Michael Sereinigg

JTV519 (K201) reduces sarcoplasmic reticulum Ca2+ leak and improves diastolic function in vitro in murine and human non-failing myocardium

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4‐benzothiazepine derivative and multi‐channel blocker, stabilizes RyR2s and decrease SR Ca2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non‐failing human myocardium and explored underlying mechanisms.

Early Remodeling of Perinuclear Ca2+ Stores and Nucleoplasmic Ca2+ Signaling During the Development of Hypertrophy and Heart Failure

Background— A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

Oxidative stress and apoptosis in a pig model of brain death (BD) and living donation (LD)

BackgroundAs organ shortage is increasing, the acceptance of marginal donors increases, which might result in poor organ function and patient survival. Mostly, organ damage is caused during brain death (BD), cold ischemic time (CIT) or after reperfusion due to oxidative stress or the induction of apoptosis. The aim of this study was to study a panel of genes involved in oxidative stress and apoptosis and compare these findings with immunohistochemistry from a BD and living donation (LD) pig model and after cold ischemia time (CIT).MethodsBD was induced in pigs; after 12 h organ retrieval was performed; heart, liver and kidney tissue specimens were collected in the BD (n = 6) and in a LD model (n = 6). PCR analysis for NFKB1, GSS, SOD2, PPAR-alpha, OXSR1, BAX, BCL2L1, and HSP 70.2 was performed and immunohistochemistry used to show apoptosis and nitrosative stress induced cell damage.ResultsIn heart tissue of BD BAX, BCL2L1 and HSP 70.2 increased significantly after CIT. Only SOD2 was over-expressed after CIT in BD liver tissue. In kidney tissue, BCL2L1, NFKB, OXSR1, SOD2 and HSP 70.2 expression was significantly elevated in LD. Immunohistochemistry showed a significant increase in activated Caspase 3 and nitrotyrosine positive cells after CIT in BD in liver and in kidney tissue but not in heart tissue.ConclusionThe up-regulation of protective and apoptotic genes seems to be divergent in the different organs in the BD and LD setting; however, immunohistochemistry revealed more apoptotic and nitrotyrosine positive cells in the BD setting in liver and kidney tissue whereas in heart tissue both BD and LD showed an increase.

Myeloperoxidase and carbonyl proteins: Promising markers for non-invasive monitoring of graft rejection after heart transplantation

BACKGROUND After heart transplantation (HTx), endomyocardial biopsy (EMB) is currently the standard method to diagnose acute graft rejection. A non-invasive marker of rejection would be desirable as an alternative or to permit more selective use of the costly and invasive EMB. METHODS In this retrospective study, outcomes of routinely taken EMBs were used to select 28 patients after HTx EMB Grade 0R (8 patients), 1R (9 patients) or 2R (11 patients). For these patients, myeloperoxidase (MPO) and carbonyl proteins (CP) in serum were measured using enzyme-linked immunoassay (ELISA). RESULTS MPO and CP levels in post-HTx patients with Grade 2R rejection were significantly (MPO: p < 0.01; CP: p < 0.001) elevated at the time of rejection compared with levels 1 month earlier. MPO and CP levels predicted Grade 2R rejection and the best cut-off point was 237.5 μg/l for MPO and 222.5 pmol/mg for CP, respectively. Clinically most important was the marked increase (doubling of basic values within 1 month) of MPO and CP levels in cases of Grade 2R rejection in post-HTx patients. CONCLUSIONS MPO and CP seem to be appropriate parameters to monitor rejection events non-invasively and to minimize the application of EMBs after HTx.

A 10 min “no-touch” time – is it enough in DCD? A DCD Animal Study

Summary Donation after cardiac death (DCD) is under investigation because of the lack of human donor organs. Required times of cardiac arrest vary between 75 s and 27 min until the declaration of the patients’ death worldwide. The aim of this study was to investigate brain death in pigs after different times of cardiac arrest with subsequent cardiopulmonary resuscitation (CPR) as a DCD paradigm. DCD was simulated in 20 pigs after direct electrical induction of ventricular fibrillation. The “no‐touch” time varied from 2 min up to 10 min; then 30 min of CPR were performed. Brain death was determined by established clinical and electrophysiological criteria. In all animals with cardiac arrest of at least 6 min, a persistent loss of brainstem reflexes and no reappearance of bioelectric brain activity occurred. Reappearance of EEG activity was found until 4.5 min of cardiac arrest and subsequent CPR. Brainstem reflexes were detectable until 5 min of cardiac arrest and subsequent CPR. According to our experiments, the suggestion of 10 min of cardiac arrest being equivalent to brain death exceeds the minimum time after which clinical and electrophysiological criteria of brain death are fulfilled. Therefore shorter “no‐touch” times might be ethically acceptable to reduce warm ischemia time.

Case of Paracoccus yeei infection documented in a transplanted heart

M. Schweiger, P. Stiegler, M. Scarpatetti, A. Wasler, M. Sereinigg, G. Prenner, K. Tscheliessnigg. Case of Paracoccus yeei infection documented in a transplanted heart.
Transpl Infect Dis 2011: 13: 200–203. All rights reserved

Knowledge and attitude of ICU nurses, students and patients towards the Austrian organ donation law

BackgroundA survey on the knowledge and attitudes towards the Austrian organ donation legislation (an opt-out solution) of selected groups of the Austrian population taking into account factors such as age, gender, level of education, affiliation to healthcare professions and health related studies was conducted.MethodsAn online survey among 3 target groups (ICU nurses, health science students and non health science students) was performed and results were compared to the answers from transplantation patients to a paper questionnaire. A total of 8415 persons were asked to participate in the survey and 2025 (24%) persons correctly completed the questionnaire. 1945 online responses (ICU nurses n = 185; students of health sciences n = 1277; students of non-health science related courses n = 483) were analysed and data were compared to 80 manually filled-in responses from patients from a previous study.Results84% of participants state that they know the Austrian organ donation legislation; this percentage varies significantly (p < 0.05) within the target groups and is influenced by demographic variables of the participants. 74% think that the law is good and 79% do not favour a change. Opinions and attitudes towards the legal situation are positively influenced by the affiliation to healthcare professions and health-related fields of study. Interviewed persons who were aware of the legislation before the survey had a more positive attitude towards the existing legislation (77% versus 74%, p < 0.05).ConclusionsThe information level on Austrian organ donation legislation is high. ICU nurses and those who did not know the law before were most critical towards the existing legislation. Therefore education to increase knowledge in the general population and goal-oriented efforts to increase awareness in the target groups should be emphasized.

Massive Diaphragmatic Herniation Following Orthotopic Liver Transplantation in an Adult

Four years after orthotopic liver transplantation wasperformed for cirrhosis due to chronic hepatitis C, a58-year-old woman presented with nausea and vomit-ing, abdominal pain, a feeling of fullness and tensionin the upper abdomen, fatigue, and a loss of 10 kg ofbody weight during the preceding 8 weeks. She hadfevers up to 38.5 C (101 F). Abdominal X-rays, ab-dominal and cardiac ultrasound examinations, andgastroscopy indicated no abnormalities. However, theresults of serum biochemical liver tests were as fol-lows: aspartate aminotransferase, 483 U/L (normallevel < 30 U/L); alanine aminotransferase, 200 U/L(normal level < 35 U/L); gamma-glutamyl transferase,742 U/L (normal level < 38 U/L); bilirubin, 2.36 mg/dL (normal range ¼ 0.1-1.2 mg/dL); alkaline phos-phatase, 161 U/L (normal range ¼ 35-105 U/L); andlactate dehydrogenase, 325 U/L (normal range ¼ 120-240 U/L). A liver biopsy sample exhibited changesthat suggested allograft rejection rather than a recur-rence of hepatitis C. In addition to baseline immuno-suppression with everolimus (trough level ¼ 4.8 ng/mL) and mycophenolate mofetil (trough level ¼ 1.1lg/mL), the patient received corticosteroids to treatallograft rejection. Although the serum biochemicalliver tests gradually improved, her symptoms did not.Subjective indices of the patient’s health and well-being did not correlate with objective findings. Theadministration of analgesics and antidepressants wasassociated with only minor improvements in hersymptoms. However, abdominal computed tomogra-phy revealed a large diaphragmatic herniation; itsdimensions were 15 cm 10 cm. The entire left he-patic lobe had shifted into the mediastinum (Fig. 1).Subsequent thoracic magnetic resonance imagingconfirmed the magnitude of the herniation. The infe-rior vena cava was severely compressed, and the rightventricle was displaced by the herniated liver.The herniation was closed completely during a com-bined thoracotomy-laparotomy procedure. Intraopera-tively, most of the liver was found to have herniatedinto the thorax (Fig. 2). The liver had displaced theright atrium and the right ventricle (Fig. 1). In addi-tion, the displaced liver tightly adhered to the entire

Docosahexaenoic acid (DHA)-induced heme oxygenase-1 attenuates cytotoxic effects of DHA in vascular smooth muscle cells.

OBJECTIVE Docosahexaenoic acid (DHA), a member of n-3 polyunsaturated fatty acids (n-3 PUFA) is a potent regulator of molecular events implicated in cardiovascular health. In a previous study we found that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of unfolded protein response (UPR), cell cycle arrest and apoptosis in DHA treated primary human smooth muscle cells isolated from small pulmonary artery (hPASMC). In the present study we examined the impact of heme oxygenase (HO)-1, induced by DHA, on DHA-induced oxidative stress, UPR, cell proliferation and apoptosis in hPASMC. METHODS & RESULTS DHA led to a time- and concentration-dependent increase in HO-1 mRNA and protein levels in hPASMC. The DHA-induced HO-1 upregulation could be attenuated by preincubation of cells with a strong antioxidant Tempol or by siRNA-mediated depletion of nuclear factor erythroid 2-related factor-2 (Nrf2). In DHA-treated hPASMC, depletion of HO-1 by siRNA-mediated silencing resulted in increased levels of reactive oxygen species (ROS) and increased duration of UPR, the latter revealed by monitoring of spliced X-box binding protein 1 (XBP-1) variant. Moreover, HO-1 silencing augmented apoptosis in DHA-treated hPASMC as found by increased numbers of cleaved caspase-3-positive cells. HO-1 silencing did not affect proliferation of hPASMC exposed to DHA. CONCLUSION Our results indicate that DHA-induced, ROS-dependent upregulation of HO-1 attenuates oxidative stress, UPR and apoptosis in DHA-treated hPASMC.

Establishing a brain-death donor model in pigs.

INTRODUCTION An animal model that imitates human conditions might be useful not only to monitor pathomechanisms of brain death and biochemical cascades but also to investigate novel strategies to ameliorate organ quality and functionality after multiorgan donation. METHODS Brain death was induced in 15 pigs by inserting a catheter into the intracranial space after trephination of the skull and augmenting intracranial pressure until brain stem herniation. Intracranial pressure was monitored continuously; after 60 minutes, brain death diagnostics were performed by a neurologist including electroencephalogram (EEG) and clinical examinations. Clinical examinations included testing of brain stem reflexes as well as apnoe testing; then intensive donor care was performed according to standard guidelines until 24 hours after confirmation of brain death. Intensive donor care was performed according to standard guidelines for 24 hours after brain death. RESULTS Sixty minutes after brain-death induction, neurological examination and EEG examination confirmed brain death. Intracranial pressure increased continuously, remaining stable after the occurrence of brain death. All 15 animals showed typical signs of brain death such as diabetes insipidus, hypertensive and hypotensive periods, as well as tachycardia. All symptoms were treated with standard medications. After 24 hours of brain death we performed successful multiorgan retrieval. DISCUSSION Brain death can be induced in a pig model by inserting a catheter after trephination of the skull. According to standard guidelines the brain-death diagnosis was established by a flat-line EEG, which occurred in all animals at 60 minutes after induction.