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Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

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Principle of the SICM/FRET technique and its use to study βAR localization in cardiomyocytes. (a) SICM image (32 × 32 µm) of an adult rat cardiomyocyte acquired using a nanopipette from the top of the cell. The sample is positioned on an inverted epifluorescent microscope, so that recordings of cellular fluorescence can be performed. (b) Inset shows a 10 × 10 µm scan of the cardiomyocyte surface with characteristic structural features (cell crests, Z-lines and T-tubule openings). Effective pixel width was 156 nm, scan duration 4 min. The cells are expressing a FRET-based cAMP sensor Epac2-camps, which reports changes in intracellular cAMP levels after local cell surface stimulation via an SICM nanopipette with β1AR or β2AR selective ligands applied either into a T-tubule opening or onto the cell crest. Binding of cAMP to the sensor causes a change in its conformations, which results in a longer distance between the fluorophores (CFP and YFP) and lower FRET signal. (c) Stimulation of β1ARs in both T-tubular (red line) and cell crest region (black line) results in a decrease of FRET, which reflects an increase in cAMP levels. In contrast, β2AR induces cAMP signals only when stimulated in the T-tubule, but not on the cell crest (n = 9). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Nikolaev et al. [18] with permission. (Online version in colour.)
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RSIF20100597F9: Principle of the SICM/FRET technique and its use to study βAR localization in cardiomyocytes. (a) SICM image (32 × 32 µm) of an adult rat cardiomyocyte acquired using a nanopipette from the top of the cell. The sample is positioned on an inverted epifluorescent microscope, so that recordings of cellular fluorescence can be performed. (b) Inset shows a 10 × 10 µm scan of the cardiomyocyte surface with characteristic structural features (cell crests, Z-lines and T-tubule openings). Effective pixel width was 156 nm, scan duration 4 min. The cells are expressing a FRET-based cAMP sensor Epac2-camps, which reports changes in intracellular cAMP levels after local cell surface stimulation via an SICM nanopipette with β1AR or β2AR selective ligands applied either into a T-tubule opening or onto the cell crest. Binding of cAMP to the sensor causes a change in its conformations, which results in a longer distance between the fluorophores (CFP and YFP) and lower FRET signal. (c) Stimulation of β1ARs in both T-tubular (red line) and cell crest region (black line) results in a decrease of FRET, which reflects an increase in cAMP levels. In contrast, β2AR induces cAMP signals only when stimulated in the T-tubule, but not on the cell crest (n = 9). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Nikolaev et al. [18] with permission. (Online version in colour.)

Mentions: G-protein-coupled receptors such as β-adrenergic receptors (βARs) and M2 muscarinic receptors play a central role in regulating cardiac function and disease. We recently developed a novel functional approach that combines SICM with local ligand application and fluorescence resonance energy transfer (FRET)-based measurements of cAMP production by locally activated receptors (figure 9b) [18]. Using this hybrid SICM/FRET technique, we showed that β2AR are selectively localized in the T-tubules of healthy adult rat cardiomyocytes (figure 9b,c), while β1AR are evenly distributed across the cell membrane. Importantly, cells isolated from rats after myocardial infarction revealed a redistribution of β2AR, which now appeared in non-tubular areas of detubulated failing cardiomyocytes [18]. Redistribution of this receptor also resulted in changes of subcellular compartmentation of cAMP signals, which might play an important role in the development of cardiac disease. Figure 9 shows that one can combine SICM with FRET to analyse the precise distribution of various membrane receptors with a few-hundred nanometre resolution and to correlate disease-driven changes in cell surface morphology with alterations in intracellular signalling. We believe that this approach provides another multi-parametric possibility to study functionally relevant signalling compartments in cardiac cells and to investigate how receptor distribution and the subcellular mechanisms of receptor-mediated downstream signalling are changed in cardiac disease.Figure 9.


Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Principle of the SICM/FRET technique and its use to study βAR localization in cardiomyocytes. (a) SICM image (32 × 32 µm) of an adult rat cardiomyocyte acquired using a nanopipette from the top of the cell. The sample is positioned on an inverted epifluorescent microscope, so that recordings of cellular fluorescence can be performed. (b) Inset shows a 10 × 10 µm scan of the cardiomyocyte surface with characteristic structural features (cell crests, Z-lines and T-tubule openings). Effective pixel width was 156 nm, scan duration 4 min. The cells are expressing a FRET-based cAMP sensor Epac2-camps, which reports changes in intracellular cAMP levels after local cell surface stimulation via an SICM nanopipette with β1AR or β2AR selective ligands applied either into a T-tubule opening or onto the cell crest. Binding of cAMP to the sensor causes a change in its conformations, which results in a longer distance between the fluorophores (CFP and YFP) and lower FRET signal. (c) Stimulation of β1ARs in both T-tubular (red line) and cell crest region (black line) results in a decrease of FRET, which reflects an increase in cAMP levels. In contrast, β2AR induces cAMP signals only when stimulated in the T-tubule, but not on the cell crest (n = 9). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Nikolaev et al. [18] with permission. (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104336&req=5

RSIF20100597F9: Principle of the SICM/FRET technique and its use to study βAR localization in cardiomyocytes. (a) SICM image (32 × 32 µm) of an adult rat cardiomyocyte acquired using a nanopipette from the top of the cell. The sample is positioned on an inverted epifluorescent microscope, so that recordings of cellular fluorescence can be performed. (b) Inset shows a 10 × 10 µm scan of the cardiomyocyte surface with characteristic structural features (cell crests, Z-lines and T-tubule openings). Effective pixel width was 156 nm, scan duration 4 min. The cells are expressing a FRET-based cAMP sensor Epac2-camps, which reports changes in intracellular cAMP levels after local cell surface stimulation via an SICM nanopipette with β1AR or β2AR selective ligands applied either into a T-tubule opening or onto the cell crest. Binding of cAMP to the sensor causes a change in its conformations, which results in a longer distance between the fluorophores (CFP and YFP) and lower FRET signal. (c) Stimulation of β1ARs in both T-tubular (red line) and cell crest region (black line) results in a decrease of FRET, which reflects an increase in cAMP levels. In contrast, β2AR induces cAMP signals only when stimulated in the T-tubule, but not on the cell crest (n = 9). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Nikolaev et al. [18] with permission. (Online version in colour.)
Mentions: G-protein-coupled receptors such as β-adrenergic receptors (βARs) and M2 muscarinic receptors play a central role in regulating cardiac function and disease. We recently developed a novel functional approach that combines SICM with local ligand application and fluorescence resonance energy transfer (FRET)-based measurements of cAMP production by locally activated receptors (figure 9b) [18]. Using this hybrid SICM/FRET technique, we showed that β2AR are selectively localized in the T-tubules of healthy adult rat cardiomyocytes (figure 9b,c), while β1AR are evenly distributed across the cell membrane. Importantly, cells isolated from rats after myocardial infarction revealed a redistribution of β2AR, which now appeared in non-tubular areas of detubulated failing cardiomyocytes [18]. Redistribution of this receptor also resulted in changes of subcellular compartmentation of cAMP signals, which might play an important role in the development of cardiac disease. Figure 9 shows that one can combine SICM with FRET to analyse the precise distribution of various membrane receptors with a few-hundred nanometre resolution and to correlate disease-driven changes in cell surface morphology with alterations in intracellular signalling. We believe that this approach provides another multi-parametric possibility to study functionally relevant signalling compartments in cardiac cells and to investigate how receptor distribution and the subcellular mechanisms of receptor-mediated downstream signalling are changed in cardiac disease.Figure 9.

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus