Dana Dlouha

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors offer a novel approach for reducing low‐density lipoprotein cholesterol (LDL‐C) levels in patients with familial hypercholesterolemia (FH). In our study, we aimed to compare the plasma proteome profiles of heterozygous FH (HeFH) patients and non‐FH patients treated with PCSK9 inhibitors (PCSK9i). Methods: We analyzed 132 plasma samples from 38 patients. Samples were collected before the start of the PCSK9i treatment and then again at the third, sixth, and 12 ^th months of treatment. Using LC‒MS technology, we compared two approaches: depleted and nondepleted samples. Results: Across all time points of treatment, numerous differentially expressed proteins were detected regardless of the FH status compared to baseline. We identified only three proteins with downregulated expression—PCSK9, CRTAC1, and PON1—with p _adj p <0.05). Conclusions: Our study suggests that there might be differences in the proteomes between individuals with HeFH and those without FH in response to PCSK9i treatment. Furthermore, PCSK9i treatment is likely to affect the plasma proteome, regardless of FH status.

Background: Following acute myocardial infarction (AMI), patients face an approximately 20% risk of experiencing another cardiovascular event within the first year. Engaging in physical activity (PA) aids in the management of risk factors, improves quality of life, and reduces the risk of cardiac recurrence and mortality. MicroRNAs (miRNAs) are post-transcriptional gene regulators that play a crucial role in the development of atherosclerosis. We aimed to monitor levels of atherogenic cell-free circulating miRNAs in response to PA in patients after AMI. Methods: A total of 29 patients diagnosed with AMI were enrolled in our randomized crossover study (10% women; mean age 52.3 ± 11 years). Patients were assigned to either a control or intervention group, crossing over after three months. Blood plasma was collected during hospitalization after AMI diagnosis, at the end of the first month, and after each three-month intervention period. Nine selected atherogenic circulating miRNAs were measured using quantitative PCR. Results: Most of the analyzed miRNAs declined during the year following AMI (all P < 0.0005), except for miR-142-5p, which increased. PA did not impact the plasma levels of any measured miRNAs, except for a slight reduction in miR-146a-5p ( P = 0.08). Multiple linear regression indicated an inverse correlation between miR-146a-5p and VO ₂ max ( P = 0.02), and between miR-130a-3p and active body mass ( P = 0.006) and basal metabolism ( P < 0.05). Conclusion: PA in post-AMI patients may affect circulating miRNAs related to inflammation and adipogenesis.

Journal of Applied Biomedicine promotes translation of basic biomedical research into clinical investigation, conversion of clinical evidence into practice in all medical fields, and publication of new ideas for conquering human health problems across disciplines.

Introduction: Plasma concentrations of cell-free DNA (cfDNA) serve as markers of overtraining or muscle injury. We have examined, if nuclear (n) and mitochondrial (mt) cfDNA has the potential as a marker of muscle burden or damage.Material and methods: Ten healthy, physically active volunteers (6 females, aged 27.1±6.8 years) performed a downhill running test. Samples for cfnDNA and cfmtDNA analysis were collected before, 30 minutes, 1 hour, and 14 days after the downhill run. CfnDNA and cfmtDNA (two markers for both) were analysed using qPCR.Results: There was an extreme (~ 40-times) increase in cfnDNA at the 30-min time-point against the baseline (p<0.00001 for both markers), followed by a quick drop to baseline levels after 1 hour after the end of the downhill run for all subjects. In contrast, plasma levels of cfmtDNA did not increase significantly (p=0.27 and 0.12). It reflects the fact, that in 6 subjects, the pattern was similar as for cfnDNA but in 4 subjects rather a decrease of cfmtDNA concentration was observed at the 30-mi time-point. These differences correlate with age, BMI, and sex of the participants. Plasma cfnDNA significantly (p<0.01 for all) correlated with concentrations of muscle damage markers such as AST, ALT, and lactate dehydrogenase, and chemokines MIP-1α and IP-10 (positive). No homogenous correlation between cfmtDNA and biomarkers was detected.Conclusions: Our study confirms the extreme release and clearance of cfnDNA in physically active subjects after strenuous exercise. In contrast, the trajectory of cfmtDNA concentrations seems to have much higher inter-individual variability than cfnDNA concentrations.

Plasma circulating donor‐derived cell‐free DNA (ddcfDNA) can be used to noninvasively monitor acute rejection of heart transplants (HTx). This study utilized digital droplet PCR to analyze ddcfDNA concentrations (measured in copies per milliliter) and the fractional abundance (%ddcfDNA) to differentiate between donor and recipient DNA on the basis of single nucleotide polymorphism (SNP) homozygosity. Seventy‐seven patients participated in a study, providing 300 plasma samples. Both markers, mean ddcfDNA (cp/mL) and %ddcfDNA, showed similar decreasing trends following the HTx, ( R ² < 0.2; p < 0.001). Significantly higher levels of ddcfDNA (cp/mL) and %ddcfDNA were observed during episodes of acute rejection (AR) compared to non‐rejection samples ( p < 0.001). Additionally, antibody‐mediated rejection (AMR) was associated with increased %ddcfDNA levels compared to non‐rejection and to acute cellular rejection samples ( p < 0.001 and p < 0.01). A logistic regression model identified %ddcfDNA as an early predictor of AMR risk 10–19 days post‐heart transplant (odds ratio 158, p < 0.02). Performance analysis established an optimal %ddcfDNA threshold of 0.125% for AMR detection, correctly identifying all patients without subsequent AMR. These findings suggest that early %ddcfDNA measurements post‐HTx can accurately identify individuals unlikely to develop AMR during the first posttransplant year.

Background: Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease (CAD) that is characterized by concentric fibrous intimal hyperplasia along the length of coronary vessels, and is recognized as long-term complication after heart transplantation. The chromosomal loci 9p21, 6q25.1, and 2q36.3, represented by their respective leading variants rs10757274, rs6922269 and rs2943634, have been linked with a history of CAD by genome-wide association studies. We aimed to investigate the associations of genetic variants at the loci 9p21, 6q25.1, and 2q36.3 with CAV as genetic risk factors for early prediction. Methods: Genomic DNA was extracted from paired aortic samples of 727 heart recipients (average age 50.8 ± 12.2 years; 21.3% women) and corresponding donors (average age 39.7 ± 12.0 years; 26.1% women). The variants within the loci 9p21, 6q25.1, and 2q36.3 were genotyped using PCR-RFLP. Results: The recipients’ variants of 9p21 (OR 1.97; 95% CI, 1.21-3.19 for GG vs. +A comparison, p = 0.0056) and 2q36.3 (OR 2.46; 95% CI, 1.12–6.17 for +C vs. AA comparison, p = 0.0186) were associated with higher incidence of CAV during the first year following heart transplantation. No such association was found for donor genotypes. Conclusions: Our data suggest that variants at the locus 9p21 (rs10757274) and 2q36.3 (rs2943634) are associated with early CAV development.

The current standard for graft rejection surveillance is endomyocardial biopsy (EMB), an invasive procedure with rare but potentially serious complications. Detection of circulating donor‑derived cell‑free DNA (ddcfDNA) is an option for noninvasive monitoring of graft injury and rejection. A 63‑year‑old man and a 65‑year‑old woman were monitored by EMB for allograft rejection. A total of 48 single‑nucleotide polymorphisms with a minor allele frequency range of 0.4‑0.5 were screened to distinguish donor and recipient DNA based on homozygosity, and digital droplet PCR was used to analyze ddcfDNA concentrations. Both subjects suffered rejection within the first 6 months after transplantation. The maximal ddcfDNA level of 270 copies (cp)/ml during EMB‑confirmed acute cellular rejection (ACR; mild grade 1R/2, patient 1), and the maximal concentration of 1,846 cp/ml in the case of EMB‑confirmed antibody‑mediated rejection (AMR; grade 1+; patient 2), was detected. Individual monitoring of ddcfDNA dynamics from the 1st to the 6th month posttransplant reflected cardiac graft injury in patients suffering ACR or AMR, meaning that ddcfDNA may serve as a noninvasive biomarker.

Lipoprotein apheresis (LA) is a therapeutic option for patients with severe hypercholesterolemia who have persistently elevated LDL-C levels despite attempts at drug therapy. MicroRNAs (miRNAs), important posttranscriptional gene regulators, are involved in the pathogenesis of atherosclerosis. Our study aimed to monitor the dynamics of twenty preselected circulating miRNAs in patients under long-term apheresis treatment. Plasma samples from 12 FH patients (men = 50%, age = 55.3 ± 12.2 years; mean LA overall treatment time = 13.1 ± 7.8 years) were collected before each apheresis therapy every sixth month over the course of four years of treatment. Eight complete follow-up (FU) samples were measured in each patient. Dynamic changes in the relative quantity of 6 miRNAs (miR-92a, miR-21, miR-126, miR-122, miR-26a, and miR-185; all p < 0.04) during FU were identified. Overall apheresis treatment time influenced circulating miR-146a levels (p < 0.04). In LDLR mutation homozygotes (N = 5), compared to heterozygotes (N = 7), we found higher plasma levels of miR-181, miR-126, miR-155, and miR-92a (all p < 0.03). Treatment with PCSK9 inhibitors (N = 6) affected the plasma levels of 7 miRNAs (miR-126, miR-122, miR-26a, miR-155, miR-125a, miR-92a, and miR-27a; all p < 0.04). Long-term monitoring has shown that LA in patients with severe familial hypercholesterolemia influences plasma circulating miRNAs involved in endothelial dysfunction, cholesterol homeostasis, inflammation, and plaque development. The longer the treatment using LA, the better the miRNA milieu depicting the potential cardiovascular risk.

Background: The hypocretin/orexin system has been shown to play a role in heart failure. Whether it also influences myocardial infarction (MI) outcomes is unknown. We evaluated the effect of the rs7767652 minor allele T associated with decreased transcription of the hypocretin/orexin receptor‐2 and circulating orexin A concentrations on mortality risk after MI. Methods and Results: Data from a single‐center, prospectively designed registry of consecutive patients hospitalized for MI at a large tertiary cardiology center were analyzed. Patients without previous history of MI or heart failure were included. A random population sample was used to compare allele frequencies in the general population. Out of 1009 patients (aged 64±12 years, 74.6% men) after MI, 6.1% were homozygotes (TT) and 39.4% heterozygotes (CT) for minor allele. Allele frequencies in the MI group did not differ from 1953 subjects from general population (χ ² P =0.62). At index hospitalization, MI size was the same, but ventricular fibrillation and the need for cardiopulmonary resuscitation were more prevalent in the TT allele variant. Among patients with ejection fraction ≤40% at discharge, the TT variant was associated with a lower increase in left ventricular ejection fraction during follow‐up ( P =0.03). During the 27‐month follow‐up, there was a statistically significant association of the TT variant with increased mortality risk (hazard ratio [HR], 2.83; P =0.001). Higher circulating orexin A was associated with a lower mortality risk (HR, 0.41; P <0.05). Conclusions: Attenuation of hypocretin/orexin signaling is associated with increased mortality risk after MI. This effect may be partially explained by the increased arrhythmic risk and the effect on the left ventricular systolic function recovery.

Reaching critically short telomeres induces cellular senescence and ultimately cell death. Cellular senescence contributes to the loss of tissue function. We aimed to determine the association between variants within genes involved in telomere length maintenance, posttransplant events, and aortic telomere length in heart transplant patients. DNA was isolated from paired aortic samples of 383 heart recipients (age 50.7 ± 11.9 years) and corresponding donors (age 38.7 ± 12.0 years). Variants within the TERC (rs12696304), TERF2IP (rs3784929 and rs8053257), and OBCF1 (rs4387287) genes were genotyped, and telomere length was measured using qPCR. We identified similar frequencies of genotypes in heart donors and recipients. Antibody-mediated rejection (AMR) was more common (p < 0.05) in carriers of at least one G allele within the TERF2IP locus (rs3784929). Chronic graft dysfunction (CGD) was associated with the TERC (rs12696304) GG donor genotype (p = 0.05). The genetic risk score did not determine posttransplant complication risk prediction. No associations between the analyzed polymorphisms and telomere length were detected in either donor or recipient DNA. In conclusion, possible associations between donor TERF2IP (rs3784929) and AMR and between TERC (rs12696304) and CGD were found. SNPs within the examined genes were not associated with telomere length in transplanted patients.