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.
Highlights
Cardiovascular disease is recognized as the foremost cause of global mortality, and a goal of modern medical research is to uncover the complex mechanisms of this pathology in its natural context
We show that SICM is a suitable technique for the (i) identification of contractile cell phenotype, i.e. a cluster of human embryonic stem cell-derived cardiomyocytes among other cells that derived from stem cells and (ii) simultaneous investigation of inotropy and Ca2þ transient in neonatal ventricular myocytes, in combination with optical recording using a fast video camera
Using SICM, we found that in human embryonic stem cell-derived cardiomyocytes (hESCMs) clusters only a small fraction of cells are contracting, a characteristic of differentiated cardiomyocytes [53] (figure 6a(ii))
Summary
Cardiovascular disease is recognized as the foremost cause of global mortality, and a goal of modern medical research is to uncover the complex mechanisms of this pathology in its natural context. In the context of the complex nature of cardiovascular disease, in addition to the use of multiple conventional methods that address individual questions, it would be extremely useful to develop a novel universal technique capable of correlating cell function with morphology, macroscopic structural remodelling in intact tissue, and spatio-temporal aspects of intracellular signalling or ion channel activity measured in single cells and subcellular compartments. The aim of this review is to describe the SICM technique alone or in combination with other optical and electrical methods to perform highly resolved dynamic and integrative analysis of cardiac structure, physiology and mechanisms of cardiovascular disease at the subcellular, cellular and tissue levels (figure 1)
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