Dual effect of polyaromatic hydrocarbons on sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity of a teleost fish (Oncorhynchus mykiss).

Polycyclic aromatic hydrocarbons (PAHs) are embryo- and cardiotoxic to fish that might be associated with improper intracellular Ca2+ management. Since sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of intracellular Ca2+, the SERCA activity and the contractile properties of rainbow trout (Oncorhynchus mykiss) ventricle were measured in the presence of 3- and 4-cyclic PAHs. In unfractionated ventricular homogenates, acute exposure of SERCA to 0.1-1.0 µM phenanthrene (Phe), retene (Ret), fluoranthene (Flu), or pyrene (Pyr) resulted in concentration-dependent increase in SERCA activity, except for the Flu exposure, with maximal effects of 49.7-83 % at 1 µM. However, PAH mixture did not affect the contractile parameters of trout ventricular strips. Similarly, all PAHs, except Ret, increased the myotomal SERCA activity, but with lower effect (27.8-40.8 % at 1 µM). To investigate the putative chronic effects of PAHs on SERCA, the atp2a2a gene encoding trout cardiac SERCA was expressed in human embryonic kidney (HEK) cells. Culture of HEK cells in the presence of 0.3-1.0 µM Phe, Ret, Flu, and Pyr for 4 days suppressed SERCA expression in a concentration-dependent manner, with maximal inhibition of 49 %, 65 %, 39 % (P < 0.05), and 18 % (P > 0.05), respectively at 1 µM. Current findings indicate divergent effects of submicromolar PAH concentrations on SERCA: stimulation of SERCA activity in acute exposure and inhibition of SERCA expression in chronic exposure. The depressed expression of SERCA is likely to contribute to the embryo- and cardiotoxicity of PAHs by depressing muscle function and altering gene expression.

Transcript expression of inward rectifier potassium channels of Kir2 subfamily in Arctic marine and freshwater fish species.

Inward rectifier K+ (Kir2) channels are critical for electrical excitability of cardiac myocytes. Here, we examine expression of Kir2 channels in the heart of three Gadiformes species, polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and burbot (Lota lota) of the temperate lakes to find out the role of Kir2 channels in cardiac adaptation to cold. Five boreal freshwater species: brown trout (Salmo trutta fario), arctic char (Salvelinus alpinus), roach (Rutilus rutilus), perch (Perca fluviatilis) and pike (Esox lucius), and zebrafish (Danio rerio), were included for comparison. Transcript expression of genes encoding Kir2.1a, - 2.1b, - 2.2a, - 2.2b and - 2.4 was studied from atrium and ventricle of thermally acclimated or acclimatized fish by quantitative PCR. Kir2 composition in the polar cod was more diverse than in other species in that all Kir2 isoforms were relatively highly expressed. Kir2 composition of navaga and burbot differed from that of the polar cod as well as from those of other species. The relative expression of Kir2.2 transcripts, especially Kir2.2b, was higher in both atrium and ventricle of navaga and burbot (56-89% from the total Kir2 pool) than in other species (0.1-11%). Thermal acclimation induced only small changes in cardiac Kir2 transcript expression in Gadiformes species. However, Kir2.2b transcripts were upregulated in cold-acclimated navaga and burbot hearts. All in all, the cardiac Kir2 composition seems to be dependent on both phylogenetic position and thermal preference of the fish.

Cardiac voltage-gated sodium channel expression and electrophysiological characterization of the sodium current in the zebrafish (Danio rerio) ventricle.

Na+ channel α-subunit composition of the zebrafish heart and electrophysiological properties of Na+ current (INa) of zebrafish ventricular myocytes were examined. Eight Na+ channel α-subunits were expressed in both atrium and ventricle of the zebrafish heart. Nav1.5Lb, an orthologue to the human Nav1.5, was clearly the predominant isoform in both chambers representing 65.2 ±â€¯4.1% and 83.1 ±â€¯2.1% of all Na+ channel transcripts in atrium and ventricle, respectively. Nav1.4b, an orthologue to human Nav1.4, formed 34.1 ±â€¯4.1 and 16.2 ±â€¯2.0% of the Na+ channel transcripts in atrium and ventricle, respectively. The density of INa and the rate of action potential upstroke in zebrafish ventricular myocytes at 28 °C were similar to those of human ventricles at the comparable temperature. Na+ channel isoforms and the main electrophysiological characteristics of the INa are largely similar in zebrafish and human hearts indicating evolutionary conservation of Na+ channel composition and function. The zebrafish INa differs from the human cardiac INa in terms of higher tetrodotoxin sensitivity (IC50-value = 5.3 ±â€¯0.1 nM) and slower inactivation kinetics. The zebrafish INa was inhibited with tricaine (MS-222) with an IC50-value of 1.2 ±â€¯0.18 mM (336 mg l-1), suggesting some care in the use of MS-222 as an anesthetic.

Effects of seasonal acclimatization on thermal tolerance of inward currents in roach (Rutilus rutilus) cardiac myocytes.

To test the hypothesis of temperature-dependent deterioration of electrical excitability (TDEE) (Vornanen, J Exp Biol 219:1941-1952, 2016), the role of sodium (I Na) and calcium (I Ca) currents in heat tolerance of cardiac excitability was examined in a eurythermic fish, the roach (Rutilus rutilus). Densities of cardiac I Ca and I Na and their acute heat tolerance were measured in winter-acclimatized (WiR) and summer-acclimatized (SuR) fish maintained in the laboratory at 4 ± 1 and 18 ± 1 °C, respectively. A robust L-type Ca2+ current (I CaL), but no T-type Ca2+ current, was present in roach atrial and ventricular myocytes. Peak density of I CaL was smaller in atrial (- 1.97 ± 0.14 and - 1.75 ± 0.19 pA/pF for WiR and SuR, respectively) than ventricular myocytes (- 4.00 ± 0.59 and - 2.88 ± 0.47 pA/pF for WiR and SuR, respectively) (p < 0.05), but current density and heat tolerance of I CaL did not change between seasons in either cell type. In contrast to I Ca, marked differences appeared in I Na between WiR and SuR. I Na density was 38% higher in WiR than SuR atrial myocytes (- 80.03 ± 5.92 vs. - 49.77 ± 4.72 pA/pF; p < 0.05) and 48% higher in WiR than SuR ventricular myocytes (- 39.25 ± 3.06 vs. - 20.03 ± 1.79 pA/pF; p < 0.05). The winter increase in I Na density was associated with 55% (1.70 ± 0.27 vs. 0.77 ± 0.12) and 54% (1.08 ± 0.19 vs. 0.50 ± 0.10) up-regulation of the total Na+ channel (scn4 + scn5 + scn8) transcripts in atrium and ventricle, respectively (p < 0.05). Heat tolerance of atrial I Na was lower in WiR with a breakpoint temperature of 20.3 ± 1.2 °C than in SuR (23.8 ± 0.7 °C) (p < 0.05). The response of I Na to seasonal acclimatization conforms to the TDEE hypothesis. The lower heat tolerance of I Na in WiR is consistent with the lower heat tolerance of in vivo heart rate in WiR in comparison to SuR, but the match is not quantitatively perfect, suggesting that other factors in addition to I Na may be involved.

Species- and chamber-specific responses of 12 kDa FK506-binding protein to temperature in fish heart.

The sarcoplasmic reticulum (SR) Ca(2+) release channel or ryanodine receptor (RyR) of the vertebrate heart is regulated by the FK506-binding proteins, FKBP12 and FKBP12.6. This study examines whether temperature-related changes in the SR function of fish hearts are associated with changes in FKBP12 expression. For this purpose, a polyclonal antibody against trout FKBP12 was used to compare FKPB12 expression in cold-acclimated (4 °C, CA) and warm-acclimated (18 °C, WA) rainbow trout (Oncorhynchus mykiss), burbot (Lota lota) and crucian carp (Carassius carassius) hearts. FKBP12 expression was modulated in a species- and tissue-specific manner. Temperature acclimation affected FKBP12 expression only in atrial tissue. Changes in the ventricular FKBP12 expression were not detected in any of the fish species. In the atria of rainbow trout and crucian carp, temperature acclimation produced opposite thermal responses: FKBP12 increased in the trout atrium and decreased in the crucian carp atrium under cold acclimation. In the burbot heart, chronic temperature changes did not affect cardiac FKBP12 levels. Expression of FKBP12 mRNA in rainbow trout and crucian carp hearts suggests that the transcript levels are higher in the ventricle than in the atrium and are elevated by cold acclimation in trout, but not in crucian carp. Since FKBP12 is known to increase the Ca(2+) sensitivity of cardiac RyRs and thereby the opening frequency of the Ca(2+) release channels, temperature-related changes in FKBP12 expression may modify the SR function in excitation-contraction coupling. The cold-induced increase in FKBP12 in the trout atrium and decrease in the crucian carp atrium are consistent with the previously noted increase and decrease, respectively, of SR Ca(2+) stores in cardiac contraction in these species.

Temperature dependence of sarco(endo)plasmic reticulum Ca2+ ATPase expression in fish hearts.

Cardiac function in fish acclimates to long-term temperature shifts by generating compensatory changes in structure and function of sarcoplasmic reticulum (SR) including the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2). The current study compares temperature responses of the cardiac SERCA in two fish species, burbot (Lota lota) and crucian carp (Carassius carassius), which differ in regard to thermal tolerance and activity pattern. Burbot are cold stenothermal and cold-active, while crucian carp are eurythermal and cold-dormant. The fish were acclimated at 4 °C (cold-acclimation, CA) or 18 °C (warm-acclimation, WA) and expression of SERCA proteins and transcript was measured from atrium and ventricle. Burbot heart expresses one major isoform of SERCA (110 kDa), while crucian carp heart expresses two isoforms (110 and 93 kDa). Expression of SERCA proteins was about four times higher (P < 0.05) in the heart of CA burbot than WA burbot, in both cardiac chambers. In the heart of crucian carp, thermal acclimation did not affect SERCA proteins, in either chamber (P > 0.05). The expression of SERCA transcripts did not follow the expression pattern of SERCA protein in either species, suggesting that SERCA expression is mainly regulated posttranscriptionally. These findings show that the stenothermal and cold-active burbot compensates for the decrease in ambient temperature by increasing the expression of SERCA. In the eurythermal and cold-dormant crucian carp SERCA expression is independent of temperature, while the presence of two SERCA isoforms may provide some thermal independence in SR Ca(2+) pumping.

Expression of SERCA and phospholamban in rainbow trout (Oncorhynchus mykiss) heart: comparison of atrial and ventricular tissue and effects of thermal acclimation.

In the heart of rainbow trout (Oncorhynchus mykiss), the rate of contraction and Ca(2+) uptake into the sarcoplasmic reticulum (SR) are faster in atrial than ventricular muscle, and contraction force relies more on SR Ca(2+) stores after acclimation to cold. This study tested the hypothesis that differences in contractile properties and Ca(2+) regulation between atrial and ventricular muscle, and between warm-(WA) and cold-acclimated (CA) trout hearts, are associated with differences in expression of sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) and/or phospholamban (PLN), an inhibitor of the cardiac SERCA. Quantitative PCR (SERCA only) and antibodies raised against SERCA and PLN were used to determine abundances of SERCA2 transcripts and SERCA and PLN proteins, respectively, in atrium and ventricle of trout acclimated to cold (+4°C, CA) and warm (+18°C, WA) temperatures. Expression of SERCA2 transcripts was 1.6 and 2.1 times higher in atrium than ventricle of WA and CA trout, respectively (P<0.05). At the protein level, differences in SERCA expression between atrium and ventricle were 6.1- and 23-fold for WA and CA trout, respectively (P<0.001). Acclimation to cold increased SERCA2 transcripts 2.6- and 2.0-fold in atrial and ventricular muscle, respectively (P<0.05). At the protein level, cold-induced elevation of SERCA (4.6-fold) was noted only in atrial (P<0.05) but not in ventricular tissue (P>0.05). The expression pattern of PLN was similar to that of the SERCA protein, but chamber-specific and temperature-induced differences were much smaller than in the case of SERCA. In the ventricle, PLN/SERCA ratio was 2.1 and 7.0 times higher than in the atrium for WA and CA fish, respectively. These findings are consistent with the hypothesis that low PLN/SERCA ratio in atrial tissue enables faster SR Ca(2+) reuptake and thus contributes to faster kinetics of contraction in comparison with ventricular muscle. Similarly, cold-induced decrease in PLN/SERCA ratio may be associated with faster contraction kinetics of the CA trout heart, in particular in the atrial muscle.

Expression of calsequestrin in atrial and ventricular muscle of thermally acclimated rainbow trout.

Calsequestrin (CASQ) is the main Ca(2+) binding protein within the sarcoplasmic reticulum (SR) of the vertebrate heart. The contribution of SR Ca(2+) stores to contractile activation is larger in atrial than ventricular muscle, and in ectothermic fish hearts acclimation to low temperatures increases the use of SR Ca(2+) in excitation-contraction coupling. The hypotheses that chamber-specific and temperature-induced differences in SR function are due to the increased SR CASQ content were tested in rainbow trout (Oncorhynchus mykiss) acclimated at either 4 degrees C (cold acclimation, CA) or 18 degrees C (warm acclimation, WA). To this end, the trout cardiac CASQ (omCASQ2) was cloned and sequenced. The omCASQ2 consists of 1275 nucleotides encoding a predicted protein of 425 amino acids (54 kDa in molecular mass, MM) with a high (75-87%) sequence similarity to other vertebrate cardiac CASQs. The transcript levels of the omCASQ2 were 1.5-2 times higher in CA than WA fish and about 2.5 times higher in the atrium than ventricle (P<0.001). The omCASQ2 protein was measured from western blots using a polyclonal antibody against the amino acid sequence 174-315 of the omCASQ2. Unlike the omCASQ2 transcripts, no differences were found in the abundance of the omCASQ2 protein between CA and WA fish, nor between the atrium and ventricle (P>0.05). However, a prominent qualitative difference appeared between the acclimation groups: two CASQ isoforms with apparent MMs of 54 and 59 kDa, respectively, were present in atrial and ventricular muscle of the WA trout whereas only the 54 kDa protein was clearly expressed in the CA heart. The 59 kDA isoform was a minor CASQ component representing 22% and 13% of the total CASQ proteins in the atrium and ventricle of the WA fish, respectively. In CA hearts, the 59 kDa protein was present in trace amounts (1.5-2.4%). Collectively, these findings indicate that temperature-related and chamber-specific differences in trout cardiac SR function are not related to the abundance of luminal Ca(2+) buffering by cardiac CASQ.