Markus B. Sikkel

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.

Respiratory syncytial virus persistence in chronic obstructive pulmonary disease.

Respiratory syncytial virus (RSV) is predominantly recognized as a pediatric pathogen although sensitive molecular diagnostic techniques have led to its more frequent detection in some adult settings. In some studies RSV has been detected just as frequently in stable chronic obstructive pulmonary disease (COPD) patients as in those suffering disease exacerbations, leading to the suggestion that RSV may persist in COPD. Although some studies have found negligible RSV in stable COPD, others have detected RSV in one-quarter to one-third of stable COPD samples. Possible reasons for this discrepancy are explored within the article. A relationship between RSV detection and increased disease severity, including rate of decline in lung function and systemic/airway inflammation, has been found on both occasions it has been sought. Susceptibility to persistent RSV infection could involve both host and viral factors. Cigarette smoking and COPD are likely to result in impaired antiviral immunity, and RSV is capable of evading immune responses by inducing skewed type 2 T-helper cell responses, antagonizing antiviral cytokines, mimicking chemokines, inhibiting apoptosis, and entering immune-privileged cells such as pulmonary neurons. It can also escape an established immune response through antigenic drift. This article examines current evidence regarding persistence of RSV in COPD and its possible mechanisms. We also discuss various roles for RSV persistence in COPD pathogenesis. Further elucidation of the contribution of persistent RSV to the pathogenesis of COPD requires interventional studies. Persistence of RSV in COPD may have direct relevance to the pathogenesis of childhood diseases such as postbronchiolitic wheeze and asthma.

Plasticity of Surface Structures and β2-Adrenergic Receptor Localization in Failing Ventricular Cardiomyocytes During Recovery from Heart Failure

Background— Cardiomyocyte surface morphology and T-tubular structure are significantly disrupted in chronic heart failure, with important functional sequelae, including redistribution of sarcolemmal &bgr;2-adrenergic receptors (&bgr;2AR) 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 &bgr;2AR-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 &bgr;2AR redistribution back into the z-groove and T-tubular network, whereas minimal cAMP responses were initiated after local &bgr;2AR stimulation of crest membrane, as observed in failing cardiomyocytes. Improvement of &bgr;2AR localization was associated with recovery of &bgr;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 &bgr;2AR location and signaling responses.

Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

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.

Flecainide reduces Ca2+ spark and wave frequency via inhibition of the sarcolemmal sodium current

Aims Ca2+ waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca2+ waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism. Methods and results We imaged spontaneous sarcoplasmic reticulum (SR) Ca2+ release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca2+ sparks or waves. Spark frequency, wave frequency, and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca2+ content. Inhibition of INa via alternative pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced similar changes. To assess the contribution of INa to spark and wave production, voltage clamping was used to activate contraction from holding potentials of −80 or −40 mV. This confirmed that reducing Na+ influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca2+ wave reduction when INa is active. It was found that Na+/Ca2+-exchanger (NCX)-mediated Ca2+ efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na+]o, suggesting an important downstream role for NCX function. Conclusion Flecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves INa reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced INa results in increased Ca2+ efflux via NCX across the sarcolemma, reducing Ca2+ concentration in the vicinity of the RyR2.

The Mechanism of Flecainide Action in CPVT Does Not Involve a Direct Effect on RyR2

RATIONALE Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to β-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. OBJECTIVE To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. METHODS AND RESULTS Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

Myocardial MiR-30 downregulation triggered by doxorubicin drives alterations in β -adrenergic signaling and enhances apoptosis

The use of anthracyclines such as doxorubicin (DOX) has improved outcome in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF) but our understanding of molecular mechanisms underlying this and essential for development of cardioprotective strategies remains largely obscure. As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in DOX-induced HF and the alteration of cellular processes downstream. Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes or via chronic exposure in vivo, and compared with miRNA profiles from remodeled hearts following myocardial infarction. The miR-30 family was downregulated in all three models. We describe here that miR-30 act regulating the β-adrenergic pathway, where preferential β1- and β2-adrenoceptor (β1AR and β2AR) direct inhibition is combined with Giα-2 targeting for fine-tuning. Importantly, we show that miR-30 also target the pro-apoptotic gene BNIP3L/NIX. In aggregate, we demonstrate that high miR-30 levels are protective against DOX toxicity and correlate this in turn with lower reactive oxygen species generation. In addition, we identify GATA-6 as a mediator of DOX-associated reductions in miR-30 expression. In conclusion, we describe that DOX causes acute and sustained miR-30 downregulation in cardiomyocytes via GATA-6. miR-30 overexpression protects cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective and anti-tumorigenic strategy for anthracyclines.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

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. Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Microdomain-Specific Modulation of L-type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure

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