Duncan Horlock

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.

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.

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.

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 antioxidant N‐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 administer N‐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 and III were quantified in heart tissues. Cardiac perivascular and interstitial fibrosis were greater by 114% and 209%, respectively, in MST‐1 compared to wild type (P ≤ 0.001). In MST‐1 mice administered N‐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 in MST‐1 than in wild type (P < 0.001) and N‐acetylcysteine attenuated oxidative stress in MST‐1 by 42% (P = 0.005). These data indicate that N‐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 of N‐acetylcysteine in chronic heart failure.

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.

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.

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.

[BP.10.03] DETERMINING THE ROLE OF FIBRE, THROUGH CHANGES IN THE GUT MICROBIOTA, IN BLOOD PRESSURE REGULATION

Objective: Fibre intake has been associated with lower incidence of hypertension, but the mechanisms involved are not clear. Fibre feeds the commensal gut microbiota, and release short-chain fatty acids (SCFAs) as a result of fibre fermentation. Here we aimed to determine the role of fibre and SCFAs, through modulation of the gut microbiota, in lowering blood pressure (BP) using the angiotensin II (Ang II) minipump model of hypertension. Design and method: Mice were fed a diet without fibre (‘no fibre’) or high in fibre (72% resistant starches) for 3-weeks prior to sham surgery or Ang II minipump implantation (n = 6–10/group), and were followed for 4-weeks post-surgery. Germ-free (gnotobiotic) mice received a faecal transplant from ‘no fibre’ Ang II mice (n = 6). Cardiorenal function and BP were examined. P < 0.05 was considered significant, and multiple comparison analyses were adjusted by false discovery rate (q < 0.05). Gut microbiota by 16S is currently being determined. Results: Ang II mice in the ‘no fibre’ diet had significantly higher systolic and diastolic BP than sham mice in either diet or high fibre Ang II (systolic mean ± SD: 145.6 ± 3.2 vs 128.0 ± 5.3, q = 0.0009; diastolic: 101.5 ± 2.2 vs 88.6 ± 3.7, q = 0.0008). Similarly, Ang II mice in the ‘no fibre’ diet had significantly larger lung and heart to body weight, and the high fibre diet was able to prevent both lung (5.7 ± 0.4 vs 5.2 ± 0.4, q = 0.01) and cardiac hypertrophy (5.2 ± 0.4 vs 4.8 ± 0.4, q = 0.036) in Ang II mice. Germ-free mice that received a faecal transplant from ‘no fibre’ Ang II mice had significantly higher end-diastolic pressure (8.1 ± 2.3 vs 5.0 ± 1.1, q = 0.013), left ventricular contractility (10452 ± 897 vs 8043 ± 631, q = 0.036) and heart to body weight ratio (5.3 ± 0.2 vs 4.5 ± 0.5, q = 0.001) than ‘no fibre’ sham mice. These measurements were similar to the ones of the faecal donor mice. Conclusions: To date our study has shown that a diet poor in fibre contributes to the development of hypertension, while high dietary intake of fibre may prevent the development of hypertension and cardiac hypertrophy in an Ang II model. Evidence from germ-free studies support that the gut microbiota is likely to have a role in CVD in this setting.