Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the 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 usedependent. 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.

Effect of Carvacrol, TRP Channels Modulator, on Cardiac Electrical Activity.

Despite the wide application of carvacrol (CAR) in medicines, dietary supplements, and foods, there is still insufficient electrophysiological data on the mechanisms of action of CAR, particularly with regard to heart function. Therefore, in this study, we attempted to elucidate whether CAR, whose inhibitory effect on both cardiac and vascular TRPM7 and L-type Ca2+ currents has been demonstrated previously, could modify cardiac electrical activity. We used a combination of optical mapping and microelectrode techniques to track the action potentials (APs) and the spread of electrical activity in a Langendorff-perfused rabbit heart model during atrial/endo/epicardial pacing. Simultaneously, ECG recordings were acquired. Because human trials on CAR are still lacking, we tested the action of CAR on human ventricular preparations obtained from explanted hearts. Activation time (AT), AP duration (APD), and conduction velocity maps were constructed. We demonstrated that at a low concentration (10 µM) of CAR, only marginal changes in the AP parameters were observed. At higher concentrations (≥100 µM), a decrease in AP upstroke velocity (dV/dt max), suggesting inhibition of Na+ current, and APD (at 50 and 90% repolarization) was detected; also slowing in the spread of electrical signals via the atrioventricular node was observed, suggesting impaired functioning of Ca2+ channels. In addition, a decrease in the T-wave amplitude was seen on the ECG, suggesting an impaired repolarization process. Nevertheless, those changes occurred without a significant impact on the resting membrane potential and were reversible. We suggest that CAR might play a role in modulating cardiac electrical activity at high concentrations.

Spectral characteristics of voltage-sensitive indocyanine green fluorescence in the heart.

Indocyanine green (ICG) fluorescent dye has been approved by the FDA for use in medical diagnostics. Recently, we demonstrated that ICG dye has voltage-sensitive properties with a dual-component (fast and slow) response in the Langendorff-perfused rabbit heart. Here, we extended our studies by showing the different spectral properties of both components for analysis of the fractional change in ICG fluorescence in response to voltage changes. We used light from four LEDs to obtain excitation; emission was measured using an EMCCD camera with band-pass filters and a spectrometer. We applied a graphical model with Gaussian functions to construct and evaluate the individual emission curves and calculated the voltage-sensitive portion of each component of the ICG fluorescence in the rabbit heart. The results revealed that each isolated component (fast and slow) emanates from a unique ICG pool in a different environment within the cell membrane and that each component is also composed of two constituents (ICG-monomeric and ICG-aggregated). We propose the existence of different voltage-sensitive mechanisms for the components: (I) electrochromism and field-induced reorientation for the fast component; and (II) field-induced dye squeezing that amplifies intermolecular interactions, resulting in self-quenching of the dye fluorescence, for the slow component.

Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations.

TRPM7 channels participate in a variety of physiological/pathological processes. TRPM7 currents are modulated by protons but opposing effects of external pH (pHo) (potentiation vs inhibition) have been reported. TRPM7 has been less studied in human cardiomyocytes than in heart-derived non-cardiomyocyte cells. We used the whole-cell patch-clamp technique on isolated human atrial cardiomyocytes to investigate the impact of an acidic pHo on the TRPM7 current. With voltage-dependent and other ion channels inhibited, cardiomyocytes were challenged with external acidification in either the presence or the absence of extracellular divalent cations. TRPM7 outward and inward currents were increased by acidic pHo in extracellular medium containing Ca2+ and Mg2+, but suppressed by acidic pHo in the absence of extracellular Ca2+ and Mg2+. The potentiating effect in the presence of extracellular divalents occurred at pHo below 6 and was voltage-dependent. The inhibitory effect in the absence of extracellular divalents was already marked at pHo of 6 and was practically voltage-independent. TRPM7 current density was higher in cardiomyocytes from patients with history of coronary vascular disease and the difference compared to cardiomyocytes from patients without history of myocardial ischemia increased with acidic pHo. We demonstrate that proton-induced modification of TRPM7 currents depends on the presence of extracellular Ca2+ and Mg2+. Variability of the TRPM7 current density in human cardiomyocytes is related to the clinical history, being higher in atrial fibrillation and in ischemic cardiomyopathy.

Voltage-Sensitive Fluorescence of Indocyanine Green in the Heart.

So far, the optical mapping of cardiac electrical signals using voltage-sensitive fluorescent dyes has only been performed in experimental studies because these dyes are not yet approved for clinical use. It was recently reported that the well-known and widely used fluorescent dye indocyanine green (ICG), which has FDA approval, exhibits voltage sensitivity in various tissues, thus raising hopes that electrical activity could be optically mapped in the clinic. The aim of this study was to explore the possibility of using ICG to monitor cardiac electrical activity. Optical mapping experiments were performed on Langendorff rabbit hearts stained with ICG and perfused with electromechanical uncouplers. The residual contraction force and electrical action potentials were recorded simultaneously. Our research confirms that ICG is a voltage-sensitive dye with a dual-component (fast and slow) response to membrane potential changes. The fast component of the optical signal (OS) can have opposite polarities in different parts of the fluorescence spectrum. In contrast, the polarity of the slow component remains the same throughout the entire spectrum. Separating the OS into these components revealed two different voltage-sensitivity mechanisms for ICG. The fast component of the OS appears to be electrochromic in nature, whereas the slow component may arise from the redistribution of the dye molecules within or around the membrane. Both components quite accurately track the time of electrical signal propagation, but only the fast component is suitable for estimating the shape and duration of action potentials. Because ICG has voltage-sensitive properties in the entire heart, we suggest that it can be used to monitor cardiac electrical behavior in the clinic.