Matti Vornanen

Plasticity of excitation-contraction coupling in fish cardiac myocytes

Ultrastructure, molecular composition and electrophysiological properties of cardiac myocytes and functional characteristics of the fish heart suggest that cycling of extracellular Ca(2+) is generally more important than intracellular cycling of Ca(2+) stores of the sarcoplasmic reticulum (SR) in activating contraction of fish cardiac myocytes. This is especially true for the ventricle. However, prominent species-specific differences exist in cardiac excitation-contraction coupling and in the relative roles of extracellular and intracellular Ca(2+) sources among the teleostean fish. In fact, in some fish species (tunas, burbot) the SR of atrial myocytes, under certain circumstances, may act as the major source of systolic Ca(2+). These interspecific differences are obviously an outcome of evolutionary adaptation to different habitats and modes of activity in these habitats. There is also substantial intraspecific variation in the SR Ca(2+)-release-to-SL-Ca(2+) influx ratio depending on acute and chronic temperature changes. Consequently excitation-contraction coupling of the fish cardiac myocytes is not a fixed entity, but rather a highly variable and malleable process that enables fish to have an appropriate cardiac scope to exploit a diverse range of environments.

The force-frequency relationship in fish hearts—a review

1. IntroductionThe relationship between the strength of con-traction and the interval between contractions wasfirst recognised as an important intrinsic controlsystem of the heart when Bowditch (1871 ) notedthat ‘the interval between a contraction of the heartand the proceeding beat is of such importance forthe strength of the contraction that the study of thiseffect is a prime necessity’. This relationship, oftenreferred to as the ‘force–frequency relationship’,the ‘force–interval relationship’ or the ‘staircase-effect’, is studied in multicellular and single cellpreparations to understand a fundamental propertyof the heart, namely, its ability to develop force atdifferent frequencies. Thus, with the aid of phar-macological agents and by extrapolation of theresults to in vivo contraction frequencies, theforce–frequency relationship continues to provideuseful information to cardiac physiologists.In most fish hearts, contractile force decreasesas contraction frequency increases, resulting in a

Responses of Action Potential and K+ Currents to Temperature Acclimation in Fish Hearts: Phylogeny or Thermal Preferences?

Electrical activity of the heart is assumed to be one of the key factors that set thermal tolerance limits for ectothermic vertebrates. Therefore, we hypothesized that in thermal acclimation—the duration of cardiac action potential and the repolarizing K+ currents that regulate action potential duration (APD)—the rapid component of the delayed rectifier K+ current (IKr) and the inward rectifier K+ current (IK1) are more plastic in eurythermal than in stenothermal fish species. The hypothesis was tested in six freshwater teleosts representing four different fish orders (Cadiformes, Cypriniformes, Perciformes, Salmoniformes) acclimated at +4°C (cold acclimation) or +18°C (warm acclimation). In cold acclimation, a compensatory shortening of APD occurred in all species regardless of thermal tolerances, life styles, or phylogenies of the fish, suggesting that this response is a common characteristic of the teleost heart. The strength of the response did not, however, obey simple eurythermy‐stenothermy gradation but differed among the phylogenetic groups. Salmoniformes fish showed the greatest acclimation capacity of cardiac electrical activity, whereas the weakest response appeared in the perch (Perciformes) heart. The underlying ionic mechanisms were also partly phylogeny dependent. Modification of the IKr current was almost ubiquitously involved in acclimation response of fish cardiac myocytes to temperature, while the ability to change the IK1 current under chronic thermal stress was absent or showed inverse compensation in Salmoniformes species. Thus, in Salmoniformes fish, the thermal plasticity of APD is strongly based on IKr, while other fish groups rely on both IKr and IK1.

Acute Temperature Change Modulates the Response of ICa to Adrenergic Stimulation in Fish Cardiomyocytes

The purpose of this study was to investigate how the endogenous catecholamine adrenaline protects sarcolemmal Ca2+ flux through the L‐type Ca2+ channel (ICa) during acute exposure to cold in the fish heart. We examined the response of ICa to adrenergic stimulation at three temperatures (7°, 14°, and 21°C) in atrial myocytes isolated from rainbow trout acclimated to 14°C. We found that ICa amplitude varied directly with test temperature and was increased by adrenergic stimulation (AD; 5 nM and 1 μM) at all temperatures. However, ICa was significantly more sensitive to adrenergic stimulation at the coldest test temperature. In fact, at 7°C in the absence of AD, ICa was extremely low. The addition of 1 μM AD increased peak ICa 7.2‐fold at 7°C, 2.6‐fold at 14°C, and 1.6‐fold at 21°C and ameliorated the temperature‐dependent difference in Ca2+ influx across the cell membrane. We suggest that this increased adrenergic sensitivity is a critical compensatory mechanism that allows the rainbow trout heart to maintain contractility during acute exposure to cold temperatures. In particular, the tonic level of adrenergic stimulation provided by circulating plasma catecholamines (i.e., in the nM concentration range) may be crucial for effective excitation‐contraction coupling in the cold cardiomyocyte.

Temperature acclimation modifies Na+ current in fish cardiac myocytes.

SUMMARY The present study was designed to test the hypothesis that temperature acclimation modifies sarcolemmal Na+ current (INa) of the fish cardiac myocytes differently depending on the animals lifestyle in the cold. Two eurythermal fish species with different physiological strategies for surviving in the cold, a cold-dormant crucian carp (Carassius carassius L.) and a cold-active rainbow trout (Oncorhynchus mykiss), were used in acclimation experiments. The INa of carp and trout were also compared with INa of a cold stenothermal burbot (Lota lota). In accordance with the hypothesis, cold-acclimation decreased the density of INa in crucian carp and increased it in rainbow trout, suggesting depression of impulse conduction in cold-acclimated carp and positive compensation of impulse propagation in cold-acclimated trout. The steady-state activation curve of trout INa was shifted by 6 mV to more negative voltages by cold acclimation, which probably lowers the stimulus threshold for action potentials and further improves cardiac excitability in the cold. In burbot myocytes, the INa density was high and the position of the steady-state activation curve on the voltage axis was even more negative than in trout or carp myocytes, suggesting that the burbot INa is adapted to maintain high excitability and conductivity in the cold. The INa of the burbot heart differed from those of carp and trout in causing four times larger charge influx per excitation, which suggests that INa may also have a significant role in cardiac excitation–contraction coupling of the burbot heart. In summary, INa of fish cardiac myocytes shows thermal plasticity that is different in several respects in cold-dormant and cold-active species and thus has a physiologically meaningful role in supporting the variable life styles and habitat conditions of each species.

Acute heat tolerance of cardiac excitation in the brown trout (Salmo trutta fario)

The upper thermal tolerance and mechanisms of heat-induced cardiac failure in the brown trout (Salmo trutta fario) was examined. The point above which ion channel function and sinoatrial contractility in vitro, and electrocardiogram (ECG) in vivo, started to fail (break point temperature, BPT) was determined by acute temperature increases. In general, electrical excitation of the heart was most sensitive to heat in the intact animal (electrocardiogram, ECG) and least sensitive in isolated cardiac myocytes (ion currents). BPTs of Ca2+ and K+ currents of cardiac myocytes were much higher (>28°C) than BPT of in vivo heart rate (23.5±0.6°C) (P<0.05). A striking exception among sarcolemmal ion conductances was the Na+ current (INa), which was the most heat-sensitive molecular function, with a BPT of 20.9±0.5°C. The low heat tolerance of INa was reflected as a low BPT for the rate of action potential upstroke in vitro (21.7±1.2°C) and the velocity of impulse transmission in vivo (21.9±2.2°C). These findings from different levels of biological organization strongly suggest that heat-dependent deterioration of Na+ channel function disturbs normal spread of electrical excitation over the heart, leading to progressive variability of cardiac rhythmicity (missed beats, bursts of fast beating), reduction of heart rate and finally cessation of the normal heartbeat. Among the cardiac ion currents INa is ‘the weakest link’ and possibly a limiting factor for upper thermal tolerance of electrical excitation in the brown trout heart. Heat sensitivity of INa may result from functional requirements for very high flux rates and fast gating kinetics of the Na+ channels, i.e. a trade-off between high catalytic activity and thermal stability.

Sarcolemmal ion currents and sarcoplasmic reticulum Ca2+content in ventricular myocytes from the cold stenothermic fish, the burbot(Lota lota)

SUMMARY The burbot (Lota lota) is a cold stenothermic fish species whose heart is adapted to function in the cold. In this study we use whole-cell voltage-clamp techniques to characterize the electrophysiological properties of burbot ventricular myocytes and to test the hypothesis that changes in membrane currents and intracellular Ca2+ cycling associated cold-acclimation in other fish species are routine for stenothermic cold-adapted species. Experiments were performed at 4°C, which is the body temperature of burbot for most of the year, and after myocytes were acutely warmed to 11°C, which is in the upper range of temperatures experienced by burbot in nature. Results on K+ channels support our hypothesis as the relative density of K-channel conductances in the burbot heart are similar to those found for cold-acclimated cold-active fish species. IK1 conductance was small (39.2±5.4 pS pF-1 at 4°C and 71.4±1.7 pS pF-1 at 11°C) and IKr was large (199±27 pS pF-1 at 4°C and 320.3±8 pS pF-1 at 11°C) in burbot ventricular myocytes. We found high Na+-Ca2+ exchange (NCX) activity (35.9±6.3 pS pF-1 at 4°C and 58.6±8.4 pS pF-1 at 11°C between -40 and 20 mV), suggesting that it may be the primary pathway for sarcolemmal (SL) Ca2+ influx in this species. In contrast, the density (ICa, 0.81±0.13 pA pF-1 at 4°C, and 1.35±0.18 pA pF-1 at 11°C) and the charge (QCa, 0.24±0.043 pC pF-1 at 4°C and 0.21±0.034 pC pF-1 at 11°C) carried by the l-type Ca2+ current was small. Our results on sarcolemmal ion currents in burbot ventricular myocytes suggest that cold stenothermy and compensative cold-acclimation involve many of the same subcellular adaptations that culminate in enhanced excitability in the cold.

Zebrafish heart as a model for human cardiac electrophysiology

ABSTRACT The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

A novel inwardly rectifying K+ channel, Kir2.5, is upregulated under chronic cold stress in fish cardiac myocytes.

SUMMARY A new member of the inward-rectifier K+ channel subfamily Kir2 was isolated and characterised from the crucian carp (Carassius carassius) heart. When expressed in COS-1 cells this 422 amino acid protein produced an inward-rectifying channel with distinct single-channel conductance, mean open time and open probability. Phylogenetic sequence comparisons indicate that it is not homologous to any known vertebrate Kir channel, yet belongs to the Kir2 subfamily. This novel crucian carp channel increases the number of vertebrate Kir2 channels to five, and has therefore been designated as ccKir2.5 (cc for Carassius carassius). In addition to the ccKir2.5 channel, the ccKir2.2 and ccKir2.1 channels were expressed in the crucian carp heart, ccKir2.1 being present only in trace amounts (<0.8% of all Kir2 transcripts). Whole-cell patch clamp in COS-1 cells demonstrated that ccKir2.5 is a stronger rectifier than ccKir2.2 or ccKir2.1, and therefore passes weakly outward current. Single-channel conductance, mean open time and open probability of ccKir2.5 were, respectively, 1.6, 4.96 and 4.17 times as large as that of ccKir2.2. ccKir2.5 was abundantly expressed in atrium and ventricle of the heart and in skeletal muscle, but was a minor component of Kir2 in brain, liver, gill and kidney. Noticeably, ccKir2.5 was strongly responsive to chronic cold exposure. In fish reared at 4°C for 4 weeks, ccKir2.5 mRNA formed 59.1±2.1% and 65.6±3.2% of all ccKir2 transcripts in atrium and ventricle, respectively, while in fish maintained at 18°C the corresponding transcript levels were only 16.2±1.7% and 23.3±1.7%. The increased expression of ccKir2.5 at 4°C occurred at the expense of ccKir2.2, which was the main Kir2 isoform in 18°C acclimated fish. A cold-induced increase in the slope conductance of the ventricular IK1 from 707±49 to 1001±59 pS pF–1 (P<0.05) was thus associated with an isoform shift from ccKir2.2 towards ccKir2.5, suggesting that ccKir2.5 is a cold-adapted and ccKir2.2 a warm-adapted isoform of the inward-rectifying K+ channel.

Effects of acute anoxia on heart function in crucian carp : importance of cholinergic and purinergic control

The objective of this study was to characterize the effects of acute anoxia on contractile and electrical activity in the heart of an anoxia-tolerant fish species, the crucian carp ( Carassius carassius L.). Responses of atrial and ventricular tissue or isolated cells to NaCN, adenosine, and carbachol were determined to examine the effects of anoxia on cardiac performance and to clarify the possible role of local purinergic modulation and parasympathetic nervous control in the function of the anoxic fish heart. The contractility of the crucian carp heart is strongly decreased by acute anoxia. A rapid reduction in cardiac contractility is attained by reflex bradycardia and suppression of atrial contractility. These responses are mediated by muscarinic cholinergic receptors through the opening of inwardly rectifying potassium channels and are likely to protect the cardiac muscle from hypoxic/anoxic damage. The depletion of tissue oxygen content also directly depresses heart rate and cardiac force. Ultimately, an increase in cytosolic Ca2+ concentration occurs that activates sarcolemmal Ca2+extrusion through the Na+-Ca2+-exchange and generates an inward exchange current with consequent depolarization of the resting membrane potential and possible cell death. At physiological concentration, the effects of adenosine on contractile and electrical activity were relatively weak, suggesting that the purinergic system is not involved in the acute anoxia response of the crucian carp heart.The objective of this study was to characterize the effects of acute anoxia on contractile and electrical activity in the heart of an anoxia-tolerant fish species, the crucian carp (Carassius carassius L.). Responses of atrial and ventricular tissue or isolated cells to NaCN, adenosine, and carbachol were determined to examine the effects of anoxia on cardiac performance and to clarify the possible role of local purinergic modulation and parasympathetic nervous control in the function of the anoxic fish heart. The contractility of the crucian carp heart is strongly decreased by acute anoxia. A rapid reduction in cardiac contractility is attained by reflex bradycardia and suppression of atrial contractility. These responses are mediated by muscarinic cholinergic receptors through the opening of inwardly rectifying potassium channels and are likely to protect the cardiac muscle from hypoxic/anoxic damage. The depletion of tissue oxygen content also directly depresses heart rate and cardiac force. Ultimately, an increase in cytosolic Ca(2+) concentration occurs that activates sarcolemmal Ca(2+) extrusion through the Na(+)-Ca(2+)-exchange and generates an inward exchange current with consequent depolarization of the resting membrane potential and possible cell death. At physiological concentration, the effects of adenosine on contractile and electrical activity were relatively weak, suggesting that the purinergic system is not involved in the acute anoxia response of the crucian carp heart.