Linlin Gao

Functional Characterization of Inward Rectifier Potassium Ion Channel in Murine Fetal Ventricular Cardiomyocytes

Aims: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. Methods: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. Results: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. Conclusions: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

Traditional Chinese Medicine Baicalin Suppresses mESCs Proliferation through Inhibition of miR-294 Expression

Background: Traditional Chinese herbal medicines (TCMs) have been widely used against a broad spectrum of biological activities, including influencing the cardiac differentiation from mouse embryonic stem cells (mESCs). However, their effects and mechanisms of action on ESCs proliferation remain to be determined. The present study aimed to determine the effect of three TCMs, baicalin, ginsenoside Rg1, and puerarin, on mESCs proliferation and to elucidate the possible mechanism of their action. Methods: Cell proliferation was examined with a cell proliferation assay Cell Counting Kit-8 (CCK-8), propidium iodide (PI) staining was used to visualize cell cycle. The mRNA expression level of c-myc, c-fos, c-jun, GAPDH and microRNAs were measured by quantitative real time RT-PCR. Results: We found that baicalin 50 μM suppressed the proliferation of mESCs as observations in more cells in G1 phase and less cells in either S phase or G2/M phase. Moreover, baicalin suppressed the expressions of c-jun and c-fos in mESCs and down-regulated the expression of miR-294. Overexpression of miR-294 in mESCs significantly reversed the effects of baicalin both on mESC proliferation and c-fos/c-jun expression. Conclusions: Baicalin down-regulation of miR-294 may be its key mechanism of action in decreasing mESCs proliferation.

Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C

Aim:To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (ICa,L) in rat ventricular myocytes.Methods:The whole-cell patch-clamp techniques were used to record ICa,L in rat ventricular myocytes.Results:Hyposmotic challenge(∼220 mosmol/L) induced biphasic changes of ICa,L, a transient increase followed by a sustained decrease. ICa,L increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased ICa,L to 78.1%±11.0% of control, caused the inactivation of ICa,L, and shifted the steady-state inactivation curve of ICa,L to the right. The decreased ICa,L induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated ICa,L by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of ICa,L, but did not affect the swelling-induced sustained decrease of ICa,L. NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of ICa,L. Protein kinase C (PKC) activator PMA decreased ICa,L and hyposmotic solution with PMA reverted the decreased ICa,L by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of ICa,L.Conclusion:Hyposmotic challenge induced biphasic changes of ICa,L, a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of ICa,L during short exposure to hyposmotic solution.

Roles of I(f) and Intracellular Ca2+ Release in Spontaneous Activity of Ventricular Cardiomyocytes During Murine Embryonic Development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca2+ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca2+ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (INa/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013.

A Single-Cell-Type Real-Time PCR Method Based on a Modified Patch-Pipette Cell Harvesting System

Real-time PCR is a powerful tool for quantifying nucleic acid expression. Real-time PCR is conventionally performed at the tissue level to guarantee an abundance of nucleic acid for detection. The precision and reliability of this method, however, is limited by usually being composed of a mixture of different cell types. Single-cell PCR, in contrast, eliminates the purity problem of the cell source. However, use of this method is usually impeded by difficulties in cell harvesting and stringent requirements for processing of very small quantities of nucleic acids. In this study, we combined the advantages of the high purity of selected cells in single-cell PCR with the greater nucleic acid quantities and thus greater ease of tissue-level PCR. The key aspect of our method is to use a modified patch-clamp pipette to harvest several selected cells of the same type. This method is therefore especially useful for cells that can be morphologically or histologically identified such as primary sensory neurons, striated muscle fibers and cells labeled with fluorescent makers.

Properties and functions of KATP during mouse perinatal development

BACKGROUND Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes. METHODS Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels. RESULTS Similar K(ATP) current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular-biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia-ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK(ATP) blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK(ATP) blocker 5-HD did not significantly influence the response to acute hypoxia-ischemia at either EDS or LDS. In summary, sarcK(ATP) played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK(ATP) subunits. MitoK(ATP) were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia-ischemia and no developmental changes of K(ATP) subunits were found in mitochondria.

Difference of acute dissociation and 1-day culture on the electrophysiological properties of rat dorsal root ganglion neurons

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6–1.9 and Cav2.1–2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6–1.9 and Cav2.1–2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences.