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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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L-type Ca2+ channel distribution in the cardiac myocytes sarcolemma: mapping of ion channels by the high-resolution scanning patch-clamp technique. (a) Experimental topographic image of a representative rat cardiomyocyte sarcolemma. Z-grooves, T-tubule opening and characteristic sarcomere units are marked. (b) Functional schematic of sarcomere units showing the position of the probed region (Z-groove, T-tubule opening and scallop crest). Probabilities of forming a gigaseal as a function of surface position shown in parentheses. (c) Statistical distribution of L-type Ca2+ channels with the highest density near the T-tubule opening. (d) Cell-attached Ba2+ current transients at voltages of +20, ±0, −20 mV. (e) Several current transients elicited at 0 mV from one patch and ensemble average of 12 transients showing typical L-type inactivation kinetics. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of approximately 100 nm. Topography was recorded in the conventional distance modulated mode with pixel number set to 1024 × 256. Scan duration was 20 min. Modified from Gu et al. [55], with permission. (Online version in colour.)
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RSIF20100597F8: L-type Ca2+ channel distribution in the cardiac myocytes sarcolemma: mapping of ion channels by the high-resolution scanning patch-clamp technique. (a) Experimental topographic image of a representative rat cardiomyocyte sarcolemma. Z-grooves, T-tubule opening and characteristic sarcomere units are marked. (b) Functional schematic of sarcomere units showing the position of the probed region (Z-groove, T-tubule opening and scallop crest). Probabilities of forming a gigaseal as a function of surface position shown in parentheses. (c) Statistical distribution of L-type Ca2+ channels with the highest density near the T-tubule opening. (d) Cell-attached Ba2+ current transients at voltages of +20, ±0, −20 mV. (e) Several current transients elicited at 0 mV from one patch and ensemble average of 12 transients showing typical L-type inactivation kinetics. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of approximately 100 nm. Topography was recorded in the conventional distance modulated mode with pixel number set to 1024 × 256. Scan duration was 20 min. Modified from Gu et al. [55], with permission. (Online version in colour.)

Mentions: Obtaining a topographical image before an intervention permits the accurate selection of the location for the cell-attach (figure 8). SICM combined with patch-clamp technique formed a unique ‘smart’ patch-clamp system [17,56] on the surface of adult cardiomyocytes, where ion channels are confined in determined regions (figure 8b,c). We demonstrate a measurement of Ca2+ L-type channels within T-tubules system by measuring Ba2+ current transient at voltage of +20, 0 and −20 mV (figure 8d) in cell-attach configuration and a typical L-type inactivation kinetics (figure 8e).Figure 8.


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)

L-type Ca2+ channel distribution in the cardiac myocytes sarcolemma: mapping of ion channels by the high-resolution scanning patch-clamp technique. (a) Experimental topographic image of a representative rat cardiomyocyte sarcolemma. Z-grooves, T-tubule opening and characteristic sarcomere units are marked. (b) Functional schematic of sarcomere units showing the position of the probed region (Z-groove, T-tubule opening and scallop crest). Probabilities of forming a gigaseal as a function of surface position shown in parentheses. (c) Statistical distribution of L-type Ca2+ channels with the highest density near the T-tubule opening. (d) Cell-attached Ba2+ current transients at voltages of +20, ±0, −20 mV. (e) Several current transients elicited at 0 mV from one patch and ensemble average of 12 transients showing typical L-type inactivation kinetics. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of approximately 100 nm. Topography was recorded in the conventional distance modulated mode with pixel number set to 1024 × 256. Scan duration was 20 min. Modified from Gu et al. [55], with permission. (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSIF20100597F8: L-type Ca2+ channel distribution in the cardiac myocytes sarcolemma: mapping of ion channels by the high-resolution scanning patch-clamp technique. (a) Experimental topographic image of a representative rat cardiomyocyte sarcolemma. Z-grooves, T-tubule opening and characteristic sarcomere units are marked. (b) Functional schematic of sarcomere units showing the position of the probed region (Z-groove, T-tubule opening and scallop crest). Probabilities of forming a gigaseal as a function of surface position shown in parentheses. (c) Statistical distribution of L-type Ca2+ channels with the highest density near the T-tubule opening. (d) Cell-attached Ba2+ current transients at voltages of +20, ±0, −20 mV. (e) Several current transients elicited at 0 mV from one patch and ensemble average of 12 transients showing typical L-type inactivation kinetics. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of approximately 100 nm. Topography was recorded in the conventional distance modulated mode with pixel number set to 1024 × 256. Scan duration was 20 min. Modified from Gu et al. [55], with permission. (Online version in colour.)
Mentions: Obtaining a topographical image before an intervention permits the accurate selection of the location for the cell-attach (figure 8). SICM combined with patch-clamp technique formed a unique ‘smart’ patch-clamp system [17,56] on the surface of adult cardiomyocytes, where ion channels are confined in determined regions (figure 8b,c). We demonstrate a measurement of Ca2+ L-type channels within T-tubules system by measuring Ba2+ current transient at voltage of +20, 0 and −20 mV (figure 8d) in cell-attach configuration and a typical L-type inactivation kinetics (figure 8e).Figure 8.

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