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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.sup.2+] content, was tested by comparing steady-state (SS) and maximal (Max) [Ca.sup.2+] loads of the SR in three teleost fish, rainbow trout (Oncorhynchus mykiss), burbot (Lota Iota), 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.sup.2+] content was released by a rapid application of l0 mM caffeine to single cardiac myocytes; its amount was determined from the [Na.sup.+] -[Ca.sup.2+] exchange current at 18[degrees]C. SS [Ca.sup.2+] load was larger in atrial (304-915 [micro]mol/l) than ventricular (224-540 [micro]mol/l) myocytes in all fish species (P < 0.05), and the same was true for Max SR [Ca.sup.2+] content: 550-1,522 [micro]mol/l and 438-840 [micro]mol/l for atrial and ventricular myocytes, respectively (P < 0.05). Consistent with the hypothesis, acclimation to cold increased [Ca.sup.2+] load of the cardiac SR in the burbot heart, but contrary to the hypothesis, temperature acclimation did not affect SR [Ca.sup.2+] content in rainbow trout and crucian carp hearts. Furthermore, there was an inverse relation between SR [Ca.sup.2+] content and Ry sensitivity of contraction force: the species with the smallest SR [Ca.sup.2+] content (burbot) is most sensitive to Ry. Collectively, these findings show that SR [Ca.sup.2+] content of fish cardiac myocytes is several times larger than that in mammalian cardiac SR. excitation-contraction coupling; sarcoplasmic reticulum calcium load; caffeine; sodium-calcium exchange current doi: 10.1152/ajpregu.00022.2009

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.sup.+] current ([I.sub.K1]) in rainbow trout acclimated to cold (4[degrees]C, CA) and warm (18[degrees]C, WA) temperature. Inward rectifier [K.sup.+] channel ([K.sub.ir])2.1 and [K.sub.ir]2.2 transcripts were expressed in atrium and ventricle of the trout heart, [K.sub.ir]2.1 being the major component in both cardiac chambers. The relative expression of [K.sub.ir]2.2 was, however, higher (P < 0.05) in atrium than ventricle. The density of ventricular [I.sub.K1] was ~25% larger (P < 0.05) in WA than CA trout. Furthermore, the [I.sub.K1] of the WA trout was 10 times more sensitive to [Ba.sup.2+] (I[C.sub.50] 0.18 [+ or -] 0.42 [micro]M) than the [I.sub.K1] of the CA trout (1.17 [+ or -] 0.44 [micro]M) (P < 0.05), and opening kinetics of single [K.sub.ir]2 channels was slower in WA than CA trout (P < 0.05). When expressed in COS-1 cells, the homomeric [K.sub.ir]2.2 channels demonstrated higher [Ba.sup.2+] sensitivity (2.88 [+ or -]- 0.42 [micro]M) than [K.sub.ir]2.1 channels (24.99 [+ or -] 7.40 [micro]M) (P < 0.05). In light of the different [Ba.sup.2+] sensitivities of rainbow trout (om)[K.sub.ir]2.1 and om[K.sub.ir]2.2 channels, it is concluded that warm acclimation increases either number or activity of the om[K.sub.ir]2.2 channels in trout ventricular myocytes. The functional changes in [I.sub.K1] are independent of om[K.sub.ir]2 transcript levels, which remained unaltered by thermal acclimation. Collectively, these findings suggest that thermal acclimation modifies functional properties and subunit composition of the trout [K.sub.ir]2 channels, which may be needed for regulation of cardiac excitability at variable temperatures. inward rectifier potassium channels; atrial myocytes; ventricular myocytes; thermal plasticity doi: 10.1152/ajpregu.00354.2006

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 [+ or -] 6 APs/min) than warm-acclimated trout (38 [+ or -] 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 [+ or -] 6 vs. 38 [+ or -] 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 [micro]M ryanodine and 1 [micro]M 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 [+ or -] 2 to 42 [+ or -] 6 APs/min (P < 0.05) at 18[degrees]C. At 11[degrees]C, a half-maximal blockade of the delayed rectifier [K.sup.+] current ([I.sub.Kr]) with 0.1 [micro]M E-4031 reduced heart rate more in warm-acclimated (from 45 [+ or -] 1 to 24 [+ or -] 5 APs/min) than cold-acclimated trout (56 [+ or -] 3 vs. 48 [+ or -] 2 APs/min), whereas [I.sub.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.sub.Kr] in the cold-acclimated trout, whereas contribution of SR [Ca.sup.2+] release to thermal compensation of heart rate is negligible. cardiac pacemaker; heart rate; sarcoplasmic reticulum; delayed rectifier potassium current

Electrical excitability (EE) is vital for cardiac function and strongly modulated by temperature and external K+ concentration ([K+]o), as formulated in the hypothesis of temperature-dependent deterioration of electrical excitability (TDEE). As little is known about EE of arctic stenothermic fishes, we tested the TDEE hypothesis on ventricular myocytes of polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and those of temperate freshwater burbot (Lota lota). Ventricular action potentials (APs) were elicited in current-clamp experiments at 3, 9 and 15°C, and AP characteristics and the current needed to elicit APs were examined. At 3°C, ventricular APs of polar cod and navaga were similar but differed from those of burbot in having a lower rate of AP upstroke and a higher rate of repolarization. EE of ventricular myocytes - defined as the ease with which all-or-none APs are triggered - was little affected by acute temperature changes between 3 and 15°C in any species. However, AP duration (APD50) was drastically reduced at higher temperatures. Elevation of [K+]o from 3 to 5.4 mmol l-1 and further to 8 mmol l-1 at 3, 9 and 15°C strongly affected EE and AP characteristics in polar cod and navaga, but had a lesser effect in burbot. In all species, ventricular excitation was resistant to acute temperature elevations, while small increases in [K+]o severely compromised EE, in particular in the marine stenotherms. This suggests that EE of the heart in these Gadiformes species is resistant against acute warming, but less so against the simultaneous temperature and exercise stresses. [ABSTRACT FROM AUTHOR]

Funny current (If), 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 (fH) 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 If 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 If activity. If 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 If currents. In contrast, HCN3 (+MiRP1) failed to produce If in the same expression system. Cs+ (2 mM), which blocked 84 ± 12% of the native If, reversibly reduced fH 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 IKr, which was also inhibited (63.5 ± 4.6%) by Cs+ These results strongly suggest that fH regulation in the brown trout heart is largely independent on If. [ABSTRACT FROM AUTHOR]

The effects of sustained anoxia on cardiac electrical excitability were examined in the anoxia-tolerant crucian carp (Carassius carassius). The electrocardiogram (ECG) and expression of excitation-contraction coupling genes were studied in fish acclimatised to normoxia in summer (+18°C) or winter (+2°C), and in winter fish after 1, 3 and 6 weeks of anoxia. Anoxia induced a sustained bradycardia from a heart rate of 10.3±0.77 beats min-1 to 4.1±0.29 beats min-1 (P<0.05) after 5 weeks, and heart rate slowly recovered to control levels when oxygen was restored. Heart rate variability greatly increased under anoxia, and completely recovered under reoxygenation. The RT interval increased from 2.8±0.34 s in normoxia to 5.8±0.44 s under anoxia (P<0.05), which reflects a doubling of the ventricular action potential (AP) duration. Acclimatisation to winter induced extensive changes in gene expression relative to summer-acclimatised fish, including depression in those genes coding for the sarcoplasmic reticulum calcium pump (Serca2a_q2) and ATP-sensitive K+ channels (Kir6.2) (P<0.05). Genes of delayed rectifier K+ (kcnh6) and Ca2+ channels (cacna1c) were up-regulated in winter fish (P<0.05). In contrast, the additional challenge of anoxia caused only minor changes in gene expression, e.g. depressed expression of Kir2.2b K+ channel gene (kcnj12b), whereas expression of Ca2+ (cacna1a, cacna1c and cacna1g) and Na+ channel genes (scn4a and scn5a) was not affected. These data suggest that low temperature pre-conditions the crucian carp heart for winter anoxia, whereas sustained anoxic bradycardia and prolongation of AP duration are directly induced by oxygen shortage without major changes in gene expression. [ABSTRACT FROM AUTHOR]

Electrophysiological properties and molecular background of the zebrafish ( Danio rerio) cardiac inward rectifier current (I) were examined. Ventricular myocytes of zebrafish have a robust (−6.7 ± 1.2 pA pF at −120 mV) strongly rectifying and Ba-sensitive (IC = 3.8 μM) I. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5 %) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0 %). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba block, drKir2.4 was the most sensitive (IC = 1.8 μM) and drKir2.1a the least sensitive channel (IC = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances. [ABSTRACT FROM AUTHOR]