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Functional cardiac imaging by random access microscopy.

Crocini C, Coppini R, Ferrantini C, Pavone FS, Sacconi L - Front Physiol (2014)

Bottom Line: Advances in the development of voltage sensitive dyes and Ca(2+) sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution.At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved.With this approach, local Ca(2+) or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored.

View Article: PubMed Central - PubMed

Affiliation: European Laboratory for Non-Linear Spectroscopy (LENS) Florence, Italy.

ABSTRACT
Advances in the development of voltage sensitive dyes and Ca(2+) sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca(2+) release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca(2+) release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca(2+) or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.

No MeSH data available.


Related in: MedlinePlus

Intercellular propagation of action potential. (A) Bright-field image of a rat ventricular trabecula. The yellow arrowhead marks the stimulation site and the yellow diamond encompasses the recording area. (Scale bar: 1 mm.) (B) TPF image of the area highlighted in yellow in (A); trabecula stained with di-4-ANE(F)PPTEA (Scale bar: 20 μm.) (C) The region in the yellow box of b shows two adjacent myocytes magnified. (D) Normalized fluorescence traces from the scanned lines indicated in (C). APs are elicited at 0.2 Hz, corresponding to the black arrowhead. The traces are the average of 10 sequential episodes. (E) Normalized fluorescence traces (average of 10 episodes) recorded from SS and TT in cell 1 during stimulation at 5 Hz. Reproduced with permission from Sacconi et al. (2012).
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Figure 2: Intercellular propagation of action potential. (A) Bright-field image of a rat ventricular trabecula. The yellow arrowhead marks the stimulation site and the yellow diamond encompasses the recording area. (Scale bar: 1 mm.) (B) TPF image of the area highlighted in yellow in (A); trabecula stained with di-4-ANE(F)PPTEA (Scale bar: 20 μm.) (C) The region in the yellow box of b shows two adjacent myocytes magnified. (D) Normalized fluorescence traces from the scanned lines indicated in (C). APs are elicited at 0.2 Hz, corresponding to the black arrowhead. The traces are the average of 10 sequential episodes. (E) Normalized fluorescence traces (average of 10 episodes) recorded from SS and TT in cell 1 during stimulation at 5 Hz. Reproduced with permission from Sacconi et al. (2012).

Mentions: As discussed above, Ca2+ waves propagation is caused by local Ca2+ release from the SR, followed by diffusion and subsequent activation of adjacent SR Calcium Release units, generating a cascade of events. Ca2+ waves are accompanied by DADs (Ter Keurs and Boyden, 2007). Since the average ventricular myocyte is directly coupled to about 11 other myocytes, an afterdepolarization would be immediately suppressed by the large “current sink” unless a sufficient number of neighboring cardiomyocytes also synchronously develop an afterdepolarization at the same beat. Propagation of Ca2+ waves from cell-to-cell has been shown to occur at a constant velocity (Vprop) that ranged from 0.1 to 15 mm/s, a speed that is too slow to be determined by active (1 m/s) or electrotonic membrane conduction (0.1 m/s) (Ter Keurs and Boyden, 2007) but is compatible with a facilitated Ca2+ diffusion. Mechanisms underlying Ca2+ waves propagation are still poorly understood. However, Vprop can increase under certain circumstances, such as increased RyR open probability or in presence of SR Ca2+ overload. The faster the Vprop the higher the probability that a large area would “nearly simultaneously” be invaded by a propagated Ca2+ wave, thus increasing the likelihood of “synchronized” DADs in adjacent cells. Exploiting the advantage of multi-photon excitation, multisite optical recordings can also be performed in multicellular preparation such as intact trabeculae and muscle bundles in which cell-to-cell conduction occurs through sarcolemmal gap junctions (Sacconi et al., 2012; Ferrantini et al., 2014). Figure 2 shows examples of APs recorded in SS and TATS of two adjacent cells. Trabeculae were locally stimulated in a region 2 mm apart from the recording area (Figure 2A), and an 15 ms delay was found between the stimulus and the rapid AP upstroke phase (Figure 2D), in agreement with the expected myocardium conduction velocity at room temperature.


Functional cardiac imaging by random access microscopy.

Crocini C, Coppini R, Ferrantini C, Pavone FS, Sacconi L - Front Physiol (2014)

Intercellular propagation of action potential. (A) Bright-field image of a rat ventricular trabecula. The yellow arrowhead marks the stimulation site and the yellow diamond encompasses the recording area. (Scale bar: 1 mm.) (B) TPF image of the area highlighted in yellow in (A); trabecula stained with di-4-ANE(F)PPTEA (Scale bar: 20 μm.) (C) The region in the yellow box of b shows two adjacent myocytes magnified. (D) Normalized fluorescence traces from the scanned lines indicated in (C). APs are elicited at 0.2 Hz, corresponding to the black arrowhead. The traces are the average of 10 sequential episodes. (E) Normalized fluorescence traces (average of 10 episodes) recorded from SS and TT in cell 1 during stimulation at 5 Hz. Reproduced with permission from Sacconi et al. (2012).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4202699&req=5

Figure 2: Intercellular propagation of action potential. (A) Bright-field image of a rat ventricular trabecula. The yellow arrowhead marks the stimulation site and the yellow diamond encompasses the recording area. (Scale bar: 1 mm.) (B) TPF image of the area highlighted in yellow in (A); trabecula stained with di-4-ANE(F)PPTEA (Scale bar: 20 μm.) (C) The region in the yellow box of b shows two adjacent myocytes magnified. (D) Normalized fluorescence traces from the scanned lines indicated in (C). APs are elicited at 0.2 Hz, corresponding to the black arrowhead. The traces are the average of 10 sequential episodes. (E) Normalized fluorescence traces (average of 10 episodes) recorded from SS and TT in cell 1 during stimulation at 5 Hz. Reproduced with permission from Sacconi et al. (2012).
Mentions: As discussed above, Ca2+ waves propagation is caused by local Ca2+ release from the SR, followed by diffusion and subsequent activation of adjacent SR Calcium Release units, generating a cascade of events. Ca2+ waves are accompanied by DADs (Ter Keurs and Boyden, 2007). Since the average ventricular myocyte is directly coupled to about 11 other myocytes, an afterdepolarization would be immediately suppressed by the large “current sink” unless a sufficient number of neighboring cardiomyocytes also synchronously develop an afterdepolarization at the same beat. Propagation of Ca2+ waves from cell-to-cell has been shown to occur at a constant velocity (Vprop) that ranged from 0.1 to 15 mm/s, a speed that is too slow to be determined by active (1 m/s) or electrotonic membrane conduction (0.1 m/s) (Ter Keurs and Boyden, 2007) but is compatible with a facilitated Ca2+ diffusion. Mechanisms underlying Ca2+ waves propagation are still poorly understood. However, Vprop can increase under certain circumstances, such as increased RyR open probability or in presence of SR Ca2+ overload. The faster the Vprop the higher the probability that a large area would “nearly simultaneously” be invaded by a propagated Ca2+ wave, thus increasing the likelihood of “synchronized” DADs in adjacent cells. Exploiting the advantage of multi-photon excitation, multisite optical recordings can also be performed in multicellular preparation such as intact trabeculae and muscle bundles in which cell-to-cell conduction occurs through sarcolemmal gap junctions (Sacconi et al., 2012; Ferrantini et al., 2014). Figure 2 shows examples of APs recorded in SS and TATS of two adjacent cells. Trabeculae were locally stimulated in a region 2 mm apart from the recording area (Figure 2A), and an 15 ms delay was found between the stimulus and the rapid AP upstroke phase (Figure 2D), in agreement with the expected myocardium conduction velocity at room temperature.

Bottom Line: Advances in the development of voltage sensitive dyes and Ca(2+) sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution.At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved.With this approach, local Ca(2+) or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored.

View Article: PubMed Central - PubMed

Affiliation: European Laboratory for Non-Linear Spectroscopy (LENS) Florence, Italy.

ABSTRACT
Advances in the development of voltage sensitive dyes and Ca(2+) sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca(2+) release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca(2+) release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca(2+) or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.

No MeSH data available.


Related in: MedlinePlus