Selective vulnerability of hippocampal CA1 and CA3 pyramidal cells: What are possible pathomechanisms and should more attention be paid to the CA3 region in future studies?

Transient ischemia and reperfusion selectively damage neurons in brain, with hippocampal pyramidal cells being particularly vulnerable. Even within hippocampus, heterogeneous susceptibility is evident, with higher vulnerability of CA1 versus CA3 neurons described for several decades. Therefore, numerous studies have focused exclusively on CA1. Pediatric cardiac surgery is increasingly focusing on studies of hippocampal structures, and a negative impact of cardiopulmonary bypass on the hippocampus cannot be denied. Recent studies show a shift in selective vulnerability from neurons of CA1 to CA3. This review shows that cell damage is increased in CA3, sometimes stronger than in CA1, depending on several factors (method, species, age, observation period). Despite a highly variable pattern, several markers illustrate greater damage to CA3 neurons than previously assumed. Nevertheless, the underlying cellular mechanisms have not been fully deciphered to date. The complexity is reflected in possible pathomechanisms discussed here, with numerous factors (NMDA, kainate and AMPA receptors, intrinsic oxidative stress potential and various radicals, AKT isoforms, differences in vascular architecture, ratio of pro- and anti-apoptotic Bcl-2 factors, vulnerability of interneurons, mitochondrial dysregulation) contributing to either enhanced CA1 or CA3 vulnerability. Furthermore, differences in expressed genome, proteome, metabolome, and transcriptome in CA1 and CA3 appear to influence differential behavior after damaging stimuli, thus metabolomics-, transcriptomics-, and proteomics-based analyses represent a viable option to identify pathways of selective vulnerability in hippocampal neurons. These results emphasize that future studies should focus on the CA3 field in addition to CA1, especially with regard to improving therapeutic strategies after ischemic/hypoxic brain injury.

Correction: Neubert et al. Effects of Normobaric Hypoxia and Adrenergic Blockade over 72 h on Cardiac Function in Rats. Int. J. Mol. Sci. 2023, 24, 11417.

In the original publication [...].

Effects of Normobaric Hypoxia and Adrenergic Blockade over 72 h on Cardiac Function in Rats.

In rats, acute normobaric hypoxia depressed left ventricular (LV) inotropic function. After 24 h of hypoxic exposure, a slight recovery of LV function occurred. We speculated that prolonged hypoxia (72 h) would induce acclimatization and, hence, recovery of LV function. Moreover, we investigated biomarkers of nitrosative stress and apoptosis as possible causes of hypoxic LV depression. To elucidate the role of hypoxic sympathetic activation, we studied whether adrenergic blockade would further deteriorate the general state of the animals and their cardiac function. Ninety-four rats were exposed over 72 h either to normal room air (N) or to normobaric hypoxia (H). The rodents received infusion (0.1 mL/h) with 0.9% NaCl or with different adrenergic blockers. Despite clear signs of acclimatization to hypoxia, the LV depression continued persistently after 72 h of hypoxia. Immunohistochemical analyses revealed significant increases in markers of nitrosative stress, adenosine triphosphate deficiency and apoptosis in the myocardium, which could provide a possible explanation for the absence of LV function recovery. Adrenergic blockade had a slightly deteriorative effect on the hypoxic LV function compared to the hypoxic group with maintained sympathetic efficacy. These findings show that hypoxic sympathetic activation compensates, at least partially, for the compromised function in hypoxic conditions, therefore emphasizing its importance for hypoxia adaptation.

Relaxin does not prevent development of hypoxia-induced pulmonary edema in rats.

Acute hypoxia impairs left ventricular (LV) inotropic function and induces development of pulmonary edema (PE). Enhanced and uneven hypoxic pulmonary vasoconstriction is an important pathogenic factor of hypoxic PE. We hypothesized that the potent vasodilator relaxin might reduce hypoxic pulmonary vasoconstriction and prevent PE formation. Furthermore, as relaxin has shown beneficial effects in acute heart failure, we expected that relaxin might also improve LV inotropic function in hypoxia. Forty-two rats were exposed over 24 h to normoxia or hypoxia (10% N2 in O2). They were infused with either 0.9% NaCl solution (normoxic/hypoxic controls) or relaxin at two doses (15 and 75 µg kg-1 day-1). After 24 h, hemodynamic measurements and bronchoalveolar lavage were performed. Lung tissue was obtained for histological and immunohistochemical analyses. Hypoxic control rats presented significant depression of LV systolic pressure by 19% and of left and right ventricular contractility by about 40%. Relaxin did not prevent the hypoxic decrease in LV inotropic function, but re-increased right ventricular contractility. Moreover, hypoxia induced moderate interstitial PE and inflammation in the lung. Contrasting to our hypothesis, relaxin did not prevent hypoxia-induced pulmonary edema and inflammation. In hypoxic control rats, PE was similarly distributed in the apical and basal lung lobes. In relaxin-treated rats, PE index was 35-40% higher in the apical than in the basal lobe, which is probably due to gravity effects. We suggest that relaxin induced exaggerated vasodilation, and hence pulmonary overperfusion. In conclusion, the results show that relaxin does not prevent but rather may aggravate PE formation.

Left ventricular depression and pulmonary edema in rats after short-term normobaric hypoxia: effects of adrenergic blockade and reduced fluid load.

Acute normobaric hypoxia may induce pulmonary injury with edema (PE) and inflammation. Hypoxia is accompanied by sympathetic activation. As both acute hypoxia and high plasma catecholamine levels may elicit PE, we had originally expected that adrenergic blockade may attenuate the severity of hypoxic pulmonary injury. In particular, we investigated whether administration of drugs with reduced fluid load would be beneficial with respect to both cardiocirculatory and pulmonary functions in acute hypoxia. Rats were exposed to normobaric hypoxia (10% O2) over 1.5 or 6 h and received 0.9% NaCl or adrenergic blockers either as infusion (1 ml/h, increased fluid load) or injection (0.5 ml, reduced fluid load). Control animals were kept in normoxia and received infusions or injections of 0.9% NaCl. After 6 h of hypoxia, LV inotropic function was maintained with NaCl injection but decreased significantly with NaCl infusion. Adrenergic blockade induced a similar LV depression when fluid load was low, but did not further deteriorate LV depression after 6 h of infusion. Reduced fluid load also attenuated pulmonary injury after 6 h of hypoxia. This might be due to an effective fluid drainage into the pleural space. Adrenergic blockade could not prevent PE. In general, increased fluid load and impaired LV inotropic function promote the development of PE in acute hypoxia. The main physiologic conclusion from this study is that fluid reduction under hypoxic conditions has a protective effect on cardiopulmonary function. Consequently, appropriate fluid management has particular importance to subjects in hypoxic conditions.

Cyclosporine A-enhanced cardioplegia preserves mitochondrial basal respiration after ischemic arrest.

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·O2·s-1·mg-1·ww) than in the HTK group (3.8 ± 1.4 pmol·O2·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.

Impact of Custodiol-N cardioplegia on acute kidney injury after cardiopulmonary bypass.

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 (Hbbaseline : 6.0 ± 0.6 mmol/L, Hbreperfusion : 6.2 ± 0.7 mmol/L, P = .12; Ca2+baseline : 1.36 ± 0.05 mmol/L, Ca2+reperfusion : 1.28 ± 0.05 mmol/L, P = .16) compared to the HTK group (Hbbaseline : 5.9 ± 0.4 mmol/L, Hbreperfusion : 4.7 ± 0.8 mmol/L, P < .01; Ca2+baseline : 1.34 ± 0.07 mmol/L, Ca2+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.

Golgi Fragmentation in Human Patients with Chronic Atrial Fibrillation: A New Aspect of Remodeling.

BACKGROUND: Atrial fibrillation (AF) is the most common chronic arrhythmia in elderly people and is accompanied by remodeling processes. While much is known about changes in ionic channels and in extracellular matrix, less is known about possible changes of intracellular structures. OBJECTIVE: We wanted to investigate, whether AF may also affect the structure of the Golgi apparatus and the microtubular network. METHODS: One-hundred fifty-three cardiac surgery patients were investigated [n = 24 in sinus rhythm (SR) and n = 129 with chronic AF of >1 year duration]. Tissue samples of the left atrial free wall were examined immunohistochemically. Golgi apparatus was detected by GM130 and its phosphorylated isoform. Furthermore, we investigated the length of the microtubules by α-tubulin staining. We also measured stathmin (phospho-S37), which is known to induce microtubule depolymerization. In addition, we investigated the cyclin-dependent kinase cdk5-activation, a typical stimulus for Golgi fragmentation, by measuring membrane-associated cdk5. RESULTS: We found significant fragmentation of the Golgi apparatus in AF together with a reduced fragment size. Significant more fragments of the Golgi were found lateral to the nucleus in AF, while the Golgi in SR was located more to the polar side of the nucleus, that is, in the longitudinal axis of the cell. This was accompanied by a significant reduction of the number of tubulin strands longer than 10 µm. These changes did not go along with an activation of stathmin, but with an increase in membrane association of cdk5. CONCLUSIONS: The present data may show that AF associated remodeling also involves intracellular remodeling of the Golgi-microtubular apparatus.

Aspects of Methamphetamine Abuse in Adolescents and Young Adults in a Thuringian County.

BACKGROUND/OBJECTIVE: Drug abuse is an increasing problem among young adults in European countries. We wanted to assess the prevalence of methamphetamine and cannabis use in young people and to ask for their socioeconomic, parental and emotional background. METHODS: All pupils (n = 1,087) in the final class of 3 school types (-general school [in Germany: Hauptschule/Regelschule mit Hauptschulabschluss] class 9, regular school [in Germany: Regelschule] class 10, high school [in Germany: Gymnasium] class 10) and the first 2 classes of the vocational school (in Germany: Berufsschule) in the entire rural district of Altenburger Land, a Thuringian county in Germany with a total of 92,344 inhabitants were asked to fill out a 2-page questionnaire. In all, 920 questionnaires could be finally evaluated (mean age: 16.6 ± 0.07 years; mode value: 16; maximum range: 13-29). RESULTS: In the entire group, 5.81% (33 females, 20 males) reported the use of methamphetamine, while 42.8% stated that they have used it only once, the remaining 57.2% specified that they have used it on a more regular basis, mostly once a month, but 14.2% had used it almost daily. With regard to the socioeconomic background, 55% of the parents of the methamphetamine users were employees and 10% of the parents were in a leading position. This was not different from the non-user group (p = 0.193). Unemployment of the parents was found in 6.5% of the methamphetamine users and in 4.7% of the non-users. Overall, 67.92% had a basic knowledge of the side effects of this drug. In 55.32% of the methamphetamine users, the parents lived alone, were divorced or widowed, while this was only 33.1% in the non-user group. Of all the methamphetamine users, 69% had friends who also consumed the drug, which may give a hint on the impact of a peer group. In the entire group, 29.84% were smokers (female group: 28.47%; male group 31.25%) and 25.5% consumed cannabis. Ecstasy was used by a total of 4.05% and cocaine by 1.53%. No participant mentioned that they have used opiates. CONCLUSION: Among pupils aged about 16 years nearly 6% reported to have consumed methamphetamine. This group has a middle socioeconomic background. Two factors were strongly associated with methamphetamine usage: a family with only a single parent and friends who were used to taking methamphetamine. The self-perception of users was generally considered to be a disadvantage as compared to the other factors.

Epigallocatechin Gallate Reduces Ischemia/Reperfusion Injury in Isolated Perfused Rabbit Hearts.

Cardioplegic arrest during heart operations is often used in cardiac surgery. During cardioplegia, the heart is subjected to a global ischemia/reperfusion-injury. (-)-epigallocatechin gallate (EGCG), one of the main ingredients of green tea, seems to be beneficial in various cardiac diseases. Therefore, the aim of our study was to evaluate EGCG in a rabbit model of cardioplegic arrest. Twenty four mature Chinchilla rabbits were examined. Rabbit hearts were isolated and perfused according to Langendorff. After induction of cardioplegia (without and with 20 µmol/L EGCG, n = 6 each) the hearts maintained arrested for 90-min. Thereafter, the hearts were re-perfused for 60 min. During the entire experiment hemodynamic and functional data were assessed. At the end of each experiment, left ventricular samples were processed for ATP measurements and for histological analysis. Directly after cessation of cardioplegia, all hearts showed the same decline in systolic and diastolic function. However, hearts of the EGCG-group showed a significantly faster and better hemodynamic recovery during reperfusion. In addition, tissue ATP-levels were significantly higher in the EGCG-treated hearts. Histological analysis revealed that markers of nitrosative and oxidative stress were significantly lower in the EGCG group. Thus, addition of EGCG significantly protected the cardiac muscle from ischemia/reperfusion injury.

Epigallocatechin gallate attenuates cardiopulmonary bypass-associated lung injury.

BACKGROUND: Lung dysfunction constitutes a severe complication after major cardiac surgery with cardiopulmonary bypass (CPB), substantially contributing to postoperative morbidity and mortality. The current possibilities of preventive and therapeutic interventions, however, remain insufficient. We, therefore, investigated the effects of intraoperative application of the antioxidant and anti-inflammatory green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on CPB-associated lung injury. MATERIALS AND METHODS: Thirty piglets (8-15 kg) were divided into four groups: sham-operated and saline-treated control group (n = 7); sham-operated and EGCG-treated control group (EGCG-control group; n = 7); CPB group (n = 10); and CPB + EGCG group (n = 6). The CPB groups underwent 120 min of CPB followed by 90 min of recovery time. In the CPB + EGCG group, EGCG (10 mg/kg body weight) was administered intravenously before and after CPB. Hemodynamic monitoring, blood gas analysis, hematoxylin-eosin staining, and immunohistochemistry of lung tissue were performed. RESULTS: Histologic examination revealed thickening of the alveolar wall and enhanced alveolar neutrophil infiltration in the CPB group (P < 0.05) compared with those in the control group, which was prevented by EGCG (P < 0.05). In the CPB group, higher formation of poly(ADP-ribose) and nuclear translocation of apoptosis-inducing factor was detected in comparison with those in the control group (P < 0.001), which were both reduced in the CPB + EGCG group (P < 0.001). Compared with the control group, the EGCG-control group showed thickening of the alveolar wall and increased neutrophil infiltration (P < 0.05). CONCLUSIONS: CPB leads to lung edema, pulmonary neutrophil infiltration, and presumably initiation of poly(ADP-ribose) polymerase-dependent cell death signaling in the lung. EGCG appears to attenuate CPB-associated lung injury, suggesting that this may provide a novel pharmacologic approach.

Neuroprotective Strategies during Cardiac Surgery with Cardiopulmonary Bypass.

Aortocoronary bypass or valve surgery usually require cardiac arrest using cardioplegic solutions. Although, in principle, in a number of cases beating heart surgery (so-called off-pump technique) is possible, aortic or valve surgery or correction of congenital heart diseases mostly require cardiopulmonary arrest. During this condition, the heart-lung machine also named cardiopulmonary bypass (CPB) has to take over the circulation. It is noteworthy that the invention of a machine bypassing the heart and lungs enabled complex cardiac operations, but possible negative effects of the CPB on other organs, especially the brain, cannot be neglected. Thus, neuroprotection during CPB is still a matter of great interest. In this review, we will describe the impact of CPB on the brain and focus on pharmacological and non-pharmacological strategies to protect the brain.

Effects of minocycline on parameters of cardiovascular recovery after cardioplegic arrest in a rabbit Langendorff heart model.

Pharmacological cardiac organ protection during cardiopulmonary bypass presents an opportunity for improvement. A number of different strategies have been established to minimize ischemia/reperfusion-induced damage to the heart. Among these, cardioplegia with histidine-tryptophan-ketoglutarate solution and hypothermia are the most frequently used regimens. The antibiotic minocycline has been used in this context for neuroprotection. The aim of the current study was to evaluate whether the application of minocycline prior to cardioplegia exerts a protective effect on cardiac muscle. For this purpose, this study investigated six rabbit hearts with minocycline treatment (1 µmol/L) and six without in a Langendorff model of 90 min cold cardioplegic arrest using Custodiol followed by a 30 min recovery phase. Histological analysis of cardiac muscle revealed that markers of apoptosis, oxidative and nitrosative stress were significantly lower in the minocycline group, whereas adenosine triphosphate (ATP)- and malondialdehyde (MDA)-levels and O2-consumption were not affected by minocycline. Functionally, recovery of dP/dt (max) and dP/dt (min) was significantly faster in the minocycline group than in control. This leads to the conclusion that adding minocycline to the cardioplegic solution may improve left ventricular recovery after cardioplegic arrest involving reduced pro-apoptotic effects.

Effects of mechanical forces and stretch on intercellular gap junction coupling.

Mechanical forces provide fundamental physiological stimulus in living organisms. Recent investigations demonstrated how various types of mechanical load, like strain, pressure, shear stress, or cyclic stretch can affect cell biology and gap junction intercellular communication (GJIC). Depending on the cell type, the type of mechanical load and on strength and duration of application, these forces can induce hypertrophic processes and modulate the expression and function of certain connexins such as Cx43, while others such as Cx37 or Cx40 are reported to be less mechanosensitive. In particular, not only expression but also subcellular localization of Cx43 is altered in cardiomyocytes submitted to cyclic mechanical stretch resulting in the typical elongated cell shape with an accentuation of Cx43 at the cell poles. In the heart both cardiomyocytes and fibroblasts can alter their GJIC in response to mechanical load. In the vasculature both endothelial cells and smooth muscle cells are subject to strain and cyclic stretch resulting from the pulsatile flow. In addition, vascular endothelial cells are mainly affected by shear stress resulting from the blood flow parallel to their surface. These mechanical forces lead to a regulation of GJIC in vascular tissue. In bones, osteocytes and osteoblasts are coupled via gap junctions, which also react to mechanical forces. Since gap junctions are involved in regulation of cell growth and differentiation, the mechanosensitivity of the regulation of these channels might open new perspectives to explain how cells can respond to mechanical load, and how stretch induces self-organization of a cell layer which might have implications for embryology and the development of organs. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Hypoxia-Induced Pulmonary Injury-Adrenergic Blockade Attenuates Nitrosative Stress, and Proinflammatory Cytokines but Not Pulmonary Edema.

Hypoxia can induce pulmonary edema (PE) and inflammation. Furthermore, hypoxia depresses left ventricular (LV) inotropy despite sympathetic activation. To study the role of hypoxic sympathetic activation, we investigated the effects of hypoxia with and without adrenergic blockade (AB) on cardiovascular dysfunction and lung injury, i.e., pulmonary edema, congestion, inflammation, and nitrosative stress. Eighty-six female rats were exposed for 72 h to normoxia or normobaric hypoxia and received infusions with NaCl, prazosin, propranolol, or prazosin-propranolol combination. We evaluated hemodynamic function and performed histological and immunohistochemical analyses of the lung. Hypoxia significantly depressed LV but not right ventricular (RV) inotropic and lusitropic functions. AB significantly decreased LV function in both normoxia and hypoxia. AB effects on RV were weaker. Hypoxic rats showed signs of moderate PE and inflammation. This was accompanied by elevated levels of tumor necrosis factor α (TNFα) and nitrotyrosine, a marker of nitrosative stress in the lungs. In hypoxia, all types of AB markedly reduced both TNFα and nitrotyrosine. However, AB did not attenuate PE. The results suggest that hypoxia-induced sympathetic activation contributes to inflammation and nitrosative stress in the lungs but not to PE. We suggest that AB in hypoxia aggravates hypoxia-induced inotropic LV dysfunction and backlog into the pulmonary circulation, thus promoting PE.

Right or left ventricular pacing in young minipigs with chronic atrioventricular block: long-term in vivo cardiac performance, morphology, electrophysiology, and cellular biology.

BACKGROUND: Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. METHODS AND RESULTS: Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104 ± 5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall-paced minipigs exhibited significantly more LV dyssynchrony than LV apex-paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154 ± 36 ms/28 ± 3%, LV apical pacing, 52 ± 19 ms/45 ± 2%, control 47 ± 14 ms/62 ± 1%; P=0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall- and LV apex-paced animals. CONCLUSIONS: The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing.

Reno-protective effects of epigallocatechingallate in a small piglet model of extracorporeal circulation.

Cardiopulmonary bypass still often is a necessary tool in cardiac surgery in particular in the correction of congenital heart defects in small infants. Nevertheless, among the complications linked to extracorporeal circulation (ECC) with cardiopulmonary bypass (CPB) in both infants and adults one of the most serious problems is renal impairment. Since this might be caused by ischemia/reperfusion injury and accumulation of free radicals, we used (-)-epigallocatechin-3-gallate (EGCG), a derivate from green tea, which is known to possess antioxidant, antiapoptotic and NO-scavenging properties in order to find out whether EGCG may protect the kidney. 23 four-week-old Angler Sattelschwein-piglets (8-15 kg) were divided into three groups: control-group (n=7), ECC-group (n=10), EGCG-group (n=6). The ECC- and EGCG-group were thoracotomized and underwent CPB for 120 min followed by a 90-min recovery-time. The EGCG-group received 10 mg/kg EGCG before and after CPB. Histology revealed that CPB led to widening of Bowman's capsule, and to vacuolization of proximal tubular cells (p<0.05) which could be prevented by EGCG (p<0.05). Using immunohistology, we found significant nuclear translocation of hypoxia-inducible-factor-1-alpha (HIF-1-alpha) and increased nitrotyrosine formation in the ECC-group. Both were significantly (p<0.05) inhibited by EGCG. ECC-induced loss of energy-rich phosphates was prevented by EGCG. In blood samples we found that CPB resulted in increases in creatinine and urea (in serum) and led to loss of total protein (p<0.05), which all was not present in EGCG-treated animals. We conclude that CPB causes damage in the kidney which can be attenuated by EGCG.

On the different roles of AT1 and AT2 receptors in stretch-induced changes of connexin43 expression and localisation.

Cyclic mechanical stretch (CMS) and angiotensin II (ATII) play an important role in cardiac remodelling. Thus, we aimed to examine how ATII affects CMS-induced changes in localisation and expression of the gap junction protein connexin43 (Cx43). Neonatal rat cardiomyocytes cultured on gelatin-coated Flexcell cell culture plates were kept static or were exposed to CMS (110 % of resting length, 1 Hz) for 24 h with or without additional ATII (0.1 µmol/L). Moreover, inhibitors of ATII receptors (AT-R) were used (for AT(1)-R: losartan 0.1 µmol/L, for AT(2)-R: PD123177 0.1 µmol/L). Thereafter, the cardiomyocytes were investigated by immunohistology, PCR and Western blot. After 24 h of CMS, cardiomyocytes were significantly elongated and orientated 75 ± 1.6° nearly perpendicular to the stretch axis. Furthermore, CMS significantly accentuated Cx43 at the cell poles (ratio Cx43 polar/lateral static: 2.32 ± 0.17; CMS: 10.08 ± 3.2). Additional ATII application significantly reduced Cx43 polarisation (ratio Cx43 polar/lateral ATII: 4.61 ± 0.42). The combined administration of ATII and losartan to CMS further reduced Cx43 polarisation to control levels, whilst the AT(2)-R blocker PD123177 restored polarisation. Moreover, CMS and ATII application resulted in a significant Cx43 protein and Cx43 mRNA up-regulation which could be blocked by losartan but not by PD123177. Thus, CMS results in a self-organisation of the cardiomyocytes leading to elongated cells orientated transversely towards the stretch axis with enhanced Cx43 expression and Cx43 accentuation at the cell poles. ATII enhances total Cx43 mRNA and protein expression probably via AT(1)-R (=inhibitory effect of losartan) and reduces Cx43 polarisation presumably via AT(2)-R, since PD123177 (but not losartan) inhibited the negative effects of ATII on polarisation.

Cyclic mechanical stretch induces cardiomyocyte orientation and polarization of the gap junction protein connexin43.

RATIONALE: Cyclic mechanical stretch (CMS) is an important physiological and pathological factor in the heart. OBJECTIVE: We examined whether CMS can affect localization of gap junctions with regard to the cell axis. METHODS AND RESULTS: Neonatal rat cardiomyocytes were cultured (7 days) on flexible 6-well plates. Thereafter, cells were kept static or stimulated with CMS (1 Hz; 0, 10, 20% elongation) for 0, 24, or 48 hours (with or without 10 micromol/L PD98059, 5 micromol/L BIM I (bisindolylmaleimide I), 2 micromol/L H8 [N-(2-methlyamino-ethyl)-5-isoquinoline-sulfonamid], or 0.1 micromol/L angiotensin II. Additionally, cells were exposed to 24 hours of CMS followed by 24 hours of static recovery. CMS (24 hour, 10%) induced elongation of the cardiomyocytes and orientation 79+/-8 degrees toward the stretch direction. Moreover, the distribution of connexin (Cx)43 together with N-cadherin changed, so that both proteins were accentuated at the cell poles, whereas in nonstretched cells, they were distributed around the cell without preferential localization. Additional angiotensin II reduced polar Cx43 accentuation. The CMS-induced changes in Cx43 were reversible within 24 hours after end of stretch, and could be completely prevented by the MEK1/2 inhibitor PD98059 but not by BIM I or H8. Moreover, stretch resulted in Cx43 protein and Cx43-mRNA upregulation and in a significant upregulation of the phosphorylated forms of ERK1/2, glycogen synthase kinase 3beta and AKT. Furthermore, CMS resulted in a significant increase of the transcription factors activator protein 1 and CREB (cAMP response element-binding protein) in the nucleus. CONCLUSIONS: CMS results in self-organization of cardiomyocytes leading to elongated cells orientated transverse to the stretch axis, enhanced Cx43 expression and Cx43 accentuation at the cell poles. The Cx43-changes seem to depend on the ERK1/2 signaling cascade.

Antihypertensive and cardioprotective effects of different monotherapies and combination therapies in young spontaneously hypertensive rats - A pilot study.

Spontaneously hypertensive rats (SHR) are an established animal model for antihypertensive treatment. The aim of this pilot study was a systematic search for two lines of antihypertensive treatment - a monotherapy and a combination of two drugs - to be applied in a future study on old SHR. Originally, representatives of three drug classes recommended for antihypertensive therapy in humans should be applied, namely captopril (CAP) as an antagonist of the renin-angiotensin-aldosterone system, nifedipine (NIF) as calcium channel blocker and propranolol (PROP) as ß-adrenergic blocker. As we observed that PROP had been poorly ingested, all groups with PROP therapy were excluded from the study. CAP (60 mg kg-1 d-1), NIF (10 mg kg-1 d-1) or both were administered orally to seven-week-old SHR over 3 weeks. A further group of SHR received no treatment (SHR/CTRL). Age-matched normotensive Wistar-Kyoto rats served as normotensive controls. We examined the effect of the antihypertensive therapies on systolic blood pressure, heart weight and on histological and biochemical markers of cardiac hypertrophy and fibrosis. CAP proved to be the most effective treatment reducing blood pressure and relative heart weight significantly compared to SHR/CTRL without reaching normotensive values. Beginning cardiac fibrosis observed in SHR/CTRL was completely abrogated with CAP treatment. Similar effects were achieved with a combination of CAP and NIF. CAP as monotherapy and CAP + NIF as combination therapy were chosen for the forthcoming study on old SHR.