Circadian Rhythm Perturbation Aggravates Gut Microbiota Dysbiosis in Dextran Sulfate Sodium-Induced Colitis in Mice.

Circadian rhythm disruption is increasingly considered an environmental risk factor for the development and exacerbation of inflammatory bowel disease. We have reported in a previous study that nychthemeral dysregulation is associated with an increase in intestinal barrier permeability and inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. To investigate the effect of circadian rhythm disruption on the composition and diversity of the gut microbiota (GM), sixty male C57BL/6J mice were initially divided to two groups, with the shifted group (n = 30) exposed to circadian shifts for three months and the non-shifted group (n = 30) kept under a normal light-dark cycle. The mice of the shifted group were cyclically housed for five days under the normal 12:12 h light-dark cycle, followed by another five days under a reversed light-dark cycle. At the end of the three months, a colitis was induced by 2% DSS given in the drinking water of 30 mice. Animals were then divided into four groups (n = 15 per group): sham group non-shifted (Sham-NS), sham group shifted (Sham-S), DSS non-shifted (DSS-NS) and DSS shifted (DSS-S). Fecal samples were collected from rectal content to investigate changes in GM composition via DNA extraction, followed by high-throughput sequencing of the bacterial 16S rRNA gene. The mouse GM was dominated by three phyla: Firmicutes, Bacteroidetes and Actinobacteria. The Firmicutes/Bacteroidetes ratio decreased in mice with induced colitis. The richness and diversity of the GM were reduced in the colitis group, especially in the group with inverted circadian rhythm. Moreover, the GM composition was modified in the inverted circadian rhythm group, with an increase in Alloprevotella, Turicibacter, Bacteroides and Streptococcus genera. Circadian rhythm inversion exacerbates GM dysbiosis to a less rich and diversified extent in a DSS-induced colitis model. These findings show possible interplay between circadian rhythm disruption, GM dynamics and colitis pathogenesis.

TRPC3 Regulates Islet Beta-Cell Insulin Secretion.

Insulin release is tightly controlled by glucose-stimulated calcium (GSCa) through hitherto equivocal pathways. This study investigates TRPC3, a non-selective cation channel, as a critical regulator of insulin secretion and glucose control. TRPC3's involvement in glucose-stimulated insulin secretion (GSIS) is studied in human and animal islets. TRPC3-dependent in vivo insulin secretion is investigated using pharmacological tools and Trpc3-/- mice. TRPC3's involvement in islet glucose uptake and GSCa is explored using fluorescent glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose and calcium imaging. TRPC3 modulation by a small-molecule activator, GSK1702934A, is evaluated in type 2 diabetic mice. TRPC3 is functionally expressed in human and mouse islet beta cells. TRPC3-controlled insulin secretion is KATP -independent and primarily mediated by diacylglycerol channel regulation of the cytosolic calcium oscillations following glucose stimulation. Conversely, glucose uptake in islets is independent of TRPC3. TRPC3 pharmacologic inhibition and knockout in mice lead to defective insulin secretion and glucose intolerance. Subsequently, TRPC3 activation through targeted small-molecule enhances insulin secretion and alleviates diabetes hallmarks in animals. This study imputes a function for TRPC3 at the onset of GSIS. These insights strengthen one's knowledge of insulin secretion physiology and set forth the TRPC3 channel as an appealing candidate for drug development in the treatment of diabetes.

Pirfenidone mitigates TGF-ß1-mediated fibrosis in an idiopathic inflammatory myositis-associated interstitial lung disease model.

Idiopathic inflammatory myositis (IIM) is a group of rare diseases of unknown etiology, with a pathognomonic muscular deficiency. Antisynthetase syndrome is a subtype of IIM with an associated interstitial lung disease (ILD), characterized by pulmonary inflammation and fibrosis mediated by TGF-ß. Pirfenidone is a new molecule with anti-inflammatory and anti-fibrotic properties, used for the treatment of idiopathic ILD, but has never been assessed in IIM. The aim of our study is to evaluate the effect of pirfenidone on IIM-associated ILD. Thirty-two BALB/c male mice were divided into three groups: Sham, IIM-untreated (IIM), and IIM pirfenidone-treated (IIM + PIR). IIM was induced by intramuscular injections of guinea pig muscle myosin extract and intraperitoneal injections of Pertussis toxin. Pirfenidone was given orally at a dose of 30 mg kg-1 day-1 for two months. Muscle force, blood and bronchoalveolar lavage fluid samples, as well as muscle and lung tissues, were analyzed. Progressive deterioration of muscle force and infiltration of the muscular tissue by inflammatory cells were observed with IIM. Auto-immune antibodies specific to the antisynthetase syndrome were also increased in IIM mice. Pirfenidone attenuated IIM-associated ILD with anti-inflammatory properties evidenced by decreased peribronchial inflammation and TGF-ß1 in bronchoalveolar lavage fluid. Likewise, pirfenidone attenuated pulmonary fibrosis by fine-tuning TGF-ß1-mediated epithelial-to-mesenchymal and fibrotic signaling pathways; pro-fibrotic SMAD3, ZEB2 and STAT1 expression and activation were decreased, whereas anti-fibrotic SMAD2 activation was increased. This study unravels for the first time that pirfenidone has the potential to fine-tune TGF-ß1 fibrotic signaling in IIM-associated ILD.

Calcitriol Supplementation Protects Against Apoptosis and Alleviates the Severity of Abdominal Aortic Aneurysm Induced by Angiotensin II and Anti-TGFß.

The present study aims to assess the effect of vitamin D deficiency (VDD) and its supplementation on the severity of AAA in mice. AAA was induced by AngII and anti-TGF-ß administration. Animals were divided into four groups: Sham, mice with AAA, mice with AAA, and VDD, and mice with AAA supplemented with calcitriol. Blood pressure, echocardiography, abdominal aortic tissues, and plasma samples were monitored for all groups. VDD was associated with enhanced activity of cleaved MMP-9 and elastin degradation and positively correlated with the severity of AAA. Calcitriol supplementation decreased the INFγ/IL-10 ratio and enhanced the Nrf2 pathway. Moreover, Cu/Zn-superoxide dismutase expression and catalase and neutral sphingomyelinase activity were exacerbated in AAA and VDD groups. Furthermore, calcitriol supplementation showed a significantly lower protein expression of caspase-8, caspase-3, Bid, and t-Bid, and prevented the apoptosis of VSMCs treated by AngII and anti-TGF-ß. Calcitriol supplementation may alleviate AAA severity and could be of great interest in the clinical management of AAA. VDD enhances antioxidant enzymes activity and expression, whereas calcitriol supplementation alleviates AAA severity by re-activating Nrf2 and inhibiting apoptotic pathways.

Reno-Protective Effect of GLP-1 Receptor Agonists in Type1 Diabetes: Dual Action on TRPC6 and NADPH Oxidases.

Diabetic kidney disease (DKD), a serious diabetic complication, results in podocyte loss and proteinuria through NADPH oxidases (NOX)-mediated ROS production. DUOX1 and 2 are NOX enzymes that require calcium for their activation which enters renal cells through the pivotal TRPC channels. Hypoglycemic drugs such as liraglutide can interfere with this deleterious mechanism imparting reno-protection. Herein, we aim to investigate the reno-protective effect of GLP1 receptor agonist (GLP1-RA), via its effect on TRPC6 and NADPH oxidases. To achieve our aim, control or STZ-induced T1DM Sprague-Dawley rats were used. Rats were treated with liraglutide, metformin, or their combination. Functional, histological, and molecular parameters of the kidneys were assessed. Our results show that treatment with liraglutide, metformin or their combination ameliorates DKD by rectifying renal function tests and protecting against fibrosis paralleled by restored mRNA levels of nephrin, DUOX1 and 2, and reduced ROS production. Treatment with liraglutide reduces TRPC6 expression, while metformin treatment shows no effect. Furthermore, TRPC6 was found to be directly interacting with nephrin, and indirectly interacting with DUOX1, DUOX2 and GLP1-R. Our findings suggest that treatment with liraglutide may prevent the progression of diabetic nephropathy by modulating the crosstalk between TRPC6 and NADPH oxidases.

Cardiac Phosphodiesterases Are Differentially Increased in Diabetic Cardiomyopathy.

AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

New insights in gut-liver axis in wild-type murine imiquimod-induced lupus.

BACKGROUND: Intestinal and hepatic manifestations of lupus seem to be underestimated in comparison to other major organ lesions. Although recent data point to gut-liver axis involvement in lupus, gut permeability dysfunction and liver inflammation need to be more investigated. OBJECTIVE: This study aims to assess fecal calprotectin, intestinal tight junction proteins and liver inflammation pathway in wild-type murine imiquimod- induced lupus. METHODS: C57BL/6 mice were topically treated on their right ears with 1.25 mg of 5% imiquimod cream, three times per week for six weeks. Fecal calprotectin was collected at day 0, 22 and 45. Renal, liver and intestinal pathology, as well as inflammatory markers, intestinal tight junction proteins, and E. coli protein in liver were assessed at sacrifice. RESULTS: At six weeks, lupus nephritis was confirmed on histopathology and NGAL and KIM-1 expression. Calprotectin rise started at day 22 and persists at day 45. Protein expression of Claudine, ZO-1 and occludin was significantly decreased. E. coli protein was significantly increased in liver with necro-inflammation and increased TLR4, TLR7, and pNFκB/NFκB liver expression. CONCLUSION: This study is the first to demonstrate early fecal calprotectin increase and liver activation of TLR4- NFκB pathway in wild-type murine imiquimod-induced lupus.

Ranolazine: An Old Drug with Emerging Potential; Lessons from Pre-Clinical and Clinical Investigations for Possible Repositioning.

Ischemic heart disease is a significant public health problem with high mortality and morbidity. Extensive scientific investigations from basic sciences to clinics revealed multilevel alterations from metabolic imbalance, altered electrophysiology, and defective Ca2+/Na+ homeostasis leading to lethal arrhythmias. Despite the recent identification of numerous molecular targets with potential therapeutic interest, a pragmatic observation on the current pharmacological R&D output confirms the lack of new therapeutic offers to patients. By contrast, from recent trials, molecules initially developed for other fields of application have shown cardiovascular benefits, as illustrated with some anti-diabetic agents, regardless of the presence or absence of diabetes, emphasizing the clear advantage of "old" drug repositioning. Ranolazine is approved as an antianginal agent and has a favorable overall safety profile. This drug, developed initially as a metabolic modulator, was also identified as an inhibitor of the cardiac late Na+ current, although it also blocks other ionic currents, including the hERG/Ikr K+ current. The latter actions have been involved in this drug's antiarrhythmic effects, both on supraventricular and ventricular arrhythmias (VA). However, despite initial enthusiasm and promising development in the cardiovascular field, ranolazine is only authorized as a second-line treatment in patients with chronic angina pectoris, notwithstanding its antiarrhythmic properties. A plausible reason for this is the apparent difficulty in linking the clinical benefits to the multiple molecular actions of this drug. Here, we review ranolazine's experimental and clinical knowledge on cardiac metabolism and arrhythmias. We also highlight advances in understanding novel effects on neurons, the vascular system, skeletal muscles, blood sugar control, and cancer, which may open the way to reposition this "old" drug alone or in combination with other medications.

Hydrocortisone mitigates ICU-AW by fine-tuning of muscle atrophic and hypertrophic signaling pathways in a sepsis model with limb immobilization.

AIMS: Intensive care unit-acquired weakness (ICU-AW) is a complex spectrum of disability that delays recovery of critically ill-immobilized patients with sepsis. Much discrepancy remain on the use of corticosteroids and their impact on muscle regeneration in critical illness management. Therefore, the aim of this study is to investigate whether hydrocortisone (HCT) modulates muscle mass turnover in ICU-AW induced by sepsis with limb immobilization (SI). MAIN METHODS: Sepsis by cecal ligation puncture (CLP) with forelimb-immobilization were performed in rats. The study consisted of four groups: Sham (left forelimb-immobilization), Sham HCT (left forelimb-immobilization + HCT), SI (CLP + left forelimb-immobilization) and SI HCT (CLP + left forelimb-immobilization + HCT). Motor force, blood and muscle sampling were assessed. KEY FINDINGS: HCT prevented body weight loss associated with SI and attenuated systemic and muscular inflammation. Besides, myosin was restituted in SI HCT group in conjunction to muscle mass and strength restoration. Pro-hypertrophic calcineurin (PP2B-Aß) and nuclear factor of activated T-cells C3 (NFATc3) but not protein kinase B (Akt) were re-activated by HCT. Finally, pro-atrophic extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinases (p38) but not nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) were inhibited in SI HCT group. SIGNIFICANCE: This study unravels new molecular events thought to control muscle protein synthesis in ICU-AW induced by sepsis and limb immobilization. HCT has a potential to fine-tune muscle-signaling pathways and to reduce the negative outcomes of ICU-AW.

Differential Gender-Dependent Patterns of Cardiac Fibrosis and Fibroblast Phenotypes in Aging Mice.

Aging is characterized by physiological changes within the heart leading to fibrosis and dysfunction even in individuals without underlying pathologies. Gender has been shown to influence the characteristics of cardiac aging; however, gender-dependent cardiac fibrosis occurring with age remains largely not elucidated. Thus, broadening our understanding of this phenomenon proves necessary in order to develop novel anti-fibrotic strategies in the elderly. In this study, we aim to characterize cardiac fibrosis and cardiac fibroblast (CF) populations in aged male and female mice. Echocardiography revealed eccentric hypertrophy with left ventricular dilatation in the aged male versus concentric hypertrophy with left posterior wall thickening in the female, with preserved cardiac function in both groups. Reactive fibrosis was evidenced in the myocardium and epicardium of the aged female mice hearts whereas perivascular and replacement ones where present in the male heart. Collagen I was predominant in the aged male heart whereas collagen III was the main component in the female heart. CFs in the aged male heart were mainly recruited from resident PDGFRα + populations but not derived from epicardium as evidenced by the absence of epicardial progenitor transcription factors Tcf21, Tbx18 and Wt1. Our results present a paradigm for gender-dependent cardiac fibrosis and the origins of CFs with age. This sets forth to revisit cardiac anti-fibrotic management according to the gender in the elderly and to explore novel therapeutic targets.

Circadian Rhythm Disruption Aggravates DSS-Induced Colitis in Mice with Fecal Calprotectin as a Marker of Colitis Severity.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic immunologically mediated pathology that remains a major health burden. Circadian rhythm disruption leads to a deregulation in the immune system which is a major risk factor for IBD. AIMS: Since fecal calprotectin (FC) has been a useful tool for monitoring IBD, we aimed to evaluate the effect of circadian rhythm alteration on gut inflammation status and whether FC is associated with the severity of colitis. METHODS: C57BL/6J mice were exposed to circadian shifts for 3 months, and then colitis was induced by 2% dextran sulfate sodium (DSS). Colitis was evaluated according to clinical symptoms and histological scoring. Plasma and intestinal inflammatory and permeability markers as well as fecal and intestinal calprotectin were assessed. RESULTS: Circadian shifts aggravated DSS-induced colitis with increased diarrhea, flatulence, and fecal blood associated with decreased colon length. In addition, intestinal cryptic architecture was lost with the presence of increased inflammation, mucosal muscle thickening, and cryptic abscesses. Plasma tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and C-reactive protein upregulations were paralleled by the deterioration of intestinal permeability. Calprotectin expression and distribution increased in the intestines and feces of shifted animals, and levels highly correlated with the increases in intestinal inflammation and permeability. CONCLUSIONS: Circadian rhythm disruption aggravates DSS-induced colitis, whereas fecal and intestinal calprotectin associates with the severity of disease. Calprotectin might be a useful marker and tool for assessing patients at risk of IBD due to lifestyles with disruptive sleep patterns.

Isolation, Purification, and Culture of Mouse Pancreatic Islets of Langerhans.

Pancreatic islets constitute an important tool for research and clinical applications in the field of diabetes. They are used for transplantation, unraveling new mechanisms in insulin secretion, studying pathophysiological pathways in diseased cells, and pharmacological research aimed at developing improved therapeutic strategies. Therefore, fine-tuning islet isolation protocols remains an important objective for reliable investigations. Here we describe a relatively simple mouse islet isolation protocol that relies on enzymatic digestion using low-activity collagenase and several sedimentation and Percoll gradient steps.

Long-term intake of phenolic compounds attenuates age-related cardiac remodeling.

With the onset of advanced age, cardiac-associated pathologies have increased in prevalence. The hallmarks of cardiac aging include cardiomyocyte senescence, fibroblast proliferation, inflammation, and hypertrophy. The imbalance between levels of reactive oxygen species (ROS) and antioxidant enzymes is greatly enhanced in aging cells, promoting cardiac remodeling. In this work, we studied the long-term impact of phenolic compounds (PC) on age-associated cardiac remodeling. Three-month-old Wistar rats were treated for 14 months till middle-age with either 2.5, 5, 10, or 20 mg kg-1  day-1 of PC. PC treatment showed a dose-dependent preservation of cardiac ejection fraction and fractional shortening as well as decreased hypertrophy reflected by left ventricular chamber diameter and posterior wall thickness as compared to untreated middle-aged control animals. Analyses of proteins from cardiac tissue showed that PC attenuated several hypertrophic pathways including calcineurin/nuclear factor of activated T cells (NFATc3), calcium/calmodulin-dependent kinase II (CAMKII), extracellular regulated kinase 1/2 (ERK1/2), and glycogen synthase kinase 3ß (GSK 3ß). PC-treated groups exhibited reduced plasma inflammatory and fibrotic markers and revealed as well ameliorated extracellular matrix remodeling and interstitial inflammation by a downregulated p38 pathway. Myocardia from PC-treated middle-aged rats presented less fibrosis with suppression of profibrotic transforming growth factor-ß1 (TGF-ß1) Smad pathway. Additionally, reduction of apoptosis and oxidative damage in the PC-treated groups was reflected by elevated antioxidant enzymes and reduced RNA/DNA damage markers. Our findings pinpoint that a daily consumption of phenolic compounds could preserve the heart from the detrimental effects of aging storm.

Association between vitamin D deficiency and lipid and non-lipid markers of cardiovascular diseases in the middle east region.

BACKGROUND: Previous studies have associated vitamin D deficiency with cardiovascular disease (CVD) markers. The underlying mechanism remains elusive. Lipid and non-lipid markers of CVD and their relationship to vitamin D deficiency have not been assessed simultaneously. OBJECTIVE: To measure the association between vitamin D deficiency and non-lipid markers of CVD after adjustment of lipid markers. METHODS: This cross-sectional study used the following biological data, which was routinely collected in a general hospital laboratory database between 2011 and 2016: 25OH vitamin D [25(OH)D], creatinine, CKD-EPI eGFR (eGFR), fasting blood glucose (FPG), glycated hemoglobin (HbA1c), uric acid, γ-glutamyl transferase (γGT), C-reactive protein (CRP), total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and a surrogate for CVD. Crude odds ratios (ORs) and ORs adjusted for lipid profile, gender and age using separate logistic regression models were derived. RESULTS: A total of 8658 subjects were included. Half had 25(OH)D < 20 ng/mL. 25(OH)D was associated with increased odds of CRP, eGFR, increased uric acid, γGT, FPG, HbA1c, male gender, CV status, and abnormal lipid markers. After adjustment for lipid markers, age, and gender, vitamin D deficiency was associated with increased odds of CRP, eGFR, γGT, FPG, HbA1c, and the surrogate for CVD. CONCLUSIONS: In this exploratory analysis, the first of its kind in the MENA region, vitamin D deficiency was associated with abnormal lipid markers, non-lipid markers of CVD, male gender, lower eGFR, and a surrogate variable for CVD. The association between vitamin D deficiency and non-lipid markers of CVD persisted after adjustment for lipid markers, age, and gender.

VEGF-C attenuates renal damage in salt-sensitive hypertension.

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca2+ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca2+ transient larger. Ca2+ cycling was modified with a RyR2 mediated Ca2+ leak from the sarcoplasmic reticulum and impaired Ca2+ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca2+. The Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca2+ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic "adaptations" of Ca2+ cycling with complex compensative interactions between Ca2+ handling partner and regulatory proteins.

Transient Receptor Potential Canonical 3 and Nuclear Factor of Activated T Cells C3 Signaling Pathway Critically Regulates Myocardial Fibrosis.

AIMS: Cardiac fibroblasts (CFs) are emerging as major contributors to myocardial fibrosis (MF), a final common pathway of many etiologies of heart disease. Here, we studied the functional relevance of transient receptor potential canonical 3 (TRPC3) channels and nuclear factor of activated T cells c3 (NFATc3) signaling in rodent and human ventricular CFs, and whether their modulation would limit MF. RESULTS: A positive feedback loop between TRPC3 and NFATc3 drove a rat ventricular CF fibrotic phenotype. In these cells, polyphenols (extract of grape pomace polyphenol [P.E.]) decreased basal and angiotensin II-mediated Ca2+ entries through a direct modulation of TRPC3 channels and subsequently NFATc3 signaling, abrogating myofibroblast differentiation, fibrosis and inflammation, as well as an oxidative stress-associated phenotype. N(ω)-nitro-l-arginine methyl ester (l-NAME) hypertensive rats developed coronary perivascular, sub-epicardial, and interstitial fibrosis with induction of embryonic epicardial progenitor transcription factors in activated CFs. P.E. treatment reduced ventricular CF activation by modulating the TRPC3-NFATc3 pathway, and it ameliorated echocardiographic parameters, cardiac stress markers, and MF in l-NAME hypertensive rats independently of blood pressure regulation. Further, genetic deletion (TRPC3-/-) and pharmacological channel blockade with N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-benzenesulfonamide (Pyr10) blunted ventricular CF activation and MF in l-NAME hypertensive mice. Finally, TRPC3 was present in human ventricular CFs and upregulated in MF, whereas pharmacological modulation of TRPC3-NFATc3 decreased proliferation and collagen secretion. Innovation and Conclusion: We demonstrate that TRPC3-NFATc3 signaling is modulated by P.E. and critically regulates ventricular CF phenotype and MF. These findings strongly argue for P.E., through TRPC3 targeting, as potential and interesting therapeutics for MF management.

FokI vitamin D receptor gene polymorphism and serum 25-hydroxyvitamin D in patients with cardiovascular risk.

INTRODUCTION: The biological actions of vitamin D are mediated through vitamin D receptor (VDR). Numerous single-nucleotide polymorphisms (SNPs) in the VDR gene have been identified, and some have been associated with cardiovascular disease (CVD) risk factors. This study aims to evaluate the association of five SNPs in the VDR gene with 25-hydroxyvitamin D (25[OH]D) levels in patients with at least one CVD risk factor. MATERIAL AND METHODS: Genomic DNA was sequenced using standard Sanger methods for five VDR SNPs (BsmI rs1544410; ApaI rs7975232; Cdx2 rs11568820; TaqI rs731236; FokI rs2228570) in 50 Mediterranean subjects having hypovitaminosis D with at least one documented CVD risk factor, aged 18 years or more. The collected variables were serum levels of (25[OH]D), HbA1c, fasting plasma glucose, triglycerides, LDL cholesterol, and total cholesterol. RESULTS: BsmI, ApaI, and TaqI were moderately to highly intercorrelated. Cdx2 was less frequent than expected. With respect to the number of mutations in FokI, levels of (25 [OH]D) were 11.2 ±5.5 ng/ml in the absence of mutations, 12.6 ±4.7 ng/ml in the presence of one mutation, and 16.5 ± 5.5 ng/ml in the presence of two mutations. CONCLUSIONS: FokI polymorphism is more frequent in subjects with cardiovascular risk factors than in the general Caucasian population.

Identification of Phenolic Compounds-Rich Grape Pomace Extracts Urine Metabolites and Correlation with Gut Microbiota Modulation.

The high diversity of phenolic compounds (PC) found in food matrices makes it challenging to analyze their bioavailability and their impact on health and functional metabolism. It is well recognized that PC do modulate the composition of the gut microbiota (GM), however, the literature still lacks significant data concerning the link between the metabolic fate of the ingested compounds and their bioactivity, mainly when considering the secondary metabolites produced. In this study, we assessed the metabolic fate of PC for a period covering 14 months of daily intake to identify the metabolites that could be responsible for the effects of PC on the GM observed in our previous work. Urinary analysis of polyphenol metabolites was performed using a high resolution mass spectrometry LC-QTOF-MS method. Among the sixteen metabolites identified, 3-hydroxyphenylacetic acid and 2-(4-hydroxyphenyl) propionic acid were detected simultaneously and, therefore, correlated with the growth of Bifidobacterium in the rat GM. In addition, Daidzedin, detected only at 14 months post-treatment, mostly interfered with the growth inhibition of Clostridium (Cluster I). In conclusion, the impact of the long-term intake of PC on rat GM seems to be related to specific metabolites produced after ingestion of the parental compounds and this may also be due to their additional synergistic effects.

The Impact of Long-Term Intake of Phenolic Compounds-Rich Grape Pomace on Rat Gut Microbiota.

The aim of this work is to evaluate the impact on the rat microbiota of long-term feeding with phenolic compounds (PC) rich grape pomace extracts. Thirty, 2-mo-old rats, were divided into 5 groups. Four groups were treated with different concentrations of PC (2.5, 5, 10, and 20 mg/kg/d diluted in 0.1% DMSO), and 1 group received 0.1% Dimethyl Sulfoxide (DMSO) alone (control group). The daily treatment lasted 14 mo. Major phenolic compounds constituents were characterized by the high-performance liquid chromatography and free radical scavenging capacity was measured by means of the DPPH assay. Fecal samples from young rats (2-mo old), and rats daily fed with PC or DMSO were collected at 6 and 14 mo posttreatment. The gut microbiota composition was analyzed by quantitative polymerase chain reaction. Bifidobacterium was significantly higher in the groups PC 2.5 and PC 5 than in control and young rats. Lactobacillus decreased with time in all treated and untreated groups. Bacteroides, Clostridium leptum subgroup (Clostridium cluster IV), and Enterococcus were not significantly changed by PC at any concentration when compared to control; nevertheless, after 14 mo of treatment all concentrations of PC abolished the increase of Clostridium sensu stricto (cluster I) (Clostridium Cluster I) observed in the control group when compared to young rats. PC do modulate selectively rat gut microbiome to a healthier phenotype in long-term feeding rats, and could counteract the adverse outcomes of aging on gut bacterial population. PRACTICAL APPLICATION: This research shows that phenolic-rich grape pomace extracts exhibiting a high antioxidant activity, selectively modulate rat gut microbiota to a healthier phenotype within age in a long-term feeding rats.