The role of the beta3-adrenergic receptor in animal models of cardiac injury

The β-adrenergic system is a key player in the regulation of the heart function. The role of the β1- and the β2- adrenergic receptors is well established and they are common targets of the medical treatment used in clinical practice. However the role of the β3-adrenergic receptor (β3AR) in the cardiovascular system is still poorly understood in both physiological and pathological conditions. It is known that β3AR expression in the heart is relatively low compared to β1 and β2 subtypes, nevertheless previous studies have shown that β3AR agonists have a cardioprotective effect in pressure overload hypertrophy, neurohormonal hypertrophic remodeling and ischemia/reperfusion injury. In the heart, β3ARs are present in cardiac myocytes and in endothelial cells but it is still unknown what is the relative contribution of these cell types in the cardioprotection afforded by the activation of the β3AR. Regarding heart failure little is known about the role of this receptor in the progression of cardiac dysfunction and contradictory results can be found in the literature. Recent results from the first-in-man clinical trial using a β3AR agonist in heart failure patients demand deeper knowledge about the role of the β3AR. The main aim of this doctoral thesis is to improve the knowledge concerning the β3AR in cardiac diseases. First, following an already existing research line in the laboratory we have investigated the cellular origin of the cardioprotection afforded by β3AR agonists administration before reperfusion. In this study we demonstrate for the first time using transgenic animal models never published before that β3AR activation in ischemia/reperfusion (IR) injury protects the heart by activating mainly the cardiomyocyte β3AR and not the endothelial β3AR. Moreover the overexpression of the receptor in cardiac myocytes amplifies the protection afforded by its activation, pointing out the β3AR overexpression as a potential therapy to reduce IR injury in patients at risk of acute myocardial infarction. Secondly, we have investigated the role of the β3AR in the progression of heart failure. Transgenic mice overexpressing the receptor in cardiac myocytes did not develop heart failure and gene therapy based overexpression of the receptor during the development of heart failure stopped its progression. We speculate that this protection involves increase in free fatty acids utilization by cardiac myocytes, inhibition of myocardial metabolism switch during heart failure and mitochondrial protection. This work confirms that β3AR stimulation is a therapy that should be considered to treat the failing heart. To conclude, this thesis increases the knowledge of the role of the β3AR in the cardiovascular system offering strong evidences of its therapeutic potential in the clinical arena as a target to decrease IR injury in patients with acute myocardial infarction and supporting the beneficial effect of its stimulation in the treatment of heart failure.Esta tesis doctoral ha recibido financiación de una Beca de Formación de Personal Investigador (FPI) del Ministerio de Economía y Competitividad con referencia BES-2012061091 asociada al proyecto SEV-2011-0052-02

Proteomic footprint of myocardial ischemia/reperfusion injury: Longitudinal study of the at-risk and remote regions in the pig model

Abstract Reperfusion alters post-myocardial infarction (MI) healing; however, very few systematic studies report the early molecular changes following ischemia/reperfusion (I/R). Alterations in the remote myocardium have also been neglected, disregarding its contribution to post-MI heart failure (HF) development. This study characterizes protein dynamics and contractile abnormalities in the ischemic and remote myocardium during one week after MI. Closed-chest 40 min I/R was performed in 20 pigs sacrificed at 120 min, 24 hours, 4days, and 7days after reperfusion (n = 5 per group). Myocardial contractility was followed up by cardiac magnetic resonance (CMR) and tissue samples were analyzed by multiplexed quantitative proteomics. At early reperfusion (120 min), the ischemic area showed a coordinated upregulation of inflammatory processes, whereas interstitial proteins, angiogenesis and cardio-renal signaling processes increased at later reperfusion (day 4 and 7). Remote myocardium showed decreased contractility at 120 min- and 24 h-CMR accompanied by transient alterations in contractile and mitochondrial proteins. Subsequent recovery of regional contractility was associated with edema formation on CMR and increases in inflammation and wound healing proteins on post-MI day 7. Our results establish for the first time the altered protein signatures in the ischemic and remote myocardium early after I/R and might have implications for new therapeutic targets to improve early post-MI remodeling

β1-Blockade Prevents Post-Ischemic Myocardial Decompensation Via β3AR-Dependent Protective Sphingosine-1 Phosphate Signaling

BACKGROUND: Although β-blockers increase survival in patients with heart failure (HF), the mechanisms behind this protection are not fully understood, and not all patients with HF respond favorably to them. We recently showed that, in cardiomyocytes, a reciprocal down-regulation occurs between β1-adrenergic receptors (ARs) and the cardioprotective sphingosine-1-phosphate (S1P) receptor-1 (S1PR1). OBJECTIVES: The authors hypothesized that, in addition to salutary actions due to direct β1AR-blockade, agents such as metoprolol (Meto) may improve post-myocardial infarction (MI) structural and functional outcomes via restored S1PR1 signaling, and sought to determine mechanisms accounting for this effect. METHODS: We tested the in vitro effects of Meto in HEK293 cells and in ventricular cardiomyocytes isolated from neonatal rats. In vivo, we assessed the effects of Meto in MI wild-type and β3AR knockout mice. RESULTS: Here we report that, in vitro, Meto prevents catecholamine-induced down-regulation of S1PR1, a major cardiac protective signaling pathway. In vivo, we show that Meto arrests post-MI HF progression in mice as much as chronic S1P treatment. Importantly, human HF subjects receiving β1AR-blockers display elevated circulating S1P levels, confirming that Meto promotes S1P secretion/signaling. Mechanistically, we found that Meto-induced S1P secretion is β3AR-dependent because Meto infusion in β3AR knockout mice does not elevate circulating S1P levels, nor does it ameliorate post-MI dysfunction, as in wild-type mice. CONCLUSIONS: Our study uncovers a previously unrecognized mechanism by which β1-blockers prevent HF progression in patients with ischemia, suggesting that β3AR dysfunction may account for limited/null efficacy in β1AR-blocker-insensitive HF subjects

Localized Acetylcholine Receptor Clustering Dynamics in Response to Microfluidic Focal Stimulation with Agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells