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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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Measurement of contraction by SICM in cluster of (i) human embryonic stem cell-derived cardiomyocytes (hESCMs) and (ii) neonatal rat ventricular myocytes. (a) (i) hESCMs stain with myosin heavy chain. (ii) Topographical 32 × 32 µm image of cluster of hESCM using SICM. (b) (i) Contraction of hESCM cluster in the presence of (i) doxorubicin and esmolol (ii) resulting in changes in pipette vertical displacement of SICM. As expected, the presence of doxorubicin affects cardiac contraction; this condition is restored by esmolol (ii). (c) Technical scheme of SICM/Ca2+ dynamics for concurrent measurement of contraction and intracellular Ca2+ transient. Simultaneously, the light emission of the stained cell loaded with Fluo-4 AM was detected by a custom-made photomultiplier tube apparatus. (d) Overlapped traces of Ca2+ transient (normalized at % dF/F) and contraction (vertical displacement). (i) Control cluster of cardiomyocytes denote spontaneous firing (approx. 60 b.p.m.). (ii) Same as (i) but in the presence of taurocholic acid that affects calcium transient amplitude and contraction (p < 0.05, Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Topography image in (a) was recorded in the conventional distance-modulated mode with pixel number set to 1024 × 256. Scan duration was 23 min. Modified from Gorelik et al. [51,52] with permission. (Online version in colour.)
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RSIF20100597F6: Measurement of contraction by SICM in cluster of (i) human embryonic stem cell-derived cardiomyocytes (hESCMs) and (ii) neonatal rat ventricular myocytes. (a) (i) hESCMs stain with myosin heavy chain. (ii) Topographical 32 × 32 µm image of cluster of hESCM using SICM. (b) (i) Contraction of hESCM cluster in the presence of (i) doxorubicin and esmolol (ii) resulting in changes in pipette vertical displacement of SICM. As expected, the presence of doxorubicin affects cardiac contraction; this condition is restored by esmolol (ii). (c) Technical scheme of SICM/Ca2+ dynamics for concurrent measurement of contraction and intracellular Ca2+ transient. Simultaneously, the light emission of the stained cell loaded with Fluo-4 AM was detected by a custom-made photomultiplier tube apparatus. (d) Overlapped traces of Ca2+ transient (normalized at % dF/F) and contraction (vertical displacement). (i) Control cluster of cardiomyocytes denote spontaneous firing (approx. 60 b.p.m.). (ii) Same as (i) but in the presence of taurocholic acid that affects calcium transient amplitude and contraction (p < 0.05, Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Topography image in (a) was recorded in the conventional distance-modulated mode with pixel number set to 1024 × 256. Scan duration was 23 min. Modified from Gorelik et al. [51,52] with permission. (Online version in colour.)

Mentions: In SICM, the electrical feedback system keeps the distance between the tip of the pipette and the cell surface constant, thereby providing information about the movement of the cell surface if it moves, as in contracting cells (figure 6c). The vertical displacement of the pipette can be recorded and analysed. Using SICM, we found that in hESCMs clusters only a small fraction of cells are actually contracting, a characteristic of differentiated cardiomyocytes [53] (figure 6a(ii)). Histochemically, those differentiated cells express cardiomyocytes markers, such as myosin heavy chain (figure 6a(i)). Application of drugs known to perturb contraction is useful to evaluate the state of differentiation of hESCM clusters. The most differentiated cells react to the arrhythmogenic action of doxorubicin and are protected from this action by esmolol (figure 6b) [51].Figure 6.


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)

Measurement of contraction by SICM in cluster of (i) human embryonic stem cell-derived cardiomyocytes (hESCMs) and (ii) neonatal rat ventricular myocytes. (a) (i) hESCMs stain with myosin heavy chain. (ii) Topographical 32 × 32 µm image of cluster of hESCM using SICM. (b) (i) Contraction of hESCM cluster in the presence of (i) doxorubicin and esmolol (ii) resulting in changes in pipette vertical displacement of SICM. As expected, the presence of doxorubicin affects cardiac contraction; this condition is restored by esmolol (ii). (c) Technical scheme of SICM/Ca2+ dynamics for concurrent measurement of contraction and intracellular Ca2+ transient. Simultaneously, the light emission of the stained cell loaded with Fluo-4 AM was detected by a custom-made photomultiplier tube apparatus. (d) Overlapped traces of Ca2+ transient (normalized at % dF/F) and contraction (vertical displacement). (i) Control cluster of cardiomyocytes denote spontaneous firing (approx. 60 b.p.m.). (ii) Same as (i) but in the presence of taurocholic acid that affects calcium transient amplitude and contraction (p < 0.05, Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Topography image in (a) was recorded in the conventional distance-modulated mode with pixel number set to 1024 × 256. Scan duration was 23 min. Modified from Gorelik et al. [51,52] with permission. (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSIF20100597F6: Measurement of contraction by SICM in cluster of (i) human embryonic stem cell-derived cardiomyocytes (hESCMs) and (ii) neonatal rat ventricular myocytes. (a) (i) hESCMs stain with myosin heavy chain. (ii) Topographical 32 × 32 µm image of cluster of hESCM using SICM. (b) (i) Contraction of hESCM cluster in the presence of (i) doxorubicin and esmolol (ii) resulting in changes in pipette vertical displacement of SICM. As expected, the presence of doxorubicin affects cardiac contraction; this condition is restored by esmolol (ii). (c) Technical scheme of SICM/Ca2+ dynamics for concurrent measurement of contraction and intracellular Ca2+ transient. Simultaneously, the light emission of the stained cell loaded with Fluo-4 AM was detected by a custom-made photomultiplier tube apparatus. (d) Overlapped traces of Ca2+ transient (normalized at % dF/F) and contraction (vertical displacement). (i) Control cluster of cardiomyocytes denote spontaneous firing (approx. 60 b.p.m.). (ii) Same as (i) but in the presence of taurocholic acid that affects calcium transient amplitude and contraction (p < 0.05, Student's t-test). Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. Topography image in (a) was recorded in the conventional distance-modulated mode with pixel number set to 1024 × 256. Scan duration was 23 min. Modified from Gorelik et al. [51,52] with permission. (Online version in colour.)
Mentions: In SICM, the electrical feedback system keeps the distance between the tip of the pipette and the cell surface constant, thereby providing information about the movement of the cell surface if it moves, as in contracting cells (figure 6c). The vertical displacement of the pipette can be recorded and analysed. Using SICM, we found that in hESCMs clusters only a small fraction of cells are actually contracting, a characteristic of differentiated cardiomyocytes [53] (figure 6a(ii)). Histochemically, those differentiated cells express cardiomyocytes markers, such as myosin heavy chain (figure 6a(i)). Application of drugs known to perturb contraction is useful to evaluate the state of differentiation of hESCM clusters. The most differentiated cells react to the arrhythmogenic action of doxorubicin and are protected from this action by esmolol (figure 6b) [51].Figure 6.

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