Trout Ventricular Myocytes Research Articles

Cardiac Toxicity of Cadmium Involves Complex Interactions Among Multiple Ion Currents in Rainbow Trout (Oncorhynchus mykiss) Ventricular Myocytes.

Cadmium (Cd2+ ) is cardiotoxic to fish, but its effect on the electrical excitability of cardiac myocytes is largely unknown. To this end, we used the whole-cell patch-clamp method to investigate the effects of Cd2+ on ventricular action potentials (APs) and major ion currents in rainbow trout (Oncorhynchus mykiss) ventricular myocytes. Trout were acclimated to +4 °C, and APs were measured at the acclimated temperature and elevated temperature (+18 °C). Cd2+ (10, 20, and 100 µM) altered the shape of the ventricular AP in a complex manner. The early plateau fell to less positive membrane voltages, and the total duration of AP prolonged. These effects were obvious at both +4 °C and +18 °C. The depression of the early plateau is due to the strong Cd2+ -induced inhibition of the L-type calcium (Ca2+ ) current (ICaL ), whereas the prolongation of the AP is an indirect consequence of the ICaL inhibition: at low voltages of the early plateau, the delayed rectifier potassium (K+ ) current (IKr ) remains small, delaying repolarization of AP. Cd2+ reduced the density and slowed the kinetics of the Na+ current (INa ) but left the inward rectifier K+ current (IK1 ) intact. These altered cellular and molecular functions can explain several Cd2+ -induced changes in impulse conduction of the fish heart, for example, slowed propagation of the AP in atrial and ventricular myocardia (inhibition of INa ), delayed relaxation of the ventricle (prolongation of ventricular AP duration), bradycardia, and atrioventricular block (inhibition of ICaL ). These findings indicate that the cardiotoxicity of Cd2+ in fish involves multiple ion currents that are directly and indirectly altered by Cd2+ . Through these mechanisms, Cd2+ may trigger cardiac arrhythmias and impair myocardial contraction. Elevated temperature (+18 °C) slightly increases Cd2+ toxicity in trout ventricular myocytes. Environ Toxicol Chem 2021;40:2874-2885. © 2021 SETAC.

Understanding the cardiac toxicity of the anthropogenic pollutant phenanthrene on the freshwater indicator species, the brown trout (Salmo trutta): From whole heart to cardiomyocytes

Freshwater systems are faced with a myriad of stressors including geomorphological alterations, nutrient overloading and pollution. Previous studies in marine fish showed polyaromatic hydrocarbons (PAHs) to be cardiotoxic. However, the cardiotoxicity of anthropogenic pollutants in freshwater fishes is unclear and has not been examined across multiple levels of cardiac organization. Here we investigated the effect of phenanthrene (Phe), a pervasive anthropogenic pollutant on a sentinel freshwater species, the brown trout (Salmo trutta). We first examined the electrical activity of the whole heart and found prolongation (∼8.6%) of the QT interval (time between ventricular depolarization and repolarization) of the electrocardiogram (ECG) and prolongation (∼13.2%) of the monophasic action potential duration (MAPD) following ascending doses of Phe. At the tissue level, Phe significantly reduced trabecular force generation by ∼24% at concentration 15 μM and above, suggesting Phe reduces cellular calcium cycling. This finding was supported by florescent microscopy showing a reduction (∼39%) in the intracellular calcium transient amplitude following Phe exposure in isolated brown trout ventricular myocytes. Single-cell electrophysiology was used to reveal the mechanism underlying contractile and electrical dysfunction following Phe exposure. A Phe-dependent reduction (∼38%) in the L-type Ca2+ current accounts, at least in part, for the lowered Ca2+ transient and force production. Prolongation of the MAPD and QT interval was explained by a reduction (∼70%) in the repolarising delayed rectifier K+ current following Phe exposure. Taken together, our study shows a direct impact of Phe across multiple levels of cardiac organization in a key freshwater salmonid.

Revealing calcium fluxes by analyzing inhibition dynamics in action potential clamp

In cardiac excitation-contraction coupling (ECC), calcium enters the cytosol via L-type Ca2+ channels (LTCC) and reverse Na+/Ca2+-exchange (NCXrev), or is released from the sarcoplasmic reticulum (SR) by Ca2+-induced Ca2+-release (CICR). The magnitude of Ca2+ influx via the different pathways varies with the state of the cell and is difficult to assess quantitatively, because changes in Ca2+ influx through one pathway affect the others. In rainbow trout ventricular myocytes, the role of the SR has been uncertain for decades. The aim of this work was therefore two-fold: 1) to develop a method to quantify the Ca2+ influx pathways, and 2) to determine the role of CICR from the SR in trout ventricular myocytes. The novelty of our developed method lies in the mathematical analysis of measured transsarcolemmal Ca2+ currents and their impact on the corresponding Ca2+ transient during gradual inhibition of the currents in action potential (AP) clamp. We tested the developed method using an excitation-contraction model and showed that the method was able to recover calcium fluxes from noisy synthetic data. We applied the approach to trout ventricular myocytes and quantified the relative contributions of different Ca2+ influx pathways in ECC and determined the kinetics of these fluxes. Under baseline conditions, NCXrev is the main transmembrane Ca2+ influx pathway contributing 29 ± 6% (of the Ca2+ influx), LTCC 18 ± 7%, and CICR 53 ± 10% to overall Ca2+ transient. Thus, NCXrev is an important regulator of contractility and probably plays a role in the negative force-frequency relationship of trout ventricular preparations. These results demonstrate that trout and neonatal mammalian cardiomyocytes resemble each other not only in terms of morphology and energetics but ECC as well. In summary, the developed method resolves the major problem how to separate highly interconnected fluxes in AP clamp and allows to study Ca2+ fluxes in cardiomyocytes under conditions close to in vivo.

Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes

Aim: The significance of the different calcium influx pathways like L-type Ca2+-channels (LTCC), reverse Na+/Ca2+-exchange (NCXrev) or Ca2+-induced Ca2+-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions.Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2+-transients were recorded using 1μM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10μM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10μM ryanodine and 2μM thapsigargin. We used 240μg/ml amphotericin B in pipette solution for the perforated-patch clamp configuration. Also, we composed a simplified mathematical model of Ca2+-dynamics. By fitting model solution to the recorded experimental data, we determined contribution of LTCC, NCX and CICR currents in trout.Results: From experiments where CICR was inhibited we determined the calcium buffering capacity in the cells that is 55μM (+/- 8 μM). With this method we quantify the contribution and kinetics of LTCC (10-20%), NCX (20-40%) and CICR (50-70%).Conclusions: We have developed a method to quantify calcium fluxes under physiological conditions in cardiomyocytes. Furthermore, we found that SR plays a significant role in trout EC coupling. This is in sharp contrast with previous estimates. Also, the method is not specific to trout and is easily applicable to different species.

The calcium stored in the sarcoplasmic reticulum acts as a safety mechanism in rainbow trout heart.

Cardiomyocyte contraction depends on rapid changes in intracellular Ca2+. In mammals, Ca2+ influx as L-type Ca2+ current (ICa) triggers the release of Ca2+ from sarcoplasmic reticulum (SR) and Ca2+-induced Ca2+ release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca2+ is unclear. Here, we characterized the role of ICa to trigger SR Ca2+ release in rainbow trout ventricular myocytes using ICa regulation by Ca2+ as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca2+ chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca2+, while with isoproterenol, inactivation was Ca2+-dependent (∼65%) and highly reliant on SR Ca2+ (∼46%). Thus, SR Ca2+ is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca2+ is an important source of cytosolic Ca2+. This was not attributed to differences in SR Ca2+ load because caffeine-induced transients were not different in both conditions. Therefore, Ca2+ stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.

Modulation of L-Type Calcium Current by Calcium-Dependent Mechanism in Trout Ventricular Myocytes

In cardiac myocytes, Ca has a dual role upon of L-type calcium current (ICa) by either inactivating it (calcium-dependent inactivation, CDI) or facilitating it (calcium-dependent facilitation, CDF). In fish cardiac myocytes, it is unclear whether both Ca modulations exist. This study addresses this point. Trout ventricular myocytes were enzymatically isolated. ICa was recorded using whole cell patch clamp with Na- and K-free solutions to avoid contaminating currents. With a low concentration of a slow Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2± 2.4 ms (mean±SEM, n=14). CDF was absent in all cells studied. When a fast Ca buffer (BAPTA 10 mM) was present in the pipette solution, ICa decay was similar to the decay in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9) and CDF was absent (n=9). We quantified the relative contribution of CDI and sarcoplasmic reticulum (SR) CDI according to our published method, and estimated that CDI represents ∼39% of total ICa inactivation, and that SR Ca release causes ∼12% of CDI. We conclude that in fish myocytes CDI play a role in ICa modulation but CDF is absent.Supported by the Welcome Trust.

Quantification Of L-type Ca Current Inactivation Mechanisms In Trout Ventricular Myocytes.

Inactivation of L-type calcium current (ICa) is due to two mechanisms: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). In fish cardiac myocytes, it is unknown whether Ca release from the sarcoplasmic reticulum (SR) participates in CDI of ICa. This study assesses the relative contribution of different inactivation mechanisms of ICa in trout ventricular myocytes. Trout ventricular myocytes were enzymatically isolated. ICa was recorded using whole cell patch clamp with Na- and K-free solutions to avoid contaminating currents. With a low concentration of a slow Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2±2.4 ms (mean±SEM, n=14). When a fast Ca buffer (BAPTA 10 mM) was present in the pipette solution ICa decay was similar to the decay in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9). When barium was used as a charge carrier, IBa inactivates mainly via VDI and T37 was significantly increased (43.7±3.1 ms, n=9, p<0.05, t-test), albeit T37 is twice faster than in mammalian cardiac myocytes. We quantified the relative contribution of VDI and CDI according to the method previously described [Brette et al. (2004). Circ Res; 95; e1-7]. We measured the fraction of current remaining 20 ms after its peak (IR20). IR20 was 0.39±0.03 in EGTA, 0.42±0.02 in BAPTA and 0.64±0.03 in barium. We estimated that CDI represents ∼39% of total fast ICa inactivation, and that SR Ca release causes only ∼12% of CDI. We conclude that the main inactivation mechanisms in the trout myocyte are due to VDI and CDI from Ca entering the cell via ICa and not SR Ca release.Supported by the Wellcome Trust and the BBSRC.

Cloning and expression of cardiac Kir2.1 and Kir2.2 channels in thermally acclimated rainbow trout

Potassium currents are plastic entities that modify electrical activity of the heart in various physiological conditions including chronic thermal stress. We examined the molecular basis of the inward rectifier K+ current (IK1) in rainbow trout acclimated to cold (4 degrees C, CA) and warm (18 degrees C, WA) temperature. Inward rectifier K+ channel (Kir)2.1 and Kir2.2 transcripts were expressed in atrium and ventricle of the trout heart, K(ir)2.1 being the major component in both cardiac chambers. The relative expression of Kir2.2 was, however, higher (P < 0.05) in atrium than ventricle. The density of ventricular IK1 was approximately 25% larger (P < 0.05) in WA than CA trout. Furthermore, the IK1 of the WA trout was 10 times more sensitive to Ba2+ (IC50 0.18 +/- 0.42 microM) than the IK1 of the CA trout (1.17 +/- 0.44 microM) (P < 0.05), and opening kinetics of single Kir2 channels was slower in WA than CA trout (P < 0.05). When expressed in COS-1 cells, the homomeric Kir2.2 channels demonstrated higher Ba2+ sensitivity (2.88 +/- 0.42 microM) than Kir2.1 channels (24.99 +/- 7.40 microM) (P < 0.05). In light of the different Ba2+ sensitivities of rainbow trout (om)Kir2.1 and omKir2.2 channels, it is concluded that warm acclimation increases either number or activity of the omK(ir)2.2 channels in trout ventricular myocytes. The functional changes in I(K1) are independent of omKir2 transcript levels, which remained unaltered by thermal acclimation. Collectively, these findings suggest that thermal acclimation modifies functional properties and subunit composition of the trout Kir2 channels, which may be needed for regulation of cardiac excitability at variable temperatures.

Three-dimensional mitochondrial arrangement in ventricular myocytes: from chaos to order

We have developed a novel method to quantitatively analyze mitochondrial positioning in three dimensions. Using this method, we compared the relative positioning of mitochondria in adult rat and rainbow trout (Oncorhynchus mykiss) ventricular myocytes. Energetic data suggest that trout, in contrast to the rat, have two subpopulations of mitochondria in their cardiomyocytes. Therefore, we speculated whether trout cardiomyocytes exhibit two types of mitochondrial patterns. Stacks of confocal images of mitochondria were acquired in live cardiomyocytes. The images were processed and mitochondrial centers were detected automatically. The mitochondrial arrangement was analyzed by calculating the three-dimensional probability density and distribution functions describing the distances between neighboring mitochondrial centers. In the rat (8 cells with a total of 7,546 mitochondrial centers), intermyofibrillar mitochondria are highly ordered and arranged in parallel strands. These strands are separated by approximately 1.8 mum and can be found in any transversal direction relative to each other. Neighboring strands exhibit the same mitochondrial periodicity. In contrast to the rat, trout ventricular myocytes (22 cells; 5,528 mitochondrial centers) exhibit a relatively chaotic mitochondrial pattern. Neighboring mitochondria can be found in any direction relative to each other. Thus, two potential subpopulations of mitochondria in trout are not distinguishable by their pattern. The developed method required minor interaction in the filtering of the mitochondrial centers. It is therefore a practical approach to describe intracellular organization and may also be used for analysis of time-dependent organizational changes. The obtained quantitative description of mitochondrial organization is a requisite for accurate mathematical analysis of mitochondrial systems biology.

Temporal and spatial properties of cellular Ca2+flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca(2+) channel agonist (-)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-)BAY K 8644 or high Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.

Electrophysiological properties of rainbow trout cardiac myocytes in serum-free primary culture.

A low-density primary culture of trout ventricular myocytes in serum-free growth medium was established and maintained for up to 10 days at 17 degrees C. The myocytes retained their normal rod shaped morphology, capacitive surface area of the sarcolemma (SL), and contractile quiescence. However, sarcolemmal cation currents changed significantly, some permanently, some transiently, after 8-10 days of culture. TTX-sensitive sodium current (I(Na)) and Ba(2+)-sensitive background inward rectifier potassium current (I(K1)) were permanently depressed to 24-28% of their control density measured in freshly isolated myocytes. In contrast, L-type calcium current (I(Ca)) was only transiently downregulated; after 2-3 days in culture, the density of the current was 32% of the control and recovered to the control value after 8-10 days in culture. The changes in membrane currents were reflected in the shape of the action potential (AP). After 2-3 days in culture, maximal overshoot potential and resting potential were significantly reduced, and the durations of the AP at 50 and 90% repolarization were significantly increased. These changes became significantly more pronounced after 8-10 days of culture, with the exception of AP duration at 50% repolarization level. The shortening of the early plateau phase may reflect an additional change to an outward current, presumably the rapid component of the delayed rectifier (I(Kr)). Although the present findings indicate that fish cardiac myocytes can be maintained in serum-free primary culture for at least 10 days at 17 degrees C, some but not all of the electrophysiological characteristics of the myocytes change markedly during culture. The changes in ion currents were not due to loss of sarcolemmal membrane and therefore are likely to represent altered expression of cation currents as an adaptive response to culture conditions.

Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes.

We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20 degreesC to 10 or 5 degreesC reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 +/- 2.8 amol Ca2+/pF or 750 microM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 +/- 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced INCX increased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 +/- 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 +/- 1.2 amol Ca2+. pF-1. s-1 or 417 microM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.