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 β₂-adrenergic receptor (β₂AR) 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_iinactivation by pertussis toxin pretreatment. β₂AR 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 β₂AR-Gi–dependent manner. Preventing epinephrine-G_ieffects increased mortality in the Takotsubo model, whereas β-blockers that activate β₂AR-G_iexacerbated 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 β₂AR-G_sat low concentrations and for G_iat high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β₂ARs explaining the differential regional responses. We suggest this epinephrine-specific β₂AR-G_isignaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

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.

Cardiomyocyte β₂-adrenergic receptor (β₂AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors’ subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β₂ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β₂AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown. Rat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β₂ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β₂AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β₂AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β₂AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β₂AR-mediated signal compartmentation or reduce cAMP diffusion. Although changes in T-tubule structure and β₂AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β₂AR-mediated cAMP is temporarily reversed. Complete disorganization of β₂AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

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.