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Maja-Theresa Dieterlen,&ast; Eva Katharina Messer,&ast; Kristin Klaeske, Franz Sieg, Sandra Eifert, Josephina Haunschild, Khalil Jawad, Diyar Saeed, Alexey Dashkevich,&ast; Michael Andrew Borger&ast; University Clinic of Cardiac Surgery, Leipzig Heart Center, HELIOS Clinic, Leipzig, Germany&ast;These authors contributed equally to this workCorrespondence: Maja-Theresa Dieterlen, University Department for Cardiac Surgery, Heart Center Leipzig, University Hospital, HELIOS Clinic, Strümpellstraße 39, Leipzig, 04289, Germany, Tel +49 – 341 865 1651, Fax +49 – 341 865 1452, Email mdieterlen@web.dePurpose: Infection is the most common complication after left ventricular assist device (LVAD) implantation. The immune status of LVAD patients is relevant for the incidence and severity of infection, but it is unknown if there is a predisposing immune status prior to LVAD implantation that contributes to an increased risk for infection in the post-implant period. We analyzed the pre-LVAD immune status in patients with infection within 3 months after LVAD implantation in comparison to infection-free patients.Patients and Methods: Fifty-four consecutive LVAD patients were included in this study. According to their infectious history in the first 3 months after LVAD implantation, these patients were grouped into an infection (n=23) and an infection-free group (n=31). Pre-LVAD blood samples were obtained for flow cytometric analysis of immunological parameters including B cells, subsets of T, dendritic and natural killer cells. Patient-specific, clinical and laboratory data were recorded.Results: Blood count analysis prior to LVAD implantation showed comparable counts of erythrocytes (p=0.19), platelets (p=0.33) and leukocytes (p=0.50) between patients with infection and infection-free patients in the post-implant period. Patients with infection in the first 3 months after LVAD implantation had lower concentrations of lymphocytes (p=0.02). Forty percent of the patients with infection showed more often pre-LVAD neutrophil-to-lymphocyte ratios (NLR) > 7 than patients without infection in the first 3 months after LVAD implantation (14%, p=0.05). Patients with infection already had lower percentages of CD3+ T cells (p=0.03), CD19+ B cells (p< 0.01), BDCA2+ pDCs (p=0.03) and BDCA4+ plasmacytoid DCs (pDCs) (p=0.05) prior to LVAD implantation than infection-free patients.Conclusion: Our results demonstrated that patients with infection in the early post-implant period showed lower concentrations of lymphocytes, especially of CD3+ T cells and CD19+ B cells, decreased percentages of BDCA2+ and BDCA4+ pDCs, and had more often NLRs > 7 indicating moderate-to-severe inflammation. Thus, we identified specific immunological changes pre-LVAD that could help to identify patients at risk for infection in the early post-implant period.Keywords: LVAD, immune system, T cells, B cells, plasmacytoid dendritic cells, neutrophil–lymphocyte ratio

Non-surgical bleeding (NSB) remains the most critical complication in patients under left ventricular assist device (LVAD) support. It is well known that blood exposed to non-physiological shear stress result in platelet dysfunction. Compared to patients without NSB, decreased surface expression of platelet receptor GPIbα was observed in LVAD patients with NSB. In addition to the mechanism of receptor shedding, understanding the functional changes in platelets contributing to NSB is crucial for preventing this adverse event. In the present study, we aimed to compare the expression level of glycoprotein (GP)Ib-IX-V platelet receptor complex in HeartMate 3 (HM 3) patients with and without bleeding complications to investigate the alterations of the platelet transcriptomic profile on platelet damage and increased bleeding risk. Blood samples were obtained from HM 3 patients with NSB (bleeder group, n = 27) and without NSB (non-bleeder group, n = 55). The bleeder group was further divided into patients with NSB within the first 3 months (bleeder ≤ 3 mo, n=19) and patients with NSB after 3 months (bleeder > 3 mo, n=8). The mRNA and protein expression of the platelet receptor complex subunits GPIbα, GPIX and GPV were quantified by real-time qPCR and enzyme-linked immunosorbent assay. Demographic and clinical characteristics as well as LVAD-related parameters were documented for each patient. Non-bleeder, bleeder ≤ 3 mo and bleeder > 3 mo were comparable regarding demographic and clinical characteristics of age, BMI, gender, etiology of heart disease, pump parameters, INR and von Willebrand factor diagnostics (p > 0.05). The three study groups were comparable regarding the mRNA expression of GPIbα, GPIX and GPV (p>0.05). The protein analysis revealed a significantly reduced expression level of the main receptor subunit GPIbα in bleeders ≤ 3 mo (p=0.04). We suggest, that the observed reduction of platelet receptor GPIbα protein expression in patients experienced their first bleeding event within 3 months after LVAD implantation may influence platelet physiology under pathologic shear stress conditions. The alterations of functional GPIbα potentially reduce the platelet adhesion capacities, which may lead to an impaired haemostatic process and elevated propensity of bleeding in HM 3 patients. [ABSTRACT FROM AUTHOR]

Purpose: Mitral valve prolapse (MVP) is associated with left ventricle (LV) fibrosis, including the papillary muscles (PM), which is in turn linked to malignant arrhythmias. This study aims to evaluate comprehensive tissue characterization of the PM by cardiovascular magnetic resonance (CMR) imaging and its association with LV fibrosis observed by intraoperative biopsies., Methods: MVP patients with indication for surgery due to severe mitral regurgitation (n = 19) underwent a preoperative CMR with characterization of the PM: dark-appearance on cine, T1 mapping, conventional bright blood (BB) and dark blood (DB) late gadolinium enhancement (LGE). CMR T1 mapping was performed on 21 healthy volunteers as controls. LV inferobasal myocardial biopsies were obtained in MVP patients and compared to CMR findings., Results: MVP patients (54 ± 10 years old, 14 male) had a dark-appearance of the PM with higher native T1 and extracellular volume (ECV) values compared with healthy volunteers (1096 ± 78ms vs. 994 ± 54ms and 33.9 ± 5.6% vs. 25.9 ± 3.1%, respectively, p < 0.001). Seventeen MVP patients (89.5%) had fibrosis by biopsy. BB-LGE + in LV and PM was identified in 5 (26.3%) patients, while DB-LGE + was observed in LV in 9 (47.4%) and in PM in 15 (78.9%) patients. DB-LGE + in PM was the only technique that showed no difference with detection of LV fibrosis by biopsy. Posteromedial PM was more frequently affected than the anterolateral (73.7% vs. 36.8%, p = 0.039) and correlated with biopsy-proven LV fibrosis (Rho 0.529, p = 0.029)., Conclusions: CMR imaging in MVP patients referred for surgery shows a dark-appearance of the PM with higher T1 and ECV values compared with healthy volunteers. The presence of a positive DB-LGE at the posteromedial PM by CMR may serve as a better predictor of biopsy-proven LV inferobasal fibrosis than conventional CMR techniques., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Mitochondrial permeability transition pore (mPTP) opening plays a crucial role in cell death during ischemia-reperfusion injury (IRI). Cyclosporine A (CsA) inhibits mPTP opening. This study aimed to investigate the effects of CsA treatment during cardioplegia on the mitochondrial function and cardiac IRI., Methods: Landrace pigs (52.9 ± 3.7 kg) were subjected to midline sternotomy, cardiopulmonary bypass at 34°C and 90 minutes of cardiac arrest. They received either a single shot of standard 4°C cold histidine-tryptophan-α-ketoglutarate (HTK)-Bretschneider solution ( n  = 11) or HTK-Bretschneider plus 1.2 mg/L CsA (histidine-tryptophan-α-ketoglutarate plus cyclosporine A (HTK/CsA); n  = 11). During reperfusion global left-ventricular function was assessed and myocardial biopsies were harvested at baseline, during ischemia and 45 minutes following reperfusion. High-resolution respirometry and hydrogen peroxide production were measured. Immunohistochemical stainings for apoptosis-inducing factor and hypoxia-inducible factor-1α as well as a flow cytometry-based JC-1 mitochondrial membrane potential assay were performed., Results: Hemodynamic parameters were comparable between both groups. The cytochrome C release (HTK: 930.3 ± 804.4 pg/mg, HTK/CsA: 699.7 ± 394.0 pg/mg, p = 0.457) as well as PGC1α content (HTK: 66.7%, HTK/CsA: 33.3%, p = 0.284) was lower in the HTK/CsA group. Respiratory measurements revealed that the oxygen flux under basal respiration was higher in the HTK/CsA group (8.2 ± 1.3 pmol·O 2 ·s -1 ·mg -1 ·ww) than in the HTK group (3.8 ± 1.4 pmol·O 2 ·s -1 ·mg -1 ·ww, p = 0.045). There were no significant differences regarding histological surrogates of apoptosis and necrosis., Conclusions: Supplementing cardioplegic solutions with CsA enhances the basal mitochondrial respiration thereby exerting a cardioprotective effect and diminishing IRI-induced damage. CsA seems to preserve mitochondrial function via non-ROS related pathways., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Infection is a common complication following left ventricular assist device (LVAD) implantation. Patients with obesity are particularly at risk due to their high percentage of adipose tissue and the resulting chronic inflammatory state and resulting immunological changes. This study investigated changes of immunological parameters in relation to body mass index (BMI) during the first year after LVAD implantation., Methods: Blood samples were obtained prior to LVAD implantation and at 3 (1 st FU), 6 (2 nd FU) and 12 mo (3 rd FU) after LVAD implantation. Patients were divided into three groups (normal weight: BMI of 18.5-24.9 kg/m 2 ; n=12; pre-obesity: 25.0-29.9 kg/m 2 ; n=15; obesity: ≥ 30.0 kg/m 2 ; n=17) based on their BMI at the time of LVAD implantation. Flow cytometric analyses for CD4 + and CD8 + T cells, regulatory T cells (T regs ), B cells as well as dendritic cells (DCs) were performed., Results: After LVAD implantation, obese patients (0.51 ± 0.20%) showed a higher proportion of overall DCs than normal-weight (0.28 ± 0.10%) and pre-obese patients (0.32 ± 0.11%, p<0.01) at 3 rd FU. The proportion of BDCA3 + myeloid DCs was lower in obese patients (64.3 ± 26.5%) compared to normal-weight patients (82.7 ± 10.0%, p normal-weight vs. obesity =0.05) at 2 nd FU after LVAD implantation. The analysis of BDCA4 + plasmacytoid DCs revealed a reduced proportion in pre-obese (21.1 ± 9.8%, p normal-weight vs. pre-obesity =0.01) and obese patients (23.7 ± 10.6%, p normal-weight vs. obesity =0.05) compared to normal-weight patients (33.1 ± 8.2%) in the 1 st FU. T cell analysis showed that CD4 + T cells of obese patients (62.4 ± 9.0%) significantly increased in comparison to pre-obese patients (52.7 ± 10.0%, p pre-obesity vs. obesity =0.05) and CD8 + T cells were lower in obese patients (31.8 ± 8.5%) than in normal-weight patients (42.4 ± 14.2%; p normal-weight vs. obesity =0.04) at the 3 rd FU. Furthermore, we observed significantly reduced proportions of T regs in pre-obese patients compared to normal-weight and obese patients at 2 nd FU (p=0.02) and 3 rd FU (p=0.01) after LVAD implantation., Conclusion: This study reported changes of the innate and adaptive immune system of pre-obese and obese compared to normal-weight patients one year after LVAD implantation. DCs and their subsets, CD8 + T cells and T regs were affected immune cell populations that indicate immunological changes which might increase the incidence of postoperative infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Klaeske, Messer, Klein, Sieg, Eifert, Haunschild, Jawad, Saeed, Dashkevich, Borger and Dieterlen.)

Background: The guidelines for mechanical circulatory support of the International Society for Heart and Lung Transplantation do not recommend the routine replacement or repair of the mitral valve at the time point of left ventricular assist device (LVAD) implantation. We investigated different parameters of health status including exercise capacity, anxiety and depression after LVAD implantation in patients with different preoperative grades of mitral regurgitation (MR)., Methods: A single-center analysis of health status was performed including 45 patients with HeartMate 3 (HM 3) implantation using the 12-items Short Form Health Survey (SF-12) and the Hospital Anxiety and Depression Score (HADS) questionnaires. The study groups were classified according to echocardiographically defined preoperative grade of MR. The group without severe MR comprised 33 patients; the group with severe MR comprised 12 patients., Results: Demographic and preclinical characteristics as well as LVAD complications such as thrombosis and bleeding events were comparable between LVAD patients with severe and not severe MR (p > 0.05). Severe MR resolved in all patients after LVAD implantation and improved to moderate, mild or no MR in both groups in a period ranging from 6 months until 2 years. The analyses of SF-12 questionnaire revealed that the physical (p = 0.44) and mental health (p = 0.64) was comparable. The grade of anxiety (p = 0.34) and depression (p = 0.44) was comparable between the groups. Exercise capacity measured by the 6 min walk test correlated positively with the SF-12-determined physical health (p < 0.01, r = 0.518) and negatively with the HADS anxiety (p = 0.01, r = -0.399) and depression (p < 0.01, r = -0.570) scores., Conclusions: Our data showed that the health status is comparable in HM 3 patients with different preoperative MR severities in the post-LVAD period. Preoperative severe MR resolves in the majority of patients early after LVAD implantation and is not associated with concomitant mitral valve repair or replacement at the time of LVAD implantation., (© 2023. International Society for Quality of Life Research (ISOQOL).)

The bicuspid aortic valve (BAV) is the most common cardiovascular congenital abnormality and is frequently associated with proximal aortopathy. We analyzed the tissues of patients with bicuspid and tricuspid aortic valve (TAV) regarding the protein expression of the receptor for advanced glycation products (RAGE) and its ligands, the advanced glycation end products (AGE), as well as the S100 calcium-binding protein A6 (S100A6). Since S100A6 overexpression attenuates cardiomyocyte apoptosis, we investigated the diverse pathways of apoptosis and autophagic cell death in the human ascending aortic specimen of 57 and 49 patients with BAV and TAV morphology, respectively, to identify differences and explanations for the higher risk of patients with BAV for severe cardiovascular diseases. We found significantly increased levels of RAGE, AGE and S100A6 in the aortic tissue of bicuspid patients which may promote apoptosis via the upregulation of caspase-3 activity. Although increased caspase-3 activity was not detected in BAV patients, increased protein expression of the 48 kDa fragment of vimentin was detected. mTOR as a downstream protein of Akt was significantly higher in patients with BAV, whereas Bcl-2 was increased in patients with TAV, assuming a better protection against apoptosis. The autophagy-related proteins p62 and ERK1/2 were increased in patients with BAV, assuming that cells in bicuspid tissue are more likely to undergo apoptotic cell death leading to changes in the wall and finally to aortopathies. We provide first-hand evidence of increased apoptotic cell death in the aortic tissue of BAV patients which may thus provide an explanation for the increased risk of structural aortic wall deficiency possibly underlying aortic aneurysm formation or acute dissection.

Non-surgical bleeding (NSB) remains the most critical complication in patients under left ventricular assist device (LVAD) support. It is well known that blood exposed to high shear stress results in platelet dysfunction. Compared to patients without NSB, decreased surface expression of platelet receptor GPIbα was observed in LVAD patients with NSB. In this study, we aimed to compare the expression level of glycoprotein (GP)Ib-IX-V platelet receptor complex in HeartMate 3 (HM 3) patients with and without bleeding complications to investigate the alterations of the platelet transcriptomic profile on platelet damage and increased bleeding risk. Blood samples were obtained from HM 3 patients with NSB (bleeder group, n = 27) and without NSB (non-bleeder group, n = 55). The bleeder group was further divided into patients with early NSB (bleeder ≤ 3 mo, n = 19) and patients with late NSB (bleeder > 3 mo, n = 8). The mRNA and protein expression of GPIbα, GPIX and GPV were quantified for each patient. Non-bleeder, bleeder ≤ 3 mo and bleeder > 3 mo were comparable regarding the mRNA expression of GPIbα, GPIX and GPV ( p > 0.05). The protein analysis revealed a significantly reduced expression level of the main receptor subunit GPIbα in bleeders ≤ 3 mo ( p = 0.04). We suggest that the observed reduction of platelet receptor GPIbα protein expression in patients who experienced their first bleeding event within 3 months after LVAD implantation may influence platelet physiology. The alterations of functional GPIbα potentially reduce the platelet adhesion capacities, which may lead to an impaired hemostatic process and the elevated propensity of bleeding in HM 3 patients.

Mitral valve prolapse (MVP) is a cardiac valve disease that not only affects the mitral valve (MV), provoking mitral regurgitation, but also leads to maladaptive structural changes in the heart. Such structural changes include the formation of left ventricular (LV) regionalized fibrosis, especially affecting the papillary muscles and inferobasal LV wall. The occurrence of regional fibrosis in MVP patients is hypothesized to be a consequence of increased mechanical stress on the papillary muscles and surrounding myocardium during systole and altered mitral annular motion. These mechanisms appear to induce fibrosis in valve-linked regions, independent of volume-overload remodeling effects of mitral regurgitation. In clinical practice, quantification of myocardial fibrosis is performed with cardiovascular magnetic resonance (CMR) imaging, even though CMR has sensitivity limitations in detecting myocardial fibrosis, especially in detecting interstitial fibrosis. Regional LV fibrosis is clinically relevant because even in the absence of mitral regurgitation, it has been associated with ventricular arrhythmias and sudden cardiac death in MVP patients. Myocardial fibrosis may also be associated with LV dysfunction following MV surgery. The current article provides an overview of current histopathological studies investigating LV fibrosis and remodeling in MVP patients. In addition, we elucidate the ability of histopathological studies to quantify fibrotic remodeling in MVP and gain deeper understanding of the pathophysiological processes. Furthermore, molecular changes such as alterations in collagen expression in MVP patients are reviewed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CB declared a past co-authorship with the authors MAB, RAL, RAN to the handling editor., (© 2023 Dieterlen, Klaeske, Spampinato, Marin-Cuartas, Wiesner, Morningstar, Norris, Melnitchouk, Levine, van Kampen and Borger.)

Introduction: Infections are a major problem after left ventricular assist device (LVAD) implantation that affects morbidity, mortality, and the quality of life. Obesity often increases the risk for infection. In the cohort of LVAD patients, it is unknown if obesity affects the immunological parameters involved in viral defense. Therefore, this study investigated whether overweight or obesity affects immunological parameters such as CD8+ T cells and natural killer (NK) cells., Methods: Immune cell subsets of CD8+ T cells and NK cells were compared between normal-weight (BMI 18.5-24.9 kg/m2, n = 17), pre-obese (BMI 25.0-29.9 kg/m2, n = 24), and obese (BMI ≥30 kg/m2, n = 27) patients. Cell subsets and cytokine serum levels were quantified prior to LVAD implantation and at 3, 6, and 12 months after LVAD implantation., Results: At the end of the first postoperative year, obese patients (31.8% ± 2.1%) had a lower proportion of CD8+ T cells than normal-weight patients (42.4% ± 4.1%; p = 0.04), and the percentage of CD8+ T cells was negatively correlated with BMI (p = 0.03; r = -0.329). The proportion of circulating NK cells increased after LVAD implantation patients in normal-weight (p = 0.01) and obese patients (p &lt; 0.01). Patients with pre-obesity showed a delayed increase (p &lt; 0.01) 12 months after LVAD implantation. Further, obese patients showed an increase in the percentage of CD57+ NK cells after 6 and 12 months (p = 0.01) of treatment, higher proportions of CD56bright NK cells (p = 0.01), and lower proportions of CD56dim/neg NK cells (p = 0.03) 3 months after LVAD implantation than normal-weight patients. The proportion of CD56bright NK cells positively correlated with BMI (p &lt; 0.01, r = 0.403) 1 year after LVAD implantation., Conclusions: This study documented that obesity affects CD8+ T cells and subsets of NK cells in patients with LVAD in the first year after LVAD implantation. Lower proportions of CD8+ T cells and CD56dim/neg NK cells and higher proportion of CD56bright NK cells were detected in obese but not in pre-obese and normal-weight LVAD patients during the first year after LVAD implantation. The induced immunological imbalance and phenotypic changes of T and NK cells may influence viral and bacterial immunoreactivity., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Disturbances in the ubiquitin proteasome system, and especially changes of the E3 ligases, are subjects of interest when searching for causes and therapies for cardiomyopathies. The aim of this study was to clarify whether the myostatin/AKT/forkhead box O (FOXO) pathway, which regulates the expression of the E3 ligases muscle atrophy F-box gene (MAFbx) and muscle ring-finger protein-1 (MuRF1), is changed in dilated cardiomyopathy of ischemic origin (IDCM) and dilated cardiomyopathy of non-ischemic origin (NIDCM). The mRNA and protein expression of myostatin, AKT, FOXO1, FOXO3, MAFbx and MuRF1 were quantified by real-time polymerase chain reaction and ELISA, respectively, in myocardial tissue from 26 IDCM and 23 NIDCM patients. Septal tissue from 17 patients undergoing Morrow resection served as a control. MAFbx and FOXO1 mRNA and protein expression (all p < 0.05), AKT mRNA (p < 0.01) and myostatin protein expression (p = 0.02) were decreased in NIDCM patients compared to the control group. Apart from decreases of AKT and MAFbx mRNA expression (both p < 0.01), no significant differences were detected in IDCM patients compared to the control group. Our results demonstrate that the myostatin/AKT/FOXO pathway is altered in NIDCM but not in IDCM patients. FOXO1 seems to be an important drug target for regulating the expression of MAFbx in NIDCM patients.

Non-surgical bleeding (NSB) is one of the major clinical complications in patients under continuous-flow left ventricular assist device (LVAD) support. The increased shear stress leads to an altered platelet receptor composition. Whether these changes increase the risk for NSB is unclear. Thus, we compared the platelet receptor composition of patients with (bleeder group, n = 18) and without NSB (non-bleeder group, n = 18) prior to LVAD implantation. Blood samples were obtained prior to LVAD implantation and after bleeding complications in the post-implant period. Platelet receptor expression of GPIbα, GPIIb/IIIa, P-selectin and CD63 as well as intra-platelet oxidative stress levels were quantified by flow cytometry. Bleeders and non-bleeders were comparable regarding clinical characteristics, von Willebrand factor diagnostics and the aggregation capacity before and after LVAD implantation (p > 0.05). LVAD patients in the bleeder group suffered from gastrointestinal bleeding (33%; n = 6), epistaxis (22%; n = 4), hematuria or hematoma (17%; n = 3, respectively) and cerebral bleeding (11%; n = 2). Prior to LVAD implantation, a restricted surface expression of the platelet receptors P-selectin and GPIIb/IIIa was observed in the bleeder group (P-selectin: 7.2 ± 2.6%; GPIIb/IIIa: 26,900 ± 13,608 U) compared to non-bleeders (P-selectin: 12.4 ± 8.1%, p = 0.02; GPIIb/IIIa: 36,259 ± 9914 U; p = 0.02). We hypothesized that the reduced platelet receptor expression of P-selectin and GPIIb/IIIa prior to LVAD implantation may be linked to LVAD-related NSB.

The pathological changes of ubiquitination and deubiquitination following myocardial infarction (MI) and chronic heart failure (CHF) have been sparsely examined. We investigated the expression of muscle-specific E3 ubiquitin ligases and deubiquitinases in MI and CHF. Therefore, mice were assigned to coronary artery ligation for 3 days or 10 weeks as well as for sham operation (each n = 10). Expression of E3 ligases (MAFBX, MURF1, CHIP, ITCH, MDM2) and deubiquitinases (A20, CYLD, UCH-L1, USP14, USP19) was determined. After MI and in CHF, the mRNA expression of MURF1, CHIP and MDM2 (all p < 0.05) was decreased. Protein expression analyses revealed that ITCH expression decreased in CHF ( p = 0.01), whereas MDM2 expression increased in MI ( p = 0.02) and decreased in CHF ( p = 0.02). Except for USP19 mRNA expression that decreased at 3 days and 10 weeks (both p < 0.01), the expression of other deubiquitinases remained unaffected after MI and CHF. The expression of myocardial E3 ligases is differentially regulated following MI, raising the question of whether an upstream regulation exists that is activated by MI for tissue protection or whether the downregulation of E3 ligases enables myocardial hypertrophy following MI.

Background: Patients treated with an inhibitor of the mechanistic target of rapamycin (mTORI) in a calcineurin inhibitor (CNI)-free immunosuppressive regimen after heart transplantation (HTx) show a higher risk for transplant rejection. We developed an immunological monitoring tool that may improve the identification of mTORI-treated patients at risk for rejection., Methods: Circulating dendritic cells (DCs) and regulatory T cells (T regs ) were analysed in 19 mTORI- and 20 CNI-treated HTx patients by flow cytometry. Principal component and cluster analysis were used to identify patients at risk for transplant rejection., Results: The percentages of total T regs ( p = 0.02) and CD39 + T regs ( p = 0.05) were higher in mTORI-treated patients than in CNI-treated patients. The principal component analysis revealed that BDCA1 + , BDCA2 + and BDCA4 + DCs as well as total T regs could distinguish between non-rejecting and rejecting mTORI-treated patients. Most mTORI-treated rejectors showed higher levels of BDCA2 + and BDCA4 + plasmacytoid DCs and lower levels of BDCA1 + myeloid DCs and T regs than mTORI non-rejectors., Conclusion: An mTORI-based immunosuppressive regimen induced a sufficient, tolerance-promoting reaction in T regs , but an insufficient, adverse effect in DCs. On the basis of patient-specific immunological profiles, we established a flow cytometry-based monitoring tool that may be helpful in identifying patients at risk for rejection.

Background: Extracorporeal photopheresis (ECP) induces immunological changes that lead to a reduced risk of transplant rejection. The aim of the present study was to determine optimum conditions for ECP treatment by analyzing a variety of tolerance-inducing immune cells to optimize the treatment., Methods: Ten ECP treatments were applied to each of 17 heart-transplant patients from month 3 to month 9 post-HTx. Blood samples were taken at baseline, three times during treatment, and four months after the last ECP treatment. The abundance of subsets of tolerance-inducing regulatory T cells (T regs ) and dendritic cells (DCs) in the samples was determined by flow cytometry. A multivariate statistical model describing the immunological status of rejection-free heart transplanted patients was used to visualize the patient-specific immunological improvement induced by ECP., Results: All BDCA + DC subsets (BDCA1 + DCs: p < 0.01, BDCA2 + DCs: p < 0.01, BDCA3 + DCs: p < 0.01, BDCA4 + DCs: p < 0.01) as well as total T regs ( p < 0.01) and CD39 + T regs ( p < 0.01) increased during ECP treatment, while CD62L + T regs decreased (p < 0.01). The cell surface expression level of BDCA1 (p < 0.01) and BDCA4 (p < 0.01) on DCs as well as of CD120b (p < 0.01) on T regs increased during the study period, while CD62L expression on T regs decreased significantly (p = 0.04). The cell surface expression level of BDCA2 (p = 0.47) and BDCA3 (p = 0.22) on DCs as well as of CD39 (p = 0.14) and CD147 (p = 0.08) on T regs remained constant during the study period. A cluster analysis showed that ECP treatment led to a sustained immunological improvement., Conclusions: We developed an immune monitoring assay for ECP treatment after heart transplantation by analyzing changes in tolerance-inducing immune cells. This assay allowed differentiation of patients who did and did not show immunological improvement. Based on these results, we propose classification criteria that may allow optimization of the duration of ECP treatment., Competing Interests: FA declared the following conflict of interest: Therakos: Honoraria, Research funding. MJB declared the following conflict of interest: Therakos: Honoraria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dieterlen, Klaeske, Bernhardt, Borger, Klein, Garbade, Lehmann, Ayuk, Reichenspurner and Barten.)

Background: Non-surgical bleeding (NSB) is a major complication after left ventricular assist device (LVAD) implantation. It has been reported that non-physiological shear stress caused by LVADs could alter platelet receptor expression, which leads to bleeding disorders caused by coagulation dysfunctions., Objectives: Because bleeding diathesis could be multifactorial, we focused on the combined characterization of platelet receptor expression patterns and oxidative stress to compare patients with NSB and patients without coagulation disorder in a monocentric, prospective study., Methods: Blood samples were obtained from LVAD patients with NSB (bleeder group, n = 19) and without NSB (non-bleeder group, n = 20). The platelet receptors platelet endothelial cell adhesion molecule-1 (PECAM-1), glycoprotein (GP)Ibα, P-selectin, CD63, and GPIIb/IIIa, as well as the production of intraplatelet reactive oxygen species (ROS) were quantified by flow cytometry. Aggregation capacity was evaluated by aggregometry., Results: The surface expression level of P-selectin and GPIbα on platelets was decreased in bleeders (P-selectin: 465 ± 72 U; GPIbα: 435 ± 41 U) compared to non-bleeders (P-selectin: 859 ± 115 U, P < .01; GPIbα: 570 ± 49 U, p = .04). Additionally, the mean fluorescence intensity of ADP-stimulated P-selectin and PECAM-1 expressing platelets were reduced in bleeders (P-selectin: 944 ± 84 U; PECAM-1: 6722 ± 419 U) compared to non-bleeders (P-selectin: 1269 ± 130 U, P = .04; PECAM-1: 8542 ± 665 U, P = .03). Bleeders showed a higher amount of ROS formation in platelets (88.0 ± 2.6%) than non-bleeders (81.5 ± 2.1%, P = .05)., Conclusions: These findings suggested that changes of three platelet receptors (GPIbα, P-selectin, and PECAM-1) and elevated oxidative stress may play a role in patients with bleeding complications following LVAD implantation. These results might help to explain the high incidence of spontaneous hemorrhage during LVAD support through an altered platelet function., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Myocardial protection during cardiopulmonary bypass (CPB) can be achieved using cardioplegic solutions. Although, acute kidney injury (AKI) is a common complication following CPB, the effects of cardioplegic solutions on AKI have rarely been investigated. Within this study, the effects of the cardioplegic solutions histidine-tryptophan-ketoglutarate (HTK; Custodiol) and HTK-N (Custodiol-N) on AKI in a large animal model were compared. Therefore, Landrace pigs underwent median sternotomy, CPB at 34°C, 90 minutes of cardiac arrest and 120 minutes of reperfusion. Animals were randomized for single-shot cardioplegia with either HTK (n = 10) or HTK-N (n = 10). Renal biopsies and sera were analyzed to determine AKI biomarkers and apoptosis. Compared to HTK, HTK-N induced a decreased extent of proximal tubule swelling (48.3 ± 1.6 µm vs 52.3 ± 1.1 µm, P = .05) and decreased cytochrome c release (0.26 ± 0.04 vs 0.46 ± 0.08, P = .04) without reaching statistical significance due to Bonferroni correction. Comparing baseline and postreperfusion levels, the hemoglobin (Hb) and blood calcium levels were lower in HTK-N (Hb baseline : 6.0 ± 0.6 mmol/L, Hb reperfusion : 6.2 ± 0.7 mmol/L, P = .12; Ca 2+ baseline : 1.36 ± 0.05 mmol/L, Ca 2+ reperfusion : 1.28 ± 0.05 mmol/L, P = .16) compared to the HTK group (Hb baseline : 5.9 ± 0.4 mmol/L, Hb reperfusion : 4.7 ± 0.8 mmol/L, P < .01; Ca 2+ baseline : 1.34 ± 0.07 mmol/L, Ca 2+ reperfusion : 1.24 ± 0.06 mmol/L, P < .01). The present study showed that HTK-N could positively affect the kidney during CPB. Hb and calcium levels were stabilized. A statistical trend was found showing that AKI-related proximal tubule swelling and cytochrome c release were diminished., (© 2019 John Wiley & Sons Australia, Ltd.)

Background: Tolerance induction following organ transplantation can be achieved by adoptive cell transfer of regulatory T-cells (T regs ) or dendritic cells (DCs). However, the target immunological profile is unknown. The present study aimed to identify an immunological profile connected to tolerance induction following heart transplantation (HTx)., Methods: Blood samples of long-term rejection-free HTx patients (LT-HTx, n = 20) and patients on the HTx waiting list (pre-HTx, n = 20) were compared. Flow cytometric and multiplex analyses of DCs, T regs , subsets of both cell types and serum cytokines were performed. Furthermore, principle component and cluster analysis was used to identify a target immunological profile using a multiparametric dataset., Results: Plasmacytoid DCs expressing blood DC antigen (BDCA) 2 and BDCA4 were significantly increased in LT-HTx patients (BDCA2 + : 29.4 ± 10.1%, p = .022; BDCA4 + : 26.4 ± 9.3%, p = .008) compared to pre-HTx patients (BDCA2 + : 22.8 ± 7.2%; BDCA4 + : 18.9 ± 7.4%). The percentage of total T regs and of their CD62L + subset was reduced in LT-HTx patients (%T regs : 9.1 ± 3.7%, p = .026; %CD62L + : 85.1 ± 11.9%, p = .009) compared to pre-HTx patients (%T regs : 11.8 ± 3.6%; %CD62L + : 93.3 ± 4.5%). LT-HTx patients showed different cytokine levels than pre-HTx patients. Principle component and cluster analysis revealed that the total DCs, BDCA2 + and BDCA4 + DCs and CD147 + T regs had the strongest influence to distinguish among long-term rejection-free and pre-HTx patients., Conclusion: In conclusion, we defined the immune status of pre-HTx patients and the target immunological profile of LT-HTx patients. These data may help to establish a monitoring tool that is based on a multiparametric dataset., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: The acquired von Willebrand syndrome (AvWS), which is associated with left ventricular assist device support, is caused by the loss of the von Willebrand factor (vWF) high molecular weight multimers (HMWMs). We investigated whether the implantation of the left ventricular assist device HeartMate 3 (HM 3) is superior to the HeartWare ventricular assist device (HVAD) in preserving the multimeric structure of vWF., Methods: In total, 70 patients with implanted HM 3 (n = 35) or HVAD (n = 35) were retrospectively investigated. HMWMs, intermediate molecular weight multimers and low molecular weight multimers were quantified by using a densitometric methodology. vWF antigen, vWF activity and vWF collagen-binding activity, as well as demographic and clinical data, were analysed., Results: AvWS, which is characterized by a decrease in vWF HMWMs, was found in 97.1% of patients in the HM 3 group and 100% of patients in the HVAD group. Compared to normal pooled plasma, HM 3 induced a reduction in HMWMs (40.7 ± 8.2% vs 26.7 ± 7.5%, P < 0.01) and an increase in low molecular weight multimers (31.3 ± 11.8% vs 42.7 ± 9.8%, P < 0.01), whereas HVAD patients exhibited an increase in the percentage of intermediate molecular weight multimers (28.0 ± 5.0% vs 38.4 ± 7.7%, P < 0.01) in addition to a decrease in the percentage of HMWM (23.0 ± 11.0%, P < 0.01). A comparison of both left ventricular assist device types showed a difference in vWF multimeric structure (HMWMs: P < 0.01, intermediate molecular weight multimer: P = 0.05, low molecular weight multimer: P = 0.03). Furthermore, vWF activity was elevated in patients with an implanted HM 3 device (153.7 ± 54.4%) compared to those with an HVAD device (126.3 ± 39.7%, P = 0.02)., Conclusions: Patients with an implanted HM 3 had more intact HMWMs and a higher vWF activity during device support. This may reduce the manifestation of AvWS in HM 3 patients and could thus lead to a lower bleeding complication rate., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Introduction: Cardiopulmonary bypass surgery is accompanied by an inflammatory response and pulmonary dysfunction that renders patients vulnerable to postoperative complications. The majority of studies investigating the inflammatory response in cardiopulmonary bypass focus on cytokine measurements. This study investigated the early response of peripheral blood cell types and early changes in lung tissue in on-pump versus off-pump cardiopulmonary bypass surgery., Methods: Landrace pigs were assigned to the following groups (n = 6 per group): 1. off-pump cardiopulmonary bypass, 2. conventional cardiopulmonary bypass, 3. heparin-coated cardiopulmonary bypass, 4. surface-reduced cardiopulmonary bypass, and 5. surface-reduced cardiopulmonary bypass plus lung perfusion. Surgery was performed under mild hyperthermia (32°C), with 90-minute ischemia and 180-minute reperfusion. Histological and flow cytometric analyses were performed., Results: Lung water content increased during reperfusion in heparin-coated (84.63 ± 2.99%) compared to conventional cardiopulmonary bypass (76.33 ± 4.56%, p = 0.04). Alveolar septal thickness increased during ischemia at heparin-coated (p < 0.01) and surface-reduced cardiopulmonary bypass plus lung perfusion (p = 0.05). Tumor necrosis factor expression increased significantly (p < 0.01) in peribronchial, perivascular, and peripheral lung areas in all on-pump groups, but not in off-pump cardiopulmonary bypass. The usage of heparin-coated cardiopulmonary bypass led to increased percentages of CD3 + CD4 + (p = 0.03) and CD3 + CD8 + (p = 0.01) T cells compared to an uncoated device. Natural killer and mature B lymphocytes decreased at conventional and surface-reduced cardiopulmonary bypass plus lung perfusion. Activated granulocytes and macrophages increased at conventional cardiopulmonary bypass and heparin-coated cardiopulmonary bypass., Conclusion: Off-pump cardiopulmonary bypass induces less immunological response and lung injury than on-pump surgery. The reduction of cardiopulmonary bypass surface reduces the inflammatory immune response induced by cardiopulmonary bypass. Lung perfusion of surface-reduced cardiopulmonary bypass diminished the extravasation caused by surface reduction of the cardiopulmonary bypass.

Objectives: Cardioplegic solutions induce cardiac arrest and protect cardiac tissue from ischaemia-reperfusion injury. However, the effects on the brain, which is vulnerable to cardiopulmonary bypass (CPB) surgery and ischaemia-reperfusion injury, mostly remain unknown. We investigated if cardioplegic solutions differ in their effects in altered oxygen conditions and in their ability to induce cerebral inflammation., Methods: Thirty pigs were subjected to a midline sternotomy and CPB at 34°C with 90 min cardiac arrest followed by 120 min reperfusion. Following randomization on a 1:1:1 basis, they received either a single shot of histidine-tryptophan-α-ketoglutarate (HTK)-Bretschneider solution (n = 10), histidine-tryptophan-α-ketoglutarate-N (HTK-N; n = 10) or HTK plus 1.2 mg/l cyclosporine A (HTK/CsA; n = 10). Brain regions of interest (frontal cortex, cerebellum, brain stem, diencephalon, colliculus superior) were analysed by real time quantitative reverse transcriptase polymerase chain reaction for hypoxia-inducible factor-1α (HIF-1α), tumour necrosis factor-α, interleukin (IL)-10, IL-1β and IL-1β receptor as well as by immunohistochemical analysis for HIF-1α. Blood gas and electrolyte analyses were performed., Results: Comparisons between baseline and reperfusion period levels revealed that HTK-N cardioplegia induced a smaller reduction of the haemoglobin content and blood calcium concentrations (hbbaseline: 5.97 ± 0.63 mmol/l; hbreperfusion: 6.16 ± 0.66 mmol/l; P = 0.428; Cabaseline2+: 1.36 ± 0.05 mmol/l; Careperfusion2+: 1.28 ± 0.05 mmol/l; P < 0.001) compared to HTK (hbbaseline: 5.93 ± 0.45 mmol/l; hbreperfusion: 4.72 ± 0.79 mmol/l; P = 0.001; Cabaseline2+: 1.34 ± 0.07 mmol/l; Careperfusion2+: 1.24 ± 0.06 mmol/l; P = 0.004) and HTK/CsA cardioplegia (hbbaseline: 5.88 ± 0.44 mmol/l; hbreperfusion: 5.14 ± 0.87 mmol/l; P = 0.040; Cabaseline2+: 1.38 ± 0.04 mmol/l; Careperfusion2+: 1.20 ± 0.14 mmol/l; P = 0.001). Brain region-specific regulation of the HIF-1α expression, no general HIF-1α activation and a lower tumour necrosis factor-α expression (pto HTK = 0.050, pto HTK/CsA = 0.013) were documented for HTK-N cardioplegia., Conclusions: HTK-N (Custodiol-N) induced fewer cerebral effects and less inflammation during CPB surgery than HTK and HTK/CsA cardioplegia. These data suggest that HTK-N exerts brain protective effects during and after CPB surgery., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Chronic pressure overload due to aortic valve stenosis leads to pathological cardiac hypertrophy and heart failure. Hypertrophy is accompanied by an increase in myocyte surface area, which requires a proportional increase in the number of cell-cell and cell-matrix contacts to withstand enhanced workload. In a proteomic analysis we identified nerve injury-induced protein 1 (Ninjurin1), a 16kDa transmembrane cell-surface protein involved in cell adhesion and nerve repair, to be increased in hypertrophic hearts from patients with aortic stenosis. We hypothesised that Ninjurin1 is involved in myocyte hypertrophy. We analyzed cardiac biopsies from aortic-stenosis patients and control patients undergoing elective heart surgery. We studied cardiac hypertrophy in mice after transverse aortic constriction and angiotensin II infusions, and performed mechanistic analyses in cultured myocytes. We assessed the physiological role of ninjurin1 in zebrafish during heart and skeletal muscle development. Ninjurin1 was increased in hearts of aortic stenosis patients, compared to controls, as well as in hearts from mice with cardiac hypertrophy. Besides the 16kDa Ninjurin1 (Ninjurin1-16) we detected a 24kDa variant of Ninjurin1 (Ninjurin1-24), which was predominantly expressed during myocyte hypertrophy. We disclosed that the higher molecular weight of Ninjurin1-24 was caused by N-glycosylation. Ninjurin1-16 was contained in the cytoplasm of myocytes where it colocalized with stress-fibers. In contrast, Ninjurin1-24 was localized at myocyte membranes. Gain and loss-of-function experiments showed that Ninjurin1-24 plays a role in myocyte hypertrophy and myogenic differentiation in vitro. Reduced levels of ninjurin1 impaired cardiac and skeletal muscle development in zebrafish. We conclude that Ninjurin1 contributes to myocyte growth and differentiation, and that these effects are mainly mediated by N-glycosylated Ninjurin1-24., Competing Interests: The authors have declared that no competing interests exist.

Background: Oxidative stress due to reactive oxygen species (ROS) production is a key factor in the development of heart failure (HF). This study investigated the thioredoxin (Trx) system, which plays a major role in antioxidant defense, in patients suffering from ischemic (ICM) or dilated (DCM) cardiomyopathy., Methods and Results: Myocardial tissue from ICM (n = 13) and DCM (n = 13) patients, as well as septal tissue of patients with aortic stenosis but without diagnosed hypertrophic cardiomyopathy or subaortic stenosis (control; n = 12), was analyzed for Trx1, Trx-interacting protein (TXNIP) and E3 ligase ITCH (E3 ubiquitin-protein ligase Itchy homolog) expression. Trx-reductase 1 (TXNRD1) amount and activity, cytosolic cytochrome C content, and apoptosis markers were quantified by means of enzyme-linked immunosorbent assay and multiplexing. Compared with control samples, ITCH and Trx1 expression, TXNRD1 amount and activity were reduced and TXNIP expression was increased in ICM (ITCH: P = .013; Trx1: P = .028; TXNRD1 amount: P = .035; TXNRD1 activity: P = .005; TXNIP: P = .014) but not in DCM samples. A higher level of the downstream apoptosis marker caspase-9 (ICM: 582 ± 262 MFI [P = .995]; DCM: 1251 ± 548 MFI [P = .002], control: 561 ± 214 MFI) was detected in DCM tissue. A higher expression of Bcl-2 was found in DCM (P = .011)., Conclusion: The Trx system was impaired in ICM but not in DCM. ITCH appeared to be responsible for the down-regulation of the Trx system. ROS-induced mitochondrial instability appeared to play a role in DCM., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The clinical use of extracorporeal photopheresis (ECP) is based on its ability to induce cell-mediated immune tolerance towards foreign and self-antigens. Up-to-date, no clear consensus consists on how to use ECP after heart transplantation (HTx). In this pilot study, we evaluated the stimulatory effects of ECP on immune cells in HTx patients., Methods: HTx patients received ECP therapy as prophylaxis of rejection (PRX, n = 15), to treat acute cellular rejection (ACR, n = 13) or cardiac allograft vasculopathy (CAV, n = 5). Three ECP cycles with monthly frequency were performed. Blood samples were taken before every ECP cycle and 2 months after the last ECP cycle and were analyzed for cytokines and the tolerance-inducing cell subsets regulatory T cells (T regs ), myeloid (mDCs), and plasmacytoid dendritic cells (pDCs)., Results: While ECP treatment induced first an increase of pDCs in the CAV group (baseline: 22.0% ± 9.6%, prior third ECP cycle: 8.6% ± 3.2%, follow-up: 31.5% ± 8.4%, P = .009), no significant changes of DC subsets and T regs were observed in the ACR- and in the PRX group. Furthermore, analysis of the immune balance showed different response profiles of pro- and anti-inflammatory cytokines among prophylactically ECP-treated patients and ECP-treated patients suffering from CAV or ACR., Conclusions: In our pilot study, we showed different stimulatory effects of ECP on pDCs and cytokines among prophylactic and therapeutic ECP therapy after HTx. Immunological monitoring should be included in a larger clinical study of ECP treatment following HTx and to identify predictable parameters for ECP efficacy., (© 2018 Wiley Periodicals, Inc.)

Objectives: The treatment of patients with extensive thoracic aortic disease involving the arch and descending aorta is often performed using the frozen elephant trunk technique (FET). Spinal cord blood flow (SCBF) in cervical, thoracic and lumbar sections prior, during and after aortic arch surgery were compared in conventional elephant trunk (cET) and FET technique in a pig model., Methods: German Landrace pigs (75-85 kg) underwent aortic arch surgery using the FET (n = 8) or cET (n = 8) techniques. The E-vita Open hybrid stent graft was applied in all FET animals. Regional SCBF was measured 4 times: (i) before cardiopulmonary bypass, (ii) after 1 h, (iii) after 3 h, and (iv) after 6 h of reperfusion using fluorescence microspheres. Spinal cord segments were examined histopathologically and by immunohistochemistry., Results: SCBF in FET decreased significantly from 0.13 ± 0.03 to 0.05 ± 0.02 ml/min/g after 1 h (P = 0.047). While at 3 h of reperfusion, SCBF increased and was comparable to baseline (0.09 ± 0.01 ml/min/g), beyond this time SCBF decreased again (0.05 ± 0.02 ml/min/g). A similar trend was found for SCBF in the cET group (baseline: 0.16 ± 0.04 ml/min/g, 1 h reperfusion: 0.02 ± 0.01 ml/min/g, 3 h reperfusion: 0.03 ± 0.01 ml/min/g and 6 h reperfusion: 0.02 ± 0.01 ml/min/g, P = 0.019). Cervical, thoracic and lumbar SCBF were also comparable in both groups. Histological analyses of spinal cord showed no differences in necrosis between cET and FET, while no differences were found for hypoxia-inducible factor-1α and apoptosis-inducing factor. In contrast, oxidative stress and caspase-induced apoptosis were higher in cET versus FET., Conclusions: The SCBF changed significantly during extensive aortic arch surgery with circulatory arrest and moderate hypothermia, but such changes were comparable between the FET and cET groups. The implantation of hybrid stent graft did not influence SCBF in thoracic and lumbar segments of the spinal cord. The immunohistological examination showed no differences between cET and FET regarding ischaemic damage and hypoxia-induced effects in spinal cord segments., (© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)