H uman e ther-à-go-go- r elated g ene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (I Kr ) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K + channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R -roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R -roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R -roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC 50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R -roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R- roscovitine’s relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.
14Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that 15 conducts the delayed rectifier potassium current (I Kr ) during the repolarization phase of cardiac 16 action potentials. hERG channels have a larger pore than other K + channels and can trap many 17 unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine, a cyclin-dependent 18 kinase (CDK) inhibitor that also inhibits L-type calcium channels, inhibits open hERG channels 19 but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus 20 oocytes expressing wild-type (WT) or mutant (T623A, S624A, Y652A, F656A) hERG channels 21 demonstrated that, compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 µM R-22 roscovitine was ~ 48 %, 29 %, and 73 % weaker, respectively. In contrast, S624A hERG was 23 inhibited more potently than WT hERG, with an ~ 34 % stronger inhibition. These findings were 24 further supported by the IC 50 values, which were increased for T623A, Y652A and F656A (by 25~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data 26 suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 27 may not be. This relatively unique feature, coupled with R-roscovitine's tolerance in clinical 28 trials, could guide future drug screens. We discuss our findings and how they lend support for the 29 recent Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines on the re-evaluation of 30 potentially useful drugs that had failed testing due to unintended interactions with hERG. 31 32 Introduction 33 Human ether-à-go-go-related gene, or hERG [Kv11.1], is a voltage-gated potassium 34 channel critical for nerve and cardiac function [1,2]. In the heart, hERG channels initially open 3 35 during the depolarization phase of the cardiac action potential (cAP) but immediately inactivate. 36 Upon cAP repolarization, hERG channels de-inactivate and reopen, which allows the ensuing 37 large K + efflux to speed cAP repolarization [1], limit cardiac excitability, and maintain normal 38 QT intervals [3]. Consequently, mutations in hERG are one of the leading causes of congenital 39 long QT syndrome (cLQTS), with a neonatal incidence rate of up to 1 in 2,500 [4]; abnormal 40 cardiac phenotypes are usually triggered during exercise, arousal, or rest [5].41 hERG channels are tetrameric proteins, with each monomer consisting of six 42 transmembrane alpha helices (S1-S6) and cytoplasmic amino and carboxy termini [6]. Similar to 43 other voltage-gated channels, S1-S4 is considered the primary voltage-sensing region, with S4 44 containing positively-charged residues that move slowly outward to induce the characteristically 45 slow activation kinetics of hERG [7,8]. The four S5 and S6 helices and their intervening 46 sections, including the P-loops and P-helices, form the pore and selectivity filter of the channel 47 [9,10]. The pore is thought to have a region between the pore helix and the S6 segments that may 48 provide p...
BACKGROUND: Our earlier studies showed that inhibiting prolyl-4-hydroxylase enzymes (PHD-1 and PHD-3) improves angiogenesis, heart function, and limb perfusion in mouse models via stabilizing hypoxia-inducible transcription factor-alpha (HIF-1α). The present study explored the effects of the prolyl-4-hydroxylase enzyme, PHD-2, on ischemic heart failure using cardiac-specific PHD-2 gene knockout (KO) mice (PHD2−/−). STUDY DESIGN: Adult wild-type (WT) and PHD2−/− mice, 8–12 weeks old, were subjected to myocardial infarction (MI) by irreversibly ligating the left anterior descending (LAD) coronary artery. All sham group mice underwent surgery without LAD ligation. Animals were divided into 4 groups: (1) wild-type sham (WTS); (2) wild-type myocardial infarction (WTMI); (3) PHD2KO sham (PHD2−/−S); (4) PHD2KO myocardial infarction (PHD2−/−MI). Left ventricular tissue samples collected at various time points after surgery were used for microRNA expression profiling, Western blotting, and immunohistochemical analysis. RESULTS: Volcano plot analysis revealed 19 differentially-expressed miRNAs in the PHD2−/−MI group compared with the WTMI group. Target analysis using Ingenuity Pathway Analysis showed several differentially regulated miRNAs targeting key signaling pathways such as Akt, VEGF, Ang-1, PTEN, apoptosis, and hypoxia pathways. Western blot analysis showed increased HIF-1α, VEGF, phospho-AKT, β-catenin expression and reduced Bax expression for the PHD2−/−MI group compared with the WTMI group. Echocardiographic analysis showed preserved heart functions, and picrosirius red staining revealed decreased fibrosis in PHD2−/−MI compared with the WTMI group. CONCLUSIONS: PHD2 inhibition showed preserved heart function, enhanced angiogenic factor expression, and decreased apoptotic markers after MI. Overall, cardiac PHD2 gene inhibition is a promising candidate for managing cardiovascular diseases.
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