Microtubule-Dependent Mitochondria Alignment Regulates Calcium Release in Response to Nanomechanical Stimulus in Heart Myocytes.

Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart.

Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy.

Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle-cell interactions.

The scanning ion conductance microscope for cellular physiology.

The quest for nonoptical imaging methods that can surmount light diffraction limits resulted in the development of scanning probe microscopes. However, most of the existing methods are not quite suitable for studying biological samples. The scanning ion conductance microscope (SICM) bridges the gap between the resolution capabilities of atomic force microscope and scanning electron microscope and functional capabilities of conventional light microscope. A nanopipette mounted on a three-axis piezo-actuator, scans a sample of interest and ion current is measured between the pipette tip and the sample. The feedback control system always keeps a certain distance between the sample and the pipette so the pipette never touches the sample. At the same time pipette movement is recorded and this generates a three-dimensional topographical image of the sample surface. SICM represents an alternative to conventional high-resolution microscopy, especially in imaging topography of live biological samples. In addition, the nanopipette probe provides a host of added modalities, for example using the same pipette and feedback control for efficient approach and seal with the cell membrane for ion channel recording. SICM can be combined in one instrument with optical and fluorescent methods and allows drawing structure-function correlations. It can also be used for precise mechanical force measurements as well as vehicle to apply pressure with precision. This can be done on living cells and tissues for prolonged periods of time without them loosing viability. The SICM is a multifunctional instrument, and it is maturing rapidly and will open even more possibilities in the near future.

Functional interaction between charged nanoparticles and cardiac tissue: a new paradigm for cardiac arrhythmia?

AIM: To investigate the effect of surface charge of therapeutic nanoparticles on sarcolemmal ionic homeostasis and the initiation of arrhythmias. MATERIALS & METHODS: Cultured neonatal rat myocytes were exposed to 50 nm-charged polystyrene latex nanoparticles and examined using a combination of hopping probe scanning ion conductance microscopy, optical recording of action potential characteristics and patch clamp. RESULTS: Positively charged, amine-modified polystyrene latex nanoparticles showed cytotoxic effects and induced large-scale damage to cardiomyocyte membranes leading to calcium alternans and cell death. By contrast, negatively charged, carboxyl-modified polystyrene latex nanoparticles (NegNPs) were not overtly cytotoxic but triggered formation of 50-250-nm nanopores in the membrane. Cells exposed to NegNPs revealed pro-arrhythmic events, such as delayed afterdepolarizations, reduction in conduction velocity and pathological increment of action potential duration together with an increase in ionic current throughout the membrane, carried by the nanopores. CONCLUSION: The utilization of charged nanoparticles is a novel concept for targeting cardiac excitability. However, this unique nanoscopic investigation reveals an altered electrophysiological substrate, which sensitized the heart cells towards arrhythmias.

An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy.

Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.

A protective antiarrhythmic role of ursodeoxycholic acid in an in vitro rat model of the cholestatic fetal heart.

UNLABELLED: Intrahepatic cholestasis of pregnancy may be complicated by fetal arrhythmia, fetal hypoxia, preterm labor, and, in severe cases, intrauterine death. The precise etiology of fetal death is not known. However, taurocholate has been demonstrated to cause arrhythmia and abnormal calcium dynamics in cardiomyocytes. To identify the underlying reason for increased susceptibility of fetal cardiomyocytes to arrhythmia, we studied myofibroblasts (MFBs), which appear during structural remodeling of the adult diseased heart. In vitro, they depolarize rat cardiomyocytes via heterocellular gap junctional coupling. Recently, it has been hypothesized that ventricular MFBs might appear in the developing human heart, triggered by physiological fetal hypoxia. However, their presence in the fetal heart (FH) and their proarrhythmogenic effects have not been systematically characterized. Immunohistochemistry demonstrated that ventricular MFBs transiently appear in the human FH during gestation. We established two in vitro models of the maternal heart (MH) and FH, both exposed to increasing doses of taurocholate. The MH model consisted of confluent strands of rat cardiomyocytes, whereas for the FH model, we added cardiac MFBs on top of cardiomyocytes. Taurocholate in the FH model, but not in the MH model, slowed conduction velocity from 19 to 9 cm/s, induced early after depolarizations, and resulted in sustained re-entrant arrhythmias. These arrhythmic events were prevented by ursodeoxycholic acid, which hyperpolarized MFB membrane potential by modulating potassium conductance. CONCLUSION: These results illustrate that the appearance of MFBs in the FH may contribute to arrhythmias. The above-described mechanism represents a new therapeutic approach for cardiac arrhythmias at the level of MFB.

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

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.

The Renin-Angiotensin system mediates the effects of stretch on conduction velocity, connexin43 expression, and redistribution in intact ventricle.

UNLABELLED: Effect of Stretch on Conduction and Cx43. INTRODUCTION: In disease states such as heart failure, myocardial infarction, and hypertrophy, changes in the expression and location of Connexin43 (Cx43) occur (Cx43 remodeling), and may predispose to arrhythmias. Stretch may be an important stimulus to Cx43 remodeling; however, it has only been investigated in neonatal cell cultures, which have different physiological properties than adult myocytes. We hypothesized that localized stretch in vivo causes Cx43 remodeling, with associated changes in conduction, mediated by the renin-angiotensin system (RAS). METHODS AND RESULTS: In an open-chest canine model, a device was used to stretch part of the right ventricle (RV) by 22% for 6 hours. Activation mapping using a 312-electrode array was performed before and after stretch. Regional stretch did not change longitudinal conduction velocity (post-stretch vs baseline: 51.5 ± 5.2 vs 55.3 ± 8.1 cm/s, P = 0.24, n = 11), but significantly reduced transverse conduction velocity (28.7 ± 2.5 vs 35.4 ± 5.4 cm/s, P < 0.01). It also reduced total Cx43 expression, by Western blotting, compared with nonstretched RV of the same animal (86.1 ± 32.2 vs 100 ± 19.4%, P < 0.02, n = 11). Cx43 labeling redistributed to the lateral cell borders. Stretch caused a small but significant increase in the proportion of the dephosphorylated form of Cx43 (stretch 9.95 ± 1.4% vs control 8.74 ± 1.2%, P < 0.05). Olmesartan, an angiotensin II blocker, prevented the stretch-induced changes in Cx43 levels, localization, and conduction. CONCLUSION: Myocardial stretch in vivo has opposite effects to that in neonatal myocytes in vitro. Stretch in vivo causes conduction changes associated with Cx43 remodeling that are likely caused by local stretch-induced activation of the RAS.

Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart.

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

Nanoscale live-cell imaging using hopping probe ion conductance microscopy.

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.

Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study.

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with approximately 200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis.

Imaging single virus particles on the surface of cell membranes by high-resolution scanning surface confocal microscopy.

We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 +/- 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell.

Mechanosensitive-mediated interaction, integration, and cardiac control.

This review covers aspects of the cardiac mechanotransduction field at different levels, and advocates the possibility that mechanoelectro-chemical transduction forms part of a network of mechanically linked integration in heart-mechanically mediated integration (MMI). It assembles evidence and observations in the literature to promote this hypothesis. Mechanical components can provide the bond between interactions at molecular, cellular, and macro levels to enable the integration. Stretch-activated channels (SACs) exist in the heart, but stresses and strains can affect other membrane channels or receptors. A cellular mechanical change can thus promote several ionic or downstream changes. Cell signal cascades have been implicated and can affect membrane electrophysiology. MMI could shape intracellular and downstream signals using the cytoskeleton and intracellular Ca(2+). MMI also spans other regulatory systems and processes such as the autonomic nervous system (ANS) and operates throughout the whole heart as an integrative system. Finally, supporting the hypothesis, if elements of the normal integration become deranged it contributes to cardiovascular disease and, potentially, lethal arrhythmia.

The use of scanning ion conductance microscopy to image A6 cells.

BACKGROUND: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Stretch-induced regional mechanoelectric dispersion and arrhythmia in the right ventricle of anesthetized lambs.

Regional mechanical and electrophysiological changes accompany most ventricular arrhythmias and, it has been suggested, by mechanoelectric feedback. We hypothesized that an intervention producing regional mechanical dispersion was associated with regional, proarrhythmic electrical dispersion and studied the regional mechanoelectric feedback in the right ventricle (RV) of anesthetized lambs. Ten lambs were deeply anesthetized, and their hearts were exposed. Three tripodal devices, each incorporating three monophasic action potential electrodes and an integrated strain-gauge system, were placed on the RV apex outflow and inflow regions. Measurements were made before, during, and after 10-s pulmonary arterial occlusion. Pulmonary arterial occlusion increased RV pressure and overall regional segment length. Length excursion became out of phase with RV pressure beats immediately after occlusion, and the strain patterns were different in the three regions at the peak of occlusion. The occlusion resulted in different alterations in regional monophasic action potential morphology, including reduction in monophasic action potential amplitude and duration by different amounts and early afterdepolarizations that were unevenly distributed in the monophasic action potential recordings. This was associated with dispersion of repolarization and recovery time. The combination of electromechanical events precipitated a variety of arrhythmias. Acute RV distension is proarrhythmic, possibly through a causal relationship among mechanically induced afterdepolarizations, dispersion (heterogeneity) of mechanical strain, and dispersion of electrical recovery. The relationship among the different wall motions, the dispersion of repolarization, and arrhythmia underscored mechanoelectric feedback as an important part of arrhythmogenesis in pulmonary embolism and commotio cordis.

Right ventricular myocardial responses to chronic pulmonary regurgitation in lambs: disturbances of activation and conduction.

Patients after repair of tetralogy of Fallot are at increased risk of arrhythmic death. Clinical data suggest that pulmonary regurgitation predisposes to these arrhythmias, although the cellular electrophysiologic effects of pulmonary regurgitation are unknown. We induced pulmonary regurgitation in lambs, and 3 mo later, having quantified the pulmonary regurgitant (PR) fraction, studied right ventricular mechanical and electrophysiologic properties in vivo and in vitro. The PR fraction was greater in PR (75 +/- 10%) than in sham-operated animals (8 +/- 4%; p < 0.01). In vivo, monophasic action potential duration and activation time, at rest and during acute right ventricular stretch, were similar in both groups. However, the dispersion of activation time was greater in PR animals at rest (13 +/- 1.1 versus 8 +/- 1.1 ms; p < 0.05). Furthermore, the dispersion of activation increased during right ventricular stretch in PR, but not in sham-operated animals. In vitro, myocardial force-frequency responses were similar in both groups, indicating preserved systolic performance, but mechanical restitution studies showed a prolonged refractory period (447 +/- 22 versus 370 +/- 26 ms; p < 0.05) and a decreased recovery time constant (184 +/- 19 versus 265 +/- 20 ms; p < 0.001) in PR animals, indicating altered calcium cycling. Furthermore, the myocardial conduction velocity was reduced in PR animals (31 +/- 3.58 versus 47.9 +/- 5.1 cm/s; p < 0.01), resulting from a 2-fold increase in intracellular resistance (437.25 +/- 125.93 versus 194 +/- 43.27 Omega. cm; p = 0.025). Chronic PR leads to inhomogeneity of right ventricular activation, alters myocardial calcium cycling, reduces conduction velocity, and increases intracellular resistivity. These may contribute to the development of arrhythmias associated with PR, including those in patients after tetralogy repair.

Esmolol is antiarrhythmic in doxorubicin-induced arrhythmia in cultured cardiomyocytes - determination by novel rapid cardiomyocyte assay.

Cardiac toxicity is an uncommon but potentially serious complication of cancer therapy, especially with anthracyclines. One of the most effective anticancer drugs is doxorubicin, but its value is limited by the risk of developing cardiomyopathy and ventricular arrhythmia. When applied to a network of periodically contracting cardiomyocytes in culture, doxorubicin induces rhythm disturbances. Using a novel rapid assay based on non-invasive ion-conductance microscopy we show that the beta-antagonist esmolol can restore rhythm in doxorubicin-treated cultures of cardiomyocytes. Moreover, esmolol pre-treatment can protect the culture from doxorubicin-induced arrhythmia.

Dynamic assembly of surface structures in living cells.

Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary "building blocks" for the assembly of specialized structures on the cell surface.

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.