Expression Of Ion Channel Proteins Research Articles

Androgens and erectile dysfunction: from androgen deficiency to treatment.

Androgens play important roles in regulating the growth and development of the male reproductive system and maintaining libido and erectile function. The specific mechanisms by which androgen deficiency leads to erectile dysfunction (ED) are not yet fully understood. To understand the mechanisms and treatment of androgen deficiency-related ED. A literature search in the past 10years was conducted in PubMed and Google Scholar to determine the effects of androgen deficiency on erectile function and the treatment of androgen deficiency. Androgen deficiency can be caused by hypothalamic-pituitary lesions and injuries, testicular-related diseases and injuries, endocrine and metabolic disorders, the side effects of medication, and age. Androgen deficiency can lead to ED by inhibiting the NOS/NO/cGMP pathway (nitric oxide synthase/nitric oxide/cyclic guanosine monophosphate) and altering the expression of ion channel proteins, as well as by inducing oxidative stress, death, and fibrosis in penile corpus cavernosum cells. Testosterone replacement therapy is effective at improving the serum testosterone levels and erectile function in patients with androgen deficiency. For patients who need to maintain a low androgenic state, erectile function can be improved by lifestyle changes, treatment with phosphodiesterase type 5 inhibitors, low-intensity extracorporeal shock wave therapy, and stem cell therapy. Androgen deficiency can affect the structure and function of the penile corpus cavernosum, leading to ED. Areas of further study include how androgen replacement therapy can improve erectile function and how to improve the maintenance of erectile function in patients with hypoandrogenic status.

Impact of isorhamnetin for suppression of action potential aberrations associated with atrial fibrillation

Abstract Background Isorhamnetin, a natural flavonoid, has potent antioxidant, antifibrotic and L-type Cav1.2 channel modulation effects, but its antiarrhythmic effect has not been reported. Purpose The purpose of this study is to investigate the effect of isorhamnetin on angiotensin II (Ang II)-induced atrial fibrillation (AF) in mice. Methods Wild-type male mice (C57BL/6J, 8 weeks old) were randomly divided into the following three groups: 1) control group, 2) Ang II treated group, 3) Ang II+isorhamnetin treated group. Ang II (1000 ng/kg/min) was administered by an implantable osmotic pump for two consecutive weeks, and isorhamnetin (5 mg/kg) was administered intraperitoneally for three weeks, one week before the initiation of Ang II administration. HL-1 cells were also assigned three groups. Ang II (1 μM) was treated for one day, and isorhamnetin (10 μM) was pretreated one hour before the beginning of Ang II treatment. To evaluate the effect of isorhamnetin on AF, electrophysiologic study including AF induction and action potential duration (APD) measurement with atrial tissue and HL-1 cells was performed. The expression of ion channel genes and proteins, including Cav1.2, were also evaluated. Results AF induction rate was markedly increased by Ang II and significantly decreased by isorhamnetin. AF duration was remarkably prolonged by Ang II and significantly shortened by isorhamnetin. Atrial effective refractory period (AERP) shortened by Ang II and reversed by isorhamnetin. Ang II administration significantly prolonged APD; that was alleviated by isorhamnetin. In addition, Ang II increased the frequency of delayed afterdepolarization (DAD), whereas isorhamnetin reduced its frequency. Cav1.2 channel expression was enhanced by Ang II and normalized by isorhamnetin. On the other hand, Ang II and isorhamnetin had no effect on potassium channels. Conclusion The findings in this study suggest that isorhamnetin prevents APD prolongation via the modulation of Cav1.2 channel and reduced the frequency of DAD, which is one of the mechanisms to prevent AF vulnerability.Effect of isorhamnetin on APD

Vericiguat alleviates ventricular remodeling and arrhythmias in mouse models of myocardial infarction via CaMKII signaling

AimsMaladaptive ventricular remodeling is a major cause of ventricular arrhythmias following myocardial infarction (MI) and adversely impacts the quality of life of affected patients. Vericiguat is a new soluble guanylate cyclase (sGC) activator with cardioprotective properties. However, its effects on MI-induced ventricular remodeling and arrhythmias are not fully comprehended; hence, our research evaluated the effect of vericiguat on mice post-MI. Materials and methodsMice were divided into four treatment groups: Sham, Sham+Veri, MI, and MI + Veri. For the MI groups and MI + Veri groups, the left anterior descending (LAD) coronary artery was occluded to induce MI. Conversely, the Sham group underwent mock surgery. Vericiguat was administered orally daily for 28 days to the Sham+Veri and MI + Veri groups. Additionally, H9c2 cells were cultured for further mechanistic studies. Assessment methods included echocardiography, pathological analysis, electrophysiological analysis, and Western blotting. Key findingsVericiguat reduced cardiac dysfunction and infarct size after MI. It also mitigated MI-induced left ventricular fibrosis and cardiomyocyte apoptosis. Vericiguat normalized the expression of ion channel proteins (Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv7.1, KCNH2, Cav1.2) and the gap junction protein connexin 43, reducing the susceptibility to ventricular arrhythmia. Vericiguat significantly inhibited MI-induced calcium/calmodulin-dependent protein kinase II (CaMKII) pathway activation in mice. SignificanceVericiguat alleviated MI-induced left ventricular adverse remodeling and arrhythmias through modulation of the CamkII signaling pathway.

Abscisic Acid and Its Receptors LANCL1 and LANCL2 Control Cardiomyocyte Mitochondrial Function, Expression of Contractile, Cytoskeletal and Ion Channel Proteins and Cell Proliferation via ERRα.

The cross-kingdom stress hormone abscisic acid (ABA) and its mammalian receptors LANCL1 and LANCL2 regulate the response of cardiomyocytes to hypoxia by activating NO generation. The overexpression of LANCL1/2 increases transcription, phosphorylation and the activity of eNOS and improves cell vitality after hypoxia/reoxygenation via the AMPK/PGC-1α axis. Here, we investigated whether the ABA/LANCL system also affects the mitochondrial oxidative metabolism and structural proteins. Mitochondrial function, cell cycle and the expression of cytoskeletal, contractile and ion channel proteins were studied in H9c2 rat cardiomyoblasts overexpressing or silenced by LANCL1 and LANCL2, with or without ABA. Overexpression of LANCL1/2 significantly increased, while silencing conversely reduced the mitochondrial number, OXPHOS complex I, proton gradient, glucose and palmitate-dependent respiration, transcription of uncoupling proteins, expression of proteins involved in cytoskeletal, contractile and electrical functions. These effects, and LANCL1/2-dependent NO generation, are mediated by transcription factor ERRα, upstream of the AMPK/PGC1-α axis and transcriptionally controlled by the LANCL1/2-ABA system. The ABA-LANCL1/2 hormone-receptor system controls fundamental aspects of cardiomyocyte physiology via an ERRα/AMPK/PGC-1α signaling axis and ABA-mediated targeting of this axis could improve cardiac function and resilience to hypoxic and dysmetabolic conditions.

Amelioration of Cyclosporine A-Induced Acute Nephrotoxicity by Cordyceps cicadae Mycelia via Mg+2 Reabsorption and the Inhibition of GRP78-IRE1-CHOP Pathway: In Vivo and In Vitro.

Fruiting bodies of Cordyceps cicadae (CC) have been reported to have a therapeutic effect in chronic kidney disease. Due to the rare and expensive resources from natural habitats, artificially cultivated mycelia using submerged liquid cultivation of CC (CCM) have been recently developed as an alternative to scarce sources of CC. However, little is known regarding potential protective effects of CCM against cyclosporine A (CsA)-induced acute nephrotoxicity in vivo and in vitro. In this study, male Sprague-Dawley rats were divided into six groups: control, CCM (40 mg and 400 mg/kg, orally), CsA (10 mg/kg, oral gavage), and CsA + CCM (40 mg and 400 mg/kg, orally). At the end of the study on day 8, all rats were sacrificed, and the blood and kidneys retrieved. CsA-induced acute nephrotoxicity was evident by increased levels of blood urea nitrogen (BUN). Levels of the endoplasmic reticulum (ER) resident chaperone glucose regulated protein 78 (GRP 78) were increased significantly in rats with acute nephrotoxicity. BUN and GRP 78 were significantly ameliorated in synchronous oral groups of CCM (40 or 400 mg/kg) plus CsA. Examination of hematoxylin and eosin stained kidney tissues revealed that the combined treatment of CCM slightly improved vacuolization in renal tubules upon CsA-induced damage. CsA-induced down-regulation of protein expression of magnesium ion channel proteins and transient receptor potential melastatin 6 and 7 were abolished by the combined treatment of CCM. CCM has the potential to protect the kidney against CsA-induced nephrotoxicity by reducing magnesium ion wasting, tubular cell damage, and ER stress demonstrated further by human renal proximal tubular epithelial cell line HK-2. Our results contribute to the in-depth understanding of the role of polysaccharides and nucleobases as the main secondary metabolites of CCM in the defense system of renal functions in CsA-induced acute nephrotoxicity.

Effects of 25(OH)VD3 on Growth Performance, Pork Quality and Calcium Deposit in Growing-Finishing Pigs.

The study was conducted to evaluate the effects of 25(OH)VD3 with different inclusion levels of 0, 25, 50 and 75 μg/kg in the diet on growth performance, nutrient digestibility, bone properties and pork quality in growing-finishing pigs. The results showed that the average daily gain (p < 0.05) and body weight (p < 0.10) of pigs showed a trend of increasing quadratically as inclusion levels of 25(OH)VD3 increased. Dietary supplementation of 50 μg/kg 25(OH)VD3 increased calcium digestibility compared with the 0 μg/kg group (p < 0.05), and calcium and phosphorus digestibility increased quadratically as inclusion levels of 25(OH)VD3 increased (p < 0.05). Dietary supplementation of 50 μg/kg 25(OH)VD3 increased concentrations of polyunsaturated fatty acids, and decreased contents of saturated and monounsaturated fatty acids in the longissimus dorsi of pigs (p < 0.05). The addition of 25, 50 and 75 μg/kg 25(OH)VD3 to the diet increased breaking strength and bone stiffness in the tibia compared with the 0 μg/kg group (p < 0.05). Dietary supplementation of 50 μg/kg 25(OH)VD3 improved the activities of superoxide dismutase (SOD) and catalase (CAT), and increased the messenger RNA (mRNA) expression of Cu/Zn SOD in the longissimus dorsi compared with the 0 μg/kg group (p < 0.05). Supplementing 50 μg/kg 25(OH)VD3 improved the mRNA expression of calcium-binding protein D9k (CaBP-D9k) and D28k (CaBP-D28K) in the liver compared with the 0 μg/kg 25(OH)D3 group (p < 0.05). In conclusion, a diet with an added dose of 50 μg/kg 25(OH)VD3 showed a greatest growth performance of growing-finishing pigs, and 25(OH)VD3 enhanced calcium deposition and antioxidant capacity in the longissimus dorsi, which may be associated with improved expression of calcium ion channel proteins.

In-situ and amplification-free imaging of hERG ion channels at single-cell level using a unique core-molecule-shell-secondary antibody SERS nanoprobe

Research on ion channels and their monoclonal antibodies plays a critical role in drug development and disease diagnosis. The current ion channel researches are often not conducted in the microenvironment for cells survival, which restricts the mechanism study of the links between the cell structure and the ion channel function. In this work, we synthesized gold core-4-mercaptobenzonitrile-sliver shell-goat anti-rabbit immunoglobulin G (Au@4-MBN@Ag@IgG) nanoparticles as surface-enhanced Raman scattering (SERS) nanoprobes for investigating the human ether-a-go-go related gene (hERG) potassium ion channel in cell membranes. This is the first attempt to study ion channels using SERS. Due to the unique core-molecule-shell structure and the silver shell of nanoprobes, strong and stable SERS signal was obtained. With the help of antibodies, the Au@4-MBN@Ag@IgG nanoprobes were captured by hERG antibodies and then bonded with hERG ion channels based on the sandwich immune response. The reporter molecule, 4-MBN, displayed a strong and sharp characteristic peak at 2233 cm-1 in the Raman silent region. The intensity of this peak denoted the concentration of antibodies and the expression of ion channel proteins. We successfully applied this amplification-free method for in-situ imaging the distribution of the hERG ion channel on the transfected HEK293 cell surface at the single-cell level. This hERG ion channel profiling strategy promises a maneuverable tool for ion channel research.

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients.

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.

Nephrotoxicity and genotoxicity of silver nanoparticles in juvenile rats and possible mechanisms of action.

Because of their widespread use and potential adverse effects in young developing organism, this study focused on the nephrotoxicity and genotoxicity of chronic low-dose exposure to silver nanoparticles (AgNPs) in 32 14-day-old male Wistar rats, randomly divided into three groups receiving AgNP solution (3 mg/kg body weight) intraperitoneally for one, two, or three weeks and the untreated control group (eight animals per group). When the rats were eight weeks old, blood creatinine and urine microalbumin were tested, followed by haematoxylin and eosin (H&E) staining. Proteinuria was found in the animals treated with AgNP for three weeks, and H&E staining revealed pathological changes in the kidney sections of this group. DNA damage was detected with the alkaline comet assay in the groups treated for two and three weeks. All results indicate that chronic exposure, even at a low dose, may affect animal health. The main culprit might be increased and time-dependent reactive oxygen species (ROS) production. Highly reactive ROS could cause a major structural damage to proteins and DNA, change the expression of ion channel proteins, and trigger inflammation. The findings of our in vivo experiment raise concern about nephrotoxic and genotoxic effects of silver nanoparticles in young organisms and call for further investigation of nanoparticle properties that can be modified to minimise the risks.

Molecular characterization, expression analysis of 14-3-3 beta/alpha and the effect of RNA interference on ion transporter protein Na+-K+-ATPase, Na+-H+-exchanger and CFTR in turbot (Scophthalmus maximus).

To understand the role of 14-3-3 beta/alpha in hypoosmotic regulation of turbot (Scophthalmus maximus), we characterized the 14-3-3 beta/alpha gene and analyzed the tissue distribution and its gene transcriptional patterns in the main expressed tissues under low salt stress. The 14-3-3 beta/alpha cDNA is 892bp in length, incorporating an ORF of 774bp with a putative primary structure of 257 residues. The deduced amino acid sequences shared highly conserved structures with other eukaryotes. Quantitative real-time PCR results showed that the 14-3-3 beta/alpha transcripts were widely expressed in various tissues of turbot, with most abundant in the gill (P<.05), to a lesser extent in the kidney, intestine, brain and spleen, and at low levels in the pituitary and other tissues examined. And the expression of turbot 14-3-3 beta/alpha exhibited a trend of increasing first and then decreasing with the time of stress under low salt stress, and the highest value appeared in 12h (P<.05). After injecting different concentrations of dsRNA, the mRNA expression of 14-3-3 gene decreased significantly during the monitoring period, and the best interference effect was achieved 12h after injecting 4μg/g dsRNA. For the first time, the gene was silenced in fish by intramuscular injection of dsRNA. It also provides a new and effective way to study gene function at the individual level. Moreover, the mRNA interference of 14-3-3 beta/alpha would cause changes in the expression of several ion channel proteins, for example, the decrease of Na+-K+-ATPase and Na+-H+-exchanger and the increase of CFTR. As a result, 14-3-3 beta/alpha appears to be an important molecular regulator for osmosensory signal transduction in gill of turbot.

Neuropilin 1 ameliorates electrical remodeling at infarct border zones in rats after myocardial infarction.

Electrical remodeling at infarct border zone (IBZ) has been shown to contribute to the occurrence of ventricular arrhythmias after myocardial infarction (MI). Sema3A has been demonstrated to reduce the inducibility of ventricular arrhythmias. Neuropilin 1 (NRP1) is the receptor of Sema3A. In the present study, we investigated whether treatment with NRP1 can ameliorate electrical remodeling at IBZ after MI. Wistar rats underwent sham operation (n = 20), the ligation of left coronary artery (MI group, n = 30), MI with control adenovirus (Ad group, n = 30), and MI with NRP1 adenovirus (NRP1 group, n = 30). Eight weeks after treatment, electrophysiological properties including heart rate variability (HRV), monophasic action potential duration (MAPD), effective refractory period (ERP) and the inducibility of ventricular arrhythmias and the expression of arrhythmia-related ion channel proteins including Kv4.2, Kv4.3, KChIP2 and Kir2.1 at the IBZ of the left ventricle were examined. Compared with the MI or Ad group, NRP1 significantly increased HRV and shortened MAPD and ERP (all P < 0.05). Inducibility of VT by electrophysiological study was significantly lower in the NRP1 group than in the MI or Ad group (P < 0.05). The expression levels of Kv4.2, Kv4.3, KChIP2 and Kir2.1 proteins were significantly decreased in MI group and Ad group. In contrast, the expression levels of these proteins were restored in NRP1 group, which may represent the molecular basis of the NRP1-mediated inhibition of electrical remodeling. NRP1 can ameliorate electrical remodeling at IBZ after MI.

Pharmacological Modulation of Kir3.1 and Kir3.4 Cell Surface Expression

BackgroundSmall molecules have been shown to pharmacologically modulate the cell surface expression of ion channel proteins. We thus investigated the effects of the antimalarial aminoquinolines Chloroquine and Primaquine on the cell surface density of Kir3.1/Kir3.4 proteins which mediate the acetylcholine activated inward rectifier potassium current (IKACh) in the heart. We compared the effects of Chloroquine and Primaquine to those of known lysosomal inhibitors‐ ammonium chloride (NH4Cl) and Leupeptin‐ on the surface density of the IKACh proteins. We hypothesized that long term Chloroquine treatment increases the surface expression of Kir3.1/Kir3.4 independently of the lysosomal pathway.MethodsExperiments were performed in human embryonic kidney (HEK293) cells stably transfected with Kir3.1 and Kir3.4. ATTO‐488 conjugated TertiapinQ (a potent blocker of IKACh) was used to label the IKACh proteins in live cells, and flow cytometry was used to quantify their expression on the cell surface after 24 hours treatment with 1, 10 and 50 μM Chloroquine, 10 μM Primaquine, 10 mM NH4Cl, and 300 μM Leupeptin. Endosomal/lysosomal pH was also measured in flow cytometry, using the pH‐sensitive pHrodo Green Dextran 10,000 MW dye conjugate. Western blotting was done after overnight treatment with Chloroquine and Leupeptin to determine the total levels of Kir3.1 and Kir3.4, and of MAP LC3α/β, a protein known to undergo lysosomal degradation. Results are reported as averages ± SD.ResultsIn flow cytometry experiments, Chloroquine at 1 μM, 10 μM and 50 μM showed a dose dependent increase in Kir3.1/Kir3.4 surface expression (5.6 % ± 6 vs. control, n.s.; 31.7% ± 18, p&lt;0.01 vs. control, and 54.4% ± 18, p&lt;0.01 vs. control). Moreover, treatment with 10 μM Primaquine, a compound structurally similar to Chloroquine, did not show a significant increase compared to vehicle treatment. The lysosomal inhibitor Leupeptin (300 μM) did not significantly increase cell surface IKACh channels expression compared to control. On the other hand, disruption of lysosomal pH with 10 mM NH4Cl significantly increased cell surface expression of Kir3.1/Kir3.4, but the increase was less pronounced compared to that of Chloroquine (24% ± 18 vs 32.7% ± 11.7, p&lt;0.05). We subsequently examined the influence of Chloroquine, Primaquine, NH4Cl and Leupeptin on the endosomal/lysosomal pH. Endosomal/lysosomal pH was significantly increased (indicated by a decrease in pH‐sensitive pHrodo Green Dextran fluorescence) by NH4Cl and Primaquine versus control (1003 A.U. ± 133 and 1063 A.U. ± 115, vs 1368 A.U. ± 165, p&lt;0.05) but not by Chloroquine and Leupeptin (1520 A.U. ± 199 and 1424 A.U. ± 151, vs 1368 A.U. ± 165, n.s.). Finally, in western blots, Chloroquine and Leupeptin did not significantly affect Kir3.1 and Kir3.4 total protein levels, however, Chloroquine significantly increased MAP LC3α/β levels.ConclusionLong term Chloroquine treatment promotes cell surface expression of Kir3.1 and Kir3.4. Accumulation of the IKACh mediating proteins at the cell surface appears to be independent of the possible effects of Chloroquine on lysosomal function.Support or Funding InformationFunded in part by NIH grant R01HL129136 to SFN

Mechanisms of persistent atrial fibrillation

Atrial fibrillation is the most common sustained arrhythmia, but its mechanisms are poorly understood. In particular, little is known about the factors that contribute to the establishment of persistent or permanent atrial fibrillation. This review addresses possible common signaling pathways that might promote both structural and electrical remodeling of the atria, thus contributing to atrial fibrillation perpetuation. Sustained atrial fibrillation may trigger an inflammatory response leading to activation of myofibroblasts and to the release of cytokines such as transforming growth factor-β and platelet-derived growth factor, as well as profibrotic proteins such as galectin-3. Activation of signaling cascades involving such proteins is critical for the development of fibrosis and may also lead to ion channel dysfunction, which, along with myocyte apoptosis and extracellular matrix generation and turnover, likely contributes to both electrical and structural remodeling and predisposes to atrial fibrillation. Identifying upstream strategies targeting molecular pathways that are common to fibrosis and electrical remodeling leading to atrial fibrillation perpetuation is highly desirable. This would facilitate finding new target genes with pleiotropic effects on the expression of ion channel proteins in myocytes and profibrotic molecules in nonmyocyte cells that are important for pathologic remodeling, which could become an important goal in persistent atrial fibrillation therapy.

Ion selectivity of pore-forming peptides and ion channels measured in Xenopus oocytes.

The Xenopus laevis oocyte is a widely used system for heterologous expression of exogenous ion channel proteins. They are easy to obtain, mechanically and electrically stable, have a large size, enabling multiple types of electrophysiological recordings: two-electrode voltage clamp, single cell-attached or cell-free patch-clamp and macropatch recordings. The size of an oocyte (1 mm in diameter) also allows for the use of additional electrodes (from 1 to 3) for injection of diverse materials (Ca(2+) chelators, peptides, chemicals, antibodies, proteic-partners, etc.) before or during the course of the electrophysiological experiment.We have successfully used this system to analyze the biophysical properties of pore-forming peptides. Simple extracellular perfusion of these peptides induced the formation of channels in the oocyte plasma membrane; these channels can then be studied and characterized in diverse ionic conditions. The ease of the perfusion and the stability of the voltage-clamped oocyte make it a powerful tool for such analyses. Compared to artificial bilayers, oocytes offer a real animal plasma membrane where biophysical properties and toxicity can be studied in the stable environment.

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Non genetic risk factors of long-QT syndrome

The purpose of the present study is to provide guidelines regarding risk factors that may worsen the Long-QT Syndrome (LQTS), based on available literature. This review evaluates the current knowledge on these risk factors of acquired LQTS, with an emphasis on non genetic risk factors, including environmental factors. PubMed was searched for literature in English from 1999 to 2011 on the molecular and clinical studies of Long-QT syndrome. We agree, with recent investigations described in the literature, that variety of factors, inherited or environmental, can influence expression of ion channel proteins with impact on repolarization.

Age-dependent changes in ion channel mRNA expression in canine cardiac tissues

The expression pattern of cardiac ion channels displays marked changes during ontogeny. This study was designed to follow the developmental changes in the expression of major ventricular and atrial ion channel proteins (including both pore forming and regulatory subunits) in canine cardiac tissues at the mRNA level using competitive reverse transcription polymerase chain reaction. Therefore, the corresponding mRNA levels were compared in myocardial tissues excised from embryonic (25-60 days of gestation) and adult (2-3 years old) canine hearts. Expression level of Kv4.3, Kv1.4, KChIP2, KvLQT1, and Cav3.2 mRNAs were higher in the adult than in the embryonic hearts, while expression of Nav1.5 and minK mRNAs were higher in the embryonic than in the adult myocardium. No change in Kir2.1, HERG, Kv1.5, and Cav1.2 mRNA was observed during ontogeny. Direction of the developmental change in the mRNA level, determined for any specific channel protein, was identical in the atrial and ventricular samples. The age-dependent increase observed in the expression of Kv4.3, Kv1.4, KChIP2, and KvLQT1 is congruent with the greater repolarization reserve of the adult myocardium, associated with higher densities of Ito and IKs. The results indicate that age-dependent changes in the expression pattern of many ion channels are similar in canine and healthy human myocardium, therefore, canine cardiac muscle can be considered as a good model of studying developmental changes in the human heart.

Epithelial-to-mesenchymal transformation alters electrical conductivity of human epicardial cells

The myocardium of the developing heart tube is covered by epicardium. These epicardial cells undergo a process of epithelial-to-mesenchymal transformation (EMT) and develop into epicardium-derived cells (EPDCs). The ingrowing EPDCs differentiate into several celltypes of which the cardiac fibroblasts form the main group. Disturbance of EMT of the epicardium leads to serious hypoplasia of the myocardium, abnormal coronary artery differentiation and Purkinje fibre paucity. Interestingly, the electrophysiological properties of epicardial cells and whether EMT influences electrical conductivity of epicardial cells is not yet known. We studied the electrophysiological aspects of epicardial cells before and after EMT in a dedicated in vitro model, using micro-electrode arrays to investigate electrical conduction across epicardial cells. Therefore, human adult epicardial cells were placed between two neonatal rat cardiomyocyte populations. Before EMT the epicardial cells have a cobblestone (epithelium-like) phenotype that was confirmed by staining for the cell-adhesion molecule β-catenin. After spontaneous EMT in vitro the EPDCs acquired a spindle-shaped morphology confirmed by vimentin staining. When comparing both types we observed that the electrical conduction is influenced by EMT, resulting in significantly reduced conductivity of spindle-shaped EPDCs, associated with a conduction block. Furthermore, the expression of both gap junction (connexins 40, Cx43 and Cx45) and ion channel proteins (SCN5a, CACNA1C and Kir2.1) was down-regulated after EMT. This study shows for the first time the conduction differences between epicardial cells before and after EMT. These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.

Gap junctions and arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy is the most arrhythmogenic form of human heart disease and a major cause of sudden death in the young.1,2 It was first described as a right ventricular disease (arrhythmogenic right ventricular cardiomyopathy or ARVC), but is now recognized to include biventricular and left dominant forms which may be misdiagnosed as dilated cardiomyopathy or myocarditis. Arrhythmias occur early in the natural history of arrhythmogenic cardiomyopathy, often preceding structural remodeling of the myocardium.1, 2 This so-called “concealed phase” of the disease is unique among the primary myocardial disorders. In hypertrophic cardiomyopathy, for example, arrhythmic risk is related to the underlying substrate of myocyte disarray, hypertrophy, fibrosis and small-vessel disease. And in dilated cardiomyopathy, arrhythmias generally arise in the context of significant ventricular dilatation and contractile dysfunction accompanied by changes in the expression, activity and spatial distribution of ion channel proteins and currents. In contrast, there is something fundamentally arrhythmogenic about arrhythmogenic cardiomyopathy particularly in its early stage in which frequent arrhythmias arise in otherwise apparently normal hearts. In this sense, arrhythmogenic cardiomyopathy is more reminiscent of the ion channelopathies than other forms of non-ischemic cardiomyopathy. Arrhythmogenic cardiomyopathy has been linked to mutations in genes encoding desmosomal proteins (PKP2, DSG2, DSC2, DSP and JUP).1, 2 Desmosomes are cell-cell adhesion organelles. They are particularly abundant in heart and skin, tissues that normally experience mechanical stress. Not surprisingly therefore, clinical phenotypes in patients with desmosomal mutations take the form of myocardial and cutaneous diseases. In fact, patients with arrhythmogenic cardiomyopathy often exhibit disease flares in response to stress or exercise, emphasizing the importance of biomechanical determinants of disease. However, the relationship between changes in biomechanical properties of the myocardium produced by desmosomal mutations and heart rhythm abnormalities in arrhythmogenic cardiomyopathy is poorly characterized. Advances in understanding this relationship could reveal important basic mechanisms of arrhythmogenesis.

Semaphorin 3A Attenuates Electrical Remodeling at Infarct Border Zones in Rats after Myocardial Infarction

Electrical remodeling at infarct border zone has been shown to contribute to the occurrence of ventricular arrhythmias after myocardial infarction (MI). Electrical remodeling is causally associated with sympathetic neural remodeling in MI. Semaphorin 3A (Sema3A), a potent neural chemorepellent for sympathetic axons, has been demonstrated to suppress sympathetic neural remodeling after MI. In the present study, we investigated whether treatment with Sema3A can ameliorate electrical remodeling at infarct border zones using a rat model of MI. Wistar rats underwent sham operation (n = 20), the ligation of left coronary artery (MI group, n = 30), MI with control adenovirus (Ad group, n = 30), and MI with Sema3A adenovirus (Sema3A group, n = 30). Eight weeks after treatment, electrophysiological properties including heart rate variability (HRV), monophasic action potential duration (MAPD) and effective refractory period (ERP) and the expression of arrhythmia-related ion channel proteins including Kv4.2, KChIP2 and Kir2.1 at the infarcted border of the left ventricle were examined. These channel proteins may be required for maintaining normal heart rhythm. Compared with the Ad group, Sema3A significantly increased HRV and shortened MAPD and ERP (all p < 0.05). The expression levels of Kv4.2, KChIP2 and Kir2.1 proteins were significantly decreased in MI group and Ad group, compared to sham control. In contrast, the expression levels of these proteins were restored in Sema3A group, which may represent the molecular basis of the Sema3A-mediated inhibition of electrical remodeling. In conclusion, Sema3A can ameliorate electrical remodeling at infarct border zones after MI.