Deficiency of Prebiotic Fiber and Insufficient Signaling Through Gut Metabolite-Sensing Receptors Leads to Cardiovascular Disease.

BACKGROUND: High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Among key Western lifestyle factors, a diet poor in fiber is associated with prevalence of high BP. The impact of lack of prebiotic fiber and the associated mechanisms that lead to higher BP are unknown. Here we show that lack of prebiotic dietary fiber leads to the development of a hypertensinogenic gut microbiota, hypertension and its complications, and demonstrate a role for G-protein coupled-receptors (GPCRs) that sense gut metabolites. METHODS: One hundred seventy-nine mice including C57BL/6J, gnotobiotic C57BL/6J, and knockout strains for GPR41, GPR43, GPR109A, and GPR43/109A were included. C57BL/6J mice were implanted with minipumps containing saline or a slow-pressor dose of angiotensin II (0.25 mg·kg-1·d-1). Mice were fed diets lacking prebiotic fiber with or without addition of gut metabolites called short-chain fatty acids ([SCFA)] produced during fermentation of prebiotic fiber in the large intestine), or high prebiotic fiber diets. Cardiac histology and function, BP, sodium and potassium excretion, gut microbiome, flow cytometry, catecholamines and methylation-wide changes were determined. RESULTS: Lack of prebiotic fiber predisposed mice to hypertension in the presence of a mild hypertensive stimulus, with resultant pathological cardiac remodeling. Transfer of a hypertensinogenic microbiota to gnotobiotic mice recapitulated the prebiotic-deprived hypertensive phenotype, including cardiac manifestations. Reintroduction of SCFAs to fiber-depleted mice had protective effects on the development of hypertension, cardiac hypertrophy, and fibrosis. The cardioprotective effect of SCFAs were mediated via the cognate SCFA receptors GPR43/GPR109A, and modulated L-3,4-dihydroxyphenylalanine levels and the abundance of T regulatory cells regulated by DNA methylation. CONCLUSIONS: The detrimental effects of low fiber Westernized diets may underlie hypertension, through deficient SCFA production and GPR43/109A signaling. Maintaining a healthy, SCFA-producing microbiota is important for cardiovascular health.

High-Fiber Diet and Acetate Supplementation Change the Gut Microbiota and Prevent the Development of Hypertension and Heart Failure in Hypertensive Mice.

BACKGROUND: Dietary intake of fruit and vegetables is associated with lower incidence of hypertension, but the mechanisms involved have not been elucidated. Here, we evaluated the effect of a high-fiber diet and supplementation with the short-chain fatty acid acetate on the gut microbiota and the prevention of cardiovascular disease. METHODS: Gut microbiome, cardiorenal structure/function, and blood pressure were examined in sham and mineralocorticoid excess-treated mice with a control diet, high-fiber diet, or acetate supplementation. We also determined the renal and cardiac transcriptome of mice treated with the different diets. RESULTS: We found that high consumption of fiber modified the gut microbiota populations and increased the abundance of acetate-producing bacteria independently of mineralocorticoid excess. Both fiber and acetate decreased gut dysbiosis, measured by the ratio of Firmicutes to Bacteroidetes, and increased the prevalence of Bacteroides acidifaciens. Compared with mineralocorticoid-excess mice fed a control diet, both high-fiber diet and acetate supplementation significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and left ventricular hypertrophy. Acetate had similar effects and markedly reduced renal fibrosis. Transcriptome analyses showed that the protective effects of high fiber and acetate were accompanied by the downregulation of cardiac and renal Egr1, a master cardiovascular regulator involved in cardiac hypertrophy, cardiorenal fibrosis, and inflammation. We also observed the upregulation of a network of genes involved in circadian rhythm in both tissues and downregulation of the renin-angiotensin system in the kidney and mitogen-activated protein kinase signaling in the heart. CONCLUSIONS: A diet high in fiber led to changes in the gut microbiota that played a protective role in the development of cardiovascular disease. The favorable effects of fiber may be explained by the generation and distribution of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate. Acetate effected several molecular changes associated with improved cardiovascular health and function.

Serelaxin attenuates renal inflammation and fibrosis in a mouse model of dilated cardiomyopathy.

NEW FINDINGS: What is the central question of this study? The aim was to determine the renoprotective effects of serelaxin in the setting of chronic heart failure. What are the main findings and its importance? Our data indicate that serelaxin can reduce renal fibrosis and inflammation in experimental heart failure. Currently, there are no effective treatments to rescue renal function in heart failure patients, and our data suggest that serelaxin might have the potential to reduce renal fibrosis and inflammation in heart failure. ABSTRACT: Serelaxin has been demonstrated to attenuate renal fibrosis and inflammation in cardiorenal disease. In the present study, we tested the hypothesis that serelaxin can prevent the decline in renal function in dilated cardiomyopathy (DCM) by targeting renal fibrosis and inflammation. Male transgenic mice with DCM (n = 16) and their wild-type littermates (WT; n = 20) were administered either vehicle or serelaxin (500 µg kg-1  day-1 ; subcutaneous minipumps; 8 weeks). Cardiac function was assessed via echocardiography before and during the eighth week of serelaxin treatment. Renal function and inflammation as well as cardiac and renal fibrosis were assessed at the end of the study. Serelaxin had minimal effect on cardiac function (P ≥ 0.99). Tubulointerstitial and glomerular fibrosis were ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.001). Renal mRNA expression of Tnfα and Il1α were ∼4- and ∼3-fold greater, respectively, in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.05). Tubulointerstitial and glomerular fibrosis were 46 and 45% less, respectively, in serelaxin-treated DCM mice than in vehicle-treated DCM mice (P ≤ 0.01). Renal cortical mRNA expression of Tnfα and Il1α were 56 and 58% less, respectively, in the former group compared with the latter (P ≤ 0.05). The urinary albumin:creatinine ratio was ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P = 0.02). The urinary albumin:creatinine ratio was not significantly different between vehicle-treated DCM mice and serelaxin-treated DCM mice (P = 0.38). These data suggest that serelaxin can attenuate renal fibrosis and inflammation and has the potential to exert renoprotective effects in DCM.

Old Drug, New Trick: Tilorone, a Broad-Spectrum Antiviral Drug as a Potential Anti-Fibrotic Therapeutic for the Diseased Heart.

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-ß induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson's trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Manipulation of the gut microbiota by the use of prebiotic fibre does not override a genetic predisposition to heart failure.

Increasing evidence supports a role for the gut microbiota in the development of cardiovascular diseases such as hypertension and its progression to heart failure (HF). Dietary fibre has emerged as a modulator of the gut microbiota, resulting in the release of gut metabolites called short-chain fatty acids (SCFAs), such as acetate. We have shown previously that fibre or acetate can protect against hypertension and heart disease in certain models. HF is also commonly caused by genetic disorders. In this study we investigated whether the intake of fibre or direct supplementation with acetate could attenuate the development of HF in a genetic model of dilated cardiomyopathy (DCM) due to overexpression of the cardiac specific mammalian sterile 20-like kinase (Mst1). Seven-week-old male mice DCM mice and littermate controls (wild-type, C57BL/6) were fed a control diet (with or without supplementation with 200 mM magnesium acetate in drinking water), or a high fibre diet for 7 weeks. We obtained hemodynamic, morphological, flow cytometric and gene expression data. The gut microbiome was characterised by 16S rRNA amplicon sequencing. Fibre intake was associated with a significant shift in the gut microbiome irrespective of mouse genotype. However, neither fibre or supplementation with acetate were able to attenuate cardiac remodelling or cardiomyocyte apoptosis in Mst1 mice. Furthermore, fibre and acetate did not improve echocardiographic or hemodynamic parameters in DCM mice. These data suggest that although fibre modulates the gut microbiome, neither fibre nor acetate can override a strong genetic contribution to the development of heart failure in the Mst1 model.

CXCR4 Antagonism Reduces Cardiac Fibrosis and Improves Cardiac Performance in Dilated Cardiomyopathy.

Background: Myocardial fibrosis is a key pathologic finding in the failing heart and is implicated as a cause of increased ventricular stiffness and susceptibility to ventricular arrhythmia. Neurohormonal mediators such as aldosterone and angiotensin II are known to cause fibrosis in experimental models, however, clinical evidence for the reversal of fibrosis with relevant antagonists is limited. Recent studies suggest that inflammatory mediators may contribute to fibrosis. In dilated cardiomyopathy the mechanism for myocardial fibrosis is unclear and its implications on systolic function are not known. Methods and Results: We studied the effect of a highly selective antagonist of SDF-1/CXCR4 signaling, AMD3100, on the development of cardiac fibrosis and cardiac function in mice with dilated cardiomyopathy due to cardiac-specific transgenic overexpression of the stress-kinase, Mst1. AMD3100 significantly attenuated the progression of myocardial fibrosis and this was accompanied by significant improvements in diastolic and systolic performance as evaluated in isolated Langendorff perfused hearts. AMD3100 reduced BNP mRNA expression but did not alter the expression of Ca2+ handling genes. CXCR4 antagonism also reduced the abundance of splenic CD4+ T cells. Conclusion: This study demonstrates that CXCR4 pathway contributes to pathogenesis of cardiac fibrosis in dilated cardiomyopathy, and it represents a new potential therapeutic target in heart failure. The data also demonstrate that anti-fibrotic strategies can improve systolic performance.

Cardioprotective Actions of the Annexin-A1 N-Terminal Peptide, Ac2-26, Against Myocardial Infarction.

The anti-inflammatory, pro-resolving annexin-A1 protein acts as an endogenous brake against exaggerated cardiac necrosis, inflammation, and fibrosis following myocardial infarction (MI) in vivo. Little is known, however, regarding the cardioprotective actions of the N-terminal-derived peptide of annexin A1, Ac2-26, particularly beyond its anti-necrotic actions in the first few hours after an ischemic insult. In this study, we tested the hypothesis that exogenous Ac2-26 limits cardiac injury in vitro and in vivo. Firstly, we demonstrated that Ac2-26 limits cardiomyocyte death both in vitro and in mice subjected to ischemia-reperfusion (I-R) injury in vivo (Ac2-26, 1 mg/kg, i.v. just prior to post-ischemic reperfusion). Further, Ac2-26 (1 mg/kg i.v.) reduced cardiac inflammation (after 48 h reperfusion), as well as both cardiac fibrosis and apoptosis (after 7-days reperfusion). Lastly, we investigated whether Ac2-26 preserved cardiac function after MI. Ac2-26 (1 mg/kg/day s.c., osmotic pump) delayed early cardiac dysfunction 1 week post MI, but elicited no further improvement 4 weeks after MI. Taken together, our data demonstrate the first evidence that Ac2-26 not only preserves cardiomyocyte survival in vitro, but also offers cardioprotection beyond the first few hours after an ischemic insult in vivo. Annexin-A1 mimetics thus represent a potential new therapy to improve cardiac outcomes after MI.

N-Acetylcysteine Attenuates the Development of Renal Fibrosis in Transgenic Mice with Dilated Cardiomyopathy.

Mechanisms underlying the renal pathology in cardiorenal syndrome (CRS) type 2 remain elusive. We hypothesised that renal glutathione deficiency is central to the development of CRS type 2. Glutathione precursor, N-acetylcysteine (NAC;40 mg/kg/day; 8 weeks) or saline were administered to transgenic mice with dilated cardiomyopathy (DCM) and wild-type (WT) controls. Cardiac structure, function and glutathione levels were assessed at the end of this protocol. Renal fibrosis, glutathione content, expression of inflammatory and fibrotic markers, and function were also evaluated. In both genotypes, NAC had minimal effect on cardiac glutathione, structure and function (P ≥ 0.20). In NAC treated DCM mice, loss of glomerular filtration rate (GFR), tubulointerstitial and glomerular fibrosis and renal oxidised glutathione levels were attenuated by 38%, 99%, 70% and 52% respectively, compared to saline treated DCM mice (P ≤ 0.01). Renal expression of PAI-1 was greater in saline treated DCM mice than in WT mice (P < 0.05). Renal PAI-1 expression was less in NAC treated DCM mice than in vehicle treated DCM mice (P = 0.03). Renal IL-10 expression was greater in the former cohort compared to the latter (P < 0.01). These data indicate that normalisation of renal oxidized glutathione levels attenuates PAI-1 expression and renal inflammation preventing loss of GFR in experimental DCM.

Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.

Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.

N-acetylcysteine attenuates the development of cardiac fibrosis and remodeling in a mouse model of heart failure.

Oxidative stress plays a central role in the pathogenesis of heart failure. We aimed to determine whether the antioxidantN-acetylcysteine can attenuate cardiac fibrosis and remodeling in a mouse model of heart failure. Minipumps were implanted subcutaneously in wild-type mice (n = 20) and mice with cardiomyopathy secondary to cardiac specific overexpression of mammalian sterile 20-like kinase 1 (MST-1;n = 18) to administerN-acetylcysteine (40 mg/kg per day) or saline for a period of 8 weeks. At the end of this period, cardiac remodeling and function was assessed via echocardiography. Fibrosis, oxidative stress, and expression of collagen types I andIIIwere quantified in heart tissues. Cardiac perivascular and interstitial fibrosis were greater by 114% and 209%, respectively, inMST-1 compared to wild type (P ≤ 0.001). InMST-1 mice administeredN-acetylcysteine, perivascular and interstitial fibrosis were 40% and 57% less, respectively, compared to those treated with saline (P ≤ 0. 03). Cardiac oxidative stress was 119% greater inMST-1 than in wild type (P < 0.001) andN-acetylcysteine attenuated oxidative stress inMST-1 by 42% (P = 0.005). These data indicate thatN-acetylcysteine can blunt cardiac fibrosis and related remodeling in the setting of heart failure potentially by reducing oxidative stress. This study provides the basis to investigate the role ofN-acetylcysteine in chronic heart failure.

CXCR4 Antagonism Attenuates the Development of Diabetic Cardiac Fibrosis.

Heart failure (HF) is an increasingly recognized complication of diabetes. Cardiac fibrosis is an important causative mechanism of HF associated with diabetes. Recent data indicate that inflammation may be particularly important in the pathogenesis of cardiovascular fibrosis. We sought to determine the mechanism by which cardiac fibrosis develops and to specifically investigate the role of the CXCR4 axis in this process. Animals with type I diabetes (streptozotocin treated mice) or type II diabetes (Israeli Sand-rats) and controls were randomized to treatment with a CXCR4 antagonist, candesartan or vehicle control. Additional groups of mice also underwent bone marrow transplantation (GFP+ donor marrow) to investigate the potential role of bone marrow derived cell mobilization in the pathogenesis of cardiac fibrosis. Both type I and II models of diabetes were accompanied by the development of significant cardiac fibrosis. CXCR4 antagonism markedly reduced cardiac fibrosis in both models of diabetes, similar in magnitude to that seen with candesartan. In contrast to candesartan, the anti-fibrotic actions of CXCR4 antagonism occurred in a blood pressure independent manner. Whilst the induction of diabetes did not increase the overall myocardial burden of GFP+ cells, it was accompanied by an increase in GFP+ cells expressing the fibroblast marker alpha-smooth muscle actin and this was attenuated by CXCR4 antagonism. CXCR4 antagonism was also accompanied by increased levels of circulating regulatory T cells. Taken together the current data indicate that pharmacological inhibition of CXCR4 significantly reduces diabetes induced cardiac fibrosis, providing a potentially important therapeutic approach.

Role of mitochondrial dysfunction in hyperglycaemia-induced coronary microvascular dysfunction: Protective role of resveratrol.

Microvascular complications are now recognized to play a major role in diabetic complications, and understanding the mechanisms is critical. Endothelial dysfunction occurs early in the course of the development of complications; the precise mechanisms remain poorly understood. Mitochondrial dysfunction may occur in a diabetic rat heart and may act as a source of the oxidative stress. However, the role of endothelial cell-specific mitochondrial dysfunction in diabetic vascular complications is poorly studied. Here, we studied the role of diabetes-induced abnormal endothelial mitochondrial function and the resultant endothelial dysfunction. Understanding the role of endothelial mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular endothelial cells, and that mitochondrial inhibition induces endothelial dysfunction. Additionally, we show that resveratrol acts as a protective agent; resveratrol-mediated mitochondrial protection may be used to prevent long-term diabetic cardiovascular complications.

Abnormal mitochondrial L-arginine transport contributes to the pathogenesis of heart failure and rexoygenation injury.

BACKGROUND: Impaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury. METHODS AND RESULTS: In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress. CONCLUSION: These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.