Cardiometabolic effects of sacubitril/valsartan in a rat model of heart failure with preserved ejection fraction.

The promising results obtained in the PARADIGM-HF trial prompted the approval of sacubitril/valsartan (SAC/VAL) as a first-in-class treatment for heart failure with reduced ejection fraction (HFrEF) patients. The effect of SAC/VAL treatment was also studied in patients with heart failure with preserved ejection fraction (HFpEF) and, although improvements in New York Heart Association (NYHA) class, HF hospitalizations, and cardiovascular deaths were observed, these results were not so promising. However, the demand for HFpEF therapies led to the approval of SAC/VAL as an alternative treatment, although further studies are needed. We aimed to elucidate the effects of a 9-week SAC/VAL treatment in cardiac function and metabolism using a preclinical model of HFpEF, the Zucker Fatty and Spontaneously Hypertensive (ZSF1) rats. We found that SAC/VAL significantly improved diastolic function parameters and modulated respiratory quotient during exercise. Ex-vivo studies showed that SAC/VAL treatment significantly decreased heart, liver, spleen, and visceral fat weights; cardiac hypertrophy and percentage of fibrosis; lipid infiltration in liver and circulating levels of cholesterol and sodium. Moreover, SAC/VAL reduced glycerophospholipids, cholesterol, and cholesteryl esters while increasing triglyceride levels in cardiac tissue. In conclusion, SAC/VAL treatment improved diastolic and hepatic function, respiratory metabolism, reduced hypercholesterolemia and cardiac fibrosis and hypertrophy, and was able to modulate cardiac metabolic profile. Our findings might provide further insight into the therapeutic benefits of SAC/VAL treatment in obese patients with HFpEF.

Injectable Carrageenan/Green Graphene Oxide Hydrogel: A Comprehensive Analysis of Mechanical, Rheological, and Biocompatibility Properties.

In this work, physically crosslinked injectable hydrogels based on carrageenan, locust bean gum, and gelatin, and mechanically nano-reinforced with green graphene oxide (GO), were developed to address the challenge of finding materials with a good balance between injectability and mechanical properties. The effect of GO content on the rheological and mechanical properties, injectability, swelling behavior, and biocompatibility of the nanocomposite hydrogels was studied. The hydrogels' morphology, assessed by FE-SEM, showed a homogeneous porous architecture separated by thin walls for all the GO loadings investigated. The rheology measurements evidence that G' > G″ over the whole frequency range, indicating the dominant elastic nature of the hydrogels and the difference between G' over G″ depends on the GO content. The GO incorporation into the biopolymer network enhanced the mechanical properties (ca. 20%) without appreciable change in the injectability of the nanocomposite hydrogels, demonstrating the success of the approach described in this work. In addition, the injectable hydrogels with GO loadings ≤0.05% w/v exhibit negligible toxicity for 3T3-L1 fibroblasts. However, it is noted that loadings over 0.25% w/v may affect the cell proliferation rate. Therefore, the nano-reinforced injectable hybrid hydrogels reported here, developed with a fully sustainable approach, have a promising future as potential materials for use in tissue repair.

Human recombinant relaxin-2 (serelaxin) regulates the proteome, lipidome, lipid metabolism and inflammatory profile of rat visceral adipose tissue.

Recombinant human relaxin-2 (serelaxin) has been widely proven as a novel drug with myriad effects at different cardiovascular levels, which support its potential therapeutic efficacy in several cardiovascular diseases (CVD). Considering these effects, together with the influence of relaxin-2 on adipocyte physiology and adipokine secretion, and the connection between visceral adipose tissue (VAT) dysfunction and the development of CVD, we could hypothesize that relaxin-2 may regulate VAT metabolism. Our objective was to evaluate the impact of a 2-week serelaxin treatment on the proteome and lipidome of VAT from Sprague-Dawley rats. We found that serelaxin increased 1 polyunsaturated fatty acid and 6 lysophosphatidylcholines and decreased 4 triglycerides in VAT employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) based platforms, and that regulates 47 phosphoproteins using SWATH/MS analysis. Through RT-PCR, we found that serelaxin treatment also caused an effect on VAT lipolysis through an increase in the mRNA expression of hormone-sensitive lipase (HSL) and a decrease in the expression of adipose triglyceride lipase (ATGL), together with a reduction in the VAT expression of the fatty acid transporter cluster of differentiation 36 (Cd36). Serelaxin also caused an anti-inflammatory effect in VAT by the decrease in the mRNA expression of tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), chemerin, and its receptor. In conclusion, our results highlight the regulatory role of serelaxin in the VAT proteome and lipidome, lipolytic function, and inflammatory profile, suggesting the implication of several mechanisms supporting the potential benefit of serelaxin for the prevention of obesity and metabolic disorders.

Plasma miR-486-5p Expression Is Upregulated in Atrial Fibrillation Patients with Broader Low-Voltage Areas.

Atrial fibrillation (AF) is the most common arrhythmia worldwide, affecting 1% of the population over 60 years old. The incidence and prevalence of AF are increasing globally, representing a relevant health problem, suggesting that more advanced strategies for predicting risk stage are highly needed. miRNAs mediate several processes involved in AF. Our aim was to identify miRNAs with a prognostic value as biomarkers in patients referred for AF ablation and its association with LVA extent, based on low-voltage area (LVA) maps. In this study, we recruited 44 AF patients referred for catheter ablation. We measured the expression of 84 miRNAs in plasma from peripheral blood in 3 different groups based on LVA extent. Expression analysis showed that miR-486-5p was significantly increased in patients with broader LVA (4-fold, p = 0.0002; 5-fold, p = 0.0001). Receiver operating characteristic curve analysis showed that miR-486-5p expression could predict atrium LVA (AUC, 0.8958; p = 0.0015). Also, miR-486-5p plasma levels were associated with AF-type (AUC, 0.7137; p = 0.0453). In addition, miR-486-5p expression was positively correlated with LVA percentage, left atrial (LA) area, and LA volume (r = 0.322, p = 0.037; r = 0.372, p = 0.015; r = 0.319, p = 0.045, respectively). These findings suggest that miR-486-5p expression might have prognostic significance in LVA extent in patients with AF.

Altered MicroRNA Maturation in Ischemic Hearts: Implication of Hypoxia on XPO5 and DICER1 Dysregulation and RedoximiR State.

Ischemic cardiomyopathy (ICM) is associated with abnormal microRNA expression levels that involve an altered gene expression profile. However, little is known about the underlying causes of microRNA disruption in ICM and whether microRNA maturation is compromised. Therefore, we focused on microRNA maturation defects analysis and the implication of the microRNA biogenesis pathway and redox-sensitive microRNAs (redoximiRs). Transcriptomic changes were investigated via ncRNA-seq (ICM, n = 22; controls, n = 8) and mRNA-seq (ICM, n = 13; control, n = 10). The effect of hypoxia on the biogenesis of microRNAs was evaluated in the AC16 cell line. ICM patients showed a reduction in microRNA maturation compared to control (4.30 ± 0.94 au vs. 5.34 ± 1.07 au, p ˂ 0.05), accompanied by a deregulation of the microRNA biogenesis pathway: a decrease in pre-microRNA export (XPO5, FC = -1.38, p ˂ 0.05) and cytoplasmic processing (DICER, FC = -1.32, p ˂ 0.01). Both processes were regulated by hypoxia in AC16 cells (XPO5, FC = -1.65; DICER1, FC = -1.55; p ˂ 0.01; Exportin-5, FC = -1.81; Dicer, FC = -1.15; p ˂ 0.05). Patients displayed deregulation of several redoximiRs, highlighting miR-122-5p (FC = -2.41, p ˂ 0.001), which maintained a good correlation with the ejection fraction (r = 0.681, p ˂ 0.01). We evidenced a decrease in microRNA maturation mainly linked to a decrease in XPO5-mediated pre-microRNA export and DICER1-mediated processing, together with a general effect of hypoxia through deregulation of biogenesis pathway and the redoximiRs.

The lipidomic and inflammatory profiles of visceral and subcutaneous adipose tissues are distinctly regulated by the SGLT2 inhibitor empagliflozin in Zucker diabetic fatty rats.

The pharmacological inhibition of sodium-glucose cotransporter 2 (SGLT2) has emerged as a treatment for patients with type 2 diabetes mellitus (T2DM), cardiovascular disease and/or other metabolic disturbances, although some of the mechanisms implicated in their beneficial effects are unknown. The SGLT2 inhibitor (SGLT2i) empagliflozin has been suggested as a regulator of adiposity, energy metabolism, and systemic inflammation in adipose tissue. The aim of our study was to evaluate the impact of a 6-week-empagliflozin treatment on the lipidome of visceral (VAT) and subcutaneous adipose tissue (SAT) from diabetic obese Zucker Diabetic Fatty (ZDF) rats using an untargeted metabolomics approach. We found that empagliflozin increases the content of diglycerides and oxidized fatty acids (FA) in VAT, while in SAT, it decreases the levels of several lysophospholipids and increases 2 phosphatidylcholines. Empagliflozin also reduces the expression of the cytokines interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNFα), monocyte-chemotactic protein-1 (MCP-1) and IL-10, and of Cd86 and Cd163 M1 and M2 macrophage markers in VAT, with no changes in SAT, except for a decrease in IL-1ß. Empagliflozin treatment also shows an effect on lipolysis increasing the expression of hormone-sensitive lipase (HSL) in SAT and VAT and of adipose triglyceride lipase (ATGL) in VAT, together with a decrease in the adipose content of the FA transporter cluster of differentiation 36 (CD36). In conclusion, our data highlighted differences in the VAT and SAT lipidomes, inflammatory profiles and lipolytic function, which suggest a distinct metabolism of these two white adipose tissue depots after the empagliflozin treatment.

Circulating miR-451a Expression May Predict Recurrence in Atrial Fibrillation Patients after Catheter Pulmonary Vein Ablation.

Atrial fibrillation is the most prevalent tachyarrhythmia in clinical practice, with very high cardiovascular morbidity and mortality with a high-cost impact in health systems. Currently, it is one of the main causes of stroke and subsequent heart failure and sudden death. miRNAs mediate in several processes involved in cardiovascular disease, including fibrosis and electrical and structural remodeling. Several studies suggest a key role of miRNAs in the course and maintenance of atrial fibrillation. In our study, we aimed to identify the differential expression of circulating miRNAs and their predictive value as biomarkers of recurrence in atrial fibrillation patients undergoing catheter pulmonary vein ablation. To this effect, 42 atrial fibrillation patients were recruited for catheter ablation. We measured the expression of 84 miRNAs in non-recurrent and recurrent groups (45.2%), both in plasma from peripheral and left atrium blood. Expression analysis showed that miRNA-451a is downregulated in recurrent patients. Receiver operating characteristic curve analysis showed that miR-451a in left atrium plasma could predict atrial fibrillation recurrence after pulmonary vein isolation. In addition, atrial fibrillation recurrence is positively associated with the increment of scar percentage. Our data suggest that miRNA-451a expression plays an important role in AF recurrence by controlling fibrosis and progression.

Injectable hybrid hydrogels physically crosslinked based on carrageenan and green graphene for tissue repair.

Injectable and biocompatible novel hybrid hydrogels based on physically crosslinked natural biopolymers and green graphene for potential use in tissue engineering are reported. Kappa and iota carrageenan, locust bean gum and gelatin are used as biopolymeric matrix. The effect of green graphene content on the swelling behavior, mechanical properties and biocompatibility of the hybrid hydrogels is investigated. The hybrid hydrogels present a porous network with three-dimensionally interconnected microstructures, with lower pore size than that of the hydrogel without graphene. The addition of graphene into the biopolymeric network improves the stability and the mechanical properties of the hydrogels in phosphate buffer saline solution at 37 °C without noticeable change in the injectability. The mechanical properties of the hybrid hydrogels were enhanced by varying the dosage of graphene between 0.025 and 0.075 w/v%. In this range, the hybrid hydrogels preserve their integrity during mechanical test and recover the initial shape after removing the applied stress. Meanwhile, hybrid hydrogels with graphene content of up to 0.05 w/v% exhibit good biocompatibility for 3T3-L1 fibroblasts; the cells proliferate inside the gel structure and show higher spreading after 48 h. These injectable hybrid hydrogels with graphene have promising future as materials for tissue repair.

Relaxin-2 plasma levels in atrial fibrillation are linked to inflammation and oxidative stress markers.

Relaxin-2 exerts many favourable cardiovascular effects in pathological circumstances such as atrial fibrillation (AF) and heart failure, but the mechanisms underlying its actions are not completely understood. Since inflammation and fibrosis are pivotal processes in the pathogenesis of AF, our aim was to study the relationship between relaxin-2 plasma levels in left atrium (LA) and peripheral vein with molecules implicated in fibrosis, inflammation and oxidative stress in AF patients, and to evaluate the anti-fibrotic ability of relaxin-2 in normal human atrial cardiac fibroblasts (NHCF-A). Peripheral vein relaxin-2 plasma levels were higher than LA relaxin-2 plasma levels in men while, in women, peripheral vein relaxin-2 levels were increased compared to men. AF patients with higher levels of relaxin-2 exhibited a reduction in H2O2 plasma levels and in mRNA levels of alpha-defensin 3 (DEFA3) and IL-6 in leucocytes from LA plasma. Relaxin-2-in-vitro treatment inhibited NHCF-A migration and decreased mRNA and protein levels of the pro-fibrotic molecule transforming growth factor-ß1 (TGF-ß1). Our results support an association between relaxin-2 and molecules involved in fibrosis, inflammation and oxidative stress in AF patients, and reinforce an anti-fibrotic protective role of this hormone in NHCF-A; strengthening the relevance of relaxin-2 in AF physiopathology, diagnosis and treatment.

Cardiac Sodium/Hydrogen Exchanger (NHE11) as a Novel Potential Target for SGLT2i in Heart Failure: A Preliminary Study.

Despite the reduction of cardiovascular events, including the risk of death, associated with sodium/glucose cotransporter 2 inhibitors (SGLT2i), their basic action remains unclear. Sodium/hydrogen exchanger (NHE) has been proposed as the mechanism of action, but there are controversies related to its function and expression in heart failure (HF). We hypothesized that sodium transported-related molecules could be altered in HF and modulated through SGLT2i. Transcriptome alterations in genes involved in sodium transport in HF were investigated in human heart samples by RNA-sequencing. NHE11 and NHE1 protein levels were determined by ELISA; the effect of empagliflozin on NHE11 and NHE1 mRNA levels in rats' left ventricular tissues was studied through RT-qPCR. We highlighted the overexpression of SLC9C2 and SCL9A1 sodium transport genes and the increase of the proteins that encode them (NHE11 and NHE1). NHE11 levels were correlated with left ventricular diameters, so we studied the effect of SGLT2i on its expression, observing that NHE11 mRNA levels were reduced in treated rats. We showed alterations in several sodium transports and reinforced the importance of these channels in HF progression. We described upregulation in NHE11 and NHE1, but only NHE11 correlated with human cardiac dysfunction, and its levels were reduced after treatment with empagliflozin. These results propose NHE11 as a potential target of SGLT2i in cardiac tissue.

Relaxin-2 as a Potential Biomarker in Cardiovascular Diseases.

The pleiotropic hormone relaxin-2 plays a pivotal role in the physiology and pathology of the cardiovascular system. Relaxin-2 exerts relevant regulatory functions in cardiovascular tissues through the specific receptor relaxin family peptide receptor 1 (RXFP1) in the regulation of cardiac metabolism; the induction of vasodilatation; the reversion of fibrosis and hypertrophy; the reduction of inflammation, oxidative stress, and apoptosis; and the stimulation of angiogenesis, with inotropic and chronotropic effects as well. Recent preclinical and clinical outcomes have encouraged the potential use of relaxin-2 (or its recombinant form, known as serelaxin) as a therapeutic strategy during cardiac injury and/or in patients suffering from different cardiovascular disarrangements, especially heart failure. Furthermore, relaxin-2 has been proposed as a promising biomarker of cardiovascular health and disease. In this review, we emphasize the relevance of the endogenous hormone relaxin-2 as a useful diagnostic biomarker in different backgrounds of cardiovascular pathology, such as heart failure, atrial fibrillation, myocardial infarction, ischemic heart disease, aortic valve disease, hypertension, and atherosclerosis, which could be relevant in daily clinical practice and could contribute to comprehending the specific role of relaxin-2 in cardiovascular diseases.

Role of Sodium-Glucose Co-Transporter 2 Inhibitors in the Regulation of Inflammatory Processes in Animal Models.

Sodium-glucose co-transporter 2 inhibitors, also known as gliflozins, were developed as a novel class of anti-diabetic agents that promote glycosuria through the prevention of glucose reabsorption in the proximal tubule by sodium-glucose co-transporter 2. Beyond the regulation of glucose homeostasis, they resulted as being effective in different clinical trials in patients with heart failure, showing a strong cardio-renal protective effect in diabetic, but also in non-diabetic patients, which highlights the possible existence of other mechanisms through which gliflozins could be exerting their action. So far, different gliflozins have been approved for their therapeutic use in T2DM, heart failure, and diabetic kidney disease in different countries, all of them being diseases that have in common a deregulation of the inflammatory process associated with the pathology, which perpetuates and worsens the disease. This inflammatory deregulation has been observed in many other diseases, which led the scientific community to have a growing interest in the understanding of the biological processes that lead to or control inflammation deregulation in order to be able to identify potential therapeutic targets that could revert this situation and contribute to the amelioration of the disease. In this line, recent studies showed that gliflozins also act as an anti-inflammatory drug, and have been proposed as a useful strategy to treat other diseases linked to inflammation in addition to cardio-renal diseases, such as diabetes, obesity, atherosclerosis, or non-alcoholic fatty liver disease. In this work, we will review recent studies regarding the role of the main sodium-glucose co-transporter 2 inhibitors in the control of inflammation.

The Treatment With the SGLT2 Inhibitor Empagliflozin Modifies the Hepatic Metabolome of Male Zucker Diabetic Fatty Rats Towards a Protective Profile.

The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial evidenced the potential of sodium-glucose cotransporter 2 (SGLT2) inhibitors for the treatment of patients with diabetes and cardiovascular disease. Recent evidences have shown the benefits of the SGLT2 inhibitor empagliflozin on improving liver steatosis and fibrosis in patients with T2DM. Metabolomic studies have been shown to be very useful to improve the understanding of liver pathophysiology during the development and progression of metabolic hepatic diseases, and because the effects of empagliflozin and of other SGLT2 inhibitors on the complete metabolic profile of the liver has never been analysed before, we decided to study the impact on the liver of male Zucker diabetic fatty (ZDF) rats of a treatment for 6 weeks with empagliflozin using an untargeted metabolomics approach, with the purpose to help to clarify the benefits of the use of empagliflozin at hepatic level. We found that empagliflozin is able to change the hepatic lipidome towards a protective profile, through an increase of monounsaturated and polyunsaturated glycerides, phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylinositols and lysophosphatidylcholines. Empagliflozin also induces a decrease in the levels of the markers of inflammation IL-6, chemerin and chemerin receptor in the liver. Our results provide new evidences regarding the molecular pathways through which empagliflozin could exert hepatoprotector beneficial effects in T2DM.

Relaxin has beneficial effects on liver lipidome and metabolic enzymes.

Relaxin is an insulin-like hormone with pleiotropic protective effects in several organs, including the liver. We aimed to characterize its role in the control of hepatic metabolism in healthy rats. Sprague-Dawley rats were treated with human recombinant relaxin-2 for 2 weeks. The hepatic metabolic profile was analyzed using UHPLC-MS platforms. Hepatic gene expression of key enzymes of desaturation (Fads1/Fads2) of n-6 and n-3 polyunsaturated fatty acids (PUFAs), of phosphatidylethanolamine (PE) N-methyltransferase (Pemt), of fatty acid translocase Cd36, and of glucose-6-phosphate isomerase (Gpi) were quantified by Real Time-PCR. Activation of 5'AMP-activated protein kinase (AMPK) was analyzed by Western Blot. Relaxin-2 significantly modified the hepatic levels of 19 glycerophospholipids, 2 saturated (SFA) and 1 monounsaturated (MUFA) fatty acids (FA), 3 diglycerides, 1 sphingomyelin, 2 aminoacids, 5 nucleosides, 2 nucleotides, 1 carboxylic acid, 1 redox electron carrier, and 1 vitamin. The most noteworthy changes corresponded to the substantially decreased lysoglycerophospholipids, and to the clearly increased FA (16:1n-7/16:0) and MUFA + PUFA/SFA ratios, suggesting enhanced desaturase activity. Hepatic gene expression of Fads1, Fads2, and Pemt, which mediates lipid balance and liver health, was increased by relaxin-2, while mRNA levels of the main regulator of hepatic FA uptake Cd36, and of the essential glycolysis enzyme Gpi, were decreased. Relaxin-2 augmented the hepatic activation of the hepatoprotector and master regulator of energy homeostasis AMPK. Relaxin-2 treatment also rised FADS1, FADS2, and PEMT gene expression in cultured Hep G2 cells. Our results bring to light the hepatic metabolic features stimulated by relaxin, a promising hepatoprotective molecule.

Adipokines and Inflammation: Focus on Cardiovascular Diseases.

It is well established that adipose tissue, apart from its energy storage function, acts as an endocrine organ that produces and secretes a number of bioactive substances, including hormones commonly known as adipokines. Obesity is a major risk factor for the development of cardiovascular diseases, mainly due to a low grade of inflammation and the excessive fat accumulation produced in this state. The adipose tissue dysfunction in obesity leads to an aberrant release of adipokines, some of them with direct cardiovascular and inflammatory regulatory functions. Inflammation is a common link between obesity and cardiovascular diseases, so this review will summarise the role of the main adipokines implicated in the regulation of the inflammatory processes occurring under the scenario of cardiovascular diseases.

Empagliflozin reduces the levels of CD36 and cardiotoxic lipids while improving autophagy in the hearts of Zucker diabetic fatty rats.

The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial made evident the potentiality of pharmacological sodium-glucose cotransporter 2 (SGLT2) inhibition for treating patients with diabetes and cardiovascular disease. Since the effect of empagliflozin or other SGLT2 inhibitors on the whole cardiac metabolic profile was never analysed before, and with the purpose to contribute to elucidate the benefits at cardiac level of the use of empagliflozin, we explored the effect of the treatment with empagliflozin for six weeks on the cardiac metabolomic profile of Zucker diabetic fatty rats, a model of early stage T2DM, using untargeted metabolomics approach. Empagliflozin reduced significantly the cardiac content of sphingolipids (ceramides and sphingomyelins) and glycerophospholipids (major bioactive contributing factors linking insulin resistance to cardiac damage) and decreased the cardiac content of the fatty acid transporter cluster of differentiation 36 (CD36); induced significant decreases of the cardiac levels of essential glycolysis intermediaries 2,3-bisphosphoglycerate and phosphoenolpyruvate, and regulated the abundance of several amino acids of relevance as tricarboxylic acid suppliers and/or in the metabolic control of the cardiac function as glutamic acid, gamma-aminobutyric acid and sarcosine. Empagliflozin treatment activated the cardioprotective master regulator of cellular energyhomeostasis AMP-activatedproteinkinase (AMPK) and enhanced autophagy at cardiac level, while it decreased significantly the cardiac mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6), chemerin, TNF-α and MCP-1, reinforcing the hypothesis of a direct role for empagliflozin in attenuating cardiac inflammation. Our results provide an advancement on the knowledge of the mechanisms linking the therapy with empagliflozin with protective effects on the development of cardiometabolic diseases whose course is associated with remarkable cardiac bioenergetics dysregulation and disarrangement in cardiac metabolome and lipidome.

Serelaxin (recombinant human relaxin-2) treatment affects the endogenous synthesis of long chain poly-unsaturated fatty acids and induces substantial alterations of lipidome and metabolome profiles in rat cardiac tissue.

BACKGROUND AND PURPOSE: Recombinant human relaxin-2, serelaxin, is being proved as a novel drug with therapeutic efficacy in some cardiovascular diseases, especially heart failure, a disease whose physiopathology and course are firmly correlated with important alterations in cardiac metabolism. The aim of our present work was to investigate changes in the cardiac metabolome following relaxin-2 treatment. EXPERIMENTAL APPROACH: Sprague-Dawley rats were treated with human recombinant relaxin-2 using osmotic minipumps at a dose of 0.4 mg/kg/day for 2 weeks. Body composition was measured with a nuclear magnetic resonance imaging system seven days after surgery and on the final day of the experiment. The last two days of treatment, respiratory quotient, locomotor activity and energy expenditure were measured with a calorimetric system. The plasma levels of relaxin-2, total cholesterol, high- and low- density lipoproteins (HDL, LDL), triglycerides and the hepatic enzymes glutamic-pyruvic transaminase (GTP) and gamma-glutamyltransferase (GGT) levels were analyzed. The metabolic profiling of both atria from relaxin-2-treated and control rats was carried out using two separate ultra-high performance liquid chromatography (UHPLC)-Time of Flight-MS based platforms analyzing methanol and chloroform/methanol extracts combined with a UHPLC-single quadrupole-MS based platform used to analyze aminoacids and with a methanol/water extract platform that covered polar metabolites. Identified ion features in the methanol extract platform included fatty acids, acyl carnitines, bile acids, monoacylglycerophospholipids, monoetherglycerophospholipids, free sphingoid bases, and oxidized fatty acids. The chloroform / methanol extract platform provided coverage over glycerolipids, cholesterol esters, sphingolipids, diacylglycerophospholipids, and acyl-ether-glycerophospholipids. Gene expression levels of the adipokines adiponectin, leptin and nesfatin-1 in visceral adipose tissue and cardiac gene expression levels of key enzymes of desaturation and elongation of n-6 and n-3 PUFAs were assessed by Real Time-PCR. KEY RESULTS: Twenty-eight metabolites out of three hundred sixty-two were significantly altered by human relaxin-2. These included fifteen glycerophospholipids: three phosphatidylethanolamines (PE) and twelve phosphatidylcholines (PC); eight sphingolipids: three ceramides (Cer) and five sphingomyelins (SM); and also five aminoacids and one carboxylic acid. Interestingly, the majority of changes correspond to lipid classes, twelve of them polyunsaturated diacylglycerophosphatidylcholines with long acyl chains, containing mainly docosahexaenoic acid (22:6) and arachidonic acid (20:4). Atrial levels of Elovl5 (Elongation of very long chain fatty acids protein 5), Fads1 (Δ5-fatty acid desaturase) and Fads2 (Δ6-fatty acid desaturase), key enzymes of elongation and desaturation of n-6 and n-3 PUFAs like arachidonic acid and DHA, respectively, were significantly increased by relaxin-2 treatment. Atrial tissues from rats treated with relaxin-2 showed a significant increase in the mRNA levels of Srebf1, a transcription factor that activates the gene expression of Elovl5, Fads1 and Fads2. The treatment with relaxin-2 significantly decreased the visceral fat mRNA expression levels of adiponectin, leptin and nesfatin-1, adipokines known to exert an important influence on the regulation of cardiovascular function. CONCLUSION AND IMPLICATIONS: Serelaxin (human recombinant relaxin-2) treatment induces significant changes in cardiac major components of the membrane lipid bilayer such as glycerophospholipids and sphingolipids, known to have structural roles but also very relevant regulatory effects in cardiac function. Serelaxin induced also modifications in several aminoacids of high influence in cardiac energy metabolism regulation. Our results highlight the need to further understand the role of relaxin-2 in the regulation of cardiac energy metabolism, in the context of the therapeutic strategies for the treatment of cardiometabolic pathologies as heart failure.

Relaxin-2 in Cardiometabolic Diseases: Mechanisms of Action and Future Perspectives.

Despite the great effort of the medical community during the last decades, cardiovascular diseases remain the leading cause of death worldwide, increasing their prevalence every year mainly due to our new way of life. In the last years, the study of new hormones implicated in the regulation of energy metabolism and inflammation has raised a great interest among the scientific community regarding their implications in the development of cardiometabolic diseases. In this review, we will summarize the main actions of relaxin, a pleiotropic hormone that was previously suggested to improve acute heart failure and that participates in both metabolism and inflammation regulation at cardiovascular level, and will discuss its potential as future therapeutic target to prevent/reduce cardiovascular diseases.

Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level.

Two-pore channels (TPCs or TPCNs) are novel voltage-gated ion channels that have been postulated to act as Ca2+ and/or Na+ channels expressed exclusively in acidic organelles such as endosomes and lysosomes. TPCNs participate in the regulation of diverse biological processes and recently have been proposed to be involved in the pathophysiology of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Due to the importance of these pathologies in the development of cardiovascular diseases, we aimed to study the possible role of two-pore channel 1 (TPCN1) in the regulation of cardiac metabolism. To explore the cardiac function of TPCN1, we developed proteomic approaches as 2-DE-MALDI-MS and LC-MALDI-MS in the cardiac left ventricle of TPCN1 KO and WT mice, and found alterations in several proteins implicated in glucose and fatty acid metabolism in TPCN1 KO vs. WT mice. The results confirmed the altered expression of HFABP, a key fatty acid transport protein, and of enolase and PGK1, the key enzymes in the glycolytic process. Finally, in vitro experiments performed in neonatal rat cardiomyocytes, in which TPCN1 was silenced using siRNAs, confirmed that the downregulation of TPCN1 gene expression increased 2-deoxy-D-[3H]-glucose uptake and GLUT4 mobilization into cell peripherals in cardiac cells. Our results are the first to suggest a potential role for TPCNs in cardiac metabolism regulation.