Julia Gorelik

β2-Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation

Heart Cell Signaling in 3D A healthy heart relies on the proper transduction of cellular signals through the β1- and β2-adrenergic receptors (βARs), which are located on the surface of the hearts muscle cells (cardiomyocytes). The surface of these cells resembles a highly organized series of hills and valleys and it has been unclear whether this topography plays a role in the βAR signaling events that are critical to cell function. Nikolaev et al. (p. 1653, published online 25 February; see Perspective by Dorn) monitored the cyclic adenosine monophosphate (cAMP) signals generated by the βARs in living cardiomyocytes. In cells from healthy rats and from rats with heart failure, the β1ARs were localized across the entire cell surface. In contrast, the spatial localization of the β2ARs differed in healthy and failing cells. In healthy cardiomyocytes, the β2ARs resided exclusively within surface invaginations called transverse tubules, thereby producing spatially confined cAMP signals, whereas in failing cardiomyocytes, the β2ARs redistributed to other cell surface areas, thereby producing diffuse cAMP signals. Thus, changes in the spatial localization of β2AR-induced cAMP signaling may contribute to heart failure. A change in the distribution of a signaling molecule on the surface of heart muscle cells may contribute to heart failure. The β1- and β2-adrenergic receptors (βARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these βARs, which are coupled to heterotrimeric guanine nucleotide–binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined β2AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional β1ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, β2ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of β2ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.

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.

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.

High Levels of Circulating Epinephrine Trigger Apical Cardiodepression in a β2-Adrenergic Receptor/Gi–Dependent Manner

Background— Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic &bgr;2-adrenergic receptor (&bgr;2AR) from canonical stimulatory G-protein–activated cardiostimulant to inhibitory G-protein–activated cardiodepressant pathways. Methods and Results— We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via Gi inactivation by pertussis toxin pretreatment. &bgr;2AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a &bgr;2AR-Gi–dependent manner. Preventing epinephrine-Gi effects increased mortality in the Takotsubo model, whereas &bgr;-blockers that activate &bgr;2AR-Gi exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. Conclusions— We suggest that biased agonism of epinephrine for &bgr;2AR-Gs at low concentrations and for Gi at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in &bgr;2ARs explaining the differential regional responses. We suggest this epinephrine-specific &bgr;2AR-Gi signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

High levels of circulating epinephrine trigger apical cardiodepression in a β2-adrenergic receptor/Gi-dependent manner: a new model of Takotsubo cardiomyopathy.

Background— Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic &bgr;2-adrenergic receptor (&bgr;2AR) from canonical stimulatory G-protein–activated cardiostimulant to inhibitory G-protein–activated cardiodepressant pathways. Methods and Results— We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via Gi inactivation by pertussis toxin pretreatment. &bgr;2AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a &bgr;2AR-Gi–dependent manner. Preventing epinephrine-Gi effects increased mortality in the Takotsubo model, whereas &bgr;-blockers that activate &bgr;2AR-Gi exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. Conclusions— We suggest that biased agonism of epinephrine for &bgr;2AR-Gs at low concentrations and for Gi at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in &bgr;2ARs explaining the differential regional responses. We suggest this epinephrine-specific &bgr;2AR-Gi signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5–100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5–3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

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.

Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 microm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 microm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-microm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.