Multi-Omics Profiling of Hypertrophic Cardiomyopathy Reveals Altered Mechanisms in Mitochondrial Dynamics and Excitation-Contraction Coupling.

Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.

Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation.

Introduction: Chagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. Methods: To investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. Results: Analyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1α signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1α signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1α activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. Conclusion: These findings suggest that T. cruzi exploits a HIF-1α-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.

Genome-Wide Association of Proprotein Convertase Subtilisin/Kexin Type 9 Plasma Levels in the ELSA-Brasil Study.

Pharmacological inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9) is an established therapeutic option to treat hypercholesterolemia, and plasma PCSK9 levels have been implicated in cardiovascular disease incidence. A number of genetic variants within the PCSK9 gene locus have been shown to modulate PCSK9 levels, but these only explain a very small percentage of the overall PCSK9 interindividual variation. Here we present data on the genetic association structure between PCSK9 levels and genom-wide genetic variation in a healthy sample from the general population. We performed a genome-wide association study of plasma PCSK9 levels in a sample of Brazilian individuals enrolled in the Estudo Longitudinal de Saude do Adulto cohort (n=810). Enrolled individuals were free from cardiovascular disease, diabetes and were not under lipid-lowering medication. Genome-wide genotyping was conducted using the Axiom_PMRA.r3 array, and imputation was performed using the TOPMED multi-ancestry sample panel as reference. Total PCSK9 plasma concentrations were determined using the Quantikine SPC900 ELISA kit. We observed two genome-wide significant loci and seven loci that reached the pre-defined value of p threshold of 1×10-6. Significant variants were near KCNA5 and KCNA1, and LINC00353. Genetic variation at the PCSK9 locus was able to explain approximately 4% of the overall interindividual variations in PCSK9 levels. Colocalization analysis using eQTL data suggested RWDD3, ATXN7L1, KCNA1, and FAM177A1 to be potential mediators of some of the observed associations. Our results suggest that PCSK9 levels may be modulated by trans genetic variation outside of the PCSK9 gene and this may have clinical implications. Understanding both environmental and genetic predictors of PCSK9 levels may help identify new targets for cardiovascular disease treatment and contribute to a better assessment of the benefits of long-term PCSK9 inhibition.

Kefir metabolites in a fly model for Alzheimer's disease.

Alzheimer's Disease (AD) is the most common cause of dementia among elderly individuals worldwide, leading to a strong motor-cognitive decline and consequent emotional distress and codependence. It is traditionally characterized by amyloidogenic pathway formation of senile plaques, and recent studies indicate that dysbiosis is also an important factor in AD's pathology. To overcome dysbiosis, probiotics-as kefir-have shown to be a great therapeutic alternative for Alzheimer's disease. In this present work, we explored kefir as a probiotic and a metabolite source as a modulator of microbiome and amyloidogenic pathway, using a Drosophila melanogaster model for AD (AD-like flies). Kefir microbiota composition was determined through 16S rRNA sequencing, and the metabolome of each fraction (hexane, dichloromethane, ethyl acetate, and n-butanol) was investigated. After treatment, flies had their survival, climbing ability, and vacuolar lesions accessed. Kefir and fraction treated flies improved their climbing ability survival rate and neurodegeneration index. In conclusion, we show that kefir in natura, as well as its fractions may be promising therapeutic source against AD, modulating amyloidogenic related pathways.

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in TNNI3 and TNNT2 That Are Common in Chinese Patients.

BACKGROUND: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. METHODS: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. RESULTS: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P<0.0001) but an excess of variants of uncertain significance (24%, P<0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, P=0.0057, genome aggregation database-East Asian AF=0.0062, P=0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, P<0.0001, genome aggregation database-East Asian AF=0.0009, P<0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35-0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83-0.97]) support the likely pathogenicity of TNNT2:p.R286H. CONCLUSIONS: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.

Potential Biomarkers of the Turnover, Mineralization, and Volume Classification: Results Using NMR Metabolomics in Hemodialysis Patients.

Bone biopsy is still the gold standard to assess bone turnover (T), mineralization (M), and volume (V) in CKD patients, and serum biomarkers are not able to replace histomorphometry. Recently, metabolomics has emerged as a new technique that could allow for the identification of new biomarkers useful for disease diagnosis or for the understanding of pathophysiologic mechanisms, but it has never been assessed in the chronic kidney disease-mineral and bone disorder (CKD-MBD) scenario. In this study, we investigated the association between serum metabolites and the bone TMV classification in patients with end-stage renal disease by using serum NMR spectroscopy and bone biopsy of 49 hemodialysis patients from a single center in Brazil. High T was identified in 21 patients and was associated with higher levels of dimethylsulfone, glycine, citrate, and N-acetylornithine. The receiver-operating characteristic curve for the combination of PTH and these metabolites provided an area under the receiver-operating characteristic curve (AUC) of 0.86 (0.76 to 0.97). Abnormal M was identified in 30 patients and was associated with lower ethanol. The AUC for age, diabetes mellitus, and ethanol was 0.83 (0.71 to 0.96). Low V was identified in 17 patients and was associated with lower carnitine. The association of age, phosphate, and carnitine provided an AUC of 0.83 (0.70 to 0.96). Although differences among the curves by adding selected metabolites to traditional models were not statistically significant, the accuracy of the diagnosis according to the TMV classification seemed to be improved. This is the first study to evaluate the TMV classification system in relation to the serum metabolome assessed by NMR spectroscopy, showing that selected metabolites may help in the evaluation of bone phenotypes in CKD-MBD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Integrated proteomics and metabolomics analysis reveals differential lipid metabolism in human umbilical vein endothelial cells under high and low shear stress.

Atherosclerotic plaque development is closely associated with the hemodynamic forces applied to endothelial cells (ECs). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. ECs under low or oscillatory SS (LSS) show upregulation of inflammatory, adhesion, and cellular permeability molecules. On the contrary, cells under high or laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind SS regulation of an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes in endothelial cells (human umbilical vein endothelial cells) under in vitro LSS and HSS that promote an atheroprone or atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism, in special cholesterol metabolism, was downregulated in cells under LSS. The low-density lipoprotein receptor (LDLR) showed significant alterations both at the quantitative expression level as well as regarding posttranslational modifications. Under LSS, LDLR was seen at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of a HSS metabolic phenotype in EC under LSS. Altogether, our data suggest that there is significant modulation of lipid metabolism in endothelial cells under different SS intensities and that this could contribute to the atheroprone phenotype of LSS. Statin treatment was able to partially recover the protective profile of these cells.

Metabolomics biomarkers and the risk of overall mortality and ESRD in CKD: Results from the Progredir Cohort.

INTRODUCTION: Studies on metabolomics and CKD have primarily addressed CKD incidence defined as a decline on eGFR or appearance of albuminuria in the general population, with very few evaluating hard outcomes. In the present study, we investigated the association between metabolites and mortality and ESRD in a CKD cohort. SETTING AND METHODS: Data on 454 participants of the Progredir Cohort Study, Sao Paulo, Brazil were used. Metabolomics was performed by GC-MS (Agilent MassHunter) and metabolites were identified using Agilent Fiehn GC/MS and NIST libraries. After excluding metabolites present in <50% of participants, 293 metabolites were analyzed. An FDR q value <0.05 criteria was applied in Cox models on the composite outcome (mortality or incident renal replacement therapy) adjusted for batch effect, resulting in 34 metabolites associated with the outcome. Multivariable-adjusted Cox models were then built for the composite outcome, death, and ESRD incident events. Competing risk analysis was also performed for ESRD. RESULTS: Mean age was 68±12y, mean eGFR-CKDEPI was 38.4±14.6 ml/min/1.73m2 and 57% were diabetic. After adjustments (GC-MS batch, sex, age, DM and eGFR), 18 metabolites remained significantly associated with the composite outcome. Nine metabolites were independently associated with death: D-malic acid (HR 1.84, 95%CI 1.32-2.56, p = 0.0003), acetohydroxamic acid (HR 1.90, 95%CI 1.30-2.78, p = 0.0008), butanoic acid (HR 1.59, 95%CI 1.17-2.15, p = 0.003), and docosahexaenoic acid (HR 0.58, 95%CI 0.39-0.88, p = 0.009), among the top associations. Lactose (SHR 1.49, 95%CI 1.04-2.12, p = 0.03), 2-O-glycerol-α-D-galactopyranoside (SHR 1.76, 95%CI 1.06-2.92, p = 0.03), and tyrosine (SHR 0.52, 95%CI 0.31-0.88, p = 0.02) were associated to ESRD risk, while D-threitol, mannitol and myo-inositol presented strong borderline associations. CONCLUSION: Our results identify specific metabolites related to hard outcomes in a CKD population. These findings point to the need of further exploration of these metabolites as biomarkers in CKD and the understanding of the underlying biological mechanisms related to the observed associations.

1,5-Anhydroglucitol predicts CKD progression in macroalbuminuric diabetic kidney disease: results from non-targeted metabolomics.

INTRODUCTION: Metabolomics allows exploration of novel biomarkers and provides insights on metabolic pathways associated with disease. To date, metabolomics studies on CKD have been largely limited to Caucasian populations and have mostly examined surrogate end points. OBJECTIVE: In this study, we evaluated the role of metabolites in predicting a primary outcome defined as dialysis need, doubling of serum creatinine or death in Brazilian macroalbuminuric DKD patients. METHODS: Non-targeted metabolomics was performed on plasma from 56 DKD patients. Technical triplicates were done. Metabolites were identified using Agilent Fiehn GC/MS Metabolomics and NIST libraries (Agilent MassHunter Work-station Quantitative Analysis, version B.06.00). After data cleaning, 186 metabolites were left for analyses. RESULTS: During a median follow-up time of 2.5 years, the PO occurred in 17 patients (30.3%). In non-parametric testing, 13 metabolites were associated with the PO. In univariate Cox regression, only 1,5-anhydroglucitol (HR 0.10; 95% CI 0.01-0.63, p = .01), norvaline and L-aspartic acid were associated with the PO. After adjustment for baseline renal function, 1,5-anhydroglucitol (HR 0.10; 95% CI 0.02-0.63, p = .01), norvaline (HR 0.01; 95% CI 0.001-0.4, p = .01) and aspartic acid (HR 0.12; 95% CI 0.02-0.64, p = .01) remained significantly and inversely associated with the PO. CONCLUSION: Our results show that lower levels of 1,5-anhydroglucitol, norvaline and L-aspartic acid are associated with progression of macroalbuminuric DKD. While norvaline and L-aspartic acid point to interesting metabolic pathways, 1,5-anhydroglucitol is of particular interest since it has been previously shown to be associated with incident CKD. This inverse biomarker of hyperglycemia should be further explored as a new tool in DKD.

Proteome analysis of acute kidney injury - Discovery of new predominantly renal candidates for biomarker of kidney disease.

The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are organ and process specific. Here, we have used different tissues from a controlled porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60min) and 4, 11 and 16h post-reperfusion, and renal cortex samples after 24h of reperfusion. Peptides were analyzed on the Q-Exactive™. In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighted by transcription factors and epithelial adherens junction proteins. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that may provide a set of targets for kidney injury. Additionally, we identified 49, being 4 predominantly renal, proteins in urine. As prove of concept, we validated one of the identified biomarkers, dipeptidyl peptidase IV, in a set of patients with diabetic nephropathy. In conclusion, we identified 55 systemic proteins, some of them predominantly renal, candidates for biomarkers of renal disease. BIOLOGICAL SIGNIFICANCE: The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. Here we propose a comprehensive, sequential screening procedure able to identify and validate potential biomarkers for kidney disease, using kidney ischemia/reperfusion as a paradigm for a kidney pathological event.

Catheter-based induction of renal ischemia/reperfusion in swine: description of an experimental model.

Several techniques to induce renal ischemia have been proposed: clamp, PVA particles, and catheter-balloon. We report the development of a controlled, single-insult model of unilateral renal ischemia/reperfusion (I/R) without contralateral nephrectomy, using a suitable model, the pig. This is a balloon-catheter-based model using a percutaneous, interventional radiology procedure. One angioplasty balloon-catheter was placed into the right renal artery and inflated for 120 min and reperfusion over 24 h. Serial serums were sampled from the inferior vena cava and urine was directly sampled from the bladder throughout the experiment, and both kidneys were excised after 24 h of reperfusion. Analyses of renal structure and function were performed by hematoxylin-eosin/periodic Acid-Schiff, serum creatinine (SCr), blood urea nitrogen (BUN), fractional excretion of ions, and glucose, SDS-PAGE analysis of urinary proteins, and serum neutrophil gelatinase-associated lipocalin (NGAL). Total nitrated protein was quantified to characterize oxidative stress. Acute tubular necrosis (ATN) was identified in every animal, but only two animals showed levels of SCr above 150% of baseline values. As expected, I/R increased SCr and BUN. Fractional sodium, potassium, chloride, and bicarbonate excretion were modulated during ischemia. Serum-nitrated proteins and NGAL had two profiles: decreased with ischemia and increased after reperfusion. This decline was associated with increased protein excretion during ischemia and early reperfusion. Altogether, these data show that the renal I/R model can be performed by percutaneous approach in the swine model. This is a suitable translational model to study new early renal ischemic biomarkers and pathophysiological mechanisms in renal ischemia.