Sander Trenson

Novel microRNA regulators of atrial natriuretic peptide production

ABSTRACT Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-155 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferring resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-155 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational, genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels, which may have applications for the treatment of hypertension or heart failure.

Epidemiological and histological findings implicate matrix Gla protein in diastolic left ventricular dysfunction

Objectives A novel paradigm of diastolic left ventricular (LV) dysfunction proposes involvement of the cardiac microvasculature. Vitamin K dependent matrix Gla protein (MGP) plays a role in preserving microcirculatory integrity. We hypothesized that LV filling pressure–a measure of diastolic LV dysfunction–increases with higher plasma level of inactive desphospho-uncarboxylated MGP (dp-ucMGP). We also studied the distribution of active and inactive MGP in human myocardium. Methods We measured echocardiographic diastolic LV function and plasma dp-ucMGP (ELISA) in 668 Flemish and for replication in 386 Swiss. Results Among Flemish and Swiss, E/e’ (6.78 vs. 6.73) and dp-ucMGP (3.94 μg/L vs. 4.20 μg/L) were similarly distributed. In multivariable-adjusted models, for each doubling of dp-ucMGP, E/e’ increased by 0.26, 0.33 and 0.31 in Flemish, Swiss and both cohorts combined (P≤0.026); the odds ratios for having E/e’ ≥ 8.5 were 1.99, 3.29 and 2.36, respectively (P≤0.017). Cardiac biopsies from patients with ischemic or dilated cardiomyopathy and healthy hearts (n = 4 for each) were stained with conformation-specific MGP antibodies. In diseased compared with normal hearts, uncarboxylated inactive MGP was more prevalent (P≤0.004) in the perivascular matrix and interstitium (204.4 vs. 8.6 μm2 per field) and phosphorylated active MGP in and around capillaries and interstitial cells (31.3 vs. 6.6 number of positive capillaries and cells per field). Conclusions Our study supports a role of activated MGP in maintaining myocardial integrity and diastolic LV performance and can potentially be translated into new strategies for managing diastolic LV dysfunction and preventing its progression to heart failure.

Urinary Proteomics in Predicting Heart Transplantation Outcomes (uPROPHET)-Rationale and database description

Objectives Urinary Proteomics in Predicting Heart Transplantation Outcomes (uPROPHET; NCT03152422) aims: (i) to construct new multidimensional urinary proteomic (UP) classifiers that after heart transplantation (HTx) help in detecting graft vasculopathy, monitoring immune system activity and graft performance, and in adjusting immunosuppression; (ii) to sequence UP peptide fragments and to identify key proteins mediating HTx-related complications; (iii) to validate UP classifiers by demonstrating analogy between UP profiles and tissue proteomic signatures (TP) in diseased explanted hearts, to be compared with normal donor hearts; (iv) and to identify new drug targets. This article describes the uPROPHET database construction, follow-up strategies and baseline characteristics of the HTx patients. Methods HTx patients enrolled at the University Hospital Gasthuisberg (Leuven) collected mid-morning urine samples. Cardiac biopsies were obtained at HTx. UP and TP methods and the statistical work flow in pursuit of the research objectives are described in detail in the Data supplement. Results Of 352 participants in the UP study (24.4% women), 38.9%, 40.3%, 5.7% and 15.1% had ischemic, dilated, hypertrophic or other cardiomyopathy. The median interval between HTx and first UP assessment (baseline) was 7.8 years. At baseline, mean values were 56.5 years for age, 25.2 kg/m2 for body mass index, 142.3/84.8 mm Hg and 124.2/79.8 mm Hg for office and 24-h ambulatory systolic/diastolic pressure, and 58.6 mL/min/1.73 m2 for the estimated glomerular filtration rate. Of all patients, 37.2% and 6.5% had a history of mild (grade = 1B) or severe (grade ≥ 2) cellular rejection. Anti-body mediated rejection had occurred in 6.2% patients. The number of follow-up urine samples available for future analyses totals over 950. The TP study currently includes biopsies from 7 healthy donors and 15, 14, and 3 patients with ischemic, dilated, and hypertrophic cardiomyopathy. Conclusions uPROPHET constitutes a solid resources for UP and TP research in the field of HTx and has the ambition to lay the foundation for the clinical application of UP in risk stratification in HTx patients.

MicroRNAs promote skeletal muscle differentiation of mesodermal iPSC-derived progenitors

Muscular dystrophies (MDs) are often characterized by impairment of both skeletal and cardiac muscle. Regenerative strategies for both compartments therefore constitute a therapeutic avenue. Mesodermal iPSC-derived progenitors (MiPs) can regenerate both striated muscle types simultaneously in mice. Importantly, MiP myogenic propensity is influenced by somatic lineage retention. However, it is still unknown whether human MiPs have in vivo potential. Furthermore, methods to enhance the intrinsic myogenic properties of MiPs are likely needed, given the scope and need to correct large amounts of muscle in the MDs. Here, we document that human MiPs can successfully engraft into the skeletal muscle and hearts of dystrophic mice. Utilizing non-invasive live imaging and selectively induced apoptosis, we report evidence of striated muscle regeneration in vivo in mice by human MiPs. Finally, combining RNA-seq and miRNA-seq data, we define miRNA cocktails that promote the myogenic potential of human MiPs.Mesodermal iPSC-derived progenitors (MiPs) can regenerate both skeletal and cardiac muscle. Here, the authors show that a microRNA cocktail stimulates skeletal muscle differentiation and that human MiPs can engraft into striated muscle in mice.

Molecular signature of progenitor cells isolated from young and adult human hearts

The loss of endogenous cardiac regenerative capacity within the first week of postnatal life has intensified clinical trials to induce cardiac regeneration in the adult mammalian heart using different progenitor cell types. We hypothesized that donor age-related phenotypic and functional characteristics of cardiac progenitor cells (CPC) account for mixed results of cell-based cardiac repair. We compared expression profiles and cell turnover rates of human heart-derived c-kitpos progenitors (c-kitpos CPC) and cardiosphere-derived cells (CDC) from young and adult donor origin and studied their in vitro angiogenic and cardiac differentiation potential, which can be relevant for cardiac repair. We report that 3-dimensional CDC expansion recapitulates a conducive environment for growth factor and cytokine release from adult donor cells (aCDC) that optimally supports vascular tube formation and vessel sprouting. Transdifferentiation capacity of c-kitpos CPCs and CDCs towards cardiomyocyte-like cells was modest, however, most notable in young c-kitpos cells and adult CDCs. Progenitors isolated with different methods thus show cell- and donor-specific characteristics that may account for variable contributions in functional myocardial recovery.

Inactive matrix Gla protein is a novel circulating biomarker predicting retinal arteriolar narrowing in humans

Active matrix Gla protein (MGP), a potent inhibitor of calcification in large arteries, protects against macrovascular complications. Recent studies suggested that active MGP helps maintaining the integrity of the renal and myocardial microcirculation, but its role in preserving the retinal microcirculation remains unknown. In 935 randomly recruited Flemish participants (mean age, 40.9 years; 50.3% women), we measured plasma desphospho-uncarboxylated MGP (dp–ucMGP), a marker of poor vitamin K status using an ELISA-based assay at baseline (1996–2010) and retinal microvascular diameters using IVAN software (Vasculomatic ala Nicola, version 1.1) including the central retinal arteriolar (CRAE) and venular (CRVE) equivalent and the arteriole-to-venule ratio (AVR) at follow-up (2008–2015). CRAE (P = 0.005) and AVR (P = 0.080) at follow-up decreased across tertiles of the dp–ucMGP distribution. In unadjusted models, for a doubling of dp–ucMGP at baseline, CRAE and AVR at follow-up respectively decreased by 1.40 µm (95% confidence interval [CI], 0.32 to 2.48; P = 0.011) and 0.006 (CI, 0.001 to 0.011; P = 0.016). In multivariable-adjusted models accounting for sex, baseline characteristics and follow-up duration, these estimates were −1.03 µm (CI, −1.96 to −0.11; P = 0.028) and −0.007 (CI, −0.011 to −0.002; P = 0.007). Additional adjustment for changes from baseline to follow-up in major baseline characteristics yielded as estimates −0.91 µm (CI, −1.82 to −0.01; P = 0.048) and −0.006 (95% CI, −0.011 to −0.001; P = 0.014), respectively. Circulating inactive dp–ucMGP is a long-term predictor of smaller retinal arteriolar diameter in the general population. Our observations highlight the possibility that vitamin K supplementation might promote retinal health.

Urinary proteomic signatures associated with β-blockade and heart rate in heart transplant recipients

Objectives Heart transplant (HTx) recipients have a high heart rate (HR), because of graft denervation and are frequently started on β-blockade (BB). We assessed whether BB and HR post HTx are associated with a specific urinary proteomic signature. Methods In 336 HTx patients (mean age, 56.8 years; 22.3% women), we analyzed cross-sectional data obtained 7.3 years (median) after HTx. We recorded medication use, measured HR during right heart catheterization, and applied capillary electrophoresis coupled with mass spectrometry to determine the multidimensional urinary classifiers HF1 and HF2 (known to be associated with left ventricular dysfunction), ACSP75 (acute coronary syndrome) and CKD273 (renal dysfunction) and 48 sequenced urinary peptides revealing the parental proteins. Results In adjusted analyses, HF1, HF2 and CKD273 (p ≤ 0.024) were higher in BB users than non-users with a similar trend for ACSP75 (p = 0.06). Patients started on BB within 1 year after HTx and non-users had similar HF1 and HF2 levels (p ≥ 0.098), whereas starting BB later was associated with higher HF1 and HF2 compared with non-users (p ≤ 0.014). There were no differences in the urinary biomarkers (p ≥ 0.27) according to HR. BB use was associated with higher urinary levels of collagen II and III fragments and non-use with higher levels of collagen I fragments. Conclusions BB use, but not HR, is associated with a urinary proteomic signature that is usually associated with worse outcome, because unhealthier conditions probably lead to initiation of BB. Starting BB early after HTx surgery might be beneficial.

Urinary peptidomic biomarkers of renal function in heart transplant recipients

Abstract Background Chronic kidney disease (CKD) is common in patients after heart transplantation (HTx). We assessed whether in HTx recipients the proteomic urinary classifier CKD273 or sequenced urinary peptides revealing the parental proteins correlated with the estimated glomerular filtration rate (eGFR). Methods In 368 HTx patients, we measured the urinary peptidome and analysed CKD273 and 48 urinary peptides with a detectable signal in >95% of participants. After 9.1 months (median), eGFR and the urinary biomarkers were reassessed. Results In multivariable Bonferroni-corrected analyses of the baseline data, a 1-SD increase in CKD273 was associated with a 11.4 [95% confidence interval (CI) 7.25–15.5] mL/min/1.73 m2 lower eGFR and an odds ratio of 2.63 (1.56–4.46) for having eGFR <60 mL/min/1.73 m2. While relating eGFR category at follow-up to baseline urinary biomarkers, CKD273 had higher (P = 0.007) area under the curve (0.75; 95% CI 0.70–0.80) than 24-h proteinuria (0.64; 95% CI 0.58–0.69), but additional adjustment for baseline eGFR removed significance of both biomarkers. In partial least squares analysis, the strongest correlates of the multivariable-adjusted baseline eGFR were fragments of collagen I (positive) and the mucin-1 subunit α (inverse). Associations between the changes in eGFR and the urinary markers were inverse for CKD273 and mucin-1 and positive for urinary collagen I. Conclusions With the exception of baseline eGFR, CKD273 was more closer associated with imminent renal dysfunction than 24-h proteinuria. Fragments of collagen I and mucin-1—respectively, positively and inversely associated with eGFR and change in eGFR—are single-peptide markers associated with renal dysfunction.

URINARY PROTEOMIC SIGNATURES ASSOCIATED WITH BETA-BLOCKADE AND HEART RATE IN HEART TRANSPLANT RECIPIENTS

Objective: Heart transplant (HTx) recipients have a high heart rate (HR), because of graft denervation and are frequently started on &bgr;-blockade (BB). We assessed whether BB and HR post HTx are associated with a specific urinary proteomic signature. Design and method: In 336 HTx patients (mean age, 56.8 years; 22.3% women), we analyzed cross-sectional data obtained 7.3 years (median) after HTx. We recorded medication use, measured HR during right heart catheterization, and applied capillary electrophoresis coupled with mass spectrometry to determine the multidimensional urinary classifiers HF1 and HF2 (known to be associated with left ventricular dysfunction), ACSP75 (acute coronary syndrome) and CKD273 (renal dysfunction) and 48 sequenced urinary peptides revealing the parental proteins. Results: In adjusted analyses, HF1, HF2 and CKD273 (p <  = 0.024) were higher in BB users than non-users with a similar trend for ACSP75 (p = 0.06). Patients started on BB within 1 year after HTx and non-users had similar HF1 and HF2 levels (p >  = 0.098), whereas starting BB later was associated with higher HF1 and HF2 compared with non-users (p <  = 0.014). There were no differences in the urinary biomarkers (p >  = 0.27) according to HR. BB use was associated with higher urinary levels of collagen II and III fragments and non-use with higher levels of collagen I fragments. Conclusions: BB use, but not HR, is associated with a urinary proteomic signature that is usually associated with worse outcome, because unhealthier conditions probably lead to initiation of BB. Starting BB early after HTx surgery might be beneficial.

Diastolic left ventricular function in relation to circulating metabolic biomarkers in a population study

Aims We studied the association of circulating metabolic biomarkers with asymptomatic left ventricular diastolic dysfunction, a risk-carrying condition that affects 25% of the population. Methods and results In 570 randomly recruited people, we assessed in 2005–2010 and in 2009–2013 the multivariable-adjusted correlations of e’ (early left ventricular relaxation) and E/e’ (left ventricular filling pressure) measured by Doppler echocardiography with 43 serum metabolites, quantified by magnetic resonance spectroscopy. In 2009–2013, e’ cross-sectionally increased (Bonferroni corrected p ≤ 0.016) with the branched-chain amino acid valine (per one standard deviation increment, +0.274 cm/s (95% confidence interval, 0.057–0.491)) and glucose+the amino acid (AA) taurine (+0.258 cm/s (0.067–0.481)), while E/e’ decreased (p ≤ 0.017) with valine (–0.264 (–0.496– –0.031)). The risk of developing left ventricular diastolic dysfunction over follow-up (9.4%) was inversely associated (p ≤ 0.0059) with baseline glucose+amino acid taurine (odds ratio, 0.64 (0.44–0.94). In partial least squares analyses of all the baseline and follow-up data, markers consistently associated with better diastolic left ventricular function included the amino acids 2-aminobutyrate and 4-hydroxybutyrate and the branched-chain amino acids leucine and valine, and those consistently associated with worse diastolic left ventricular function glucose+amino acid glutamine and fatty acid pentanoate. Branched-chain amino acid metabolism (–log10 p  = 12.6) and aminoacyl-tRNA biosynthesis (9.9) were among the top metabolic pathways associated with left ventricular diastolic dysfunction. Conclusion The associations of left ventricular diastolic dysfunction with circulating amino acids and branched-chain amino acids were consistent over a five-year interval and suggested a key role of branched-chain amino acid metabolism and aminoacyl-tRNA biosynthesis in maintaining diastolic left ventricular function.