Ferritin Levels on Hospital Admission Predict Hypoxic-Ischemic Encephalopathy in Patients After Out-of-Hospital Cardiac Arrest: A Prospective Observational Single-Center Study.

AIM: Out-of-hospital cardiac arrest (OHCA) is a major health concern in Western societies. Poor outcome after OHCA is determined by the extent of hypoxic-ischemic encephalopathy (HIE). Dysregulation of iron metabolism has prognostic relevance in patients with ischemic stroke and sepsis. The aim of this study was to determine whether serum iron parameters help to estimate outcomes after OHCA. METHODS: In this prospective single-center study, 70 adult OHCA patients were analyzed. Serum ferritin, iron, transferrin (TRF), and TRF saturation (TRFS) were measured in blood samples drawn on day 0 (admission), day 2, day 4, and 6 months after the return of spontaneous circulation (ROSC). The association of 4 iron parameters with in-hospital mortality, neurological outcome (cerebral performance category [CPC]), and HIE was investigated by receiver operating characteristics and multivariate regression analyses. RESULTS: OHCA subjects displayed significantly increased serum ferritin levels on day 0 and lowered iron, TRF, and TRFS on days 2 and 4 after ROSC, as compared to concentrations measured at a 6-month follow-up. Iron parameters were not associated with in-hospital mortality or neurological outcomes according to the CPC. Ferritin on admission was an independent predictor of features of HIE on cranial computed tomography and death due to HIE. CONCLUSION: OHCA is associated with alterations in iron metabolism that persist for several days after ROSC. Ferritin on admission can help to predict HIE.

Querying a Clinical Data Warehouse for Combinations of Clinical and Imaging Data.

This study aims to show the feasibility and benefit of single queries in a research data warehouse combining data from a hospital's clinical and imaging systems. We used a comprehensive integration of a production picture archiving and communication system (PACS) with a clinical data warehouse (CDW) for research to create a system that allows data from both domains to be queried jointly with a single query. To achieve this, we mapped the DICOM information model to the extended entity-attribute-value (EAV) data model of a CDW, which allows data linkage and query constraints on multiple levels: the patient, the encounter, a document, and a group level. Accordingly, we have integrated DICOM metadata directly into CDW and linked it to existing clinical data. We included data collected in 2016 and 2017 from the Department of Internal Medicine in this analysis for two query inquiries from researchers targeting research about a disease and in radiology. We obtained quantitative information about the current availability of combinations of clinical and imaging data using a single multilevel query compiled for each query inquiry. We compared these multilevel query results to results that linked data at a single level, resulting in a quantitative representation of results that was up to 112% and 573% higher. An EAV data model can be extended to store data from clinical systems and PACS on multiple levels to enable combined querying with a single query to quickly display actual frequency data.

Secondary Prevention of Potentially Life-Threatening Arrhythmia Using Implantable Cardioverter Defibrillators in Patients with Biopsy-Proven Viral Myocarditis and Preserved Ejection Fraction.

BACKGROUND: Arrhythmia and sudden cardiac death (SCD) are known complications of acute viral myocarditis, regardless of ejection fraction (EF) at presentation. Whether such complications confer long-term risk is unknown, especially in those who present with preserved left ventricular (LV) function. No guidelines exist to the long-term reduction of arrhythmic death in such patients. METHOD: In this retrospective study, we analyzed the long-term results of implantable cardioverter defibrillator (ICD) treatment in patients after an acute phase of myocarditis with life-threatening arrhythmia. RESULTS: We identified 51 patients who had ICDs implanted following life-threatening arrhythmia presentation of confirmed acute viral myocarditis, despite preserved LVEF. Overall, 72.5% of patients had a clinical history of chest pain and viral infection with fever. Viral myocarditis was confirmed by cardiac magnetic resonance imaging (all had late enhancement) plus endomyocardial biopsies (most frequent were Epstein-Barr virus 29.4%, adenovirus 17.6%, and Coxsackie 17.6%), and 88.2% were discharged on anti-arrhythmic drugs. Overall, 12 patients (23.5%) required ICD intervention within the first 3 months, a further 7 patients (37.3% overall) between 3 and 12 months, and a further 12 patients (60.8% overall) until 58 months. During the follow-up, 3 of 51 patients (5.9%) died-deaths were due to cardiac events (n = 1), fatal infection (n = 1), and car accidents (n = 1). Of the 31 patients who had ventricular tachycardias after the acute phase of myocarditis, 11 needed radiofrequency ablation due to a high number of events or electrical storm. No baseline variables were identified that would serve as a basis for risk stratification. CONCLUSION: Malignant arrhythmic events due to viral myocarditis are potential predictors of future SCD in patients not only with a reduced but also with a preserved EF.

Unlocking the PACS DICOM Domain for its Use in Clinical Research Data Warehouses.

Clinical Data Warehouses (DWHs) are used to provide researchers with simplified access to pseudonymized and homogenized clinical routine data from multiple primary systems. Experience with the integration of imaging and metadata from picture archiving and communication systems (PACS), however, is rare. Our goal was therefore to analyze the viability of integrating a production PACS with a research DWH to enable DWH queries combining clinical and medical imaging metadata and to enable the DWH to display and download images ad hoc. We developed an application interface that enables to query the production PACS of a large hospital from a clinical research DWH containing pseudonymized data. We evaluated the performance of bulk extracting metadata from the PACS to the DWH and the performance of retrieving images ad hoc from the PACS for display and download within the DWH. We integrated the system into the query interface of our DWH and used it successfully in four use cases. The bulk extraction of imaging metadata required a median (quartiles) time of 0.09 (0.03-2.25) to 12.52 (4.11-37.30) seconds for a median (quartiles) number of 10 (3-29) to 103 (8-693) images per patient, depending on the extraction approach. The ad hoc image retrieval from the PACS required a median (quartiles) of 2.57 (2.57-2.79) seconds per image for the download, but 5.55 (4.91-6.06) seconds to display the first and 40.77 (38.60-41.63) seconds to display all images using the pure web-based viewer. A full integration of a production PACS with a research DWH is viable and enables various use cases in research. While the extraction of basic metadata from all images can be done with reasonable effort, the extraction of all metadata seems to be more appropriate for subgroups.

Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation.

We have previously demonstrated that inorganic polyphosphate (polyP) is a potent activator of the mitochondrial permeability transition pore (mPTP) in cardiac myocytes. PolyP depletion protected against Ca2+-induced mPTP opening, however it did not prevent and even exacerbated cell death during ischemia-reperfusion (I/R). The central goal of this study was to investigate potential molecular mechanisms underlying these dichotomous effects of polyP on mitochondrial function. We utilized a Langendorff-perfused heart model of I/R to monitor changes in polyP size and chain length at baseline, 20 min no-flow ischemia, and 15 min reperfusion. Freshly isolated cardiac myocytes and mitochondria from C57BL/6J (WT) and cyclophilin D knock-out (CypD KO) mice were used to measure polyP uptake, mPTP activity, mitochondrial membrane potential, respiration and ATP generation. We found that I/R induced a significant decrease in polyP chain length. We, therefore, tested, the ability of synthetic polyPs with different chain length to accumulate in mitochondria and induce mPTP. Both short and long chain polyPs accumulated in mitochondria in oligomycin-sensitive manner implicating potential involvement of mitochondrial ATP synthase in polyP transport. Notably, only short-chain polyP activated mPTP in WT myocytes, and this effect was prevented by mPTP inhibitor cyclosprorin A and absent in CypD KO myocytes. To the contrary, long-chain polyP suppressed mPTP activation, and enhanced ADP-linked respiration and ATP production. Our data indicate that 1) effect of polyP on cardiac function strongly depends on polymer chain length; and 2) short-chain polyPs (as increased in ischemia-reperfusion) induce mPTP and mitochondrial uncoupling, while long-chain polyPs contribute to energy generation and cell metabolism.

Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure.

Trimetazidine (TMZ) is used successfully for treatment of ischemic cardiomyopathy, however its therapeutic potential in heart failure (HF) remains to be established. While the cardioprotective action of TMZ has been linked to inhibition of free fatty acid oxidation (FAO) via 3-ketoacyl CoA thiolase (3-KAT), additional mechanisms have been suggested. The aim of this study was to evaluate systematically the effects of TMZ on calcium signaling and mitochondrial function in a rabbit model of non-ischemic HF and to determine the cellular mechanisms of the cardioprotective action of TMZ. TMZ protected HF ventricular myocytes from cytosolic Ca(2+) overload and subsequent hypercontracture, induced by electrical and ß-adrenergic (isoproterenol) stimulation. This effect was mediated by the ability of TMZ to protect HF myocytes against mitochondrial permeability transition pore (mPTP) opening via attenuation of reactive oxygen species (ROS) generation by the mitochondrial electron transport chain (ETC) and uncoupled mitochondrial nitric oxide synthase (mtNOS). The majority of ROS generated by the ETC in HF arose from enhanced complex II-mediated electron leak. TMZ inhibited the elevated electron leak at the level of mitochondrial ETC complex II and improved impaired activity of mitochondrial complex I, thereby restoring redox balance and mitochondrial membrane potential in HF. While TMZ decreased FAO by ~15%, the 3-KAT inhibitor 4-bromotiglic acid did not provide protection against palmitic acid-induced mPTP opening, indicating that TMZ effects were 3-KAT independent. Thus, the beneficial effect of TMZ in rabbit HF was not linked to FAO inhibition, but rather associated with reduced complex II- and uncoupled mtNOS-mediated oxidative stress and decreased propensity for mPTP opening.

A Specialized System for Arrhythmia Detection for Basic Research in Cardiology.

The detection of cardiac arrhythmias has a long history in medicine, with current developments focusing on early detection using mobile devices. In basic research, however, the use cases and data differ greatly from the experimental setup. We developed a Python-based system to ease detection and analysis of arrhythmic sections in signals measured on extracted and stimulated cardiac myocytes. Multiple algorithms were integrated into the system, tested and evaluated. The best algorithm resulted in an F1-score of 0.97 and was primarily provided in the application.

PK11195 Protects From Cell Death Only When Applied During Reperfusion: Succinate-Mediated Mechanism of Action.

Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 µM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 µM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 µM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 µM cyclosporin A and almost completely prevented by 50 µM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization. Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.

Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides.

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 µm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4-5 µm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB ß-hydroxybutyrate (ß-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where ß-OHB further increased the number of cells with giant Parkin-rich regions. This ß-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.

Information Extraction from Echocardiography Reports for a Clinical Follow-up Study-Comparison of Extracted Variables Intended for General Use in a Data Warehouse with Those Intended Specifically for the Study.

BACKGROUND: The interest in information extraction from clinical reports for secondary data use is increasing. But experience with the productive use of information extraction processes over time is scarce. A clinical data warehouse has been in use at our university hospital for several years, which also provides an information extraction of echocardiography reports developed for general use. OBJECTIVES: This study aims to illustrate the difficulties encountered, while using data from a preexisting information extraction process for a large clinical study. To compare the data from the preexisting process with the data obtained from a specially developed process designed to improve the quality and completeness of the study data. METHODS: We extracted the echocardiography variables for 440 patients from the general-use information extraction of the data warehouse (678 reports). Then we developed an information extraction process for the same variables but specifically for this study, with the aim to extract as much information as possible from the text. The extracted data of both processes were compared with a newly created gold standard defined by a cardiologist with long-standing experience in heart failure. RESULTS: Among 57 echocardiography variables considered relevant for the study, 50 were documented in the routine text reports and could be extracted. Twenty of the required variables were not provided by the general-use extraction process, some others were not provided correctly. The median macro F1-score (precision, recall) across the 30 variables for which values were extracted was 0.81 (0.94, 0.77). Across all 50 variables, as relevant for the study, median macro F1-score was only 0.49 (0.56, 0.46). Employing the study-specific approach considerably improved the quality and completeness of the variables, resulting in F1-scores of 0.97 (0.98, 0.96) across all variables. CONCLUSION: Data from information extractions can be used for large clinical studies. However, preexisting information extraction processes should be treated with caution, as the time and effort spent defining each variable in the information extraction process may not be clear.

Mitofusin 2 Is Essential for IP3-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes.

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP3R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).

Underestimated prevalence of heart failure in hospital inpatients: a comparison of ICD codes and discharge letter information.

BACKGROUND: Heart failure is the predominant cause of hospitalization and amongst the leading causes of death in Germany. However, accurate estimates of prevalence and incidence are lacking. Reported figures originating from different information sources are compromised by factors like economic reasons or documentation quality. METHODS: We implemented a clinical data warehouse that integrates various information sources (structured parameters, plain text, data extracted by natural language processing) and enables reliable approximations to the real number of heart failure patients. Performance of ICD-based diagnosis in detecting heart failure was compared across the years 2000-2015 with (a) advanced definitions based on algorithms that integrate various sources of the hospital information system, and (b) a physician-based reference standard. RESULTS: Applying these methods for detecting heart failure in inpatients revealed that relying on ICD codes resulted in a marked underestimation of the true prevalence of heart failure, ranging from 44% in the validation dataset to 55% (single year) and 31% (all years) in the overall analysis. Percentages changed over the years, indicating secular changes in coding practice and efficiency. Performance was markedly improved using search and permutation algorithms from the initial expert-specified query (F1 score of 81%) to the computer-optimized query (F1 score of 86%) or, alternatively, optimizing precision or sensitivity depending on the search objective. CONCLUSIONS: Estimating prevalence of heart failure using ICD codes as the sole data source yielded unreliable results. Diagnostic accuracy was markedly improved using dedicated search algorithms. Our approach may be transferred to other hospital information systems.

Nuclear calcineurin is a sensor for detecting Ca2+ release from the nuclear envelope via IP3R.

In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca2+ levels. We therefore hypothesize that decoding Ca2+ signals for hypertrophic signaling requires intracellular Ca2+ microdomains that are partly independent from cytosolic Ca2+. Furthermore, there is a need for a Ca2+ sensor within these microdomains that translates Ca2+ signals into hypertrophic signaling. Recent evidence suggested that the nucleus of cardiac myocytes might be a Ca2+ microdomain and that calcineurin, once translocated into the nucleus, could act as a nuclear Ca2+ sensor. We demonstrate that nuclear calcineurin was able to act as a nuclear Ca2+ sensor detecting local Ca2+ release from the nuclear envelope via IP3R. Nuclear calcineurin mutants defective for Ca2+ binding failed to activate NFAT-dependent transcription. Under hypertrophic conditions Ca2+ transients in the nuclear microdomain were significantly higher than in the cytosol providing a basis for sustained calcineurin/NFAT-mediated signaling uncoupled from cytosolic Ca2+. Measurements of nuclear and cytosolic Ca2+ transients in IP3 sponge mice showed no increase of Ca2+ levels during diastole as we detected in wild-type mice. Nuclei, isolated from ventricular myocytes of mice after chronic Ang II treatment, showed an elevation of IP3R2 expression which was dependent on calcineurin/NFAT signaling and persisted for 3 weeks after removal of the Ang II stimulus. These data provide an explanation how Ca2+ and calcineurin might regulate transcription in cardiomyocytes in response to neurohumoral signals independently from their role in cardiac contraction control. KEY MESSAGES: • Calcineurin acts as an intranuclear Ca2+ sensor to promote NFAT activity. • Nuclear Ca2+ in cardiac myocytes increases via IP3R2 upon Ang II stimulation. • IP3R2 expression is directly dependent on calcineurin/NFAT.

Inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum-mitochondrial crosstalk influences adenosine triphosphate production via mitochondrial Ca2+ uptake through the mitochondrial ryanodine receptor in cardiac myocytes.

AIMS: Elevated levels of inositol 1,4,5-trisphosphate (IP3) in adult cardiac myocytes are typically associated with the development of cardiac hypertrophy, arrhythmias, and heart failure. IP3 enhances intracellular Ca(2+ )release via IP3 receptors (IP3Rs) located at the sarcoplasmic reticulum (SR). We aimed to determine whether IP3-induced Ca(2+ )release affects mitochondrial function and determine the underlying mechanisms. METHODS AND RESULTS: We compared the effects of IP3Rs- and ryanodine receptors (RyRs)-mediated cytosolic Ca(2+ )elevation achieved by endothelin-1 (ET-1) and isoproterenol (ISO) stimulation, respectively, on mitochondrial Ca(2+ )uptake and adenosine triphosphate (ATP) generation. Both ET-1 and isoproterenol induced an increase in mitochondrial Ca(2+ )(Ca(2 +) m) but only ET-1 led to an increase in ATP concentration. ET-1-induced effects were prevented by cell treatment with the IP3 antagonist 2-aminoethoxydiphenyl borate and absent in myocytes from transgenic mice expressing an IP3 chelating protein (IP3 sponge). Furthermore, ET-1-induced mitochondrial Ca(2+) uptake was insensitive to the mitochondrial Ca(2+ )uniporter inhibitor Ru360, however was attenuated by RyRs type 1 inhibitor dantrolene. Using real-time polymerase chain reaction, we detected the presence of all three isoforms of IP3Rs and RyRs in murine ventricular myocytes with a dominant presence of type 2 isoform for both receptors. CONCLUSIONS: Stimulation of IP3Rs with ET-1 induces Ca(2+ )release from the SR which is tunnelled to mitochondria via mitochondrial RyR leading to stimulation of mitochondrial ATP production.

Distinct mPTP activation mechanisms in ischaemia-reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate.

AIMS: The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischaemia-reperfusion (I/R). We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca(2+)-induced mPTP opening, towards mPTP activation and cardiac cell death in I/R. METHODS AND RESULTS: A significant increase in mitochondrial free calcium concentration ([Ca(2+)]m), reactive oxygen species (ROS) generation, mitochondrial membrane potential depolarization (ΔΨm), and mPTP activity, but no cell death, was observed after 20 min of ischaemia. The [Ca(2+)]m increase during ischaemia was partially prevented by the mitochondrial Ca(2+) uniporter (MCU) inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene, suggesting two complimentary Ca(2+) uptake mechanisms. In the absence of Ru360 and dantrolene, mPTP closing by polyP depletion or CSA decreased mitochondrial Ca(2+) uptake, suggesting that during ischaemia Ca(2+) can enter mitochondria through mPTP. During reperfusion, a burst of endogenous polyP production coincided with a decrease in [Ca(2+)]m, a decline in superoxide generation, and an acceleration of hydrogen peroxide (H2O2) production. An increase in H2O2 correlated with restoration of mitochondrial pHm and an increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride]. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. CONCLUSIONS: Transient Ca(2+)/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca(2+) overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

Eya4 Induces Hypertrophy via Regulation of p27kip1.

BACKGROUND: E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy. METHODS AND RESULTS: In this study, we first show Eya4 and E193 alter the expression of p27(kip1) in vitro, suggesting Eya4 is a negative regulator of p27. Next, we generated transgenic mice with cardiac-specific overexpression of Eya4 or E193. Luciferase and chromatin immunoprecipitation assays confirmed Eya4 and E193 bind and regulate p27 expression in a contradictory manner. Activity and phosphorylation status of the downstream molecules casein kinase-2α and histone deacetylase 2 were significantly elevated in Eya4- but significantly reduced in E193-overexpressing animals compared with wild-type littermates. Magnetic resonance imaging and hemodynamic analysis indicate Eya4-overexpression results in an age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 animals develop onset of dilative cardiomyopathy as seen in human E193 patients. Both cardiac phenotypes were aggravated on pressure overload. Finally, we identified a new heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein as seen with E193. CONCLUSIONS: Our results implicate Eya4/Six1 regulates normal cardiac function via p27/casein kinase-2α/histone deacetylase 2 and indicate that mutations within this transcriptional complex and signaling cascade lead to the development of cardiomyopathy.

Impact of thoracic surgery on cardiac morphology and function in small animal models of heart disease: a cardiac MRI study in rats.

BACKGROUND: Surgical procedures in small animal models of heart disease might evoke alterations in cardiac morphology and function. The aim of this study was to reveal and quantify such potential artificial early or long term effects in vivo, which might account for a significant bias in basic cardiovascular research, and, therefore, could potentially question the meaning of respective studies. METHODS: Female Wistar rats (n = 6 per group) were matched for weight and assorted for sham left coronary artery ligation or control. Cardiac morphology and function was then investigated in vivo by cine magnetic resonance imaging at 7 Tesla 1 and 8 weeks after the surgical procedure. The time course of metabolic and inflammatory blood parameters was determined in addition. RESULTS: Compared to healthy controls, rats after sham surgery showed a lower body weight both 1 week (267.5±10.6 vs. 317.0±11.3 g, n<0.05) and 8 weeks (317.0±21.1 vs. 358.7±22.4 g, n<0.05) after the intervention. Left and right ventricular morphology and function were not different in absolute measures in both groups 1 week after surgery. However, there was a confined difference in several cardiac parameters normalized to the body weight (bw), such as myocardial mass (2.19±0.30/0.83±0.13 vs. 1.85±0.22/0.70±0.07 mg left/right per g bw, p<0.05), or enddiastolic ventricular volume (1.31±0.36/1.21±0.31 vs. 1.14±0.20/1.07±0.17 µl left/right per g bw, p<0.05). Vice versa, after 8 weeks, cardiac masses, volumes, and output showed a trend for lower values in sham operated rats compared to controls in absolute measures (782.2±57.2/260.2±33.2 vs. 805.9±84.8/310.4±48.5 mg, p<0.05 for left/right ventricular mass), but not normalized to body weight. Matching these findings, blood testing revealed only minor inflammatory but prolonged metabolic changes after surgery not related to cardiac disease. CONCLUSION: Cardio-thoracic surgical procedures in experimental myocardial infarction cause distinct alterations upon the global integrity of the organism, which in the long term also induce circumscribed repercussions on cardiac morphology and function. This impact has to be considered when analyzing data from respective animal studies and transferring these findings to conditions in patients.

Inorganic polyphosphate--an unusual suspect of the mitochondrial permeability transition mystery.

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (P(i)) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca(2+)-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes.

Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia-reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280±60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.

Endothelin-1 enhances nuclear Ca2+ transients in atrial myocytes through Ins(1,4,5)P3-dependent Ca2+ release from perinuclear Ca2+ stores.

Nuclear Ca2+ plays a key role in the regulation of gene expression. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3)] might be an important regulator of nuclear Ca2+ but its contribution to nuclear Ca2+ signalling in adult cardiomyocytes remains elusive. We tested the hypothesis that endothelin-1 enhances nuclear Ca2+ concentration transients (CaTs) in rabbit atrial myocytes through Ins(1,4,5)P3-induced Ca(2+) release from perinuclear stores. Cytoplasmic and nuclear CaTs were measured simultaneously in electrically stimulated atrial myocytes using confocal Ca2+ imaging. Nuclear CaTs were significantly slower than cytoplasmic CaTs, indicative of compartmentalisation of intracellular Ca2+ signalling. Endothelin-1 elicited a preferential (10 nM) or a selective (0.1 nM) increase in nuclear versus cytoplasmic CaTs. This effect was abolished by inhibition of endothelin-1 receptors, phospholipase C and Ins(1,4,5)P3 receptors. Fractional Ca2+ release from the sarcoplasmic reticulum and perinuclear stores was increased by endothelin-1 at an otherwise unaltered Ca2+ load. Comparable increases of cytoplasmic CaTs induced by beta-adrenoceptor stimulation or elevation of extracellular Ca2+ could not mimic the endothelin-1 effects on nuclear CaTs, suggesting that endothelin-1 specifically modulates nuclear Ca2+ signalling. Thus, endothelin-1 enhances nuclear CaTs in atrial myocytes by increasing fractional Ca2+ release from perinuclear stores. This effect is mediated by the coupling of endothelin receptor A to PLC-Ins(1,4,5)P3 signalling and might contribute to excitation-transcription coupling.