• CFTR Potentiator PG-01 and Corrector KM-11060 can rescue hERG mutations trafficking

      Zhang, J.; Shang, Lijun; Ma, A. (2016)
      Type II congenitalLong QT syndrome (LQT2) is due to genetic mutations in hERG channel. Genetic or pharmacological factors could potentially affect hERG channel biogenesis and contributes to LQTS, for example, disease mutations G601S and T473P result in hERG trafficking deficiency [1,2]. Various rescue strategies for hERG dysfuction are being developed. Some correctors for CFTR channel have been reported to act indirectly on proteostasis pathways to promote folding and correction on hERG trafficking deficiency [3]. In this study, we tested the hypothesis that the CFTR corrector KM-11060 and the potentiator PG-01 may correct hERG mutation trafficking diseases. We use HEK293 cell line expressing a well-studied trafficking disease mutation G601S-hERG channel [4]. We treated cells with CFTR potentiator PG-01and corrector KM-11060, which function through different cellular mechanisms, and assessed whether correction occurred via immunoblotting. Whole cell proteins from HEK 293 cells expressing hERG channels were used for analysis [5]. Proteins were separated on 8% SDS-polyacrylamide electrophoresis gels for 1 hour, transferred onto PVDF membrane, and blocked for 1 h with 5% nonfat milk. The blots were incubated with the primary antibody (Santa Cruz Biotechnology) for 12-16 h at 4C temperature and then incubated with a donkey antigoat horseradish peroxidase-conjugated secondary antibody( Santa Cruz Biotechnology). Actin expression was used for loading controls. The blots were visualized using the ECL detection kit (Genshare).Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Our results show that both KM-11060 (5, 10, 20uM) and PG-01(5, 15 uM) can correct G601S mutant alleles of hERG protein trafficking (Fig 1, 2). KM-11060 (20uM) but not PG-01(15 uM) enhance protein expression of wild type hERG channel (Fig 2). Further treatment on cells at low temperature with different drug concentration will be tested. Functional studies are also needed to test whether the drugs can correct the function of hERG mutation channel. These results could potentially provide novel insight into the correction mechanism of CFTR potentiator and also help to develop new treatment for LQT2.
    • Conjugating existing clinical drugs with gold nanoparticles for better treatment of heart diseases

      Zhang, J.; Ma, A.; Shang, Lijun (2018-05-29)
      Developing new methods to treat heart diseases is always a focus for basic research and clinical applications. Existing drugs have strong side-effects and also require lifetime administration for patients. Recent attempts of using nanoparticles (NPs) in treating atherosclerosis in animals and some heart diseases such as heart failure and endocarditis have provided hopes for better drug delivery and reducing of drug side-effects. In this mini-review, we summarize the present applications of using gold nanoparticles (GNPs) as a new drug delivery system in diseased hearts and of the assessment of toxicity in using GNPs. We suggest that conjugating existing clinical drugs with GNPs is a favorable choice to provide “new and double-enhanced” potentiality to those existing drugs in treating heart diseases. Other applications of using NPs in the treatment of heart diseases including using drugs in nano-form and coating drugs with a surface of relevant NP are also discussed.
    • Effect of gold nanoparticles on H9C2 myoblasts and rat peripheral blood mononuclear cells

      Zhang, Jingwen; Ma, A.; Shang, Lijun (2017-08)
      Recent studies have gained positive results using nanoparticles (NPs) in treating atherosclerosis on animals. But their toxicity and application in treating other heart diseases such as heart failure and endocarditis still need proper investigation. Gold nanoparticles (Au-NPs) were chosen as model substances as they have been successfully used in treating cancer. In this study, we use both H9C2 myoblasts and rat peripheral blood mononuclear cells to determine the influence of Au-NP size on their cytotoxicity and cell apoptosis. H9C2 cells were treated with Au-NPs of a diameter of 5, 20, 40 and 100nmfor 24 hrs before their cell viabilities tested by MTT assay, cell apoptosis measured by flow cytometry, and the generation of reactive oxygen species (ROS) detected by Fluorometric Intracellular ROS Kit. Distribution of the Au-NPs and their effects on the structure of mitochondria and lysosome were detected by electron microscopy. In addition, we obtained rat peripheral blood mononuclear cells and treated them with Au-NPs same with H9C2 cell line. Our results showed NPs of 5, 40, and 100 nm reduced cell viabilities on H9C2 cells while20nm showed no change on cell viability (Ctrl: 100±8.2 vs 20nm: 95.39±9.13, P>0.05, n=6) and some protect effect on ISO induced H9C2 cells apoptosis (ISO: 100±13.5 vs 20nm: 80.19±17.36, P>0.05, n=6). All size of Au-NPs reduced cell viabilities on rat peripheral blood mononuclear cells while 40nm showed the least reduction on cell viability (Ctrl: 100.0±3.0 vs 40nm: 76.31±3.68, P<0.001, n=6) and significant protect effect on ISO-induced rat peripheral monocytes apoptosis (ISO: 100±1.86 vs 40nm: 45.34±10.32, P<0.05, n=6). In addition, 20nm Au-NP showed some protect effect on ROS generation on ISO-induced H9C2 cells (ISO: 100±3.79 vs 20nm: 94.84±4.98, P>0.05, n=6), while 40nm produced more ROS (ISO: 100±3.79 vs 40nm: 141.63±42.81, P>0.05, n=6). Electron microscopy detection showed correlated results in structure. These results on H9C2 cell line are basically in agreeable to our animal study. The protective effect of 20nm may due to its ability to protect ISO-induced ROS generation. The results on rat peripheral monocytes are slightly different to those on H9C2 cells. Further investigation need to focus on the role of NPs size on cell apoptosis by detecting autophagy specific protein through western blotting.
    • Effects of Isoproterenol on IhERG during K+ changes in HEK293 cells

      Zhang, J.; Shang, Lijun; Wang, T.; Ni, Y.; Ma, A. (2017)
      Introduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86.
    • Methoxylated but not hydroxylated flavones elicit significant activity against Parp-1-mediated cell death (Parthanatos)

      Zhang, Jingwen; Marsh, J.R.; Tait, A.; Iqbal, M.M.; Pritchard, C.J.; Ma, A.; Shang, Lijun; Fatokun, Amos A. (2017-08)
      Flavonoids, of which flavones are a sub-group, are plant secondary metabolites found in a variety of natural food sources (e.g., vegetables) and wines. They elicit beneficial roles in health and disease through their antioxidant activity, but some of them have also now been found to exert specific effects on cell signalling. We recently showed that methoxylation of the flavone structure at the 4ʹ position, or additionally at the 3ʹ position, to produce 4ʹ-methoxyflavone (4MF) and 3ʹ,4ʹ-dimethoxyflavone (DMF), respectively, significantly enhanced activity against the cell death (“parthanatos”) mediated by poly (ADP-ribose) polymerase (PARP). We report here our attempt to correlate the antioxidant and parthanatos-inhibitory activities of these methoxylated flavones with those of the hydroxylated flavonoids. Cultures of HeLa and HaCaT cells were exposed to MNNG (50µM, up to 25min), which induces parthanatos, and the oxidant hydrogen peroxide (100µM – 2mM, up to 24h). The effects (up to 20µM) of the methoxylated flavones 4MF and DMF, the hydroxylated flavone luteolin (LN), and the non-flavone flavonoids quercetin (QE), naringin (NG) and epigallocatechin gallate (EGCG) on the reduction in viability (indicative of cell death) and morphological changes induced by MNNG or peroxide were then investigated. Both alamar blue and MTT assays were used to quantify viability. MNNG induced significant reduction in cell viability, which was not affected by the pan-caspase inhibitor Z-VAD-fmk but significantly blocked by DPQ, a PARP-1 inhibitor, consistent with the biochemical profile of parthanatos. Hydrogen peroxide also elicited a significant decrease in cell viability, with partial or no protection afforded by either Z-VAD-fmk or DPQ (dependent on peroxide concentration and treatment duration). 4MF and DMF demonstrated significant protection against MNNG-induced cell death but LN, QE, NG and EGCG showed little or no protection. On the other hand, 4MF and DMF elicited mostly negligible effects against hydrogen peroxide, whereas LN, QE, NG and EGCG elicited various levels of protection against it. We conclude that methoxylation at the 4ʹ or 3ʹ, 4ʹ positions of flavones favours anti-parthanatos but not antioxidant activity, whereas hydroxylation enhances antioxidant but not anti-parthanatos activity.
    • Nanoparticles for post-infarct ventricular remodeling

      Dong, C.; Ma, A.; Shang, Lijun (2018-12)
      In recent years, tremendous progress has been made in the treatment of acute myocardial infarction (AMI), but pathological ventricular remodeling often causes survivors to suffer from fatal heart failure. Currently, there is no effective therapy to attenuate ventricular remodeling. Recently, nanoparticles-based drug delivery system is widely applied in biomedicine especially in cancer and liver fibrosis, owing to its excellent physical, chemical, and biological properties. Therefore, using nanoparticles as delivery vehicles of small molecules, polypeptides, etc to improve post-infarct ventricular remodeling are expected. In this review, we summarized the updated researches in this fast-growing area and suggested further works needed.
    • Size dependent effects of gold nanoparticles in ISO-induced hyperthyroid rats

      Zhang, J.; Xue, Y.; Ni, Y.; Ning, F.; Shang, Lijun; Ma, A. (2018-07-19)
      In this study, we applied different sizes of gold nanoparticles (Au-NPs) to isoproterenol (ISO)-induced hyperthyroid heart disease rats (HHD rats). Single dose of 5, 40, 100 nm Au-NPs were injected intravenously. Cardiac safety tests were evaluated by cardiac marker enzymes in serum and cardiac accumulation of Au-NPs were measured by ICP-MS. Our results showed that size-dependent cardiac effects of Au-NPs in ISO-induced hyperthyroid rats. 5 nm Au-NPs had some cardiac protective effect but little accumulation in heart, probably due to smaller size Au-NPs can adapt to whole body easily in vivo. Histological analysis and TUNEL staining showed that Au-NPs can induce pathological alterations including cardiac fibrosis, apoptosis in control groups, however they can protect HHD groups from these harmful effects. Furthermore, transmission electron microscopy and western blotting employed on H9C2 cells showed that autophagy presented in Au-NPs treated cells and that Au-NPs can decrease LC3 II turning to LC3 I and decrease APG7 and caspase 12 in the process in HHD groups, while opposite effects on control groups were presented, which could be an adaptive inflammation reacts. As there are few animal studies about using nanoparticles in the treatment of heart disease, our in vivo and in vitro studies would provide valuable information before they can be considered for clinical use in general.