Sjoukje I. Lok

A Deep Sequencing Approach to Uncover the miRNOME in the Human Heart

MicroRNAs (miRNAs) are a class of non-coding RNAs of ∼22 nucleotides in length, and constitute a novel class of gene regulators by imperfect base-pairing to the 3′UTR of protein encoding messenger RNAs. Growing evidence indicates that miRNAs are implicated in several pathological processes in myocardial disease. The past years, we have witnessed several profiling attempts using high-density oligonucleotide array-based approaches to identify the complete miRNA content (miRNOME) in the healthy and diseased mammalian heart. These efforts have demonstrated that the failing heart displays differential expression of several dozens of miRNAs. While the total number of experimentally validated human miRNAs is roughly two thousand, the number of expressed miRNAs in the human myocardium remains elusive. Our objective was to perform an unbiased assay to identify the miRNOME of the human heart, both under physiological and pathophysiological conditions. We used deep sequencing and bioinformatics to annotate and quantify microRNA expression in healthy and diseased human heart (heart failure secondary to hypertrophic or dilated cardiomyopathy). Our results indicate that the human heart expresses >800 miRNAs, the majority of which not being annotated nor described so far and some of which being unique to primate species. Furthermore, >250 miRNAs show differential and etiology-dependent expression in human dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). The human cardiac miRNOME still possesses a large number of miRNAs that remain virtually unexplored. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in regulating human heart disease.

Incremental Prognostic Power of Novel Biomarkers (Growth-Differentiation Factor-15, High-Sensitivity C-Reactive Protein, Galectin-3, and High-Sensitivity Troponin-T) in Patients With Advanced Chronic Heart Failure

Elevated natriuretic peptides provide strong prognostic information in patients with heart failure (HF). The role of novel biomarkers in HF needs to be established. Our objective was to evaluate the prognostic power of novel biomarkers, incremental to the N-terminal portion of the natriuretic peptide (NT-proBNP) in chronic HF. Concentrations of circulating NT-proBNP, growth differentiation factor 15 (GDF-15), high-sensitivity C-reactive protein (hs-CRP), galectin-3 (Gal-3), and high-sensitivity troponin T (hs-TnT) were measured and related to all-cause long-term mortality. Of 209 patients (age 71 ± 10 years, 73% male patients, 97% New York Heart Association class III), 151 (72%) died during a median follow-up of 8.7 ± 1 year. The calculated area under the curve for NT-proBNP was 0.63, GDF-15 0.78, hs-CRP 0.66, Gal-3 0.68, and hs-TnT 0.68 (all p <0.01). Each marker was predictive for mortality in univariate analysis. In multivariate analysis, elevated concentrations of GDF-15 (hazard ratio [HR] 1.41, confidence interval [CI] 1.1 to 178, p = 0.005), hs-CRP (HR 1.38, CI 1.15 to 1.67, p = 0.001), and hs-TnT (HR 1.27, CI 1.06 to 1.53, p = 0.008) were independently related to mortality. All novel markers had an incremental value to NT-proBNP, using the integrated discrimination improvement. In conclusion, in chronic HF, GDF-15, hs-CRP, and hs-TnT are independent prognostic markers, incremental to NT-proBNP, in predicting long-term mortality. In this study, GDF-15 is the most predictive marker, even stronger than NT-proBNP.

Circulating growth differentiation factor‐15 correlates with myocardial fibrosis in patients with non‐ischaemic dilated cardiomyopathy and decreases rapidly after left ventricular assist device support

Growth differentiation factor‐15 (GDF‐15) is a stress‐responsive cytokine and is emerging as a biomarker of cardiac remodelling. Left ventricular assist devices (LVADs) provide unloading of the left ventricle, resulting in partial reverse remodelling. Our aim was to study GDF‐15 in patients with a non‐ischaemic dilated cardiomyopathy (DCM) during LVAD support.

Prognostic value of N-terminal pro C-type natriuretic peptide in heart failure patients with preserved and reduced ejection fraction.

A‐type and B‐type natriuretic peptides are established markers in chronic heart failure (HF). C‐type natriuretic peptide (CNP) belongs to the same peptide family, but is predominantly localized in the endothelium. The prognostic role of CNP in heart failure has not been established. The aim of the study was to determine the prognostic power and clinical correlates of the N‐terminal part of pro CNP (NT‐proCNP) in patients with chronic HF.

Single-centre experience of 85 patients with a continuous-flow left ventricular assist device: clinical practice and outcome after extended support

OBJECTIVES We evaluated our single-centre clinical experience with the HeartMate II (HM II) left ventricular assist device (LVAD) as a bridge to transplantation (BTT) in end-stage heart failure (HF) patients. METHODS Survival rates, echocardiographic parameters, laboratory values and adverse events of 85 consecutive patients supported with a HM II were evaluated. RESULTS Overall, mean age was 45 ± 13 years, 62 (73%) were male and non-ischaemic dilatated cardiomyopathy was present in 60 (71%) patients. The median duration of mechanical support was 387 days (IQR 150-600), with a range of 1-1835 days. The 6-month, 1-, 2-, 3- and 4-year survival rates during HM II LVAD support were 85, 81, 76, 76 and 68%, respectively. Echocardiographic parameters demonstrated effective left ventricular unloading, while laboratory results reflected adequate organ perfusion. However, HM II support was associated with adverse events, such as infections in 42 patients (49%; 0.67 events/patient-year), cardiac arrhythmia in 44 (52%; 0.86 events/patient-year), bleeding complications in 32 (38%; 0.43 events/patient-year) and neurological dysfunction in 17 (20%; 0.19 events/patient-year). CONCLUSIONS In view of the increasing shortage of donor hearts, HM II LVAD support may be considered a life-saving treatment in end-stage HF patients, with good survival. However, it is still associated with some serious adverse events, of which neurological complications are the most critical.

Functional and haemodynamic recovery after implantation of continuous-flow left ventricular assist devices in comparison with pulsatile left ventricular assist devices in patients with end-stage heart failure.

Caused by ageing of the population, better survival from ischaemic heart disease, and improved treatment of chronic heart disease, the incidence of heart failure has increased enormously. Worldwide, left ventricular assist devices (LVADs) are increasingly being used as a bridge or alternative to heart transplantation. In this study, we investigated whether there is difference in functional and haemodynamic recovery after implantation of pulsatile and continuous‐flow pumps.

Myocardial fibrosis and pro-fibrotic markers in end-stage heart failure patients during continuous-flow left ventricular assist device support

OBJECTIVES During support with a left ventricular assist device (LVAD), partial reverse remodelling takes place in which fibrosis plays an important role. In this study, we analysed the histological changes and expression of fibrotic markers in patients with advanced heart failure (HF) during continuous-flow LVAD (cf-LVAD) support. METHODS In 25 patients, myocardial tissue at the time of LVAD implantation (pre-LVAD) was compared with tissue from the explanted left ventricle (post-LVAD). Interstitial fibrosis and cardiomyocyte size were analysed pre- and post-LVAD. Plasma was obtained from all patients before and during LVAD support. Plasma levels, cardiac mRNA and protein expression of brain natriuretic peptide (BNP), galectin-3 (Gal-3), connective tissue growth factor (CTGF), osteopontin (OPN) and transforming growth factor β-1 were determined. RESULTS Fibrosis increased during cf-LVAD unloading (P < 0.05). Cardiomyocytes elongated (P < 0.05), whereas cross-sectional area did not change. BNP, Gal-3, CTGF and OPN were significantly elevated pre-LVAD in comparison with controls. BNP decreased significantly after 1 month of cf-LVAD support (P < 0.001) to near-normal levels. Pro-fibrotic markers remained elevated in comparison with controls. CONCLUSIONS cf-LVAD support is associated with lengthening of cardiomyocytes, without alterations in diameter size. Remarkably, myocardial fibrosis increased as well as circulating pro-fibrotic markers. Whether the morphological changes are a direct effect of reduced pulsatility during cf-LVAD support or due to HF progression requires further investigation.

Post-transcriptional Regulation of α-1-Antichymotrypsin by MicroRNA-137 in Chronic Heart Failure and Mechanical Support

Background—Better understanding of the molecular mechanisms of remodeling has become a major objective of heart failure (HF) research to stop or reverse its progression. Left ventricular assist devices (LVADs) are being used in patients with HF, leading to partial reverse remodeling. In the present study, proteomics identified significant changes in &agr;-1-antichymotrypsin (ACT) levels during LVAD support. Moreover, the potential role of ACT in reverse remodeling was studied in detail. Methods and Results—Expression of ACT mRNA (quantitative-polymerase chain reaction) decreased significantly in post-LVAD myocardial tissue compared with pre-LVAD tissue (n=15; P<0.01). Immunohistochemistry revealed that ACT expression and localization changed during LVAD support. Circulating ACT levels were elevated in HF patients (n=18) as compared with healthy controls (n=6; P=0.001) and normalized by 6 months of LVAD support. Because increasing evidence implicates that microRNAs (miRs) are involved in myocardial disease processes, we also investigated whether ACT is post-transcriptionally regulated by miRs. Bioinformatics analysis pointed miR-137 as a potential regulator of ACT. The miR-137 expression is inversely correlated with ACT mRNA in myocardial tissue. Luciferase activity assays confirmed ACT as a direct target for miR-137, and in situ hybridization indicated that ACT and miR-137 were mainly localized in cardiomyocytes and stromal cells. Conclusions—High ACT plasma levels in HF normalized during LVAD support, which coincides with decreased ACT mRNA in heart tissue, whereas miR-137 levels increased. MiR-137 directly targeted ACT, thereby indicating that ACT and miR-137 play a role in the pathophysiology of HF and reverse remodeling during mechanical support.

MicroRNA Expression in Myocardial Tissue and Plasma of Patients with End-Stage Heart Failure during LVAD Support: Comparison of Continuous and Pulsatile Devices

Aim Pulsatile flow left ventricular assist devices (pf-LVADs) are being replaced by continuous flow LVADs (cf-LVADs) in patients with end-stage heart failure (HF). MicroRNAs (miRs) play an important role in the onset and progression of HF. Our aim was to analyze cardiac miR expression patterns associated with each type of device, to analyze differences in the regulation of the induced cardiac changes. Methods and Results Twenty-six miRs were selected (based on micro-array data and literature studies) and validated in myocardial tissue before and after pf- (n = 17) and cf-LVAD (n = 17) support. Of these, 5 miRs displayed a similar expression pattern among the devices (miR-129*, miR-146a, miR-155, miR-221, miR-222), whereas others only changed significantly during pf-LVAD (miR-let-7i, miR-21, miR-378, miR-378*) or cf-LVAD support (miR-137). In addition, 4 miRs were investigated in plasma of cf-LVAD supported patients (n = 18) and healthy controls (n = 10). Circulating miR-21 decreased at 1, 3, and 6 months after LVAD implantation. MiR-146a, miR-221 and miR-222 showed a fluctuating time pattern post-LVAD. Conclusion Our data show a different miR expression pattern after LVAD support, suggesting that differentially expressed miRs are partially responsible for the cardiac morphological and functional changes observed after support. However, the miR expression patterns do not seem to significantly differ between pf- and cf-LVAD implying that most cardiac changes or clinical outcomes specific to each device do not relate to differences in miR expression levels.

Two young women with soft tissue tumours of the heart

Primary cardiac sarcomas often strike young, healthy patients and tend to have a dismal prognosis. Because of limited experience, the heterogeneous nature of cardiac sarcomas and different treatment results of patients with malignant primary tumours of the heart, the role of heart transplantation should be weighed on a case-by-case basis.