Acute and Sub-Chronic Intraperitoneal Toxicity Studies of the Elsholtzia ciliata Herbal Extract in Balb/c Mice.

Elsholtzia ciliata essential oil (E. ciliata) has been reported to have an impact on the cardiovascular system. However, its toxicity remains unknown. Therefore, the objective of this investigation was to evaluate the toxicological aspects of the E. ciliata extract. Male Balb/c mice were subjected to either acute (a single dose administered for 24 h) or sub-chronic (daily dose for 60 days) intraperitoneal injections of the E. ciliata extract. The mice were assessed for blood hematological/biochemical profiles, mitochondrial functions, and histopathological changes. Additionally, in vitro cytotoxicity assessments of the E. ciliata extract were performed on immobilized primate kidney cells (MARC-145, Vero) and rat liver cells (WBF344) to evaluate cell viability. The control groups received an equivalent volume of olive oil or saline. Our results demonstrated no significant detrimental effects on hematological and biochemical parameters, mitochondrial functions, cellular cytotoxicity, or pathological alterations in vital organs following the intraperitoneal administration of the E. ciliata extract over the 60-day sub-chronic toxicity study. In general, E. ciliata displayed no indications of toxicity, suggesting that the E. ciliata extract is a safe natural product with a well-defined therapeutic and protective index (found to be 90 and 54, respectively) in Balb/c mice.

Evaluation of the Cardiac Electrophysiological and Haemodynamic Effects of Elsholtzia ciliata Essential Oil on Swine.

The demand for the development of novel medicines with few side effects and no proarrhythmic properties is increasing. Extensive research on herbal extracts has been conducted with the expectation that the compounds will exert precise effects without harmful side effects. Elsholtzia ciliata (Thunb.) Hyl. essential oil (EO) possesses antiarrhythmic properties similar to those of class 1B antiarrhythmics, such as prolonging myocardial activation of the QRS complex and shortening the QT interval. In this study, we determined the kinetic profile of EO phytocompounds and the effects of EO on heart electrical activity and arterial blood pressure. For this study, we chose to use local breed pigs that were anaesthetized. The effects of an intravenous bolus of EO on ECG parameters, arterial blood pressure, heart rate variability, and blood levels of haematological and biochemical parameters were registered and evaluated. Following an intravenous injection of a bolus, EO exerted a vasodilatory effect, resulting in significant reductions in arterial blood pressure. EO also increased the heart rate and altered ECG parameters. The bolus of EO prolonged the QRS complex, shortened the QT interval, and nonmonotonically altered the PQ interval. After the administration of a bolus of EO, the activity of the autonomic nervous system was altered. This study confirms that EO possesses similar properties to class 1B antiarrhythmics and exerts a hypotensive effect; it reduces arterial blood pressure possibly by modulating peripheral vascular resistance.

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.

Optical mapping of the pig heart in situ under artificial blood circulation.

The emergence of optical imaging has revolutionized the investigation of cardiac electrical activity and associated disorders in various cardiac pathologies. The electrical signals of the heart and the propagation pathways are crucial for elucidating the mechanisms of various cardiac pathological conditions, including arrhythmia. The synthesis of near-infrared voltage-sensitive dyes and the voltage sensitivity of the FDA-approved dye Cardiogreen have increased the importance of optical mapping (OM) as a prospective tool in clinical practice. We aimed to develop a method for the high-spatiotemporal-resolution OM of the large animal hearts in situ using di-4-ANBDQBS and Cardiogreen under patho/physiological conditions. OM was adapted to monitor cardiac electrical behaviour in an open-chest pig heart model with physiological or artificial blood circulation. We detail the methods and display the OM data obtained using di-4-ANBDQBS and Cardiogreen. Activation time, action potential duration, repolarization time and conduction velocity maps were constructed. The technique was applied to track cardiac electrical activity during regional ischaemia and arrhythmia. Our study is the first to apply high-spatiotemporal-resolution OM in the pig heart in situ to record cardiac electrical activity qualitatively under artificial blood perfusion. The use of an FDA-approved voltage-sensitive dye and artificial blood perfusion in a swine model, which is generally accepted as a valuable pre-clinical model, demonstrates the promise of OM for clinical application.

Metabolic Inhibition Induces Transient Increase of L-type Ca2+ Current in Human and Rat Cardiac Myocytes.

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during ß-adrenergic stimulation by isoprenaline, a non-selective agonist of ß-adrenergic receptors. The whole-cell patch⁻clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.

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.

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome.

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl-]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

Metabolic inhibition reduces cardiac L-type Ca2+ channel current due to acidification caused by ATP hydrolysis.

Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca2+ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (ICa,L) which was more pronounced during ß-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.

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.

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.

Differences in the control of basal L-type Ca(2+) current by the cyclic AMP signaling cascade in frog, rat, and human cardiac myocytes.

ß-adrenergic receptors (ß-ARs) mediate the positive inotropic effects of catecholamines by cAMP-dependent phosphorylation of the L-type Ca(2+) channels (LTCCs), which provide Ca(2+) for the initiation and regulation of cell contraction. The overall effect of cAMP-modulating agents on cardiac calcium current (I Ca,L) and contraction depends on the basal activity of LTCCs which, in turn, depends on the basal activities of key enzymes involved in the cAMP signaling cascade. Our current work is a comparative study demonstrating the differences in the basal activities of ß-ARs, adenylyl cyclase, phosphodiesterases, phosphatases, and LTCCs in the frog and rat ventricular and human atrial myocytes. The main conclusion is that the basal I Ca,L, and consequently the contractile function of the heart, is secured from unnecessary elevation of its activity and energy consumption at the several "checking-points" of cAMP-dependent signaling cascade and the loading of these "checking-points" may vary in different species and tissues.

Evaluation of excitation propagation in the rabbit heart: optical mapping and transmural microelectrode recordings.

BACKGROUND: Because of the optical features of heart tissue, optical and electrical action potentials are only moderately associated, especially when near-infrared dyes are used in optical mapping (OM) studies. OBJECTIVE: By simultaneously recording transmural electrical action potentials (APs) and optical action potentials (OAPs), we aimed to evaluate the contributions of both electrical and optical influences to the shape of the OAP upstroke. METHODS AND RESULTS: A standard glass microelectrode and OM, using an near-infrared fluorescent dye (di-4-ANBDQBS), were used to simultaneously record transmural APs and OAPs in a Langendorff-perfused rabbit heart during atrial, endocardial, and epicardial pacing. The actual profile of the transmural AP upstroke across the LV wall, together with the OAP upstroke, allowed for calculations of the probing-depth constant (k ~2.1 mm, n = 24) of the fluorescence measurements. In addition, the transmural AP recordings aided the quantitative evaluation of the influences of depth-weighted and lateral-scattering components on the OAP upstroke. These components correspond to the components of the propagating electrical wave that are transmural and parallel to the epicardium. The calculated mean values for the depth-weighted and lateral-scattering components, whose sum comprises the OAP upstroke, were (in ms) 10.18 ± 0.62 and 0.0 ± 0.56 for atrial stimulation, 9.37 ± 1.12 and 3.01 ± 1.30 for endocardial stimulation, and 6.09 ± 0.79 and 8.16 ± 0.98 for epicardial stimulation; (n = 8 for each). For this dye, 90% of the collected fluorescence originated up to 4.83 ± 0.18 mm (n = 24) from the epicardium. CONCLUSIONS: The co-registration of OM and transmural microelectrode APs enabled the probing depth of fluorescence measurements to be calculated and the OAP upstroke to be divided into two components (depth-weighted and lateral-scattering), and it also allowed the relative strengths of their effects on the shape of the OAP upstroke to be evaluated.

Evolution of action potential alternans in rabbit heart during acute regional ischemia.

This study investigates the development of the spatiotemporal pattern of action potential alternans during acute regional ischemia. Experiments were carried out in isolated Langendorff-perfused rabbit heart using a combination of optical mapping and microelectrode recordings. The alternans pattern significantly changed over time and had a biphasic character reaching maximum at 6-9 min after occlusion. Phase I (3-11 minutes of ischemia) is characterized by rapid increase in the alternans magnitude and expansion of the alternans territory. Phase I is followed by gradual decline of alternans (Phase II) in both magnitude and territory. During both phases we observed significant beat-to-beat variations of the optical action potential amplitude (OAPA) alternans. Simultaneous microelectrode recordings from subepicardial and subendocardial layers showed that OAPA alternans coincided with intramural 2 : 1 conduction blocks. Our findings are consistent with the modeling studies predicting that during acute regional ischemia alternans can be driven by 2 : 1 conduction blocks in the ischemic region.

Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction.

Acute myocardial infarction is one of the major causes of mortality worldwide. For regeneration of the rabbit heart after experimentally induced infarction we used autologous skeletal myoblasts (SMs) due to their high proliferative potential, resistance to ischaemia and absence of immunological and ethical concerns. The cells were characterized with muscle-specific and myogenic markers. Cell transplantation was performed by injection of cell suspension (0.5 ml) containing approximately 6 million myoblasts into the infarction zone. The animals were divided into four groups: (i) no injection; (ii) sham injected; (iii) injected with wild-type SMs; and (iv) injected with SMs expressing connexin43 fused with green fluorescent protein (Cx43EGFP). Left ventricular ejection fraction (LVEF) was evaluated by 2D echocardiography in vivo before infarction, when myocardium has stabilized after infarction, and 3 months after infarction. Electrical activity in the healthy and infarction zones of the heart was examined ex vivo in Langendorff-perfused hearts by optical mapping using di-4-ANEPPS, a potential sensitive fluorescent dye. We demonstrate that SMs in the coculture can couple electrically not only to abutted but also to remote acutely isolated allogenic cardiac myocytes through membranous tunnelling tubes. The beneficial effect of cellular therapy on LVEF and electrical activity was observed in the group of animals injected with Cx43EGFP-expressing SMs. L-type Ca(2+) current amplitude was approximately fivefold smaller in the isolated SMs compared to healthy myocytes suggesting that limited recovery of LVEF may be related to inadequate expression or function of L-type Ca(2+) channels in transplanted differentiating SMs.

R-type calcium channels are crucial for semaphorin 3A-induced DRG axon growth cone collapse.

Semaphorin 3A (Sema3A) is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca2+ dependent process. Intracellular Ca2+ imaging results using Fura-2 AM showed Ca2+ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca2+ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca2+ channel blocker NiCl2 and by the selective R-type Ca2+ channel blocker SNX482. These Ca2+ channel blockers consistently decreased the Sema3A-induced intracellular Ca2+ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.

ß3-Adrenergic regulation of L-type Ca²âº current and force of contraction in human ventricle.

ß3-Adrenergic receptor (ß3-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca(2+) current (I(Ca,L)) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of ß3-AR agonist CGP12177 which also is a ß1-AR/ß2-AR antagonist on I(Ca,L) in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I(Ca,L) in HVMs with high potency but much lower efficacy than isoprenaline. The ß3-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on ß3-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I(Ca,L), while CGP12177 completely blocked the effect of isoprenaline. The activation of ß3-ARs by CGP12177 does not involve the activation of Gi proteins because CGP12177 had no effect on forskolin-induced stimulation of I(Ca,L). CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I(Ca,L) in HVMs. We conclude that in human ventricles ß3-AR activation has no inotropic effect, while it slightly increases I(Ca,L). In contrast to human atrium, the activation of ß3-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.

DRG axon elongation and growth cone collapse rate induced by Sema3A are differently dependent on NGF concentration.

Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon's regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.

Optical mapping at increased illumination intensities.

Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW/mm2, with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.

Src family protein tyrosine kinases modulate L-type calcium current in human atrial myocytes.

In the heart, L-type voltage dependent calcium channels (L-VDCC) provide Ca(2+) for the activation of contractile apparatus. The best described pathway for L-type Ca(2+) current (I(Ca,L)) modulation is the phosphorylation of calcium channels by cAMP-dependent protein kinase A (PKA), the activity of which is predominantly regulated in opposite manner by ß-adrenergic (ß-ARs) and muscarinic receptors. The role of other kinases is controversial and often depends on tissues and species used in the studies. In different studies the inhibitors of tyrosine kinases have been shown either to stimulate or inhibit, or even have a biphasic effect on I(Ca,L). Moreover, there is no clear picture about the route of activation and the site of action of cardiac Src family nonreceptor tyrosine kinases (Src-nPTKs). In the present study we used PP1, a selective inhibitor of Src-nPTKs, alone and together with different activators of I(Ca,L), and demonstrated that in human atrial myocytes (HAMs): (i) Src-nPTKs are activated concomitantly with activation of cAMP-signaling cascade; (ii) Src-nPTKs attenuate PKA-dependent stimulation of I(Ca,L) by inhibiting PKA activity; (iii) Gα(s) are not involved in the direct activation of Src-nPTKs. In this way, Src-nPTKs may provide a protecting mechanism against myocardial overload under conditions of increased sympathetic activity.

Influence of plasmid concentration on DNA electrotransfer in vitro using high-voltage and low-voltage pulses.

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 microl of CHO cell suspension containing 100, 10 or 1 microg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 microg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.