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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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(a) Typical surface topography image of a healthy adult cardiomyocyte. Well-organized striation and Z-grooves can be observed. Effective pixel width 125 nm, scan duration 4 min. (b) Surface topography image of an adult cardiomyocyte from HOCM patients shows an absence of T-tubules in this 9 × 9 µm area of the cell. (c) Z-grooves ratio index quantification demonstrates a significant difference in HOCM compared with control cells (n = 5 ± s.e. in both control and HOCM patients, p < 0.05 Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Lyon et al. [37] with permission. (Online version in colour.)
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RSIF20100597F4: (a) Typical surface topography image of a healthy adult cardiomyocyte. Well-organized striation and Z-grooves can be observed. Effective pixel width 125 nm, scan duration 4 min. (b) Surface topography image of an adult cardiomyocyte from HOCM patients shows an absence of T-tubules in this 9 × 9 µm area of the cell. (c) Z-grooves ratio index quantification demonstrates a significant difference in HOCM compared with control cells (n = 5 ± s.e. in both control and HOCM patients, p < 0.05 Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Lyon et al. [37] with permission. (Online version in colour.)

Mentions: Structural remodelling of the heart, which can lead to heart failure (HF) and cardiac arrhythmias [36], ranges from three-dimensional reorganization to redistribution of the ion channel repertoire and receptors on the cell surface. This is manifest at the tissue level typically involving structural disorganization and hypertrophy of cardiomyocytes. SICM has the capability to resolve this in live cardiomyocytes, with hypertrophic obstructive cardiomyopathy (HOCM) and dilated cardiomyopathy cardiomyocytes showing drastically reduced Z-grooves organization, which lead to the further functional abnormalities [37]. Figure 4 describes the surface characteristics of an adult human cardiomyocyte with the surface structures resolved with SICM. Recently, we introduced a new parameter that describes the integrity of the cardiomyocyte surface called the Z-groove index [21]. SICM images clearly show the surface topography of the cardiomyocyte (figure 4a). The domed crest between the Z-grooves, as well as the T-tubule openings are very clear in rat myocytes. Profile measurements showed that the spacing between Z-grooves was approximately 2 µm, corresponding to the predicted sarcomere length for quiescent ventricular myocytes. We showed that different pathological conditions in cardiomyocytes from rats and humans change this index. For example, in cardiomyocytes derived from dilated cardiomyopathy patients, the Z-groove index is reduced, compared with healthy cells [37]. Here, we further investigated surface structures of healthy and diseased cardiomyocytes. Cardiomyocytes from patients with HOCM contained fewer Z-grooves and therefore their Z-groove index was lower than in normal cells. Figure 4a shows a control human cardiomyocyte with striated pattern on the surface with T-tubule openings distributed at regular intervals. Z-grooves are pronounced, and the Z-groove index is 0.86 (figure 4c). In sharp contrast, cardiomyocytes from a patient with HOCM show dramatic changes in surface structure, with flattening and loss of Z-groove definition (figure 4b). The Z-groove index in HOCM cells was as low as 0.15.Figure 4.


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)

(a) Typical surface topography image of a healthy adult cardiomyocyte. Well-organized striation and Z-grooves can be observed. Effective pixel width 125 nm, scan duration 4 min. (b) Surface topography image of an adult cardiomyocyte from HOCM patients shows an absence of T-tubules in this 9 × 9 µm area of the cell. (c) Z-grooves ratio index quantification demonstrates a significant difference in HOCM compared with control cells (n = 5 ± s.e. in both control and HOCM patients, p < 0.05 Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Lyon et al. [37] with permission. (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSIF20100597F4: (a) Typical surface topography image of a healthy adult cardiomyocyte. Well-organized striation and Z-grooves can be observed. Effective pixel width 125 nm, scan duration 4 min. (b) Surface topography image of an adult cardiomyocyte from HOCM patients shows an absence of T-tubules in this 9 × 9 µm area of the cell. (c) Z-grooves ratio index quantification demonstrates a significant difference in HOCM compared with control cells (n = 5 ± s.e. in both control and HOCM patients, p < 0.05 Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Modified from Lyon et al. [37] with permission. (Online version in colour.)
Mentions: Structural remodelling of the heart, which can lead to heart failure (HF) and cardiac arrhythmias [36], ranges from three-dimensional reorganization to redistribution of the ion channel repertoire and receptors on the cell surface. This is manifest at the tissue level typically involving structural disorganization and hypertrophy of cardiomyocytes. SICM has the capability to resolve this in live cardiomyocytes, with hypertrophic obstructive cardiomyopathy (HOCM) and dilated cardiomyopathy cardiomyocytes showing drastically reduced Z-grooves organization, which lead to the further functional abnormalities [37]. Figure 4 describes the surface characteristics of an adult human cardiomyocyte with the surface structures resolved with SICM. Recently, we introduced a new parameter that describes the integrity of the cardiomyocyte surface called the Z-groove index [21]. SICM images clearly show the surface topography of the cardiomyocyte (figure 4a). The domed crest between the Z-grooves, as well as the T-tubule openings are very clear in rat myocytes. Profile measurements showed that the spacing between Z-grooves was approximately 2 µm, corresponding to the predicted sarcomere length for quiescent ventricular myocytes. We showed that different pathological conditions in cardiomyocytes from rats and humans change this index. For example, in cardiomyocytes derived from dilated cardiomyopathy patients, the Z-groove index is reduced, compared with healthy cells [37]. Here, we further investigated surface structures of healthy and diseased cardiomyocytes. Cardiomyocytes from patients with HOCM contained fewer Z-grooves and therefore their Z-groove index was lower than in normal cells. Figure 4a shows a control human cardiomyocyte with striated pattern on the surface with T-tubule openings distributed at regular intervals. Z-grooves are pronounced, and the Z-groove index is 0.86 (figure 4c). In sharp contrast, cardiomyocytes from a patient with HOCM show dramatic changes in surface structure, with flattening and loss of Z-groove definition (figure 4b). The Z-groove index in HOCM cells was as low as 0.15.Figure 4.

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