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Viscum coloratum has been used in the indigenous system of medicine for the treatment of various diseases such as myocardial ischemia and arrhythmia. Platelet-activating factor (PAF) is an important player in cardiovascular diseases. The aim of this study was to investigate the protective effects of Viscum coloratum flavonoids (VCF) against ischemic myocardial injuries in vivo and to further investigate its regulatory effect on PAF. Studies were performed in a rat model of myocardial infarction and in isolated myocytes. It was found that VCF relieved myocardial injuries during ischemia. PAF (10(-11) m) significantly increased the intracellular free Ca2+ concentration ([Ca2+]i) and VCF inhibited the changes induced by PAF in single cardiac myocytes. The results suggest that VCF can improve cardiac function and that VCF reduces ischemic myocardial injuries via blocking the signaling pathway of PAF. Therefore, PAF blockers may be candidate drugs for preventing cardiac injuries during ischemia/reperfusion, and subsequently improving cardiac function., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

In cardiac cells, KCNQ1 assembles with KCNE1 and forms a channel complex constituting the slow delayed rectifier current I(Ks). Expression of KCNQ1 and KCNE1 are regionally heterogeneous and changes with pathological states of the heart. The aims of this study were to decipher the molecular mechanisms for transcriptional and post-transcriptional regulation expression of KCNQ1 and KCNE1 genes and to shed light on the molecular mechanisms for their spatial heterogeneity of distribution. We cloned the 5'-flanking region and identified the transcription start sites of the KCNQ1 gene. We characterized the core promoters of KCNQ1 and KCNE1 and revealed the stimulating protein (Sp1) as a common transactivator of KCNQ1 and KCNE1 by interacting with the Sp1 cis-acting elements in the core promoter regions of these genes. We also characterized the 3' untranslated regions (3'UTRs) of the genes and experimentally established KCNQ1 and KCNE1 as targets for repression by the muscle-specific microRNAs miR-133 and miR-1, respectively. We demonstrated spatial heterogeneity of KCNQ1 and KCNE1 distributions at three axes (interventricular, transmural and apical-basal) and disparity between mRNA and protein expressions of these genes. We also found characteristic regional differences of expressions of Sp1 and miR-1/miR-133 in the heart. Our study unraveled a novel aspect of the cellular function of miRNAs and suggests that the I(Ks)-encoding genes KCNQ1 and KCNE1 expressions are dynamically balanced by transcription factor regulation and miRNA repression. The heterogeneities of Sp1 and miR-1/miR-133 offer an explanation for the well-recognized regional differences and disparity between mRNA and protein expressions of KCNQ1 and KCNE1.

Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene-specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene-specific microRNA mimic and microRNA-masking antisense approaches, to explore the possibility of using microRNA's principle of actions in a gene-specific manner. We examined the value of these strategies as rational approaches to develop heart rate-reducing agents and "biological pacemakers" by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene-specific microRNA mimics, 22-nt RNAs designed to target the 3'untranslated regions (3'UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene-specific microRNA mimics repressed protein levels, accompanied by depressed f-channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA-masking antisense based on the miR-1 and miR-133 target sites in the 3'UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and If conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR-1/miR-133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene-specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies.

The molecular mechanisms underlying the effects of nitric oxide (NO) and carbon monoxide (CO), individually and collectively, on large-conductance calcium-activated K(+) (K(Ca)) channels were investigated in rat vascular smooth muscle cells (SMCs). Both NO and CO increased the activity of native K(Ca) channels. Dehydrosoyasaponin-I, a specific agonist for beta subunit of K(Ca) channels, increased the open probability of native K(Ca) channels only when it was delivered to the cytoplasmic surface of membrane. CO, but not NO, further increased the activity of native K(Ca) channels that had been maximally stimulated by dehydrosoyasaponin-I. After treatment of SMCs with anti-K(Ca),beta subunit antisense oligodeoxynucleotides, the stimulatory effect of NO, but not of CO, on K(Ca) channels was nullified. CO, but not NO, enhanced the K(Ca) current densities of heterologously expressed cloned K(Ca),alpha subunit, showing that the presence of K(Ca),beta subunit is not a necessity for the effect of CO but essential for that of NO. Finally, pretreatment of SMCs with NO abolished the effects of subsequently applied CO or diethyl pyrocarbonate on K(Ca) channels. In summary, the stimulatory effects of CO and NO on K(Ca) channels rely on the specific interactions of these gases with K(Ca),alpha and K(Ca),beta subunits.

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.