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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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Neonatal rat ventricular myocytes were exposed to PE for 48 h to induce hypertrophy. (a) The 96 × 96 µm scan of cardiomyocyte after 48 h in culture under control conditions. The process on the right side of the cardiomyocyte appears to be cropped only owing to the angle of view. (b) Same size scan performed on a different cardiomyocyte exposed to 10 µmol l−1 PE. (c) Average cell volume in control and hypertrophic cardiomyocytes (n = 15 ± s.d., p < 0.05 Student's t-test). Asterisk denotes significant difference compared with control. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Effective pixel width in (a,b) is 375 nm over the cell body and 750 nm over the empty area. (M. Miragoli & P. Novak 2010, unpublished data.) (Online version in colour.)
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RSIF20100597F5: Neonatal rat ventricular myocytes were exposed to PE for 48 h to induce hypertrophy. (a) The 96 × 96 µm scan of cardiomyocyte after 48 h in culture under control conditions. The process on the right side of the cardiomyocyte appears to be cropped only owing to the angle of view. (b) Same size scan performed on a different cardiomyocyte exposed to 10 µmol l−1 PE. (c) Average cell volume in control and hypertrophic cardiomyocytes (n = 15 ± s.d., p < 0.05 Student's t-test). Asterisk denotes significant difference compared with control. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Effective pixel width in (a,b) is 375 nm over the cell body and 750 nm over the empty area. (M. Miragoli & P. Novak 2010, unpublished data.) (Online version in colour.)

Mentions: SICM, to our knowledge, is the most appropriate technique for studying cell hypertrophy directly in vitro, without damaging the sample [46,47]. Hopping probe SICM is a fairly simple modification and an accurate method to measure cardiac hypertrophy (figure 5). Although the large surface area (approx. 100 × 100 µm) of a typical hypertrophic cardiomyocyte limits the resolution of the image when recorded with the current implementation of HPICM to just 400–200 nm, it still allows an accurate cell volume calculation. One-day old neonatal rat ventricular cardiomyocytes, originating from 12 rats, were grown on 22 mm coverslips. After 24 h, six coverslips were kept as ‘control’ and the other six exposed to 10 µmol l−1 phenylephrine (PE). The volume of randomly selected 15 cells in both groups was analysed using the topography data recorded by HPICM. As expected, neonatal rat ventricular cardiomyocytes treated with PE showed a significant increase in volume. Figure 5a presents a control cardiomyocyte cultured for 48 h without PE. The average volume in the control group was 1388 ± 384 µm3. Culturing for 48 h in PE medium increased the total cellular volume to 3389 ± 599 µm3 (figure 5b,c). The volume of cardiomyocytes cultured in the PE medium was underestimated in few cases (three cells out of 15) owing to cell processes exceeding the scan area (figure 5b). Based on the volume of other processes included in the area, the resulting error was estimated to be no more than 4 per cent, five times less then the standard deviation of the mean volume in control (approx. 3.5% of the mean, figure 5c). The 132 per cent volume increase in cardiomyocytes cultured in the PE medium remained highly significant (p < 0.01) with as well as without the three cells affected by the volume underestimation.Figure 5.


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)

Neonatal rat ventricular myocytes were exposed to PE for 48 h to induce hypertrophy. (a) The 96 × 96 µm scan of cardiomyocyte after 48 h in culture under control conditions. The process on the right side of the cardiomyocyte appears to be cropped only owing to the angle of view. (b) Same size scan performed on a different cardiomyocyte exposed to 10 µmol l−1 PE. (c) Average cell volume in control and hypertrophic cardiomyocytes (n = 15 ± s.d., p < 0.05 Student's t-test). Asterisk denotes significant difference compared with control. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Effective pixel width in (a,b) is 375 nm over the cell body and 750 nm over the empty area. (M. Miragoli & P. Novak 2010, unpublished data.) (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSIF20100597F5: Neonatal rat ventricular myocytes were exposed to PE for 48 h to induce hypertrophy. (a) The 96 × 96 µm scan of cardiomyocyte after 48 h in culture under control conditions. The process on the right side of the cardiomyocyte appears to be cropped only owing to the angle of view. (b) Same size scan performed on a different cardiomyocyte exposed to 10 µmol l−1 PE. (c) Average cell volume in control and hypertrophic cardiomyocytes (n = 15 ± s.d., p < 0.05 Student's t-test). Asterisk denotes significant difference compared with control. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Effective pixel width in (a,b) is 375 nm over the cell body and 750 nm over the empty area. (M. Miragoli & P. Novak 2010, unpublished data.) (Online version in colour.)
Mentions: SICM, to our knowledge, is the most appropriate technique for studying cell hypertrophy directly in vitro, without damaging the sample [46,47]. Hopping probe SICM is a fairly simple modification and an accurate method to measure cardiac hypertrophy (figure 5). Although the large surface area (approx. 100 × 100 µm) of a typical hypertrophic cardiomyocyte limits the resolution of the image when recorded with the current implementation of HPICM to just 400–200 nm, it still allows an accurate cell volume calculation. One-day old neonatal rat ventricular cardiomyocytes, originating from 12 rats, were grown on 22 mm coverslips. After 24 h, six coverslips were kept as ‘control’ and the other six exposed to 10 µmol l−1 phenylephrine (PE). The volume of randomly selected 15 cells in both groups was analysed using the topography data recorded by HPICM. As expected, neonatal rat ventricular cardiomyocytes treated with PE showed a significant increase in volume. Figure 5a presents a control cardiomyocyte cultured for 48 h without PE. The average volume in the control group was 1388 ± 384 µm3. Culturing for 48 h in PE medium increased the total cellular volume to 3389 ± 599 µm3 (figure 5b,c). The volume of cardiomyocytes cultured in the PE medium was underestimated in few cases (three cells out of 15) owing to cell processes exceeding the scan area (figure 5b). Based on the volume of other processes included in the area, the resulting error was estimated to be no more than 4 per cent, five times less then the standard deviation of the mean volume in control (approx. 3.5% of the mean, figure 5c). The 132 per cent volume increase in cardiomyocytes cultured in the PE medium remained highly significant (p < 0.01) with as well as without the three cells affected by the volume underestimation.Figure 5.

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