Clinical pharmacology of ß-3 adrenergic receptor agonists for cardiovascular diseases.

INTRODUCTION: Few agonists of the third isotype of beta-adrenergic receptors, the ß3-adrenoreceptor, are currently used clinically, and new agonists are under development for the treatment of overactive bladder disease. As the receptor is expressed in human cardiac and vascular tissues, it is important to understand their beneficial (or adverse) effect(s) on these targets. AREAS COVERED: We discuss the most recent results of clinical trials testing the benefit and safety of ß3-adrenoreceptor activation on cardiovascular outcomes in light of current knowledge on the receptor biology, genetic polymorphisms, and agonist pharmacology. EXPERT OPINION: While evidence from small clinical trials is limited so far, the ß3-agonist, mirabegron seems to be safe in patients at high cardiovascular risk but produces benefits on selected cardiovascular outcomes only at higher than standard doses. Activation of cardiovascular ß3-adrenoreceptors deserves to be tested with more potent agonists, such as vibegron.

Extracellular Vesicles in Adipose Tissue Communication with the Healthy and Pathological Heart.

Adipose tissue and its diverse cell types constitute one of the largest endocrine organs. With multiple depot locations, adipose tissue plays an important regulatory role through paracrine and endocrine communication, particularly through the secretion of a wide range of bioactive molecules, such as nucleic acids, proteins, lipids or adipocytokines. Over the past several years, research has uncovered a myriad of interorgan communication signals mediated by small lipid-derived nanovesicles known as extracellular vesicles (EVs), in which secreted bioactive molecules are stably transported as cargo molecules and delivered to adjacent cells or remote organs. EVs constitute an essential part of the human adipose secretome, and there is a growing body of evidence showing the crucial implications of adipose-derived EVs in the regulation of heart function and its adaptative capacity. The adipose tissue modifications and dysfunction observed in obesity and aging tremendously affect the adipose-EV secretome, with important consequences for the myocardium. The present review presents a comprehensive analysis of the findings in this novel area of research, reports the key roles played by adipose-derived EVs in interorgan cross-talk with the heart and discusses their implications in physiological and pathological conditions affecting adipose tissue and/or the heart (pressure overload, ischemia, diabetic cardiomyopathy, etc.).

Sustained Downregulation of Vascular Smooth Muscle Acta2 After Transient Angiotensin II Infusion: A New Model of "Vascular Memory".

Background: Activation of the renin-angiotensin-aldosterone system (RAAS) plays a critical role in the development of hypertension. Published evidence on a putative "memory effect" of AngII on the vascular components is however scarce. Aim: To evaluate the long-term effects of transient exposure to AngII on the mouse heart and the arterial tissue. Methods: Blood pressure, cardiovascular tissue damage and remodeling, and systemic oxidative stress were evaluated in C57/B6/J mice at the end of a 2-week AngII infusion (AngII); 2 and 3 weeks after the interruption of a 2-week AngII treatment (AngII+2W and AngII +3W; so-called "memory" conditions) and control littermate (CTRL). RNAseq profiling of aortic tissues was used to identify potential key regulated genes accounting for legacy effects on the vascular phenotype. RNAseq results were validated by RT-qPCR and immunohistochemistry in a reproduction cohort of mice. Key findings were reproduced in a homotypic cell culture model. Results: The 2 weeks AngII infusion induced cardiac hypertrophy and aortic damage that persisted beyond AngII interruption and despite blood pressure normalization, with a sustained vascular expression of ICAM1, infiltration by CD45+ cells, and cell proliferation associated with systemic oxidative stress. RNAseq profiling in aortic tissue identified robust Acta2 downregulation at transcript and protein levels (α-smooth muscle actin) that was maintained beyond interruption of AngII treatment. Among regulators of Acta2 expression, the transcription factor Myocardin (Myocd), exhibited a similar expression pattern. The sustained downregulation of Acta2 and Myocd was associated with an increase in H3K27me3 in nuclei of aortic sections from mice in the "memory" conditions. A sustained downregulation of ACTA2 and MYOCD was reproduced in the cultured human aortic vascular smooth muscle cells upon transient exposure to Ang II. Conclusion: A transient exposure to Ang II produces prolonged vascular remodeling with robust ACTA2 downregulation, associated with epigenetic imprinting supporting a "memory" effect despite stimulus withdrawal.

The Beta3 Adrenergic Receptor in Healthy and Pathological Cardiovascular Tissues.

The third isotype of beta-adrenoreceptors (ß3-AR) has recently come (back) into focus after the observation of its expression in white and beige human adipocytes and its implication in metabolic regulation. This coincides with the recent development and marketing of agonists at the human receptor with superior specificity. Twenty years ago, however, we and others described the expression of ß3-AR in human myocardium and its regulation of contractility and cardiac remodeling. Subsequent work from many laboratories has since expanded the characterization of ß3-AR involvement in many aspects of cardiovascular physio(patho)logy, justifying the present effort to update current paradigms under the light of the most recent evidence.

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.

Beta 3 adrenoreceptors protect from hypertrophic remodelling through AMP-activated protein kinase and autophagy.

AIMS: The abundance of beta 3-adrenergic receptors (ß3-ARs) is upregulated in diseased human myocardium. We previously showed that cardiac-specific expression of ß3-AR inhibits the hypertrophic response to neurohormonal stimulation. Here, we further analysed signalling pathways involved in the anti-hypertrophic effect of ß3-AR. METHODS AND RESULTS: In vitro hypertrophic responses to phenylephrine (PE) were analysed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human ß3-AR (AdVhß3). We confirmed results in mice with cardiomyocyte-specific moderate expression of human ß3-AR (ß3-TG) and wild-type (WT) littermates submitted to thoracic transverse aortic constriction (TAC) for 9 weeks. We observed a colocalization of ß3-AR with the AMP-activated protein kinase (AMPK) both in neonatal rat and in adult mouse cardiomyocytes. Treatment of NRVM with PE induced hypertrophy and a decrease in phosphorylation of Thr172-AMPK (/2, P = 0.0487) and phosphorylation of Ser79-acetyl-CoA carboxylase (ACC) (/2.6, P = 0.0317), inducing an increase in phosphorylated Ser235/236 S6 protein (×2.5, P = 0.0367) known to be involved in protein synthesis. These effects were reproduced by TAC in WT mice but restored to basal levels in ß3-AR expressing cells/mice. siRNA targeting of AMPK partly abrogated the anti-hypertrophic effect of ß3-AR in response to PE in NRVM. Concomitant with hypertrophy, autophagy was decreased by PE, as measured by microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio (/2.6, P = 0.0010) and p62 abundance (×3, P = 0.0016) in NRVM or by TAC in WT mice (LC3-II/LC3-I ratio: /5.4, P = 0.0159), but preserved in human ß3-AR expressing cells and mice, together with reduced hypertrophy. CONCLUSIONS: Cardiac-specific moderate expression of ß3-AR inhibits the hypertrophic response in part through AMPK activation followed by inhibition of protein synthesis and preservation of autophagy. Activation of the cardiac ß3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodelling.

Nitric oxide signalling in cardiovascular health and disease.

Nitric oxide (NO) signalling has pleiotropic roles in biology and a crucial function in cardiovascular homeostasis. Tremendous knowledge has been accumulated on the mechanisms of the nitric oxide synthase (NOS)-NO pathway, but how this highly reactive, free radical gas signals to specific targets for precise regulation of cardiovascular function remains the focus of much intense research. In this Review, we summarize the updated paradigms on NOS regulation, NO interaction with reactive oxidant species in specific subcellular compartments, and downstream effects of NO in target cardiovascular tissues, while emphasizing the latest developments of molecular tools and biomarkers to modulate and monitor NO production and bioavailability.

The Function and Therapeutic Potential of Long Non-coding RNAs in Cardiovascular Development and Disease.

The popularization of genome-wide analyses and RNA sequencing led to the discovery that a large part of the human genome, while effectively transcribed, does not encode proteins. Long non-coding RNAs have emerged as critical regulators of gene expression in both normal and disease states. Studies of long non-coding RNAs expressed in the heart, in combination with gene association studies, revealed that these molecules are regulated during cardiovascular development and disease. Some long non-coding RNAs have been functionally implicated in cardiac pathophysiology and constitute potential therapeutic targets. Here, we review the current knowledge of the function of long non-coding RNAs in the cardiovascular system, with an emphasis on cardiovascular development and biology, focusing on hypertension, coronary artery disease, myocardial infarction, ischemia, and heart failure. We discuss potential therapeutic implications and the challenges of long non-coding RNA research, with directions for future research and translational focus.

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA.

Isoform 3 of the Na+-Ca2+ exchanger (NCX3) participates in the Ca2+ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca2+]i or [Na+]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca2+ uptake after a rise in [Ca2+]i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca2+ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca2+-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.

New and Emerging Therapies and Targets: Beta-3 Agonists.

While crucial for the acute physiologic response to stress, the adrenergic system may become maladaptive upon prolonged stimulation in the course of development of heart failure. This has been the basis for the development of beta-blocking therapies, targeting mainly beta1-2 adrenoreceptors (B1-2AR). The third isotype, B3AR, was more recently identified in cardiac myocytes and endothelial cells from human (and many other animal species), where its distinctive coupling to nitric oxide and antioxidant pathways suggested potential protective properties that were unexploited so far. The observation of beneficial effects of B3AR expression/activation on myocardial remodeling and the availability of specific agonists for clinical use now open the way for directly testing the hypothesis in heart failure patients. We will briefly review the specificities of B3AR signaling in the context of the cardiovascular adrenergic system, the evidence supporting its beneficial effects and outline an ongoing clinical trial using the B3AR agonist, mirabegron in patients with/at risk of developing heart failure with preserved ejection fraction (HFpEF).

Calpain-3-mediated regulation of the Na⁺-Ca²âº exchanger isoform 3.

Ca(2+) disturbances are observed when Ca(2+)-dependent cysteine proteases malfunction, causing muscle weakness and wasting. For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na(+)-Ca(2+) exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca(2+) content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca(2+) imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca(2+) uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca(2+) ([Ca(2+)]i) or Na(+) ([Na(+)]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca(2+) uptake of NCX3 might participate in the Ca(2+) refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca(2+) storage observed in LGMD2A.

Towards Understanding the Role of the Na²âº-Ca²âº Exchanger Isoform 3.

The Na²âº-Ca²âº exchanger (NCX) is critical for Ca²âº homeostasis throughout the body. Of the three isoforms in the NCX family, NCX1 has been extensively studied, providing a good basis for understanding the molecular aspects of the NCX family, including structural resemblances, stoichiometry, and mechanism of exchange. However, the tissue expression of the third isoform of the family, NCX3, together with its proposed involvement in the Ca²âº fluxes of the endoplasmic reticulum and the mitochondria suggests a distinctive role for this isoform. Investigations of the exchanger revealed the involvement of NCX3 in diverse processes such as bone formation, TNF-α production, slow-twitch muscle contraction, and long-term potentiation in the hippocampus. Furthermore, the study of its posttranslational modification, its cleavage by the Ca²âº-sensitive protease, calpain, and its upregulation in numerous stress conditions linked NCX3 to the aberrant Ca²âº influx seen during neuronal excitotoxicity in Alzheimer's disease, brain stroke, and neuronal injuries. Hence, beyond its role in calcium homeostasis, NCX3 plays an important role in stress conditions, neuronal excitotoxicity, and metabolism and is thereby a key element in many cell types. The present review aims to survey the knowledge on NCX3, focusing on the recent discoveries on its functional and structural properties, and discusses the implications of NCX3 in both physiological and pathological conditions.

Function and regulation of the Na+-Ca2+ exchanger NCX3 splice variants in brain and skeletal muscle.

Isoform 3 of the Na(+)-Ca(2+) exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca(2+)]i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca(2+) extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca(2+) imaging, we measured the capacity and regulation of the two variants during Ca(2+) extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na(+)]i), NCX3-AC has a higher [Na(+)]i sensitivity, as Ca(2+) influx is observed in the presence of extracellular Na(+). This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca(2+) extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca(2+). Strikingly, substitution of Glu(580) in NCX3-B with its NCX3-AC equivalent Lys(580) recapitulated the functional properties of NCX3-AC regarding Ca(2+) sensitivity, Lys(580) presumably acting through a structure stabilization of the Ca(2+) binding site. The higher Ca(2+) uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca(2+) levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca(2+) handling beyond that of extruding Ca(2+).