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

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

Simultaneous imaging of local [Ca2+]i and SL upon electrical stimulation at 5 Hz. (A) Time course of changes in Fyellow/Fcyan (red) and SL (blue) in a cardiomyocyte. Electrical stimulation was performed for 10 s (from 5 to 15 s on the x axis). Bar, period for electrical stimulation. See Video 3. (B) Enlarged view of the graph showing time-dependent changes in Fyellow/Fcyan and SL in A from 10 to 12 s. Note that SL changes in a reciprocal manner in response to a change in Fyellow/Fcyan (as previously demonstrated by us using Fluo-4: see Shintani et al., 2014). (C, top) FFT analysis for the change in SL. (bottom) Same as in the top, for the change in Fyellow/Fcyan. Note that only one peak is present at 5 Hz for both SL and Fyellow/Fcyan, indicating that the EC coupling (i.e., an increase in local [Ca2+]i and the ensuing actomyosin interaction) occurs locally at the single sarcomere level under the present condition. Data were averaged from four individual sarcomeres for both Fyellow/Fcyan and SL.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5037341&req=5

fig5: Simultaneous imaging of local [Ca2+]i and SL upon electrical stimulation at 5 Hz. (A) Time course of changes in Fyellow/Fcyan (red) and SL (blue) in a cardiomyocyte. Electrical stimulation was performed for 10 s (from 5 to 15 s on the x axis). Bar, period for electrical stimulation. See Video 3. (B) Enlarged view of the graph showing time-dependent changes in Fyellow/Fcyan and SL in A from 10 to 12 s. Note that SL changes in a reciprocal manner in response to a change in Fyellow/Fcyan (as previously demonstrated by us using Fluo-4: see Shintani et al., 2014). (C, top) FFT analysis for the change in SL. (bottom) Same as in the top, for the change in Fyellow/Fcyan. Note that only one peak is present at 5 Hz for both SL and Fyellow/Fcyan, indicating that the EC coupling (i.e., an increase in local [Ca2+]i and the ensuing actomyosin interaction) occurs locally at the single sarcomere level under the present condition. Data were averaged from four individual sarcomeres for both Fyellow/Fcyan and SL.

Mentions: The relatively slow kinetics of α-actinin–YC-Nano140 may limit its use as a Ca2+ sensor at high stimulation frequencies. Accordingly, we investigated whether α-actinin–YC-Nano140 enables simultaneous measurement of local [Ca2+]i and sarcomere dynamics at a physiologically relevant stimulation frequency, as performed in our previous study by using AcGFP expression in the Z disks (i.e., ∼240 beats per min [∼4 Hz] for rat neonates; Smotherman et al., 1991). Consistent with the findings of our previous studies on adult (Serizawa et al., 2011) as well as on neonatal (Shintani et al., 2014) myocytes of the rat, diastolic Fyellow/Fcyan was elevated by ∼0.7 U upon electric stimulation, with a relatively minor influence on systolic Fyellow/Fcyan (Fig. 5 A and Video 3). Approximately 2 s after the onset of electric stimulation, stable SL changes with a magnitude of ∼0.06 µm appeared and continued until the cessation of stimulation. We found that as observed during spontaneous beating (Figs. 1 and 4), SL decreased (increased) in response to a rise (fall) in Fyellow/Fcyan during the stimulation period (systolic and diastolic SLs, ∼1.80 and ∼1.86 µm, respectively; Fig. 5 B). Our FFT analyses revealed that a single peak existed at 5 Hz for both SL and Fyellow/Fcyan (Fig. 5 C), though the magnitude of the change in Fyellow/Fcyan during electrical stimulation was smaller than that during spontaneous beating. Although the rate of rise or fall of [Ca2+]i becomes reportedly faster in cardiomyocytes during the course of development (Haddock et al., 1999), the present experimental findings suggest that the expression of YC-Nano140 in the Z disks is a useful tool for analyzing EC coupling at the single sarcomere level at physiologically relevant action potential frequencies in myocytes, as well as in vivo by incorporating α-actinin–YC-Nano140 into adenoviruses (see Kobirumaki-Shimozawa et al., 2016). See Supplemental discussion IV and Fig. S5.


Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140
Simultaneous imaging of local [Ca2+]i and SL upon electrical stimulation at 5 Hz. (A) Time course of changes in Fyellow/Fcyan (red) and SL (blue) in a cardiomyocyte. Electrical stimulation was performed for 10 s (from 5 to 15 s on the x axis). Bar, period for electrical stimulation. See Video 3. (B) Enlarged view of the graph showing time-dependent changes in Fyellow/Fcyan and SL in A from 10 to 12 s. Note that SL changes in a reciprocal manner in response to a change in Fyellow/Fcyan (as previously demonstrated by us using Fluo-4: see Shintani et al., 2014). (C, top) FFT analysis for the change in SL. (bottom) Same as in the top, for the change in Fyellow/Fcyan. Note that only one peak is present at 5 Hz for both SL and Fyellow/Fcyan, indicating that the EC coupling (i.e., an increase in local [Ca2+]i and the ensuing actomyosin interaction) occurs locally at the single sarcomere level under the present condition. Data were averaged from four individual sarcomeres for both Fyellow/Fcyan and SL.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037341&req=5

fig5: Simultaneous imaging of local [Ca2+]i and SL upon electrical stimulation at 5 Hz. (A) Time course of changes in Fyellow/Fcyan (red) and SL (blue) in a cardiomyocyte. Electrical stimulation was performed for 10 s (from 5 to 15 s on the x axis). Bar, period for electrical stimulation. See Video 3. (B) Enlarged view of the graph showing time-dependent changes in Fyellow/Fcyan and SL in A from 10 to 12 s. Note that SL changes in a reciprocal manner in response to a change in Fyellow/Fcyan (as previously demonstrated by us using Fluo-4: see Shintani et al., 2014). (C, top) FFT analysis for the change in SL. (bottom) Same as in the top, for the change in Fyellow/Fcyan. Note that only one peak is present at 5 Hz for both SL and Fyellow/Fcyan, indicating that the EC coupling (i.e., an increase in local [Ca2+]i and the ensuing actomyosin interaction) occurs locally at the single sarcomere level under the present condition. Data were averaged from four individual sarcomeres for both Fyellow/Fcyan and SL.
Mentions: The relatively slow kinetics of α-actinin–YC-Nano140 may limit its use as a Ca2+ sensor at high stimulation frequencies. Accordingly, we investigated whether α-actinin–YC-Nano140 enables simultaneous measurement of local [Ca2+]i and sarcomere dynamics at a physiologically relevant stimulation frequency, as performed in our previous study by using AcGFP expression in the Z disks (i.e., ∼240 beats per min [∼4 Hz] for rat neonates; Smotherman et al., 1991). Consistent with the findings of our previous studies on adult (Serizawa et al., 2011) as well as on neonatal (Shintani et al., 2014) myocytes of the rat, diastolic Fyellow/Fcyan was elevated by ∼0.7 U upon electric stimulation, with a relatively minor influence on systolic Fyellow/Fcyan (Fig. 5 A and Video 3). Approximately 2 s after the onset of electric stimulation, stable SL changes with a magnitude of ∼0.06 µm appeared and continued until the cessation of stimulation. We found that as observed during spontaneous beating (Figs. 1 and 4), SL decreased (increased) in response to a rise (fall) in Fyellow/Fcyan during the stimulation period (systolic and diastolic SLs, ∼1.80 and ∼1.86 µm, respectively; Fig. 5 B). Our FFT analyses revealed that a single peak existed at 5 Hz for both SL and Fyellow/Fcyan (Fig. 5 C), though the magnitude of the change in Fyellow/Fcyan during electrical stimulation was smaller than that during spontaneous beating. Although the rate of rise or fall of [Ca2+]i becomes reportedly faster in cardiomyocytes during the course of development (Haddock et al., 1999), the present experimental findings suggest that the expression of YC-Nano140 in the Z disks is a useful tool for analyzing EC coupling at the single sarcomere level at physiologically relevant action potential frequencies in myocytes, as well as in vivo by incorporating α-actinin–YC-Nano140 into adenoviruses (see Kobirumaki-Shimozawa et al., 2016). See Supplemental discussion IV and Fig. S5.

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