inhibition effect of verapamil could be diminished by low concentration of ryanodine (10 nmol/L). However, nifedipine, a dihydropyridine calcium channel blocker, could not block TWA or arrhythmias. Moreover, verapamil, but not nifedipine, significantly decreased ROS production in ischemic myocytes. Collectively, our results indicate that verapamil can significantly inhibit the development of cardiac alternans and ventricular arrhythmias in acute myocardial ischemia, and the mechanism was related to the inhibition of RyR and the protective function to oxidative stress.

Ability of ambulatory ECG-based T-wave alternans to modify risk assessment of cardiac events: a systematic review.

BACKGROUND: Exercise-based spectral T-wave alternans (TWA) has been proposed as a noninvasive tool-identifying patients at risk of sudden cardiac death (SCD) and cardiac mortality. Prior studies have indicated that ambulatory electrocardiogram (AECG)-based TWA is an important alternative platform to exercise for risk stratification of cardiac events. This study sought to review data regarding 24-hour AECG-based TWA and to discuss its potential role in risk stratification of fatal cardiac events across a series of patient risk profiles. METHODS: Prospective clinical studies of the predictive value of AECG-based TWA obtained with daily activity published between January 1990 and November 2014 were retrieved. Major endpoints included composite endpoint of SCD, cardiac mortality, and severe arrhythmic events. RESULTS: Data were accumulated from 5 studies involving a total of 1,588 patients, including 317 positive and 1,271 negative TWA results. Compared with the negative group, positive group showed increased rates of SCD (hazard ratio [HR]: 7.49, 95% confidence interval [CI]: 2.65 to 21.15), cardiac mortality (HR: 4.75, 95% CI: 0.42 to 53.55), and composite endpoint (SCD, cardiac mortality, and severe arrhythmic events, HR: 5.94, 95% CI: 1.80 to 19.63). For the 4 studies evaluating TWA measured using the modified moving average method, the HR associated with a positive versus negative TWA result was 9.51 (95% CI: 4.99 to 18.11) for the composite endpoint. CONCLUSIONS: The positive group of AECG-based TWA has a nearly six-fold risk of severe outcomes compared with the negative group. Therefore, AECG-based TWA provides an accurate means of predicting fatal cardiac events.

[Progress on pluripotency factors in zebrafish].

The mammalian pluripotency factors, including transcription factors such as Pou5f1/Oct4, Sox2, Klf4 and Nanog, play critical roles in maintaining pluripotency of embryonic stem cells and inducing reprogramming of differentiated cells. However, the functions of vertebrate pluripotency factors in vivo have not been elucidated. Zebrafish(Danio rerio H.) is an excellent model for studying vertebrates' early embryo development. It allows functional studies of pluripotency factors to be conducted in an in vivo environment and therefore provides more accurate information on their roles. Nowadays, several homologs of mammalian pluripotency factors including oct4, nanog etc. have been identified in zebrafish. This review aimed at introducing the progress of the functional study on zebrafish pluripotency factors and comparing to those of other vertebrates.

U6 promoter-driven siRNA injection has nonspecific effects in zebrafish.

RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), which causes degradation of homologous mRNAs. RNAi has been observed in a wide range of eukaryotes, including fungi, plants and animals. In vertebrates, long dsRNA activates the interferon response and yields nonspecific degradation of mRNA. In contrast, small interference RNA (siRNA) duplexes with a length of 21-23 nucleotides trigger specific gene silencing and thus are widely used in gene function studies. The use of siRNA for gene silencing in zebrafish has rarely been reported. In this report, we studied mammalian U6 promoter-driven siRNA-mediated RNA interference in zebrafish. The well characterized genes Myf5, Dlg3 and Nacre were selected as targets. Two to four target siRNAs were synthesized with incorporation of the U6 promoter. Constructs were introduced into early zebrafish embryos through microinjection, followed by in situ hybridization and embryonic development was monitored to determine whether U6 promoter-driven siRNAs could efficiently suppress specific gene expression. We showed that these siRNAs could partially suppress endogenous gene expression and that the siRNA efficiency varied at different targeted positions. However, the U6 promoter-driven siRNAs may also have induced nonspecific gene suppression (off-target effects). It appears that, despite the findings of previous reports, the current methodology of siRNA interference is not practical for studying gene function during early zebrafish development.

Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.

DAXX interacts with phage PhiC31 integrase and inhibits recombination.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

Slit-like 2, a novel zebrafish slit homologue that might involve in zebrafish central neural and vascular morphogenesis.

Nervous and vascular systems grow as parallel networks, indicating common cues in distal targets. We have identified a novel zebrafish gene slit-like 2 (slitl2) that might involve in zebrafish central neural and vascular morphogenesis. Whole-mount in situ hybridization of zebrafish embryo detected distinct signals of slitl2 transcripts in zebrafish midline structure of central nervous system similar to that of slits. Strong expression is also observed in zebrafish vasculature. Zebrafish slitl2 shares amino acid sequence identity of 41% with Homo sapiens slitl2 (vasorin) and Mus musculus slitl2, and 35%, 33% with Danio rerio slit3, slit2. Analysis of zebrafish slitl2 cripto growth factor domain, extracellular matrix protein slit domain, and putative signal peptide confirms that as a secreted and cell-surface protein slitl2 may be essential in axon guidance, vessel development, and axis patterning. These results provide evidence that slitl2 may play important roles in zebrafish central nervous system and vascular morphogenesis.

Enhancement of naked FIX minigene expression by chloroquine in mice.

AIM: To study the effect of chloroquine on the expression of human clotting factor IX (hFIX) in mice. METHODS: Hydrodynamics-based naked DNA plasmid administration was performed by tail vein injection of 10 microg of pCMV- hFIX and chloroquine (0, 100, 200, and 500 micromol/L) in 2.2 mL of Ringer's solution within 6-7 s, the level and stability of hFIX expression, liver damage and toxicity were then examined. RESULTS: The maximum expression of hFIX level was 4.4+/-1.8 mg/L at 8 h after injection, 9.7+/-1.6 mg/L at 24 h only existed in 200 micromol/L chloroquine-treated animals, which is 3-4 fold higher than that of control (P<0.01). There is no significant difference observed among all the treated groups, 3 d later. Transaminase level and liver histological study showed the damage of liver was not related to chloroquine (P>0.05). CONCLUSION: Chloroquine can enhance and sustain exogenous gene expression in vivo without side effect under our experimental conditions.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration.

AIM: Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice. METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure. Glucose levels, body weight of treated mice, insulin levels, immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration. Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver. RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result. CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Molecular cloning and expression of a smooth muscle-specific gene SM22alpha in zebrafish.

SM22alpha is a kind of 22-kDa protein which is exclusively expressed in smooth muscle containing tissues of the vertebrates. Here we report molecular cloning of a novel zebrafish SM22alpha gene. The full length of zebrafish SM22alpha cDNA is 1296bp and it encodes a polypeptide of 201 amino acids which shares 69.2%, 69.7%, 69.2%, 67.2%, and 61.2% overall identity with human, mouse, rat, chicken, and bovine SM22alpha, respectively. Characterization of zebrafish SM22alpha genomic sequence reveals that it spans 7.7kb and contains five exons and four introns. The expression pattern of SM22alpha in zebrafish embryonic development is studied by whole-mount in situ hybridization. Strong expression is observed in vascular, gut, swim bladder, branchial arches, and fin epidermis. Furthermore, we carry out gene knock-down by antisense morpholino oligonucleotide, which results in disappearance of yolk extension, caudal fin aberrance, and deficiency of circulation system in zebrafish embryo. Cross-section of SM22alpha-deficient embryo suggests that SM22alpha may play roles in smooth muscle cell morphology transform.

[Whole amount in situ hybridization and transgene via microinjection in zebrafish].

Zebrafish is new model organism to study development of the vertebrate. In this report, we chose a fragment of gene GATA-1 which specifically expressed in zebrafish hematopoiesis system as probe to carry on whole amount in situ hybridization. Then we transported reporter gene driven by GATA-1 promoter into the embryos via microinjection approach and observed the spatial and temporal expression pattern of GFP. Our results demonstrated that efficient and reliable technology of whole amount in situ hybridization and microinjection in zebrafish was established.

Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.

AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.