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.


Changes in SL and local [Ca2+] during spontaneous beating at different temperatures. (A) Time course of changes in SL and local Fyellow/Fcyan measured with α-actinin–YC-Nano140 during spontaneous beating at 37°C. Data were averaged from 10 sarcomeres. (B) Same as in A at 22°C. Data were averaged from 13 sarcomeres. (C) Graph summarizing time to peak values of changes in SL and Fyellow/Fcyan at 22°C and 37°C. *, P < 0.05; and ***, P < 0.001 (Tukey-Kramer test). (D) Graph comparing delay time (difference in time between SL and Fyellow/Fcyan) at 22°C and 37°C. ***, P < 0.001 (Mann-Whitney U test). Bars in each graph indicate mean values.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Changes in SL and local [Ca2+] during spontaneous beating at different temperatures. (A) Time course of changes in SL and local Fyellow/Fcyan measured with α-actinin–YC-Nano140 during spontaneous beating at 37°C. Data were averaged from 10 sarcomeres. (B) Same as in A at 22°C. Data were averaged from 13 sarcomeres. (C) Graph summarizing time to peak values of changes in SL and Fyellow/Fcyan at 22°C and 37°C. *, P < 0.05; and ***, P < 0.001 (Tukey-Kramer test). (D) Graph comparing delay time (difference in time between SL and Fyellow/Fcyan) at 22°C and 37°C. ***, P < 0.001 (Mann-Whitney U test). Bars in each graph indicate mean values.

Mentions: Next, we analyzed the time course of changes in Fyellow/Fcyan and SL at different temperatures (i.e., 22°C and 37°C; Fig. 4, A and B). As in the case for Fyellow/Fcyan (see above), time to peak for SL change became abbreviated upon an increase in temperature (Fig. 4 C), indicating enhanced actomyosin interaction (see Bers [2001] and references therein). The magnitude of abbreviation was greater for SL (∼60%) than Fyellow/Fcyan (∼45%; Fig. 4 C), suggesting that because sarcomere shortening simply reflects actomyosin interaction (with ATPase), it is more sensitive to a change in temperature (as in Bers, 2001) than a rise in Fyellow/Fcyan (which may be uncoupled with an ATPase reaction, e.g., Ca2+ influx through sarcolemmal Ca2+ channels; see discussion above). We found that the peak of SL shortening preceded that of Fyellow/Fcyan at both temperatures, with the difference (noted as “delay time” in Fig. 4 D) significantly larger at 37°C (92 ± 53 ms) than at 22°C (31 ± 50 ms). Given these findings, one may point out that the relative slow response of α-actinin–YC-Nano140 to Ca2+ may be a disadvantage in the analysis of cardiac EC coupling, despite its beneficial capability of the simultaneous analysis of local Ca2+ and SL displacement via expression at a particular region in a cell. We consider that this issue will be technically solved by calculation. As generally known, the koff and kon values of the currently available Ca2+ indicators of any type (including Fluo-4) are too slow to obtain physiologically relevant changes in CaT (or Ca2+ sparks) in cardiomyocytes. Therefore, in order to accurately quantify the dynamics of [Ca2+]i, occurring either locally or globally, or both, in cardiomyocytes, the fluorescence signals of α-actinin–YC-Nano140 need to be calibrated to true signals, based on the temperature-dependent values of its kon and koff (as demonstrated by others on adult ventricular myocytes; Kaestner et al., 2014; Shang et al., 2014). See Supplemental discussion III and Fig. S4.


Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140
Changes in SL and local [Ca2+] during spontaneous beating at different temperatures. (A) Time course of changes in SL and local Fyellow/Fcyan measured with α-actinin–YC-Nano140 during spontaneous beating at 37°C. Data were averaged from 10 sarcomeres. (B) Same as in A at 22°C. Data were averaged from 13 sarcomeres. (C) Graph summarizing time to peak values of changes in SL and Fyellow/Fcyan at 22°C and 37°C. *, P < 0.05; and ***, P < 0.001 (Tukey-Kramer test). (D) Graph comparing delay time (difference in time between SL and Fyellow/Fcyan) at 22°C and 37°C. ***, P < 0.001 (Mann-Whitney U test). Bars in each graph indicate mean values.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Changes in SL and local [Ca2+] during spontaneous beating at different temperatures. (A) Time course of changes in SL and local Fyellow/Fcyan measured with α-actinin–YC-Nano140 during spontaneous beating at 37°C. Data were averaged from 10 sarcomeres. (B) Same as in A at 22°C. Data were averaged from 13 sarcomeres. (C) Graph summarizing time to peak values of changes in SL and Fyellow/Fcyan at 22°C and 37°C. *, P < 0.05; and ***, P < 0.001 (Tukey-Kramer test). (D) Graph comparing delay time (difference in time between SL and Fyellow/Fcyan) at 22°C and 37°C. ***, P < 0.001 (Mann-Whitney U test). Bars in each graph indicate mean values.
Mentions: Next, we analyzed the time course of changes in Fyellow/Fcyan and SL at different temperatures (i.e., 22°C and 37°C; Fig. 4, A and B). As in the case for Fyellow/Fcyan (see above), time to peak for SL change became abbreviated upon an increase in temperature (Fig. 4 C), indicating enhanced actomyosin interaction (see Bers [2001] and references therein). The magnitude of abbreviation was greater for SL (∼60%) than Fyellow/Fcyan (∼45%; Fig. 4 C), suggesting that because sarcomere shortening simply reflects actomyosin interaction (with ATPase), it is more sensitive to a change in temperature (as in Bers, 2001) than a rise in Fyellow/Fcyan (which may be uncoupled with an ATPase reaction, e.g., Ca2+ influx through sarcolemmal Ca2+ channels; see discussion above). We found that the peak of SL shortening preceded that of Fyellow/Fcyan at both temperatures, with the difference (noted as “delay time” in Fig. 4 D) significantly larger at 37°C (92 ± 53 ms) than at 22°C (31 ± 50 ms). Given these findings, one may point out that the relative slow response of α-actinin–YC-Nano140 to Ca2+ may be a disadvantage in the analysis of cardiac EC coupling, despite its beneficial capability of the simultaneous analysis of local Ca2+ and SL displacement via expression at a particular region in a cell. We consider that this issue will be technically solved by calculation. As generally known, the koff and kon values of the currently available Ca2+ indicators of any type (including Fluo-4) are too slow to obtain physiologically relevant changes in CaT (or Ca2+ sparks) in cardiomyocytes. Therefore, in order to accurately quantify the dynamics of [Ca2+]i, occurring either locally or globally, or both, in cardiomyocytes, the fluorescence signals of α-actinin–YC-Nano140 need to be calibrated to true signals, based on the temperature-dependent values of its kon and koff (as demonstrated by others on adult ventricular myocytes; Kaestner et al., 2014; Shang et al., 2014). See Supplemental discussion III and Fig. S4.

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.