Die Rolle von STAT3 bei der Regulation mitochondrialer reaktiver Sauerstoffspezies bei der Peripartalen Kardiomyopathie

Die Peripartale Kardiomyopathie (PPCM) entwickelt sich vorwiegend im letzten Monat der Schwangerschaft, während des Geburtsvorgangs oder den ersten Wochen nach der Entbindung bei zumeist herzgesunden Frauen und manifestiert sich insbesondere durch eine linksventrikuläre systolische Dysfunktion. Die Expression des signal transducer and activator of transcription 3 (STAT3) ist im Myokard von Patientinnen mit PPCM erniedrigt. Als Transkriptionsfaktor ist STAT3 in zahlreiche protektive Signalkaskaden über den JAK/STAT-Signaltransduktionsweg involviert. Dabei konnte in der Vergangenheit bereits gezeigt werden, dass es bei der chronischen Herzinsuffizienz im Rahmen der Dilatativen Kardiomyopathie (DCM) zu einer Herunterregulierung dieses Signaltransduktionswegs kam. Darüber hinaus reguliert STAT3 die Transkription der mitochondrialen manganhaltigen Superoxid-Dismutase (Mn-SOD) und spielt somit eine zentrale Rolle in der antioxidativen Abwehr gegenüber reaktiven Sauerstoffspezies (ROS), welche maßgeblich am Progress der Herzinsuffizenz beteiligt sind. Superoxidanionen- Radikale fallen durch unvollständige Reduktion von O2 an der mitochondrialen Atmungskette an und werden durch die Mn-SOD zu H2O2 dismutiert. H2O2 wird in der Folge durch NADPHabhängige Enzymsysteme zu H2O entgiftet. Oxidativer Stress stellt in der Pathogenese der PPCM einen bedeutenden Faktor dar, da er die Sekretion des proteolytischen Enzyms Cathepsin D triggert. Cathepsin D wandelt das im Zuge der Schwangerschaft ausgeschüttete Stillhormon Prolaktin in das maladaptive 16-kDa Prolaktin (16-kDa-PRL) Spaltprodukt um. Dieses 16-kDa- PRL soll durch seine nachweislich angiostatische und proapoptotische Wirkung in Endothelzellen kausal am Progress der PPCM beteiligt sein, da es die myokardiale Mikrozirkulation kritisch beeinträchtigt. Die Behandlung mit Bromocriptin, welche die Prolaktinsekretion durch Agonismus am Dopamin-D2-Rezeptor inhibiert, konnte das Entstehen der PPCM in einem Tiermodell vollständig verhindern. Auch in ersten klinischen Studien konnte man bei Patientinnen, die zusätzlich zur leitliniengerechten Herzinsuffizienz-Therapie Bromocriptin erhalten haben, eine deutlich verbesserte Herzfunktion und höhere Überlebensraten erzielen. Das Ziel dieser Arbeit war es die mitochondriale Energetik, die ROS-Emission und insbesondere die Aktivität der Mn-SOD an isolierten Mitochondrien von Mäusen mit einem Kardiomyozytenspezifischen STAT3-Knockout zu analysieren, wobei wir eine unbehandelte und eine mit Bromocriptin behandelte Versuchsgruppe miteinander verglichen. Darüber hinaus untersuchten wir die Kontraktilität, den Redoxstatus anhand der Autofluoreszenz von NAD(P)H/FAD und die Superoxid-Emission mithilfe des fluoreszierenden Farbstoffs MitoSOX-Red bei elektrischer Feldstimulation an intakten Kardiomyozyten aus diesen beiden Versuchsgruppen, um potentielle Auswirkungen auf die elektromechanische Kopplung (EC) zu identifizieren. Für unsere Experimente wurden Mäuse mit einer Kardiomyozyten-spezifischen Deletion von STAT3 mit Wildtyp-Mäusen, jeweils nach 2 Schwangerschaften, jeweils mit und ohne Bromocriptin- Therapie, verglichen. Unsere Ergebnisse zeigen eine signifikant reduzierte mitochondriale Atmung bei deutlich höheren Superoxid-Konzentrationen in den isolierten Mitochondrien der STAT3-KO Mäuse. Die H2O2-Emission war hingegen reduziert, sodass wir daraus eine verminderte Aktivität der Mn-SOD, die sich aus dem H2O2/·O2-Verhältnis ergibt, schlussfolgern. Auf zellulärer Ebene konnten wir keinen Unterschied in den Zellverkürzungskinetiken, dem Redoxstatus von NAD(P)H/FAD und der Superoxid-Emission, selbst nicht nach Arbeitslasterhöhung (durch Hinzugabe von Isoprenalin und Erhöhung der elektrischen Feldstimulation von 0,5 Hz auf 5 Hz), feststellen. Die Behandlung mit Bromocriptin konnte bei den STAT3-KO Mäusen eine deutliche Verbesserung bzw. Wiederherstellung der mitochondrialen Atmungskapazität erzielen. Wir schlussfolgern aus unseren Ergebnissen, dass die verminderte Aktivität der Mn-SOD im Zuge des STAT3-Verlusts bei der PPCM die Akkumulation von Superoxidanionen-Radikalen in den Mitochondrien begünstigt und dadurch die Entstehung des 16-kDa-PRL und die damit assoziierten maladaptiven Prozesse weiterhin gefördert werden. Doch auch unabhängig davon könnten ROS die Kardiomyopathie durch Begünstigung von Apoptose durch Öffnung des mitochondrial permeability transition pore (mPTP), Beteiligung am Remodeling und direkten Schäden an der Elektronentransportkette (ETC) den Krankheitsprogress unterhalten. Das Ansprechen auf Bromocriptin suggeriert, dass das 16-kDa-PRL in der Tat kausal mit der Entstehung der PPCM im Zusammenhang steht und bestätigt die bisherigen Hypothesen hierzu. Neben dem Einsatz von Bromocriptin könnten allerdings auch ROS reduzierende Substanzen von Nutzen sein.Peripartum cardiomyopathy is characterized as an idiopathic cardiomyopathy presenting with heart failure due to left-ventricular systolic dysfunction in previously healthy women and mainly emerges towards the end of pregnancy, during delivery or within the first month after delivery. Myocardial protein levels of signal transducer and activator of transcription 3 (STAT3) are reduced in patients with PPCM. STAT3 mediates its protective actions via the myocardial JAK/STAT signal transduction pathway and plays a crucial role in protecting the heart from oxidative stress by upregulating the antioxidative enzyme manganese superoxide dismutase (MnSOD). In previous studies on failing hearts, dilated cardiomyopathy (DCM) was associated with the downregulation of STAT3 and the JAK/STAT signal transduction pathway, which may be a maladaptive process in light of the cardio-protective role of this pathway. Superoxide is produced at the respiratory chain due to incomplete reduction of oxygen and gets dismutated to hydrogen peroxide (H2O2) by the MnSOD. H2O2 subsequently gets detoxified to H2O by NADPH-dependent enzymes. An increased production of reactive oxygen species (ROS) is considered as an important upstream event, since it leads to the cleavage of Prolactin, the nursing hormone from the pituary gland, into its maladaptive 16-kDa form, mediated by the proteolytic enzyme Cathepsin D. This 16-kDa Prolactin (16-kDa-PRL) induces apoptosis and inhibits proliferation in endothelial cells, thereby impairing myocardial microvasculature. Treatment with the dopamine D2 agonist Bromocriptine reduces prolactin secretion and therefore also the formation of the 16-kDa-PRL. In mice with a myocardial specific deletion of STAT3 treatment with Bromocriptine fully prevented the onset of PPCM and also improved cardiac function in human patients. We performed a detailed and functional analysis of the mitochondrial function, including respiration and the formation of superoxide and H2O2, to test whether the loss of STAT3 effects mitochondrial energetics and ROS emission. Furthermore, we analyzed contractility, redox-states of NAD(P)H/FAD and superoxide-emission in intact cardiomyocytes isolated from mice with a cardiomyocyte-restricted deletion of STAT3 while being stimulated in an electric field, to check whether the STAT3 knockout in postpartum mice affects electromechanical coupling. Our results show a significantly decreased respiration in postpartum STAT3-KO mice compared to their WT littermates. In addition, mitochondrial formation of superoxide was higher in STAT3-KO mice, while H2O2 emission was reduced. Thus the ratio of H2O2 per ·O2 - was significantly decreased, indicating reduced MnSOD activity. Contractility, redox-state and superoxide emission in intact cardiomyocytes were comparable in postpartum STAT3-KO and WT, even after increased workload, which was performed by simultaneous increase of electric field stimulation (5 Hz) and the use of isoproterenol. Moreover our results indicate that treatment with Bromocriptine restores mitochondrial respiration in STAT3-KO mice, while in WT mice, Bromocriptine treatment did not affect respiration. In conclusion, postpartum mice with a cardiomyocyte-restricted deletion of STAT3 have a decreased activity of the antioxidative enzyme MnSOD, which predisposes to increased ·O2 - formation. Increased oxidative stress further enhances the cleavage of Prolactin into its maladaptive 16-kDa-PRL, which finally contributes to the development of PPCM. On the other hand, ROS are known to be involved in the progress of heart failure by promoting apoptosis via the mitochondrial permeability transition pore (mPTP), adverse myocardial remodeling and even direct ETC damage. Targeting mitochondrial ROS or STAT3-controlled signaling pathways may therefore be useful in the treatment of patients with PPCM in addition to the protective actions of Bromocriptine. Since Bromocriptine clearly improved the condition of postpartum STAT3-KO mice, we support the notion in line with previous findings that the 16-kDa-PRL plays a causative role in the development of PPCM

Identification and effect of monsoon on family Ocypodidae in the intertidal zone in Gulf of Oman, Hormozgan Province

This study was carried out in three inter tidal zones in the Iranian coast of Gulf of Oman, in 2010 to identify the species belonging to Family Ocypodidae and to investigate effect of monsoon on ecology of crabs. Two species of genus Uca (U. sindensis and U. iranica) from Jask and Khour-Khalasi study area and one species of genus Ocypode (O. rotundata) from Vanak beach were identified. The results were used to verify previous reports on species identification. Monsoon was found to affect distribution and frequency of these crabs. The maximum and minimum relative density were observed in pre-monsoon (May, 9.33 individual/m2) and monsoon (September, 6.38 individual/m2), respectively. Dense distribution of Uca was found in the muddy beaches of Khour-Khalasi and the genus Ocipode in the sandy beaches of Vanak area

MSR1 repeats modulate gene expression and affect risk of breast and prostate cancer

[Background] MSR1 repeats are a 36–38 bp minisatellite element that have recently been implicated in the regulation of gene expression, through copy number variation (CNV).[Patients and methods] Bioinformatic and experimental methods were used to assess the distribution of MSR1 across the genome, evaluate the regulatory potential of such elements and explore the role of MSR1 elements in cancer, particularly non-familial breast cancer and prostate cancer.[Results] MSR1s are predominately located at chromosome 19 and are functionally enriched in regulatory regions of the genome, particularly regions implicated in short-range regulatory activities (H3K27ac, H3K4me1 and H3K4me3). MSR1-regulated genes were found to have specific molecular roles, such as serine-protease activity (P = 4.80 × 10−7) and ion channel activity (P = 2.7 × 10−4). The kallikrein locus was found to contain a large number of MSR1 clusters, and at least six of these showed CNV. An MSR1 cluster was identified within KLK14, with 9 and 11 copies being normal variants. A significant association with the 9-copy allele and non-familial breast cancer was found in two independent populations (P = 0.004; P = 0.03). In the white British population, the minor allele conferred an increased risk of 1.21–3.51 times for all non-familial disease, or 1.7–5.3 times in early-onset disease. The 9-copy allele was also found to be associated with increased risk of prostate cancer in an independent population (odds ratio = 1.27–1.56; P =0.009).[Conclusions] MSR1 repeats act as molecular switches that modulate gene expression. It is likely that CNV of MSR1 will affect risk of development of various forms of cancer, including that of breast and prostate. The MSR1 cluster at KLK14 represents the strongest risk factor identified to date in non-familial breast cancer and a significant risk factor for prostate cancer. Analysis of MSR1 genotype will allow development of precise stratification of disease risk and provide a novel target for therapeutic agents.Prostate cancer study is supported by an National Health and Medical Research Council (NHMRC) grant and Career Development Fellowship APP1090505 to JB. The Australian Prostate Cancer BioResource is supported by the NHMRC Enabling Grant APP614296 and by a grant from the Prostate Cancer Foundation, Australia.Peer reviewe

The development of progesterone-loaded nanofibers using pressurized gyration: A novel approach to vaginal delivery for the prevention of pre-term birth

Recent evidence has continued to support the applicability of progesterone in preventing preterm birth, hence the development of an appropriate vaginal delivery system for this drug would be of considerable interest. Here, we describe the development of progesterone-loaded bioadhesive nanofibers using pressurized gyration for potential incorporation into a vaginal insert, with a particular view to addressing the challenges of incorporating a poorly water-soluble drug into a hydrophilic nanofiber carrier. Polyethylene oxide and carboxymethyl cellulose were chosen as polymers to develop the carrier systems, based on previous evidence of their yielding mucoadhesive nanofibers using the pressurized gyration technique. The fabrication parameters such as solvent system, initial drug loading and polymer composition were varied to facilitate optimisation of fiber structure and efficiency of drug incorporation. Such studies resulted in the formation of nanofibers with satisfactory surface appearance, diameters in the region of 400 nm and loading of up to 25% progesterone. Thermal and spectroscopic analyses indicated that the drug was incorporated in a nanocrystalline state. Release from the drug-loaded fibers indicated comparable rates of progesterone dissolution to that of Cyclogest, a commercially available progesterone pessary, allowing release over a period of hours. Overall, the study has shown that pressurized gyration may produce bioadhesive progesterone-loaded nanofibers which have satisfactory loading of a poorly water-soluble drug as well as having suitable structural and release properties. The technique is also capable of producing fibers at a yield commensurate with practical applicability, hence we believe that the approach shows considerable promise for the development of progesterone dosage forms for vaginal application