The beta1- and beta2-adrenergic receptors (betaARs) 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 betaARs, 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 beta2AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional beta1ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, beta2ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of beta(2)ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions.There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample but a true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been yet achieved. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, that allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . However, until now, SICM was restricted to imaging relatively flat surfaces, like all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen (Fig. 1a). Here we report a novel mode of SICM that allows imaging of even the most convoluted surface structures at the nanoscale. SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM a nanopipette is mounted on a three-dimensional piezo and automatic feedback control moves the pipette up or down to keep the pipette current at a constant level (the set point), while the sample is scanned in X-Y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In our hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features (Fig. 1b). The reference current is measured while the pipette is well away from the surface. The pipette then approaches until the current is reduced by a predefined amount, usually 0.25-1%. The Z-position when the current achieves this reduction is recorded as the height of the sample at this imaging point. Typically, even at a 1% reduction of the current, the pipette is still at a distance of about one inner pipette radius from the surface. Therefore, the probe never touches the surface of the cell. The pipette is then withdrawn away from the surface and the sample moved laterally to determine the next imaging point. By continuously upd...

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 ␤ 2 -adrenergic receptor (␤ 2 AR) 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 G i inactivation by pertussis toxin pretreatment. ␤ 2 AR 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 ␤ 2 AR-Gi-dependent manner. Preventing epinephrine-G i effects increased mortality in the Takotsubo model, whereas ␤-blockers that activate ␤ 2 AR-G i 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 ␤ 2 AR-G s at low concentrations and for G i at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in ␤ 2 ARs explaining the differential regional responses. We suggest this epinephrine-specific ␤ 2 AR-G i signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress. (Circulation. 2012;126:697-706.)Key Words: acute heart failure Ⅲ catecholamines Ⅲ receptors, adrenergic, beta Ⅲ Takotsubo syndrome T here has been a rapid increase in the recognition of a syndrome of acute and severe but reversible heart failure called Takotsubo or stress cardiomyopathy, 1-3 also known as broken heart syndrome, which usually follows within hours of an identifiable emotional, psychological, or physical stress. Takotsubo cardiomyopathy mimics symptoms of acute myocardial infarction but is distinguished by the lack of coronary occlusion and by characteristic regional wall-motion abnormalities, classically a virtual apical ballooning appearance caused by a hypercontractile base of the heart relative to hypokinetic or akinetic apical and mid left ventricular myocardium, the latter extending beyond a single coronary artery territory. Clinical Perspective on p 706The pathophysiological mechanisms for this increasingly recogn...