Limb Remote Ischemic Preconditioning Applied During Sevoflurane Anesthesia Does Not Protect the Lungs in Patients Undergoing Adult Heart Valve Surgery

Background: Two consistent overall cell protective preconditioning treatments should provide more protection. We hypothesized that limb remote ischemic preconditioning (RIPC, second preconditioning stimulus) applied during sevoflurane inhalation (first preconditioning stimulus) would provide more protection to the lungs of patients undergoing adult heart valve surgery. Methods: In this randomized, placebo-controlled, double-blind trial, 50 patients were assigned to the RIPC group or the placebo group (1:1). Patients in the RIPC group received three 5-min cycles of 300 mmHg cuff inflation/deflation of the left-side lower limb before aortic cross-clamping. Anesthesia consisted of opioids and propofol for induction and sevoflurane for maintenance. The primary end point was comparison of the postoperative arterial–alveolar oxygen tension ratio (a/A ratio) between groups. Secondary end points included comparisons of pulmonary variables, postoperative morbidity and mortality and regional and systemic inflammatory cytokines between groups. Results: In the RIPC group, the a/A ratio and other pulmonary variables exhibited no significant differences throughout the study period compared with the placebo group. No significant differences in either plasma or bronchoalveolar lavage levels of TNF- α were noted between the groups at 10 min after anesthetic induction and 1 h after cross-clamp release. The percentage of neutrophils at 12 h postoperation was significantly increased in the RIPC group compared with the placebo group (91.34±0.00 vs. 89.42±0.10, P = 0.023). Conclusions: Limb RIPC applied during sevoflurane anesthesia did not provide additional significant pulmonary protection following adult valvular cardiac surgery.

Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular composition. The data are further explored to e.g. identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2073 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile and validated these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

The adult heart requires baseline expression of the transcription factor Hand2 to withstand right ventricular pressure overload

Aims Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV). Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a ‘foetal gene programme’ contributing to pathological cardiac remodelling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodelling in response to pressure overload induced by pulmonary artery banding (PAB). Methods and results In this study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived haemodynamic parameters as well as molecular remodelling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control-banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV pressure-overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass, and impaired cardiac function. RNA-sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV. Conclusion Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressu re overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodelling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.

Chronic colitis upregulates microRNAs suppressing brain-derived neurotrophic factor in the adult heart

Ulcerative colitis and Crohn’s disease are classified as chronic inflammatory bowel diseases (IBD) with known extraintestinal manifestations. The interplay between heart and gut in IBD has previously been noted, but the mechanisms remain elusive. Our objective was to identify microRNAs mediating molecular remodeling and resulting cardiac impairment in a rat model of colitis. To induce chronic colitis, dextran sodium sulfate (DSS) was given to adult rats for 5 days followed by 9 days with normal drinking water for 4 cycles over 8 weeks. Echocardiography was performed to evaluate heart function. DSS-induced colitis led to a significant decrease in ejection fraction, increased left ventricular mass and size, and elevated B-type natriuretic protein. MicroRNA profiling showed a total of 56 miRNAs significantly increased in the heart by colitis, 8 of which are predicted to target brain-derived neurotrophic factor (BDNF). RT-qPCR validated the increases of miR-1b, Let-7d, and miR-155. Transient transfection revealed that miR-155 significantly suppresses BDNF in H9c2 cells. Importantly, DSS colitis markedly decreased BDNF in both myocardium and serum. Levels of various proteins critical to cardiac homeostasis were also altered. Functional studies showed that BDNF increases cell viability and mitigates H2O2-induced oxidative damage in H9c2 cells, demonstrating its protective role in the adult heart. Mechanistically, cellular experiments identified IL-1β as the inflammatory mediator upregulating cardiac miR-155; this effect was confirmed in adult rats. Furthermore, IL-1β neutralizing antibody ameliorated the DSS-induced increase in miR-155 and concurrent decrease in BDNF in the adult heart, showing therapeutic potential. Our findings indicate that chronic colitis impairs heart function through an IL-1β→miR-155→BDNF signaling axis.