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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.


Related in: MedlinePlus

Sarcomere dynamics and local [Ca2+]i along a myofibril in a cardiomyocyte under various conditions. (A) Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z9, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 37°C. (B, top) Time course of changes in the lengths of five sarcomeres in a myocyte in A during spontaneous beating. Black line, superimposed data of sequentially connecting eight sarcomeres. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 4. (C) Same as in A, but the experimentation was performed in the presence of 100 nM ISO. Sarcomeres in the yellow rectangular region in the epi-illumination image were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z11, numbers of the Z disks for analyzed sarcomeres). (D) As indicated in this kymograph of the yellow fluorescence signal (for 5 s), two major Ca2+ waves were observed during the course of imaging. (E) Time courses (5 s) of changes in SL and ΔR/R0. Arrows indicate the points at which Ca2+ waves (and the ensuing sarcomere contractions) occurred. (F) Time course (0.5 s) of changes in SL and Ca2+ waves. (G) Variances in SL versus ΔR/R0, resulting in the significant linear correlation between ΔR/R0 and ΔSL for both the first and second Ca2+ waves. See Video 5. (H) Same as in A, but the experimentation was performed in the presence of 2 µM OM. Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z6, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 36°C. (I, top) Time course of changes in the lengths of sequentially connecting five sarcomeres in a myocyte in H during spontaneous beating. Black line, superimposed data. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 6.
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fig7: Sarcomere dynamics and local [Ca2+]i along a myofibril in a cardiomyocyte under various conditions. (A) Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z9, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 37°C. (B, top) Time course of changes in the lengths of five sarcomeres in a myocyte in A during spontaneous beating. Black line, superimposed data of sequentially connecting eight sarcomeres. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 4. (C) Same as in A, but the experimentation was performed in the presence of 100 nM ISO. Sarcomeres in the yellow rectangular region in the epi-illumination image were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z11, numbers of the Z disks for analyzed sarcomeres). (D) As indicated in this kymograph of the yellow fluorescence signal (for 5 s), two major Ca2+ waves were observed during the course of imaging. (E) Time courses (5 s) of changes in SL and ΔR/R0. Arrows indicate the points at which Ca2+ waves (and the ensuing sarcomere contractions) occurred. (F) Time course (0.5 s) of changes in SL and Ca2+ waves. (G) Variances in SL versus ΔR/R0, resulting in the significant linear correlation between ΔR/R0 and ΔSL for both the first and second Ca2+ waves. See Video 5. (H) Same as in A, but the experimentation was performed in the presence of 2 µM OM. Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z6, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 36°C. (I, top) Time course of changes in the lengths of sequentially connecting five sarcomeres in a myocyte in H during spontaneous beating. Black line, superimposed data. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 6.

Mentions: First, we analyzed individual behaviors of sequentially connecting single sarcomeres along a myofibril, simultaneously with local CaT. Although the magnitude of shortening or lengthening of sequentially connecting eight sarcomeres varied from ∼0.1 to ∼0.2 µm (with diastolic SL varying from ∼1.65 to ∼1.92 µm) during the course of contraction, the averaged SL displacement became diminished to the level of ∼0.06 µm (Fig. 7 A and Video 4). In contrast to sarcomere dynamics, the changes in Fyellow/Fcyan in the Z disks were well synchronized, suggesting that CaT occurs in a uniform manner along a myofibril, but each sarcomere exerts its own pattern of shortening/lengthening during spontaneous beating (as in Shintani et al., 2014). Therefore, the imbalance of tug of war between sarcomeres along a myofibril (because of the individuality of each sarcomere, such as variance in structure or PKA or protein kinase C–dependent phosphorylation of thick/thin filaments and the ensuing difference in Ca2+ sensitivity), but not varying levels of local [Ca2+]i, is likely to underlie unsynchronized sarcomeric behaviors. In a recent study on the beating heart in healthy adult mice, we demonstrated that SL in left ventricular myocytes varied by as much as ∼300 nm in both diastole and systole (Kobirumaki-Shimozawa et al., 2016). Therefore, future studies should be directed to elucidating how individual dynamic properties of cardiac sarcomeres are integrated to exert rhythmic pump functions of the heart by systematically analyzing local EC coupling in myocytes at various locations of the heart.


Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140
Sarcomere dynamics and local [Ca2+]i along a myofibril in a cardiomyocyte under various conditions. (A) Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z9, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 37°C. (B, top) Time course of changes in the lengths of five sarcomeres in a myocyte in A during spontaneous beating. Black line, superimposed data of sequentially connecting eight sarcomeres. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 4. (C) Same as in A, but the experimentation was performed in the presence of 100 nM ISO. Sarcomeres in the yellow rectangular region in the epi-illumination image were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z11, numbers of the Z disks for analyzed sarcomeres). (D) As indicated in this kymograph of the yellow fluorescence signal (for 5 s), two major Ca2+ waves were observed during the course of imaging. (E) Time courses (5 s) of changes in SL and ΔR/R0. Arrows indicate the points at which Ca2+ waves (and the ensuing sarcomere contractions) occurred. (F) Time course (0.5 s) of changes in SL and Ca2+ waves. (G) Variances in SL versus ΔR/R0, resulting in the significant linear correlation between ΔR/R0 and ΔSL for both the first and second Ca2+ waves. See Video 5. (H) Same as in A, but the experimentation was performed in the presence of 2 µM OM. Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z6, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 36°C. (I, top) Time course of changes in the lengths of sequentially connecting five sarcomeres in a myocyte in H during spontaneous beating. Black line, superimposed data. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 6.
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fig7: Sarcomere dynamics and local [Ca2+]i along a myofibril in a cardiomyocyte under various conditions. (A) Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z9, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 37°C. (B, top) Time course of changes in the lengths of five sarcomeres in a myocyte in A during spontaneous beating. Black line, superimposed data of sequentially connecting eight sarcomeres. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 4. (C) Same as in A, but the experimentation was performed in the presence of 100 nM ISO. Sarcomeres in the yellow rectangular region in the epi-illumination image were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z11, numbers of the Z disks for analyzed sarcomeres). (D) As indicated in this kymograph of the yellow fluorescence signal (for 5 s), two major Ca2+ waves were observed during the course of imaging. (E) Time courses (5 s) of changes in SL and ΔR/R0. Arrows indicate the points at which Ca2+ waves (and the ensuing sarcomere contractions) occurred. (F) Time course (0.5 s) of changes in SL and Ca2+ waves. (G) Variances in SL versus ΔR/R0, resulting in the significant linear correlation between ΔR/R0 and ΔSL for both the first and second Ca2+ waves. See Video 5. (H) Same as in A, but the experimentation was performed in the presence of 2 µM OM. Epi-illumination image of a myocyte expressing α-actinin–YC-Nano140. Sarcomeres in the yellow rectangular region were used for the analyses on Fyellow/Fcyan and SL dynamics (Z1 and Z6, numbers of the Z disks for analyzed sarcomeres). Experiments were performed at 36°C. (I, top) Time course of changes in the lengths of sequentially connecting five sarcomeres in a myocyte in H during spontaneous beating. Black line, superimposed data. (bottom) Time course of changes in the mean Fyellow/Fcyan of the Z disks of each sarcomere. See Video 6.
Mentions: First, we analyzed individual behaviors of sequentially connecting single sarcomeres along a myofibril, simultaneously with local CaT. Although the magnitude of shortening or lengthening of sequentially connecting eight sarcomeres varied from ∼0.1 to ∼0.2 µm (with diastolic SL varying from ∼1.65 to ∼1.92 µm) during the course of contraction, the averaged SL displacement became diminished to the level of ∼0.06 µm (Fig. 7 A and Video 4). In contrast to sarcomere dynamics, the changes in Fyellow/Fcyan in the Z disks were well synchronized, suggesting that CaT occurs in a uniform manner along a myofibril, but each sarcomere exerts its own pattern of shortening/lengthening during spontaneous beating (as in Shintani et al., 2014). Therefore, the imbalance of tug of war between sarcomeres along a myofibril (because of the individuality of each sarcomere, such as variance in structure or PKA or protein kinase C–dependent phosphorylation of thick/thin filaments and the ensuing difference in Ca2+ sensitivity), but not varying levels of local [Ca2+]i, is likely to underlie unsynchronized sarcomeric behaviors. In a recent study on the beating heart in healthy adult mice, we demonstrated that SL in left ventricular myocytes varied by as much as ∼300 nm in both diastole and systole (Kobirumaki-Shimozawa et al., 2016). Therefore, future studies should be directed to elucidating how individual dynamic properties of cardiac sarcomeres are integrated to exert rhythmic pump functions of the heart by systematically analyzing local EC coupling in myocytes at various locations of the heart.

View Article: PubMed Central - HTML - PubMed

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.


Related in: MedlinePlus