Cardiac Optical Mapping in Situ in Swine Models: A View of the Current Situation.

Optical mapping is recognized as a promising tool for the registration of electrical activity in the heart. Most cardiac optical mapping experiments are performed in ex vivo isolated heart models. However, the electrophysiological properties of the heart are highly influenced by the autonomic nervous system as well as humoral regulation; therefore, in vivo investigations of heart activity in large animals are definitely preferred. Furthermore, such investigations can be considered the last step before clinical application. Recently, two comprehensive studies have examined optical mapping approaches for pig hearts in situ (in vivo), likely advancing the methodological capacity to perform complex electrophysiological investigations of the heart. Both studies had the same aim, i.e., to develop high-spatiotemporal-resolution optical mapping suitable for registration of electrical activity of pig heart in situ, but the methods chosen were different. In this brief review, we analyse and compare the results of recent studies and discuss their translational potential for in situ cardiac optical mapping applications in large animals. We focus on the modes of blood circulation that are employed, the use of different voltage-sensitive dyes and their loading procedures, and ways of eliminating contraction artefacts. Finally, we evaluate the possible scenarios for optical mapping (OM) application in large animals in situ and infer which scenario is optimal.

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.

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.

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.

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.

Elsholtzia ciliata Essential Oil Exhibits a Smooth Muscle Relaxant Effect.

A recent in vivo study in pigs demonstrated the hypotensive properties of essential oil extracted from the blossoming plant Elsholtzia ciliata. This study was designed to examine the effect of E. ciliata essential oil (EO) on smooth muscle contraction. Tension measurements were performed on prostate strips and intact aortic rings isolated from rats. Results showed that EO caused a concentration-dependent reduction in phenylephrine-induced contraction of both the prostate and aorta, with a more pronounced inhibitory effect in the prostate. The IC50 of EO for the prostate was 0.24 ± 0.03 µL/mL (n = 10) and for the aorta was 0.72 ± 0.11 µL/mL (n = 4, p < 0.05 vs. prostate). The chromatographic analysis identified elsholtzia ketone (10.64%) and dehydroelsholtzia ketone (86.23%) as the predominant compounds in the tested EO. Since both compounds feature a furan ring within their molecular structure, other furan ring-containing compounds, 2-acetylfuran (2AF) and 5-methylfurfural (5MFF), were examined. For the first time, our study demonstrated the relaxant effects of 2AF and 5MFF on smooth muscles. Further, results showed that EO, 2AF, and 5MFF altered the responsiveness of prostate smooth muscle cells to phenylephrine. Under control conditions, the EC50 of phenylephrine was 0.18 ± 0.03 µM (n = 5), while in the presence of EO, 2AF, or 5MFF, the EC50 values were 0.81 ± 0.3 µM (n = 5), 0.89 ± 0.11 µM (n = 5), and 0.69 ± 0.23 µM (n = 4), respectively, p < 0.05 vs. control. Analysis of the affinity of EO for α1-adrenergic receptors in the prostate suggested that EO at a certain range of concentrations has a competitive antagonistic effect on α1-adrenergic receptors. In conclusion, EO elicits a relaxant effect on smooth muscles which may be related to the inhibition of α1-adrenoreceptors.

The Effect of Potassium Nitrate Supplementation on the Force and Properties of Extensor digitorum longus (EDL) Muscles in Mice.

Adding potassium nitrate (KNO3) to the diet improves the physiological properties of mammalian muscles (rebuilds weakened muscle, improves structure and functionality). The aim of this study was to investigate the effect of KNO3 supplementation in a mouse model. BALB/c mice were fed a KNO3 diet for three weeks, followed by a normal diet without nitrates. After the feeding period, the Extensor digitorum longus (EDL) muscle was evaluated ex vivo for contraction force and fatigue. To evaluate the possible pathological changes, the histology of EDL tissues was performed in control and KNO3-fed groups after 21 days. The histological analysis showed an absence of negative effects in EDL muscles. We also analyzed 15 biochemical blood parameters. After 21 days of KNO3 supplementation, the EDL mass was, on average, 13% larger in the experimental group compared to the controls (p < 0.05). The muscle-specific force increased by 38% in comparison with the control group (p < 0.05). The results indicate that KNO3 has effects in an experimental mouse model, showing nitrate-diet-induced muscle strength. This study contributes to a better understanding of the molecular changes in muscles following nutritional intervention and may help develop strategies and products designated to treat muscle-related issues.

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.

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current.

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

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.

Effect of inhibitors of mitochondrial respiratory chain complexes on the electromechanical activity in human myocardium.

UNLABELLED: THE AIM OF THE STUDY was to investigate the effect of inhibitors of mitochondrial respiratory chain complexes I, III, and IV on the electromechanical activity in human myocardium. MATERIAL AND METHODS: The experiments were performed on the human myocardial strips obtained from patients with heart failure (NYHA class III or IV) using a conventional method of registration of myocardial electromechanical activity. Under the perfusion with physiological Tyrode solution (control), contraction force (P) was 0.94±0.12 mN (n=16), relaxation time (t50) was 173.38±5.03 ms (n=15), action potential durations measured at 50% (AP50) and 90% (AP90) repolarization were 248.96±13.38 ms and 398.59±17.93 ms, respectively (n=13). RESULTS: The inhibition of respiratory chain complex I by rotenone (3 × 10⁻5 M, the highest concentration applied) decreased contraction force of human myocardium to 48.99%±14.74% (n=3) (P<0.05); AP50, to 81.34%±15.81%; and AP90, to 87.28%±7.25% (n=3) (P>0.05) of control level, while relaxation time and resting tension remained almost unchanged. Antimycin A, an inhibitor of complex III, applied at the highest concentration (3 × 10⁻4 M) reduced P to 41.66%±8.8% (n=5) (P<0.001) and marginally increased t50 and decreased the durations of AP. Anoxia (3 mM Na2S2O4) that inhibits the activity of complex IV reduced the contraction force to 9.23%±3.56% (n=6) (P<0.001), AP50 and AP90 to 65.46%±9.95% and 71.07%±8.39% (n=5) (P<0.05) of control level, respectively; furthermore, the resting tension augmented (contracture developed). CONCLUSIONS: Our results show that the inhibition of respiratory chain complex IV had the strongest inhibitory effect on the electromechanical activity of failing human myocardium.

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.

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.

Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases.

A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.

L-type Ca2+ channels in the heart: structure and regulation.

This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

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.

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.