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Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Haustein M, Hannes T, Trieschmann J, Verhaegh R, Köster A, Hescheler J, Brockmeier K, Adelmann R, Khalil M - PLoS ONE (2015)

Bottom Line: We found an overall negative force-frequency relationship (FFR).Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05).In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatric Cardiology, Cologne Heart Centre, Medical Faculty, University of Cologne, Cologne, North Rhine-Westphalia, Germany.

ABSTRACT
Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

No MeSH data available.


Related in: MedlinePlus

Effects of Ni2+.(A) Force of contraction after application of increasing doses of NiCl2 relative to baseline. NiCl2 reduced developed force in a dose-dependent manner. (B) Averaged twitches of a single experiment after application of 5 mM NiCl2 compared to baseline. Upper panel: peak force of contraction was reduced. Lower panel (normalised to baseline): relaxation was prolonged after application of 5 mM NiCl2. (C) Contraction and relaxation kinetics relative to amplitude. Maximum contraction velocity (dF/dtmax) was not affected by NiCl2. Maximum relaxation velocity (dF/dtmin) was significantly reduced after application of 5 mM NiCl2, but not affected at lower concentrations. (D) Time after peak to 50%, 75%, and 90% relaxation after application of NiCl2. Only 5 mM, but not lower concentrations, of NiCl2 prolonged relaxation significantly. Data are represented as mean ± SEM, asterisks indicate statistically significant differences (* p < 0.05 vs. baseline).
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pone.0125654.g005: Effects of Ni2+.(A) Force of contraction after application of increasing doses of NiCl2 relative to baseline. NiCl2 reduced developed force in a dose-dependent manner. (B) Averaged twitches of a single experiment after application of 5 mM NiCl2 compared to baseline. Upper panel: peak force of contraction was reduced. Lower panel (normalised to baseline): relaxation was prolonged after application of 5 mM NiCl2. (C) Contraction and relaxation kinetics relative to amplitude. Maximum contraction velocity (dF/dtmax) was not affected by NiCl2. Maximum relaxation velocity (dF/dtmin) was significantly reduced after application of 5 mM NiCl2, but not affected at lower concentrations. (D) Time after peak to 50%, 75%, and 90% relaxation after application of NiCl2. Only 5 mM, but not lower concentrations, of NiCl2 prolonged relaxation significantly. Data are represented as mean ± SEM, asterisks indicate statistically significant differences (* p < 0.05 vs. baseline).

Mentions: To study the effects of NCX inhibition on force generation, we used NiCl2 which is known for its moderately specific inhibitory action on NCX. Application of NiCl2 decreased force of contraction to 97 ± 1% at 0.1 mM, 88 ± 3% at 1.0 mM, 83 ± 4% at 2.0 mM and 38 ± 5% at 5.0 mM (n = 5, all p < 0.05 compared to baseline) (Fig 5A). In contrast to the LTCC blocker VER and RyR inhibition by Ry, NiCl2 significantly prolonged relaxation at a concentration of 5.0 mM (time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms; 75% relaxation: 95 ± 3 ms vs. 146 ± 10 ms; 90% relaxation: 128 ± 5 vs. 193 ± 12 ms, n = 5, all p < 0.05) (Fig 5B and 5D). Consistently, maximum relaxation velocity relative to twitch amplitude was reduced from 11.1 ± 0.5 s-1 to 7.1 ± 0.5 s-1 (n = 5, p < 0.05) (Fig 5C). Relaxation was not affected by lower concentrations and maximum contraction velocity was not changed by NiCl2 at all (Fig 5C and 5D).


Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Haustein M, Hannes T, Trieschmann J, Verhaegh R, Köster A, Hescheler J, Brockmeier K, Adelmann R, Khalil M - PLoS ONE (2015)

Effects of Ni2+.(A) Force of contraction after application of increasing doses of NiCl2 relative to baseline. NiCl2 reduced developed force in a dose-dependent manner. (B) Averaged twitches of a single experiment after application of 5 mM NiCl2 compared to baseline. Upper panel: peak force of contraction was reduced. Lower panel (normalised to baseline): relaxation was prolonged after application of 5 mM NiCl2. (C) Contraction and relaxation kinetics relative to amplitude. Maximum contraction velocity (dF/dtmax) was not affected by NiCl2. Maximum relaxation velocity (dF/dtmin) was significantly reduced after application of 5 mM NiCl2, but not affected at lower concentrations. (D) Time after peak to 50%, 75%, and 90% relaxation after application of NiCl2. Only 5 mM, but not lower concentrations, of NiCl2 prolonged relaxation significantly. Data are represented as mean ± SEM, asterisks indicate statistically significant differences (* p < 0.05 vs. baseline).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418605&req=5

pone.0125654.g005: Effects of Ni2+.(A) Force of contraction after application of increasing doses of NiCl2 relative to baseline. NiCl2 reduced developed force in a dose-dependent manner. (B) Averaged twitches of a single experiment after application of 5 mM NiCl2 compared to baseline. Upper panel: peak force of contraction was reduced. Lower panel (normalised to baseline): relaxation was prolonged after application of 5 mM NiCl2. (C) Contraction and relaxation kinetics relative to amplitude. Maximum contraction velocity (dF/dtmax) was not affected by NiCl2. Maximum relaxation velocity (dF/dtmin) was significantly reduced after application of 5 mM NiCl2, but not affected at lower concentrations. (D) Time after peak to 50%, 75%, and 90% relaxation after application of NiCl2. Only 5 mM, but not lower concentrations, of NiCl2 prolonged relaxation significantly. Data are represented as mean ± SEM, asterisks indicate statistically significant differences (* p < 0.05 vs. baseline).
Mentions: To study the effects of NCX inhibition on force generation, we used NiCl2 which is known for its moderately specific inhibitory action on NCX. Application of NiCl2 decreased force of contraction to 97 ± 1% at 0.1 mM, 88 ± 3% at 1.0 mM, 83 ± 4% at 2.0 mM and 38 ± 5% at 5.0 mM (n = 5, all p < 0.05 compared to baseline) (Fig 5A). In contrast to the LTCC blocker VER and RyR inhibition by Ry, NiCl2 significantly prolonged relaxation at a concentration of 5.0 mM (time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms; 75% relaxation: 95 ± 3 ms vs. 146 ± 10 ms; 90% relaxation: 128 ± 5 vs. 193 ± 12 ms, n = 5, all p < 0.05) (Fig 5B and 5D). Consistently, maximum relaxation velocity relative to twitch amplitude was reduced from 11.1 ± 0.5 s-1 to 7.1 ± 0.5 s-1 (n = 5, p < 0.05) (Fig 5C). Relaxation was not affected by lower concentrations and maximum contraction velocity was not changed by NiCl2 at all (Fig 5C and 5D).

Bottom Line: We found an overall negative force-frequency relationship (FFR).Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05).In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatric Cardiology, Cologne Heart Centre, Medical Faculty, University of Cologne, Cologne, North Rhine-Westphalia, Germany.

ABSTRACT
Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

No MeSH data available.


Related in: MedlinePlus