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Heart failure (HF) is a growing public health burden, with high prevalence and mortality rates. In contrast to ischemic heart failure (IHF), the diagnosis of non-ischemic heart failure (NIHF) is established in the absence of coronary artery disease. Angiopoietins (ANGPTs), vascular endothelial growth factors (VEGFs) and secretory phospholipases A2 (sPLA2s) are proinflammatory mediators and key regulators of endothelial cells. In the present manuscript, we analyze the plasma concentrations of angiogenic (ANGPT1, ANGPT2, VEGF-A) and lymphangiogenic (VEGF-C, VEGF-D) factors and the plasma activity of sPLA2 in patients with IHF and NIHF compared to healthy controls. The concentrations of ANGPT1, ANGPT2 and their ratio significantly differed between HF patients and healthy controls. Similarly, plasma levels of VEGF-D and sPLA2 activity were higher in HF as compared to controls. Concentrations of ANGPT2 and the ANGPT2/ANGPT1 ratio (an index of vascular permeability) were increased in NIHF patients. VEGF-A and VEGF-C concentrations did not differ among the three examined groups. Interestingly, VEGF-D was selectively increased in IFH patients compared to controls. Plasma activity of sPLA2 was increased in IHF and NIHF patients compared to controls. Our results indicate that several regulators of vascular permeability and smoldering inflammation are specifically altered in IHF and NIHF patients. Studies involving larger cohorts of these patients will be necessary to demonstrate the clinical implications of our findings.

The mineralocorticoid hormone aldosterone (Aldo) has been intensively studied for its ability to influence both the physiology and pathophysiology of the cardiovascular system. Indeed, although research on Aldo actions for decades has mainly focused on its effects in the kidney, several lines of evidence have now demonstrated that this hormone exerts disparate extrarenal adverse effects, especially in the circulatory system. Accordingly, in the last lusters, a number of studies in preclinical models (in vitro and in vivo) and in humans have established that Aldo, following the interaction with its receptor—the mineralocorticoid receptor (MR)—is able to activate specific intracellular genomic and nongenomic pathways, thus regulating the homeostasis of the cardiovascular system. Importantly, through this mechanism of action, this hormone becomes a crucial regulator of the function and growth of different types of cells, including fibroblasts, cardiomyocytes, and vascular cells. For this main reason, it is plausible that when Aldo is present at high levels in the blood, it profoundly modifies the physiology of these cells, therefore being at the foundation of several cardiovascular disorders, such as heart failure (HF). On these grounds, in this review, we will provide an updated account on the current knowledge concerning Aldo activity in the cardiovascular system and the most recent preclinical studies and clinical trials designed to test better approaches able to counter the hyperactivity of the Aldo/MR signaling pathway in the setting of cardiovascular diseases.

Following hindlimb ischemia (HI) increased catecholamine levels within the ischemic muscle can cause dysregulation of β2-adrenergic receptor (β2AR) signaling leading to reduced revascularization. Indeed, in vivo β2AR overexpression, via gene therapy, enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of βAR signaling, and βARKct, a peptide inhibitor of GRK2, has been shown to prevent βAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury, through restoration of β2AR protective signaling (i.e. Akt/eNOS). Herein, we tested potential therapeutic effects of adenoviral-mediated βARKct gene transfer in an experimental model of HI and its effects on βAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant βAR down-regulation that was paralleled by an about 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the βARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and βAR density in the ischemic muscle, compared to control groups. The effect of βARKct expression was also assessed, in vitro in cultured ECs. Interestingly, in ECs expressing the βARKct, fenoterol, a β2AR-agonist, induced enhanced β2AR pro-angiogenic signaling and increased EC function. In conclusion, our results suggest that βARKct gene-therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR downregulation.

Rationale: It has been reported that epicardial adipose tissue (EAT) may affect myocardial autonomic function. Objective: The aim of this study was to explore the relationship between EAT and cardiac sympathetic nerve activity in patients with heart failure. Methods and Results: In 110 patients with systolic heart failure, we evaluated the correlation between echocardiographic EAT thickness and cardiac adrenergic nerve activity assessed by 123 I-metaiodobenzylguanidine ( 123 I-MIBG). The predictive value of EAT thickness on cardiac sympathetic denervation ( 123 I-MIBG early and late heart:mediastinum ratio and single-photon emission computed tomography total defect score) was tested in a multivariate analysis. Furthermore, catecholamine levels, catecholamine biosynthetic enzymes, and sympathetic nerve fibers were measured in EAT and subcutaneous adipose tissue biopsies obtained from patients with heart failure who underwent cardiac surgery. EAT thickness correlated with 123 I-MIBG early and late heart:mediastinum ratio and single-photon emission computed tomography total defect score, but not with left ventricular ejection fraction. Moreover, EAT resulted as an independent predictor of 123 I-MIBG early and late heart:mediastinum ratio and single-photon emission computed tomography total defect score and showed a significant additive predictive value on 123 I-MIBG planar and single-photon emission computed tomography results over demographic and clinical data. Although no differences were found in sympathetic innervation between EAT and subcutaneous adipose tissue, EAT showed an enhanced adrenergic activity demonstrated by the increased catecholamine levels and expression of catecholamine biosynthetic enzymes. Conclusions: This study provides the first evidence of a direct correlation between increased EAT thickness and cardiac sympathetic denervation in heart failure.

Background: Lo scompenso cardiaco è caratterizzato dall’iperattività del sistema nervoso simpatico che rappresenta inizialmente un processo di adattamento rivolto a compensare la riduzione della performance cardiaca. Il cronico incremento delle catecolamine plasmatiche si associa ad una marcata disregolazione dei recettori β-adrenergici (β-AR) cardiaci e ad una progressiva perdita delle fibre nervose cardiache sia adrenergiche che colinergiche. La restrizione calorica (RC) è un intervento dietetico capace di migliorare la funzione del sistema cardiovascolare ed il profilo metabolico con conseguenze favorevoli sulla qualità della vita. Scopo: Lo scopo del progetto è stato quello di valutare gli effetti di un trattamento dietetico con RC sulla funzione cardiaca e sullo stato di innervazione cardiaca in un modello di SC post-ischemico.Materiali e metodi: Sono stati studiati 38 ratti, sottoposti all’operazione chirurgica di induzione dell’infarto del miocardio. Animali sham-operati sono stati inclusi come controllo. Dopo 4 settimane dall’induzione dell’infarto, i ratti sono stati studiati ecocardiograficamente e quindi randomizzati ad un regime di RC o dieta ad libitum per un periodo di 12 mesi. Risultati: Come atteso un anno di dieta a regime calorico ristretto induceva una significativa diminuzione sia del peso corporeo che di quello cardiaco rispetto al gruppo di ratti a regime calorico normale. Nel gruppo di ratti scompensati la RC induceva un significativo miglioramento della funzione cardiaca sistolica, del rimodellamento ventricolare e della riserva inotropa rispetto al gruppo di ratti scompensati trattati con dieta normale. Inoltre, lo SC determinava una riduzione significativa della densità dei β-AR di membrana nel cuore rispetto agli animali sham-operati, mentre la RC era in grado di indurre un significativo aumento dei β-AR. Studi di microscopia confocale evidenziavano una marcata riduzione delle fibre nervose nei cuori dei ratti con SC rispetto ai sham-operati. Sorprendentemente, la RC era in grado di aumentare significativamente le fibre adrenergiche del cuore scompensato rispetto agli animali scompensati a dieta normale. Conclusione: I nostri dati suggeriscono che in un modello sperimentale di SC la RC, iniziata dopo l’infarto del miocardio, determina un miglioramento della densità dei recettori beta-adrenergici cardiaci ed un aumento dell’innervazione simpatica del cuore.

Introduction: In the risk stratification and selection of patients with Heart Failure (HF) eligible for Implantable Cardioverter Defibrillator (ICD) therapy, 123 I-meta-IodineBenzylGuanidine (123 I-mIBG) scintigraphy has emerged as an effective non-invasive method to assess cardiac adrenergic innervation. Similarly, clinical risk scores have been proposed to identify patients with HF at risk of all-cause mortality, for whom the net clinical benefit of device implantation would presumably be lower. Nevertheless, the association between the two classes of tools, one suggestive of arrhythmic risk, the other of all-causes mortality, needs further investigation. Objective: To test the relationship between the risk scores for predicting mortality and cardiac sympathetic innervation, assessed through myocardial 123 I-mIBG imaging, in a population of patients with HF. Methods: in HF patients undergoing 123 I-mIBG scintigraphy, eight risk stratification models were assessed: AAACC, FADES, MADIT, MADIT-ICD non-arrhythmic mortality score, PACE, Parkash, SHOCKED and Sjoblom. Cardiac adrenergic impairment was assessed by late Heart to Mediastinum ratio (H/M)

Aims: Limited data are available regarding cardiac expression of molecules involved in heart failure (HF) pathophysiology. The majority of the studies have focused on end-stage HF with reduced ejection fraction (HFrEF) without comparison with healthy subjects, while no data are available with regard to HF with preserved ejection fraction (HFpEF). HFpEF is a condition whose multiple pathophysiological mechanisms are still not fully defined, with many proposed hypotheses remaining speculative due to limited access to human heart tissue. This study aimed at evaluating cardiac expression levels of key genes of interest in human biopsy samples from patients affected with HFrEF and HFpEF in order to possibly point out distinct phenotypes. Methods and results: Total RNA was extracted from left ventricular cardiac biopsies collected from stable patients with HFrEF (n=6) and HFpEF (n=7) and healthy subjects (n=9) undergoing elective cardiac surgery for valvular replacement, mitral valvuloplasty, aortic surgery, or coronary artery bypass. Real-time PCR was performed to evaluate the mRNA expression levels of genes involved in somatotropic axis regulation [IGF-1, IGF-1 receptor (IGF-1R), and GH receptor (GHR)], in adrenergic signalling (GRK2, GRK5, ADRB1, and ADRB2), in myocardial calcium handling (SERCA2), and in TNF-α. Patients with HFrEF and HFpEF showed reduced serum IGF-1 circulating levels when compared with controls (102±35.6, 138±11.5, and 160±13.2ng/mL, P&nbsp

Heart failure (HF) is a growing public health burden, with high prevalence and mortality rates. A proportion of patients with HF have a normal ventricular ejection fraction (EF), referred to as HF with preserved EF (HFpEF), as opposed to patients with HF with reduced ejection fraction (HFrEF). HFpEF currently accounts for about 50% of all HF patients, and its prevalence is rising. Angiopoietins (ANGPTs), vascular endothelial growth factors (VEGFs) and secretory phospholipases A2 (sPLA2s) are proinflammatory mediators and key regulators of endothelial cells.

BACKGROUND: Despite severe acute respiratory syndrome (SARS)-Coronavirus (CoV-2) primarily targeting the lungs, the heart represents another critical virus target. Thus, the identification of SARS-CoV-2 disease of 2019 (COVID-19)-associated biomarkers would be beneficial to stratify prognosis and the risk of developing cardiac complications. Aldosterone and galectin-3 promote fibrosis and inflammation and are considered a prognostic biomarker of lung and adverse cardiac remodeling. Here, we tested whether galectin-3 and aldosterone levels can predict adverse cardiac outcomes in COVID-19 patients. METHODS: To this aim, we assessed galectin-3 and aldosterone serum levels in 51 patients diagnosed with COVID-19, using a population of 19 healthy subjects as controls. In in-vitro studies, we employed 3T3 fibroblasts to assess the potential roles of aldosterone and galectin-3 in fibroblast activation. RESULTS: Serum galectin-3 levels were more elevated in COVID-19 patients than healthy controls and correlated with COVID-19 severity classification and cardiac troponin-I (cTnI) serum levels. Furthermore, we observed an augmented secretion of aldosterone in COVID-19 patients. This adrenal hormone is a direct stimulator of galectin-3 secretion; therefore, we surmised that this axis could perpetrate fibrosis and adverse remodeling in these subjects. Thus, we stimulated fibroblasts with 10% of serum from COVID-19 patients. This challenge markedly rose the expression of smooth muscle alpha (α)-2 actin (ACTA2), a myofibroblast marker. CONCLUSIONS: Our study suggests that COVID-19 can affect cardiac structure and function by triggering aldosterone and galectin-3 release that may serve as prognostic and therapeutic biomarkers while monitoring the course of cardiac complications in patients suffering from COVID-19.

Hyperaldosteronism alters cardiac function, inducing adverse left ventricle (LV) remodeling either via increased fibrosis deposition, mitochondrial dysfunction, or both. These harmful effects are due, at least in part, to the activation of the G protein-coupled receptor kinase 2 (GRK2). In this context, we have previously reported that this kinase dysregulates both β-adrenergic receptor (βAR) and insulin (Ins) signaling. Yet, whether aldosterone modulates cardiac Ins sensitivity and βAR function remains untested. Nor is it clear whether GRK2 has a role in this modulation, downstream of aldosterone. Here, we show in vitro, in 3T3 cells, that aldosterone impaired insulin signaling, increasing the negative phosphorylation of insulin receptor substrate 1 (ser307pIRS1) and reducing the activity of Akt. Similarly, aldosterone prevented the activation of extracellular signal-regulated kinase (ERK) and the production of cyclic adenosine 3',5'-monophosphate (cAMP) in response to the β1/β2AR agonist, isoproterenol. Of note, all of these effects were sizably reduced in the presence of GRK2-inhibitor CMPD101. Next, in wild-type (WT) mice undergoing chronic infusion of aldosterone, we observed a marked GRK2 upregulation that was paralleled by a substantial β1AR downregulation and augmented ser307pIRS1 levels. Importantly, in keeping with the current in vitro data, we found that aldosterone effects were wholly abolished in cardiac-specific GRK2-knockout mice. Finally, in WT mice that underwent 4-week myocardial infarction (MI), we observed a substantial deterioration of cardiac function and increased LV dilation and fibrosis deposition. At the molecular level, these effects were associated with a significant upregulation of cardiac GRK2 protein expression, along with a marked β1AR downregulation and increased ser307pIRS1 levels. Treating MI mice with spironolactone prevented adverse aldosterone effects, blocking GRK2 upregulation, and thus leading to a marked reduction in cardiac ser307pIRS1 levels while rescuing β1AR expression. Our study reveals that GRK2 activity is a critical player downstream of the aldosterone signaling pathway; therefore, inhibiting this kinase is an attractive strategy to prevent the cardiac structural disarray and dysfunction that accompany any clinical condition accompanied by hyperaldosteronism.

Background: Caloric restriction (CR) has been described to have cardioprotective effects and improve functional outcomes in animal models and humans. Chronic ischemic heart failure (HF) is associated with reduced cardiac sympathetic innervation, dysfunctional β-adrenergic receptor signaling, and decreased cardiac inotropic reserve. We tested the effects of a long-term CR diet, started late after myocardial infarction on cardiac function, sympathetic innervation, and β-adrenergic receptor responsiveness in a rat model of postischemic HF. Methods and Results: Adult male rats were randomly assigned to myocardial infarction or sham operation and 4 weeks later were further randomized to a 1-year CR or normal diet. One year of CR resulted in a significant reduction in body weight, heart weight, and heart weight/tibia length ratio when compared with normal diet in HF groups. At the end of the study period, echocardiography and histology revealed that HF animals under the CR diet had ameliorated left ventricular remodeling compared with HF rats fed with normal diet. Invasive hemodynamic showed a significant improvement of cardiac inotropic reserve in CR HF rats compared with HF-normal diet animals. Importantly, CR dietary regimen was associated with a significant increase of cardiac sympathetic innervation and with normalized cardiac β-adrenergic receptor levels in HF rats when compared with HF rats on the standard diet. Conclusions: We demonstrate, for the first time, that chronic CR, when started after HF established, can ameliorate cardiac dysfunction and improve inotropic reserve. At the molecular level, we find that chronic CR diet significantly improves sympathetic cardiac innervation and β-adrenergic receptor levels in failing myocardium.

The mineralocorticoid hormone aldosterone (Aldo) has been intensively studied for its ability to influence both the physiology and pathophysiology of the cardiovascular system. Indeed, although research on Aldo actions for decades has mainly focused on its effects in the kidney, several lines of evidence have now demonstrated that this hormone exerts disparate extrarenal adverse effects, especially in the circulatory system. Accordingly, in the last lusters, a number of studies in preclinical models (in vitroandin vivo) and in humans have established that Aldo, following the interaction with its receptor—the mineralocorticoid receptor (MR)—is able to activate specific intracellular genomic and nongenomic pathways, thus regulating the homeostasis of the cardiovascular system. Importantly, through this mechanism of action, this hormone becomes a crucial regulator of the function and growth of different types of cells, including fibroblasts, cardiomyocytes, and vascular cells. For this main reason, it is plausible that when Aldo is present at high levels in the blood, it profoundly modifies the physiology of these cells, therefore being at the foundation of several cardiovascular disorders, such as heart failure (HF). On these grounds, in this review, we will provide an updated account on the current knowledge concerning Aldo activity in the cardiovascular system and the most recent preclinical studies and clinical trials designed to test better approaches able to counter the hyperactivity of the Aldo/MR signaling pathway in the setting of cardiovascular diseases.

G protein-coupled receptor (GPCR) kinase-2 (GRK2) is a regulator of GPCRs, in particular β-adrenergic receptors (ARs), and as demonstrated by decades of investigation, it has a pivotal role in the development and progression of cardiovascular disease, like heart failure (HF). Indeed elevated levels and activity of this kinase are able to promote the dysfunction of both cardiac and adrenal α- and β-ARs and to dysregulate other protective signaling pathway, such as sphingosine 1-phospate and insulin. Moreover, recent discoveries suggest that GRK2 can signal independently from GPCRs, in a 'non-canonical' manner, via interaction with non-GPCR molecule or via its mitochondrial localization. Areas covered: Based on this premise, GRK2 inhibition or its genetic deletion has been tested in several disparate animal models of cardiovascular disease, showing to protect the heart from adverse remodeling and dysfunction. Expert opinion: HF is one of the leading cause of death worldwide with enormous health care costs. For this reason, the identification of new therapeutic targets like GRK2 and strategies such as its inhibition represents a new hope in the fight against HF development and progression. Herein, we will update the readers about the 'state-of-art' of GRK2 inhibition as a potent therapeutic strategy in HF.

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 β 1 -adrenergic receptors (ARs) and the cardioprotective sphingosine-1-phosphate (S1P) receptor- 1 (S1PR 1 ). Objectives The authors hypothesized that, in addition to salutary actions due to direct β 1 AR-blockade, agents such as metoprolol (Meto) may improve post–myocardial infarction (MI) structural and functional outcomes via restored S1PR 1 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 β 3 AR knockout mice. Results Here we report that, in vitro, Meto prevents catecholamine-induced down-regulation of S1PR 1 , 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 β 1 AR-blockers display elevated circulating S1P levels, confirming that Meto promotes S1P secretion/signaling. Mechanistically, we found that Meto-induced S1P secretion is β 3 AR-dependent because Meto infusion in β 3 AR 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 β 1 -blockers prevent HF progression in patients with ischemia, suggesting that β 3 AR dysfunction may account for limited/null efficacy in β 1 AR-blocker–insensitive HF subjects.

Rationale: Sympathetic nervous system hyperactivity is associated with poor prognosis in patients with heart failure (HF), yet routine assessment of sympathetic nervous system activation is not recommended for clinical practice. Myocardial G protein-coupled receptor kinase-2 (GRK2) is upregulated in HF patients, causing dysfunctional β-adrenergic receptor signaling. Importantly, myocardial GRK2 levels correlate with levels found in peripheral lymphocytes of HF patients. Objective: The independent prognostic value of blood GRK2 measurements in HF patients has never been investigated; thus, the purpose of this study was to evaluate whether lymphocyte GRK2 levels predict clinical outcome in HF patients. Methods and Results: We prospectively studied 257 HF patients with mean left ventricular ejection fraction of 31.4±8.5%. At the time of enrollment, plasma norepinephrine, serum NT-proBNP, and lymphocyte GRK2 levels, as well as clinical and instrumental variables were measured. The prognostic value of GRK2 to predict cardiovascular (CV) death and all-cause mortality was assessed using the Cox proportional hazard model including demographic, clinical, instrumental, and laboratory data. Over a mean follow-up period of 37.5±20.2 months (range, 3–60 months), there were 102 CV deaths. Age, left ventricular ejection fraction, New York Heart Association class, chronic obstructive pulmonary disease, chronic kidney disease, N-terminal-pro brain natriuretic peptide, and lymphocyte GRK2 protein levels were independent predictors of CV mortality in HF patients. GRK2 levels showed an additional prognostic and clinical value over demographic and clinical variables. The independent prognostic value of lymphocyte GRK2 levels was also confirmed for all-cause mortality. Conclusions: Lymphocyte GRK2 protein levels can independently predict prognosis in patients with HF.

AIM: Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls. PARTICIPANTS: Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn's disease patients) were enrolled. RESULTS: Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%. CONCLUSIONS: The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.