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Funny current ( I f ), formed by hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), is supposed to be crucial for the membrane clock regulating the cardiac pacemaker mechanism. We examined the presence and activity of HCN channels in the brown trout ( Salmo trutta fario ) sinoatrial (SA) pacemaker cells and their putative role in heart rate ( f H ) regulation. Six HCN transcripts (HCN1, HCN2a, HCN2ba, HCN2bb, HCN3, and HCN4) were expressed in the brown trout heart. The total HCN transcript abundance was 4.0 and 4.9 times higher in SA pacemaker tissue than in atrium and ventricle, respectively. In the SA pacemaker, HCN3 and HCN4 were the main isoforms representing 35.8 ± 2.7 and 25.0 ± 1.5%, respectively, of the total HCN transcripts. Only a small I f with a mean current density of -1.2 ± 0.37 pA/pF at -140 mV was found in 4 pacemaker cells out of 16 spontaneously beating cells examined, despite the optimization of recording conditions for I f activity. I f was not found in any of the 24 atrial myocytes and 21 ventricular myocytes examined. HCN4 coexpressed with the MinK-related peptide 1 (MiRP1) β-subunit in CHO cells generated large I f currents. In contrast, HCN3 (+MiRP1) failed to produce I f in the same expression system. Cs + (2 mM), which blocked 84 ± 12% of the native I f , reversibly reduced f H 19.2 ± 3.6% of the excised multicellular pacemaker tissue from 53 ± 5 to 44 ± 5 beats/min ( P < 0.05). However, this effect was probably due to the reduction of I Kr , which was also inhibited (63.5 ± 4.6%) by Cs + These results strongly suggest that f H regulation in the brown trout heart is largely independent on I f ., (Copyright © 2017 the American Physiological Society.)

Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate (f H ), and at high temperatures Q̇ collapses due to heat-dependent depression of f H This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of f H in vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, f PM ), spontaneous beating rate of isolated sinoatrial preparations (f SA ), and in vivo f H of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, f PM steadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (T BP ) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum f PM at T BP was much higher [193 ± 21.0 beats per minute (bpm)] than the peak f SA (94.3 ± 6.0 bpm at 24.1°C) or peak f H (76.7 ± 2.4 at 15.7 ± 0.82°C) (P < 0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit f H at high temperatures in the brown trout in vivo., (Copyright © 2017 the American Physiological Society.)

Sturgeon (family Acipenseridae) are regarded as living fossils due to their ancient origin and exceptionally slow evolution. To extend our knowledge of fish cardiac excitability to a Chondrostei fish, we examined electrophysiological phenotype of the Siberian sturgeon (Acipenser baerii) heart with recordings of epicardial ECG, intracellular action potentials (APs), and sarcolemmal ion currents. Epicardial ECG of A. baerii had the typical waveform of the vertebrate ECG with Q-T interval (average duration of ventricular AP) of 650±30 ms and an intrinsic heart rate of 45.5±5 beats min(-1) at 20°C. Similar to other fish species, atrial AP was shorter in duration (402±33 ms) than ventricular AP (585±40) (P<0.05) at 20°C. Densities of atrial and ventricular Na+ currents were similar (-47.6±4.5 and -53.2±5.1 pA/pF, respectively) and close to the typical values of teleost hearts. Two major K+ currents, the inward rectifier K+ current (IK1), and the delayed rectifier K+ current (IKr) were found under basal conditions in sturgeon cardiomyocytes. The atrial IKr (3.3±0.2 pA/pF) was about twice as large as the ventricular IKr (1.3±0.4 pA/pF) (P<0.05) conforming to the typical pattern of teleost cardiac IKr. Divergent from other fishes, the ventricular IK1 was remarkably small (-2.5±0.07 pA/pF) and not different from that of the atrial myocytes (-1.9±0.06 pA/pF) (P>0.05). Two ligand-gated K+ currents were also found: ACh-activated inward rectifier (IKACh) was present only in atrial cells, while ATP-sensitive K+ current (IKATP) was activated by a mitochondrial blocker, CCCP, in both atrial and ventricular cells. The most striking difference to other fishes appeared in Ca2+ currents (ICa). In atrial myocytes, ICa was predominated by nickel-sensitive and nifedipine-resistant T-type ICa, while ventricular myocytes had mainly nifedipine-sensitive and nickel-resistant L-type ICa. ICaT/ICaL ratio of the sturgeon atrial myocytes (2.42) is the highest value ever measured for a vertebrate species. In ventricular myocytes, ICaT/ICaL ratio was 0.09. With the exception of the large atrial ICaT and small ventricular IK1, electrical excitability of A. baerii heart is similar to that of teleost hearts., (Copyright © 2014 the American Physiological Society.)

Ectothermic vertebrates experience acute and chronic temperature changes which affect cardiac excitability and may threaten electrical stability of the heart. Nevertheless, ectothermic hearts function over wide range of temperatures without cardiac arrhythmias, probably due to special molecular adaptations. We examine function and molecular basis of the slow delayed rectifier K(+) current (I(Ks)) in cardiac myocytes of a eurythermic fish (Carassius carassius L.). I(Ks) is an important repolarizing current that prevents excessive prolongation of cardiac action potential, but it is extremely slowly activating when expressed in typical molecular composition of the endothermic animals. Comparison of the I(Ks) of the crucian carp atrial myocytes with the currents produced by homomeric K(v)7.1 and heteromeric K(v)7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the I(Ks) are similar to those of the homomeric K(v)7.1 channels. Consistently with electrophysiological properties and homomeric K(v)7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6-1.9% of the expression level of the K(v)7.1 subunit. Since activation kinetics of the homomeric K(v)7.1 channels is much faster than activation of the heteromeric K(v)7.1/MinK channels, the homomeric K(v)7.1 composition of the crucian carp cardiac I(Ks) is thermally adaptive: the slow delayed rectifier channels can open despite low body temperatures and curtail the duration of cardiac action potential in ectothermic crucian carp. We suggest that the homomeric K(v)7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric K(v)7.1/MinK channels evolved later to adapt I(Ks) to high body temperature of endotherms.

Ryanodine (Ry) sensitivity of cardiac contraction differs between teleost species, between atrium and ventricle, and according to the thermal history of the fish. The hypothesis that variability in Ry sensitivity of contraction is due to species-specific, chamber-specific, and temperature-related differences in the sarcoplasmic reticulum (SR) Ca(2+) content, was tested by comparing steady-state (SS) and maximal (Max) Ca(2+) loads of the SR in three teleost fish, rainbow trout (Oncorhynchus mykiss), burbot (Lota lota), and crucian carp (Carassius carassius), which differ in the extent of SR contribution to excitation-contraction coupling. Fish were acclimated at 4 degrees C (cold-acclimation, CA) or 18 degrees C (warm-acclimation, WA), and SR Ca(2+) content was released by a rapid application of 10 mM caffeine to single cardiac myocytes; its amount was determined from the Na(+)-Ca(2+) exchange current at 18 degrees C. SS Ca(2+) load was larger in atrial (304-915 micromol/l) than ventricular (224-540 micromol/l) myocytes in all fish species (P < 0.05), and the same was true for Max SR Ca(2+) content: 550-1,522 micromol/l and 438-840 micromol/l for atrial and ventricular myocytes, respectively (P < 0.05). Consistent with the hypothesis, acclimation to cold increased Ca(2+) load of the cardiac SR in the burbot heart, but contrary to the hypothesis, temperature acclimation did not affect SR Ca(2+) content in rainbow trout and crucian carp hearts. Furthermore, there was an inverse relation between SR Ca(2+) content and Ry sensitivity of contraction force: the species with the smallest SR Ca(2+) content (burbot) is most sensitive to Ry. Collectively, these findings show that SR Ca(2+) content of fish cardiac myocytes is several times larger than that in mammalian cardiac SR.

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.

The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4 degrees C (cold) and 18 degrees C (warm). With electrophysiological recordings, the primary pacemaker was located at the base of the sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in cold-acclimated (46 +/- 6 APs/min) than warm-acclimated trout (38 +/- 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 +/- 6 vs. 38 +/- 6 APs/min; P < 0.05), supporting the hypothesis that thermal acclimation modifies the intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 microM ryanodine and 1 microM thapsigargin did not affect heart rate in either warm- or cold-acclimated trout at 11 degrees C but reduced heart rate in warm-acclimated trout from 74 +/- 2 to 42 +/- 6 APs/min (P < 0.05) at 18 degrees C. At 11 degrees C, a half-maximal blockade of the delayed rectifier K+ current (I(Kr)) with 0.1 microM E-4031 reduced heart rate more in warm-acclimated (from 45 +/- 1 to 24 +/- 5 APs/min) than cold-acclimated trout (56 +/- 3 vs. 48 +/- 2 APs/min), whereas I(Kr) density was higher and AP duration less in cold-acclimated trout (P > 0.05). Collectively, these findings suggest that a cold-induced increase in AP discharge frequency is at least partly due to higher density of the I(Kr) in the cold-acclimated trout, whereas contribution of SR Ca2+ release to thermal compensation of heart rate is negligible.