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Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140

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ABSTRACT

Excitation–contraction coupling results in the shortening of many individual sarcomeres along the length of a muscle fiber. Tsukamoto and colleagues develop a technique to quantitatively analyze the dynamics of intracellular calcium transients and length changes at the single sarcomere level.

No MeSH data available.


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Effects of ISO on local coupling of [Ca2+]i and SL dynamics. (A) Individual data showing time courses of changes in Fyellow/Fcyan (top) and SL (bottom) in α-actinin–YC-Nano140–expressing cardiomyocytes during spontaneous beating in the absence (left; indicated as “control”) and presence (right) of 50 nM ISO. n = 7 and 9 in the absence and presence of ISO, respectively. Experiments were performed at 37°C. (B) Individual data showing the relationship of Fyellow/Fcyan versus SL in the absence and presence of ISO. A closed loop-like trajectory was observed, regardless of the use of ISO (compare Butler et al., 2015). (C) Comparison of Fyellow/Fcyan values during relaxation (R0) and contraction (Rmax) in the absence and presence of ISO. Fyellow/Fcyan was significantly higher for both R0 and Rmax. (D) Comparison of ΔR/R0 in the absence and presence of ISO (see Fig. 2 for ΔR/R0). (E) SL values in the absence and presence of ISO, during relaxation and contraction. ISO did not significantly affect SL, regardless of the contractile state. (F) ΔSL (difference between SL at relaxation and contraction) values in the absence and presence of ISO. No significant difference was observed between groups. Likewise, the variance was similar for both groups (i.e., from ∼0.03 to ∼0.17 µm for control [difference, ∼0.14 µm] and from ∼0.03 to ∼0.15 µm for ISO [difference, ∼0.12 µm]). (G) Shortening velocity of SL dynamics in the absence and presence of ISO. The velocity became significantly faster in the presence of ISO. (H) Lengthening velocity of SL dynamics in the absence and presence of ISO. (I) Time to peak values for changes in Fyellow/Fcyan and SL in the absence and presence of ISO. The values were less for both parameters, indicating the acceleration of the EC coupling at the single sarcomere level. (J) Comparison of the decay time for Fyellow/Fcyan. The time was shorter in the presence of ISO, indicating enhanced removal of cytosolic [Ca2+]i, presumably via acceleration of SR Ca2+ pump coupled with PLN phosphorylation (see Bers [2001] and references therein). In all graphs, horizontal bars indicate mean values. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).
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fig6: Effects of ISO on local coupling of [Ca2+]i and SL dynamics. (A) Individual data showing time courses of changes in Fyellow/Fcyan (top) and SL (bottom) in α-actinin–YC-Nano140–expressing cardiomyocytes during spontaneous beating in the absence (left; indicated as “control”) and presence (right) of 50 nM ISO. n = 7 and 9 in the absence and presence of ISO, respectively. Experiments were performed at 37°C. (B) Individual data showing the relationship of Fyellow/Fcyan versus SL in the absence and presence of ISO. A closed loop-like trajectory was observed, regardless of the use of ISO (compare Butler et al., 2015). (C) Comparison of Fyellow/Fcyan values during relaxation (R0) and contraction (Rmax) in the absence and presence of ISO. Fyellow/Fcyan was significantly higher for both R0 and Rmax. (D) Comparison of ΔR/R0 in the absence and presence of ISO (see Fig. 2 for ΔR/R0). (E) SL values in the absence and presence of ISO, during relaxation and contraction. ISO did not significantly affect SL, regardless of the contractile state. (F) ΔSL (difference between SL at relaxation and contraction) values in the absence and presence of ISO. No significant difference was observed between groups. Likewise, the variance was similar for both groups (i.e., from ∼0.03 to ∼0.17 µm for control [difference, ∼0.14 µm] and from ∼0.03 to ∼0.15 µm for ISO [difference, ∼0.12 µm]). (G) Shortening velocity of SL dynamics in the absence and presence of ISO. The velocity became significantly faster in the presence of ISO. (H) Lengthening velocity of SL dynamics in the absence and presence of ISO. (I) Time to peak values for changes in Fyellow/Fcyan and SL in the absence and presence of ISO. The values were less for both parameters, indicating the acceleration of the EC coupling at the single sarcomere level. (J) Comparison of the decay time for Fyellow/Fcyan. The time was shorter in the presence of ISO, indicating enhanced removal of cytosolic [Ca2+]i, presumably via acceleration of SR Ca2+ pump coupled with PLN phosphorylation (see Bers [2001] and references therein). In all graphs, horizontal bars indicate mean values. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).

Mentions: Because α-actinin–YC-Nano140 allows us to analyze not only sarcomere dynamics but also local [Ca2+]i changes, we then investigated the effects of β-adrenergic stimulation (50 nM ISO) on EC coupling at the single sarcomere level. A multitude of data showed a tendency of an increase in the frequency of spontaneous beating upon application of ISO; i.e., 33 ± 6 and 47 ± 23 bpm (P > 0.05) in the absence and presence of ISO, respectively (Fig. 6 A). We then converted the parameters and constructed a relationship of Fyellow/Fcyan versus SL and found that the relationship showed a counter-clockwise ellipsoidal trajectory (long and short axes in the x and y directions, respectively) in the absence and presence of ISO (as reported by Butler et al. [2015] in isolated canine ventricular myocytes; Fig. 6 B).


Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140
Effects of ISO on local coupling of [Ca2+]i and SL dynamics. (A) Individual data showing time courses of changes in Fyellow/Fcyan (top) and SL (bottom) in α-actinin–YC-Nano140–expressing cardiomyocytes during spontaneous beating in the absence (left; indicated as “control”) and presence (right) of 50 nM ISO. n = 7 and 9 in the absence and presence of ISO, respectively. Experiments were performed at 37°C. (B) Individual data showing the relationship of Fyellow/Fcyan versus SL in the absence and presence of ISO. A closed loop-like trajectory was observed, regardless of the use of ISO (compare Butler et al., 2015). (C) Comparison of Fyellow/Fcyan values during relaxation (R0) and contraction (Rmax) in the absence and presence of ISO. Fyellow/Fcyan was significantly higher for both R0 and Rmax. (D) Comparison of ΔR/R0 in the absence and presence of ISO (see Fig. 2 for ΔR/R0). (E) SL values in the absence and presence of ISO, during relaxation and contraction. ISO did not significantly affect SL, regardless of the contractile state. (F) ΔSL (difference between SL at relaxation and contraction) values in the absence and presence of ISO. No significant difference was observed between groups. Likewise, the variance was similar for both groups (i.e., from ∼0.03 to ∼0.17 µm for control [difference, ∼0.14 µm] and from ∼0.03 to ∼0.15 µm for ISO [difference, ∼0.12 µm]). (G) Shortening velocity of SL dynamics in the absence and presence of ISO. The velocity became significantly faster in the presence of ISO. (H) Lengthening velocity of SL dynamics in the absence and presence of ISO. (I) Time to peak values for changes in Fyellow/Fcyan and SL in the absence and presence of ISO. The values were less for both parameters, indicating the acceleration of the EC coupling at the single sarcomere level. (J) Comparison of the decay time for Fyellow/Fcyan. The time was shorter in the presence of ISO, indicating enhanced removal of cytosolic [Ca2+]i, presumably via acceleration of SR Ca2+ pump coupled with PLN phosphorylation (see Bers [2001] and references therein). In all graphs, horizontal bars indicate mean values. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).
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fig6: Effects of ISO on local coupling of [Ca2+]i and SL dynamics. (A) Individual data showing time courses of changes in Fyellow/Fcyan (top) and SL (bottom) in α-actinin–YC-Nano140–expressing cardiomyocytes during spontaneous beating in the absence (left; indicated as “control”) and presence (right) of 50 nM ISO. n = 7 and 9 in the absence and presence of ISO, respectively. Experiments were performed at 37°C. (B) Individual data showing the relationship of Fyellow/Fcyan versus SL in the absence and presence of ISO. A closed loop-like trajectory was observed, regardless of the use of ISO (compare Butler et al., 2015). (C) Comparison of Fyellow/Fcyan values during relaxation (R0) and contraction (Rmax) in the absence and presence of ISO. Fyellow/Fcyan was significantly higher for both R0 and Rmax. (D) Comparison of ΔR/R0 in the absence and presence of ISO (see Fig. 2 for ΔR/R0). (E) SL values in the absence and presence of ISO, during relaxation and contraction. ISO did not significantly affect SL, regardless of the contractile state. (F) ΔSL (difference between SL at relaxation and contraction) values in the absence and presence of ISO. No significant difference was observed between groups. Likewise, the variance was similar for both groups (i.e., from ∼0.03 to ∼0.17 µm for control [difference, ∼0.14 µm] and from ∼0.03 to ∼0.15 µm for ISO [difference, ∼0.12 µm]). (G) Shortening velocity of SL dynamics in the absence and presence of ISO. The velocity became significantly faster in the presence of ISO. (H) Lengthening velocity of SL dynamics in the absence and presence of ISO. (I) Time to peak values for changes in Fyellow/Fcyan and SL in the absence and presence of ISO. The values were less for both parameters, indicating the acceleration of the EC coupling at the single sarcomere level. (J) Comparison of the decay time for Fyellow/Fcyan. The time was shorter in the presence of ISO, indicating enhanced removal of cytosolic [Ca2+]i, presumably via acceleration of SR Ca2+ pump coupled with PLN phosphorylation (see Bers [2001] and references therein). In all graphs, horizontal bars indicate mean values. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).
Mentions: Because α-actinin–YC-Nano140 allows us to analyze not only sarcomere dynamics but also local [Ca2+]i changes, we then investigated the effects of β-adrenergic stimulation (50 nM ISO) on EC coupling at the single sarcomere level. A multitude of data showed a tendency of an increase in the frequency of spontaneous beating upon application of ISO; i.e., 33 ± 6 and 47 ± 23 bpm (P > 0.05) in the absence and presence of ISO, respectively (Fig. 6 A). We then converted the parameters and constructed a relationship of Fyellow/Fcyan versus SL and found that the relationship showed a counter-clockwise ellipsoidal trajectory (long and short axes in the x and y directions, respectively) in the absence and presence of ISO (as reported by Butler et al. [2015] in isolated canine ventricular myocytes; Fig. 6 B).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Excitation&ndash;contraction coupling results in the shortening of many individual sarcomeres along the length of a muscle fiber. Tsukamoto and colleagues develop a technique to quantitatively analyze the dynamics of intracellular calcium transients and length changes at the single sarcomere level.

No MeSH data available.


Related in: MedlinePlus