Antibiotic-induced gut microbiota depletion exacerbates host hypercholesterolemia.

Hypercholesterolemia is a major driver of atherosclerosis, thus contributing to high morbidity and mortality worldwide. Gut microbiota have been identified as modulator of blood lipids including cholesterol levels. Few studies have already linked certain bacteria and microbial mechanisms to host cholesterol. However, in particular mouse models revealed conflicting results depending on genetics and experimental protocol. To gain further insights into the relationship between intestinal bacteria and host cholesterol metabolism, we first performed fecal 16S rRNA targeted metagenomic sequencing in a human cohort (n = 24) naïve for cholesterol lowering drugs. Here, we show alterations in the gut microbiota composition of hypercholesterolemic patients with depletion of Bifidobacteria, expansion of Clostridia and increased Firmicutes/Bacteroidetes ratio. To test whether pharmacological intervention in gut microbiota impacts host serum levels of cholesterol, we treated hypercholesterolemic Apolipoprotein E knockout with oral largely non-absorbable antibiotics. Antibiotics increased serum cholesterol, but only when mice were fed normal chow diet and cholesterol was measured in the random fed state. These elevations in cholesterol already occurred few days after treatment initiation and were reversible after stopping antibiotics with re-acquisition of intestinal bacteria. Gene expression analyses pointed to increased intestinal cholesterol uptake mediated by antibiotics in the fed state. Non-targeted serum metabolomics suggested that diminished plant sterol levels and reduced bile acid cycling were involved microbial mechanisms. In conclusion, our work further enlightens the link between gut microbiota and host cholesterol metabolism. Pharmacological disruption of the gut flora by antibiotics was able to exacerbate serum cholesterol and may impact cardiovascular disease.

Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro.

Introduction: Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways. Materials and methods and results: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP. Conclusion: Our direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.

The sodium-glucose co-transporter-2 inhibitor ertugliflozin modifies the signature of cardiac substrate metabolism and reduces cardiac mTOR signalling, endoplasmic reticulum stress and apoptosis.

AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.

Non-targeted metabolomics identify polyamine metabolite acisoga as novel biomarker for reduced left ventricular function.

AIMS: Chronic heart failure with reduced ejection fraction remains a major health issue. To date, no reliable biomarker is available to predict reduced left ventricular ejection fraction (LV-EF). We aimed to identify novel circulating biomarkers for reduced left ventricular function using untargeted serum metabolomics in two independent patient cohorts. METHODS AND RESULTS: Echocardiography and non-targeted serum metabolomics were conducted in two patient cohorts with varying left ventricular function: (1) 25 patients with type 2 diabetes with established cardiovascular disease or high cardiovascular risk (LV-EF range 20-66%) (discovery cohort) and (2) 37 patients hospitalized for myocardial infarction (LV-EF range 25-60%) (validation cohort). In the discovery cohort, untargeted metabolomics revealed seven metabolites performing better than N-terminal pro-B-type natriuretic peptide in the prediction of impaired left ventricular function shown by LV-EF. For only one of the metabolites, acisoga, the predictive value for LV-EF could be confirmed in the validation cohort (r = -0.37, P = 0.02). In the discovery cohort, acisoga did not only correlate with LV-EF (r = -60, P = 0.0016), but also with global circumferential strain (r = 0.67, P = 0.0003) and global longitudinal strain (r = 0.68, P = 0.0002). Similar results could be detected in the discovery cohort in a 6 month follow-up proofing stability of these results over time. With an area under the curve of 0.86 in the receiver operating characteristic analysis, acisoga discriminated between patients with normal EF and LV-EF < 40%. Multivariate analysis exposed acisoga as independent marker for impairment of LV-EF (Beta = -0.71, P = 0.004). CONCLUSIONS: We found the polyamine metabolite acisoga to be elevated in patients with impaired LV-EF in two independent cohorts. Our analyses suggest that acisoga may be a valuable biomarker to detect patients with heart failure with reduced ejection fraction.

Gut-Derived Metabolite Indole-3-Propionic Acid Modulates Mitochondrial Function in Cardiomyocytes and Alters Cardiac Function.

Background: The gut microbiome has been linked to the onset of cardiometabolic diseases, in part facilitated through gut microbiota-dependent metabolites such as trimethylamine-N-oxide. However, molecular pathways associated to heart failure mediated by microbial metabolites remain largely elusive. Mitochondria play a pivotal role in cellular energy metabolism and mitochondrial dysfunction has been associated to heart failure pathogenesis. Aim of the current study was to evaluate the impact of gut-derived metabolites on mitochondrial function in cardiomyocytes via an in vitro screening approach. Methods: Based on a systematic Medline research, 25 microbial metabolites were identified and screened for their metabolic impact with a focus on mitochondrial respiration in HL-1 cardiomyocytes. Oxygen consumption rate in response to different modulators of the respiratory chain were measured by a live-cell metabolic assay platform. For one of the identified metabolites, indole-3-propionic acid, studies on specific mitochondrial complexes, cytochrome c, fatty acid oxidation, mitochondrial membrane potential, and reactive oxygen species production were performed. Mitochondrial function in response to this metabolite was further tested in human hepatic and endothelial cells. Additionally, the effect of indole-3-propionic acid on cardiac function was studied in isolated perfused hearts of C57BL/6J mice. Results: Among the metabolites examined, microbial tryptophan derivative indole-3-propionic acid could be identified as a modulator of mitochondrial function in cardiomyocytes. While acute treatment induced enhancement of maximal mitochondrial respiration (+21.5 ± 7.8%, p < 0.05), chronic exposure led to mitochondrial dysfunction (-18.9 ± 9.1%; p < 0.001) in cardiomyocytes. The latter effect of indole-3-propionic acids could also be observed in human hepatic and endothelial cells. In isolated perfused mouse hearts, indole-3-propionic acid was dose-dependently able to improve cardiac contractility from +26.8 ± 11.6% (p < 0.05) at 1 µM up to +93.6 ± 14.4% (p < 0.001) at 100 µM. Our mechanistic studies on indole-3-propionic acids suggest potential involvement of fatty acid oxidation in HL-1 cardiomyocytes. Conclusion: Our data indicate a direct impact of microbial metabolites on cardiac physiology. Gut-derived metabolite indole-3-propionic acid was identified as mitochondrial modulator in cardiomyocytes and altered cardiac function in an ex vivo mouse model.

2-hydroxycaproate predicts cardiovascular mortality in patients with atherosclerotic disease.

BACKGROUND AND AIMS: We aimed to identify novel biomarkers for cardiovascular mortality through a non-targeted metabolomics approach in patients with established atherosclerotic disease from the Tor Vergata Atherosclerosis Registry (TVAR). METHODS: We compared the serum baseline metabolome of 19 patients with atherosclerosis suffering from cardiovascular death during follow-up with the baseline serum metabolome of 20 control patients matched for age, gender, body mass index (BMI) and atherosclerotic disease status, who survived during the observation period. RESULTS: Three metabolites were significantly different in the cardiovascular mortality (CVM) group compared to controls: 2-hydroxycaproate, gluconate and sorbitol. 2-hydroxycaproate (otherwise known as alpha hydroxy caproate) was also significantly correlated with time to death. The metabolites performed better when combined together rather than singularly on the identification of CVM status. CONCLUSIONS: Our analysis led to identify few metabolites potentially amenable of translation into the clinical practice as biomarkers for specific metabolic changes in the cardiovascular system in patients with established atherosclerotic disease.