Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

The cardiac Mg2+-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg2+ concentration ([Mg2+]i) to activate the Mg2+-sensitive channels, raising extracellular [Mg2+] ([Mg2+]o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg2+]i. Under voltage clamp, in cells dialyzed with zero [Mg2+]i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg2+]o and was absent in cells dialyzed with physiological [Mg2+]i. In cells dialyzed with physiological [Mg2+]i, raising [Mg2+]o decreased the L-type Ca2+ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg2+-sensitive channels, and also suggest that the cardiac Mg2+-sensitive current shortens the APD, with potential implications in arrhythmogenesis

Risk factors for vancomycin resistant enterococcus infection development

Aim of the study: Identify risk factors in patients with infections caused by vancomycin-resistant enterococci. Objectives: 1. Identify risk factors in patients with infections caused by vancomycin-resistant Enterococcus spp. 2. Determine the influence of antibiotic in patients with infections caused by vancomycin-resistant Enterococcus spp. 3. Rate differences in antibiotic resistance, depending on the isolates of Enterococcus spp. types and risk factors of Enterococcus faecalis and Enterococcus faecium infections development. Object of the work: 49 patients who in test substance was isolated Enterococcus spp. and who were treated in intensive care, surgical or therapeutic sections in Kaunas Clinics Hospital in 2015. Methods: From sourses grown bacteria were indentified on 5 percent blood agar using a mass spectrometer ,,Maldi_Tof‘‘ (BRUKER Corporation, JAV). Resistance to antibiotics has been completed by the disc diffusion method. Results: During period from clinical samples were isolated VSE (53,1 percent) and VRE (46,9 percent). 15,4 percent (n=4) of patients in VSE group and 43,5 percent of patients in VRE group (n=10) were treated in an intensive care unit (p=0,031). Peripheral venous catheter had 65,4 percent (n=17) patients of VSE group and 73,9 percent (n=17) patients of VRE group (p=0,370). Surgical procedures have been applied to 88,5 percent (n=23) patients of VSE group and to 60,9 percent (n=14) patients of VRE group (p=0,027). Antibiotics use before infection in VSE group accounted for 92,3 percent (n=24) and for 100 percent (n=23) in VRE group (p=0,276). Patient‘s creatinine average value in VSE group was 102,4 ± 89,6 µmol/l and in VRE group was 132,6 ± 92,6 µmol/l (p=0,028). Patient‘s urea average value in VSE group was 7,7 ± 7,1 mmol/l and in VRE group was 10,1 ± 7,2 mmol/l, (p=0,03). Before infection patients used these antibiotics: carbapenem (7,7 percent, n=2 in VSE group and 47,8 percent, n=11 in VRE group, p=0,002); ciprofloxacin and ofloxacin (3,8 percent, n=1 in VSE group and 26,1 percent, n=6 in VRE group, p=0,033); vancomycin (7,7 percent, n=2 in VSE group and 47,8 percent, n=11 in VRE group, p=0,002); ampicillin/sublactam (3,8 percent, n=1 in VSE group and 13,0 percent, n=3 in VRE group, p=0,259). In 20,4 percent (n=10) of samples were identified E. faecalis and 79,6 percent (n=39) E. faecium. 0 percent of patients with E. faecalis and 35,9 percent (n=14) of patients with E. faecium were treated in intensive care unit, (p=0,022). 100 percent of patients with E. faecalis and 73,7 percent of patients with E. faecium were treated in surgery unit. E. faecalis counted for 10,0 percent (n=1) and E. faecium counted for 30,8 (n=18) in patients who treated with carbapenems before infection (p=0,180). E. faecalis counted for 10,0 percent (n=1) and E. faecium counted for 15,4 (n=6) in patients who treated with ciprofloxacin and ofloxacin before infection (p=0,559). E. faecalis counted for 20,0 percent (n=2) and E. faecium counted for 28,2 (n=11) in patients who treated with vancomycin before infection (p=0,469). E. faecalis counted for 0 percent and E. faecium counted for 51,3 (n=20) in patients who treated with ampicillin/sublactam before infection (p=0,388). Conclusions: 1. More often vancomycin-resistant strains have been identified in patients who were treated in the intensive care unit and who had dysfunction of kidney – increased creatinine and urea rates. 2. More often vancomycin-resistant strains have been identified in patients who were treated with carbapenem, ciprofloxacin and vancomycin before infection. 3. E. faecium more often has resistant to ampicillin, vancomycin and teicoplanin compared with E. faecalis, but infections in intensive care unit more often caused by E. faecium

Non-homogeneous distribution of TRPM7 in human ventricular cardiomyocytes

Introduction: TRPM7 (transient receptor potential melastanin 7) gene appears to be ubiquitously expressed, with highest expression in heart tissues, and participate in a variety of physiological/ pathological processes. However the direct evidence for the presence of TRPM7 proteins in human ventricular cardiomyocytes is still lacking. Aim: To show the presence of TRPM7 protein in human ventricular cardiomyocytes, using immunofluorescence. Since the issue of antibody specificity is a very critical one, for this reason we aimed to validate this procedure as much as possible with the different antibodies. Materials and methods: We used enzymatically dissociated cardiomyocytes. After fixation, permeabilization and prevention of non-specific binding using blocking buffer, cells were incubated with primary mouse monoclonal or rabbit polyclonal anti TRPM7 antibody (1:200) in blocking buffer overnight at 4oC. For negative control, incubation with primary antibody was omitted. Further, the cells were incubated with fluorescently labelled secondary antibody AF488 (1:200), co stained with Phaloidin (1:100) and with Hoechst (25µg/mL), for labelling of the F actin cytoskeleton for contrast staining, and of the nucleus, respectively. Glass-slides were covered with Anti-fade Reagent. Results: The confocal laser scanning microscopy images prove that TRPM7 can be detected on the surface membrane of human ventricular cardiomyocytes with both monoclonal and polyclonal anti TRPM7 antibodies. Data revealed that TRPM7 is inhomogeneously expressed in the plasma membrane, with higher expression at lateral or end to end inter-connections between single ventricular cardiomyocytes. No consistent difference was revealed between the staining of cells obtained from the same patient, when using different antibodies. In addition, there was no staining visible in negative control samples indicating that TRPM7 was labelled specifically by the antibo [...]

Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart

Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the ICaL and transient outward potassium current (Ito) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg2+ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K+ current (IK1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the ICaL, Ito, and IK1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH

Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

Magnesium-sensitive transient receptor potential melastatin (TRPM) ion channels, TRPM6 and TRPM7, are present in several organs, but their roles in the heart remain unclear. Therefore, here, we studied the expression patterns of TRPM6 and TRPM7 in normal and diseased myocardium. Cardiac atrial tissue and cardiomyocytes were obtained from healthy pigs and undiseased human hearts as well as from hearts of patients with ischemic heart disease (IHD) or atrial fibrillation (AF). Immunofluorescence and ELISA were used to detect TRP proteins. TRPM6 and TRPM7 immunofluorescence signals, localized at/near the cell surface or intracellularly, were detected in pig and human atrial tissues. The TRP channel modulators carvacrol (CAR, 100 µM) or 2-aminoethoxydiphenyl borate (2-APB, 500 µM) decreased the TRPM7 signal, but enhanced that of TRPM6. At a higher concentration (2 mM), 2-APB enhanced the signals of both proteins. TRPM6 and TRPM7 immunofluorescence signals and protein concentrations were increased in atrial cells and tissues from IHD or AF patients. TRPM6 and TRPM7 proteins were both detected in cardiac atrial tissue, with relatively similar subcellular localization, but distinctive drug sensitivity profiles. Their upregulated expression in IHD and AF suggests a possible role of the channels in cardiac atrial disease

Antiarrhythmic Properties of Elsholtzia ciliata Essential Oil on Electrical Activity of the Isolated Rabbit Heart and Preferential Inhibition of Sodium Conductance

Elsholtzia ciliata essential oil (E. ciliata) has been developed in Lithuania and internationally patented as exerting antiarrhythmic properties. Here we demonstrate the pharmacological effects of this herbal preparation on cardiac electrical activity. We used cardiac surface ECG and a combination of microelectrode and optical mapping techniques to track the action potentials (APs) in the Langendorff-perfused rabbit heart model during atrial/endo-/epi-cardial pacing. Activation time, conduction velocity and AP duration (APD) maps were constructed. E. ciliata increased the QRS duration and shortened QT interval of ECG at concentrations of 0.01–0.1 μL/mL, whereas 0.3 μL/mL (0.03%) concentration resulted in marked strengthening of changes. In addition, the E. ciliata in a concentration dependent manner reduced the AP upstroke dV/dtmax and AP amplitude as well as APD. A marked attenuation of the AP dV/dtmax and a slowing spread of electrical signals suggest the impaired functioning of Na+-channels, and the effect was use-dependent. Importantly, all these changes were at least partially reversible. Our results indicate that E. ciliata modulates cardiac electrical activity preferentially inhibiting Na+ conductance, which may contribute to its effects as a natural antiarrhythmic medicine

Image_4_Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.PDF

<p>Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the I_CaL and transient outward potassium current (I_to) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg²⁺ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K⁺ current (I_K1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the I_CaL, I_to, and I_K1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.</p

Table_1_Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.PDF

<p>Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the I_CaL and transient outward potassium current (I_to) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg²⁺ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K⁺ current (I_K1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the I_CaL, I_to, and I_K1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.</p

Image_3_Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.PDF

<p>Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the I_CaL and transient outward potassium current (I_to) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg²⁺ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K⁺ current (I_K1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the I_CaL, I_to, and I_K1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.</p

Image_5_Mechanism of Action Potential Prolongation During Metabolic Inhibition in the Whole Rabbit Heart.PDF

<p>Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the I_CaL and transient outward potassium current (I_to) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg²⁺ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K⁺ current (I_K1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the I_CaL, I_to, and I_K1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.</p