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...

Rationale Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. Objective To explore microdomain-targeted remodeling of ventricular L-type Ca2+ channels (LTCCs) in HF. Methods and Results Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore distribution of single LTCCs in different membrane microdomains of non-failing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to re-distribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability (Po) was dramatically increased (0.034±0.011 vs 0.154±0.027, P<0.001). High Po was linked to enhanced calcium-calmodulin kinase II (CaMKII)-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current which contributed to the development of early afterdepolarizations (EADs). A novel model of LTCC function in HF was developed; following its validation with experimental data, the model was used to ascertain how HF–induced T-tubule loss led to altered LTCC function and EADs. The HF myocyte model was then implemented in a 3D left ventricle model, demonstrating that such EADs can propagate and initiate reentrant arrhythmias. Conclusion Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electrical remodelling in HF and adds a new dimension to cardiovascular disease.

Background— Trends in cardiovascular mortality across Europe demonstrate significant geographical variation, and an understanding of these trends has a central role in global public health. Methods and Results— Ischemic heart disease and cerebrovascular disease age-standardized death rates (as per International Classification of Diseases , ninth and tenth revisions) were collated from the World Health Organization mortality database for member states of the European Union. Trends were characterized by using Joinpoint regression analysis. An overall trend for reduction in ischemic heart disease mortality was observed, most pronounced in Western Europe (>60% for the Netherlands, United Kingdom, and Ireland) for both sexes from 1980 to 2009. Eastern European states, Romania, Croatia, and Slovakia, had modest mortality reductions. Most recently (2009), Lithuania had the highest mortality for males and females (318.1/100 000 and 166.1/100 000, respectively), followed by Latvia and Slovakia. France had the lowest mortality: 39.8/100 000 for males and 14.7/100 000 for females. Analysis of cerebrovascular disease mortality revealed that Austria had the largest reduction for both sexes (76.8% males, 76.5% females) from 1980 to 2009. The smallest improvement over this period was seen in Lithuania, Poland, and Cyprus (–5% to +20% approximately). France has the lowest present-day cerebrovascular disease mortality for both males and females (23.9/100 000 and 17.3/100 000, respectively). Conclusions— There is a growing disparity in cardiovascular mortality between Western and Eastern Europe, for which diverse explanations are discussed. The need for population-wide health promotion and primary prevention policies is emphasized.

The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

Background-Cardiomyocyte surface morphology and T-tubular structure are significantly disrupted in chronic heart failure, with important functional sequelae, including redistribution of sarcolemmal ␤ 2 -adrenergic receptors (␤ 2 AR) and localized secondary messenger signaling. Plasticity of these changes in the reverse remodeled failing ventricle is unknown. We used AAV9.SERCA2a gene therapy to rescue failing rat hearts and measured z-groove index, T-tubule density, and compartmentalized ␤ 2 AR-mediated cAMP signals, using a combined nanoscale scanning ion conductance microscopy-Förster resonance energy transfer technique. Methods and Results-Cardiomyocyte surface morphology, quantified by z-groove index and T-tubule density, was normalized in reverse-remodeled hearts after SERCA2a gene therapy. Recovery of sarcolemmal microstructure correlated with functional ␤ 2 AR redistribution back into the z-groove and T-tubular network, whereas minimal cAMP responses were initiated after local ␤ 2 AR stimulation of crest membrane, as observed in failing cardiomyocytes. Improvement of ␤ 2 AR localization was associated with recovery of ␤AR-stimulated contractile responses in rescued cardiomyocytes. Retubulation was associated with reduced spatial heterogeneity of electrically stimulated calcium transients and recovery of myocardial BIN-1 and TCAP protein expression but not junctophilin-2. Conclusions-In summary, abnormalities of sarcolemmal structure in heart failure show plasticity with reappearance of z-grooves and T-tubules in reverse-remodeled hearts. Recovery of surface topology is necessary for normalization of ␤ 2 AR location and signaling responses. (Circ Heart Fail. 2012;5:357-365.)

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Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function.Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening.Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease.Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease.

AimsIt is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests).Methods and resultsCa2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF.Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF.ConclusionCa2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses.