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Excitation-contraction coupling of the mouse embryonic cardiomyocyte.

Rapila R, Korhonen T, Tavi P - J. Gen. Physiol. (2008)

Bottom Line: One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca(2+) release from intracellular stores initiating spontaneous intracellular calcium oscillations.Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats.This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Physiology and Biocenter Oulu, University of Oulu, 90014 Oulu, Finland.

ABSTRACT
In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca(2+) release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9-11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

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Spontaneous calcium release initiates cytosolic calcium oscillations in embryonic cardiac myocytes. (A) Laser scanning confocal image from a Fluo-4–loaded, isolated E10 spontaneously active cardiomyocyte (left) with corresponding line scan (yellow line) through the cytosol surrounding the dark nuclear area (red n). Red and blue arrowheads in the line scan image denote near SR area and near SL area, respectively. Graphs above the line scan image show relative calcium signals (Fluo-4 emission, F/F0) from near SR (red line) and near SL (blue line). Insert (gray background) shows both graphs in an expanded timescale from selected area below (gray). (B) Effects of inhibitors of SR Ca2+-ATPase (10 μM thapsigargin; top), RyRs (50 μM ryanodine; middle), and IP3Rs (50 μM 2-APB; bottom) on spontaneous calcium signals from isolated E10 cardiomyocytes.
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fig2: Spontaneous calcium release initiates cytosolic calcium oscillations in embryonic cardiac myocytes. (A) Laser scanning confocal image from a Fluo-4–loaded, isolated E10 spontaneously active cardiomyocyte (left) with corresponding line scan (yellow line) through the cytosol surrounding the dark nuclear area (red n). Red and blue arrowheads in the line scan image denote near SR area and near SL area, respectively. Graphs above the line scan image show relative calcium signals (Fluo-4 emission, F/F0) from near SR (red line) and near SL (blue line). Insert (gray background) shows both graphs in an expanded timescale from selected area below (gray). (B) Effects of inhibitors of SR Ca2+-ATPase (10 μM thapsigargin; top), RyRs (50 μM ryanodine; middle), and IP3Rs (50 μM 2-APB; bottom) on spontaneous calcium signals from isolated E10 cardiomyocytes.

Mentions: To elucidate the mechanisms triggering cardiomyocyte activity we did confocal [Ca2+]i imaging and electrophysiological measurements from spontaneously beating isolated single embryonic (E9–11) myocytes. Without physical connections to other cells, isolated embryonic myocytes contract spontaneously at a rate of 0.40 ± 0.03 Hz (n = 94), accompanied by oscillations in [Ca2+]i and Vm (Table I). It was suggested earlier that spontaneous plasmalemmal APs triggered by the If initiate this activity (Nakanishi et al., 1988; Takeshima et al., 1998). If so, inhibition of this current should stop the spontaneous activity of these cells. Zeneca ZD7288 has been shown to be an efficient blocker of If in adult guinea pig sinoatrial node cells (BoSmith et al., 1993), and we wanted to test if it could also be used to block the If of the embryonic cardiomyocytes. Therefore, we measured If with whole cell patch clamp from isolated E10 cardiomyocytes by applying voltage clamps ranging from −50 to −130 mV, with 10-mV steps from a 0-mV holding potential as described previously (Yasui et al., 2001). In these conditions, Zeneca was found to be a very effective blocker of embryonic If. 10 μM Zeneca reduced If by ∼86% (Fig. 1 A), from −2.50 ± 0.26 pA/pF to −0.35 ± 0.40 pA/pF (at −130 mV; P = 0.002; n = 7). When 10 μM Zeneca was applied to spontaneously active E9–11 cardiomyocytes, the frequency of the spontaneous oscillations was slightly reduced in four out of seven myocytes and no change was observed in three out of seven. Because this effect was not consistent, the effect of If block on the frequency or the amplitude of the spontaneous calcium signals was not statistically significant (Fig. 1, B–D). We next asked if the suggested spontaneous SR calcium oscillations (Sasse et al., 2007) could provide an alternative explanation for this activity. Therefore, we did confocal calcium imaging of the E9–11 cardiomyocytes to characterize the spatio-temporal properties of the spontaneous calcium signals. We noticed that calcium signals are initiated at the vicinity of the thin perinuclear area (Fig. 2 A) corresponding to the location of the SR in developing cardiomyocytes (Mesaeli et al., 1999). After the initial release from the SR, calcium diffuses in the cytosol and the [Ca2+]i increases near the plasma membrane after a delay (Fig. 2 A and Video 1, which is available at http://www.jgp.org/cgi/content/full/jgp.200809960/DC1). The delay between [Ca2+]i rise near the SR and near the sarcolemmal (SL) varied from 8 to 68 ms, with 28.5 ± 4.9 ms (n = 12) average delay. This SR calcium release appeared to be relatively well developed; the amplitude of the spontaneous global calcium signal was 56.5 ± 0.5% (n = 23) of the amplitude of the caffeine-induced calcium transient, indicating that 56.5% of the available SR calcium is released during each spontaneous calcium release. To occur, SR calcium release seems to require intact calcium uptake as well as two types of SR calcium release channels because blocking either IP3Rs (with 2-APB) (Maruyama et al., 1997) (n = 14) or RyRs (with ryanodine; n = 27), as well as inhibiting SR calcium uptake (with thapsigargin; n = 11), initially slows down the spontaneous activity until the cells show no calcium signals (Fig. 2 B).


Excitation-contraction coupling of the mouse embryonic cardiomyocyte.

Rapila R, Korhonen T, Tavi P - J. Gen. Physiol. (2008)

Spontaneous calcium release initiates cytosolic calcium oscillations in embryonic cardiac myocytes. (A) Laser scanning confocal image from a Fluo-4–loaded, isolated E10 spontaneously active cardiomyocyte (left) with corresponding line scan (yellow line) through the cytosol surrounding the dark nuclear area (red n). Red and blue arrowheads in the line scan image denote near SR area and near SL area, respectively. Graphs above the line scan image show relative calcium signals (Fluo-4 emission, F/F0) from near SR (red line) and near SL (blue line). Insert (gray background) shows both graphs in an expanded timescale from selected area below (gray). (B) Effects of inhibitors of SR Ca2+-ATPase (10 μM thapsigargin; top), RyRs (50 μM ryanodine; middle), and IP3Rs (50 μM 2-APB; bottom) on spontaneous calcium signals from isolated E10 cardiomyocytes.
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Related In: Results  -  Collection

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fig2: Spontaneous calcium release initiates cytosolic calcium oscillations in embryonic cardiac myocytes. (A) Laser scanning confocal image from a Fluo-4–loaded, isolated E10 spontaneously active cardiomyocyte (left) with corresponding line scan (yellow line) through the cytosol surrounding the dark nuclear area (red n). Red and blue arrowheads in the line scan image denote near SR area and near SL area, respectively. Graphs above the line scan image show relative calcium signals (Fluo-4 emission, F/F0) from near SR (red line) and near SL (blue line). Insert (gray background) shows both graphs in an expanded timescale from selected area below (gray). (B) Effects of inhibitors of SR Ca2+-ATPase (10 μM thapsigargin; top), RyRs (50 μM ryanodine; middle), and IP3Rs (50 μM 2-APB; bottom) on spontaneous calcium signals from isolated E10 cardiomyocytes.
Mentions: To elucidate the mechanisms triggering cardiomyocyte activity we did confocal [Ca2+]i imaging and electrophysiological measurements from spontaneously beating isolated single embryonic (E9–11) myocytes. Without physical connections to other cells, isolated embryonic myocytes contract spontaneously at a rate of 0.40 ± 0.03 Hz (n = 94), accompanied by oscillations in [Ca2+]i and Vm (Table I). It was suggested earlier that spontaneous plasmalemmal APs triggered by the If initiate this activity (Nakanishi et al., 1988; Takeshima et al., 1998). If so, inhibition of this current should stop the spontaneous activity of these cells. Zeneca ZD7288 has been shown to be an efficient blocker of If in adult guinea pig sinoatrial node cells (BoSmith et al., 1993), and we wanted to test if it could also be used to block the If of the embryonic cardiomyocytes. Therefore, we measured If with whole cell patch clamp from isolated E10 cardiomyocytes by applying voltage clamps ranging from −50 to −130 mV, with 10-mV steps from a 0-mV holding potential as described previously (Yasui et al., 2001). In these conditions, Zeneca was found to be a very effective blocker of embryonic If. 10 μM Zeneca reduced If by ∼86% (Fig. 1 A), from −2.50 ± 0.26 pA/pF to −0.35 ± 0.40 pA/pF (at −130 mV; P = 0.002; n = 7). When 10 μM Zeneca was applied to spontaneously active E9–11 cardiomyocytes, the frequency of the spontaneous oscillations was slightly reduced in four out of seven myocytes and no change was observed in three out of seven. Because this effect was not consistent, the effect of If block on the frequency or the amplitude of the spontaneous calcium signals was not statistically significant (Fig. 1, B–D). We next asked if the suggested spontaneous SR calcium oscillations (Sasse et al., 2007) could provide an alternative explanation for this activity. Therefore, we did confocal calcium imaging of the E9–11 cardiomyocytes to characterize the spatio-temporal properties of the spontaneous calcium signals. We noticed that calcium signals are initiated at the vicinity of the thin perinuclear area (Fig. 2 A) corresponding to the location of the SR in developing cardiomyocytes (Mesaeli et al., 1999). After the initial release from the SR, calcium diffuses in the cytosol and the [Ca2+]i increases near the plasma membrane after a delay (Fig. 2 A and Video 1, which is available at http://www.jgp.org/cgi/content/full/jgp.200809960/DC1). The delay between [Ca2+]i rise near the SR and near the sarcolemmal (SL) varied from 8 to 68 ms, with 28.5 ± 4.9 ms (n = 12) average delay. This SR calcium release appeared to be relatively well developed; the amplitude of the spontaneous global calcium signal was 56.5 ± 0.5% (n = 23) of the amplitude of the caffeine-induced calcium transient, indicating that 56.5% of the available SR calcium is released during each spontaneous calcium release. To occur, SR calcium release seems to require intact calcium uptake as well as two types of SR calcium release channels because blocking either IP3Rs (with 2-APB) (Maruyama et al., 1997) (n = 14) or RyRs (with ryanodine; n = 27), as well as inhibiting SR calcium uptake (with thapsigargin; n = 11), initially slows down the spontaneous activity until the cells show no calcium signals (Fig. 2 B).

Bottom Line: One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca(2+) release from intracellular stores initiating spontaneous intracellular calcium oscillations.Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats.This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Department of Physiology and Biocenter Oulu, University of Oulu, 90014 Oulu, Finland.

ABSTRACT
In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca(2+) release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9-11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

Show MeSH
Related in: MedlinePlus