U Mehlhorn

Role of balloon occlusion for mononuclear bone marrow cell deposition after intracoronary injection in pigs with reperfused myocardial infarction

AIMS In clinical studies on cell therapy for acute myocardial infarction (MI), cells are usually applied by intracoronary infusion with balloon (IC/B). To test the utility of balloon occlusion, mononuclear bone marrow cell (MNC) retention after intracoronary infusion without balloon (IC/noB) was compared with IC/B and intramyocardial (IM) injection. METHODS AND RESULTS Four hours after LAD ligation in male pigs, reperfusion was allowed (confirmed by coronary angiography). Five days later, 1 x 10(8) autologous (111)Indium-labelled MNC were injected IC/noB (n = 4), IC/B (n = 4), or IM (n = 4). At 1 h the fraction of injected MNC that was detected in the heart was 4.1 +/- 1.1% after IC/noB injection, 6.1 +/- 2.5% after IC/B injection (P = 0.19), and 20.7 +/- 2.3% after IM injection (P < 0.001 vs. IC/noB and IC/B). At 24 h it was 3.0 +/- 0.6% (IC/noB), 3.3 +/- 0.5% (IC/B, P = 0.43), and 15.0 +/- 3.1% (IM, P < 0.001 vs. IC/noB and IC/B). Dynamic scintigrammes during each of four consecutive IC/B injections showed a rapid 19.6 +/- 8.0% cell loss during balloon inflation (no-flow period, phase 1) and a rapid 36.6 +/- 17.8% cell loss after balloon deflation (re-flow period, phase 2). After each of four consecutive IC/noB injections the peak cell deposit was lower, followed by one phase of rapid cell loss (30.9 +/- 11.0% after 6 min). After IM injection only a slow linear cell loss was observed (9.7% per h). In histology, PKH-67 labelled cells only rarely had passed the endothelial barrier after 24 h after IC injection, while they were exclusively found in the interstitium after IM injection. CONCLUSION The observation of a similar cell persistence after IC injections with and without balloon occlusion suggests that the balloon procedures currently applied in clinical studies are not necessary for cell deposit. If longer term persistence of cells plays a role for the clinical benefit of cardiac cell therapy, IM injection may be superior to IC applications.

Reevaluation of Direct True Lumen Cannulation in Surgery for Acute Type A Aortic Dissection

BACKGROUND The optimal mode of arterial cannulation in acute type A aortic dissection is controversial. We retrospectively investigated our experience with direct true lumen cannulation as an alternative to standard cannulation procedures. METHODS From April 2004 to August 2007, 29 patients (20 men, 9 women; mean age of 63.2 +/- 12.6 years) underwent emergency operation for acute type A aortic dissection with direct true lumen cannulation. After venous drainage into the venous reservoir, the ascending aorta was completely transected in the region between the sinotubular junction and innominate artery. After visual and digital identification of the true lumen, the arterial cannula was directly inserted into the true lumen and secured with a ligature. RESULTS Mean aortic cross-clamp time was 77.4 +/- 28.3 minutes, and hypothermic circulatory arrest for the distal anastomosis was 10.4 +/- 11.0 minutes. All patients survived the surgical procedure. No surgical problems were observed by applying this strategy. Mean intensive care unit stay was 4.0 +/- 3.5 days. Postoperative mean ventilation time was 43.3 +/- 41.3 hours. One patient had a prolonged postoperative course and required permanent ventilation. Two patients required temporary hemofiltration. Neurologic disorders occurred in 6 patients: 2 had severe cerebral hypoxia, and 4 had temporary hemiplegia under good regression. All patients were alive at discharge. CONCLUSIONS Direct true lumen cannulation is a promising surgical strategy for emergency operations in type A aortic dissection. It is a simple, quick, and safe method to provide antegrade flow through the true aortic lumen.

Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase α1β1, α2β1 and α3β1

Cardiac glycosides inhibit the Na(+),K(+)-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na(+),K(+)-ATPase are expressed: alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1). It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na(+),K(+)-ATPase isoforms alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1) were expressed in yeast which has no endogenous Na(+),K(+)-ATPase. Isoform specific affinities of digoxin, digitoxin, beta-acetyldigoxin, methyldigoxin and ouabain were assessed in [(3)H]-ouabain binding assays in the absence or presence of K(+) (each n=5). The subcellular localizations of the Na(+),K(+)-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K(+), methyldigoxin (alpha(1)>alpha(3)>alpha(2)) and ouabain (alpha(1)=alpha(3)>alpha(2)) showed distinct isoform specific affinities, while for digoxin, digitoxin and beta-acetyldigoxin no differences were found. In the presence of K(+), also digoxin (alpha(2)=alpha(3)>alpha(1)) and beta-acetyldigoxin (alpha(1)>alpha(3)) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only alpha(1)beta(1) and alpha(2)beta(1) are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na(+),K(+)-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.

How to do it: direct true lumen cannulation technique of the ascending aorta in acute aortic dissection type A

In acute aortic dissection type A (AADA), direct true lumen cannulation (DTLC) of the ascending aorta is a fast and safe cannulation site providing antegrade perfusion of the supraaortic and visceral vessels. An Overholt clamp is passed around the ascending aorta to place a Mersilene tape for later securing of the arterial cannula. After draining venous blood into the cardiopulmonary bypass system (CPB), the ascending aorta is transected and the aortic lumen inspected. The true lumen is identified and an arterial cannula inserted directly. Finally, the cannula is secured with the previously placed tape and CPB is initiated. DTLC can be used as arterial cannulation standard technique in operations for AADA.

Pneumocyte Apoptosis Induction during Cardiopulmonary Bypass: Effective Prevention by Radical Scavenging Using N-Acetylcysteine

Cardiopulmonary bypass (CPB) and cardioplegic arrest are associated with pulmonary dysfunction. We sought to investigate whether pulmonary ischemia/reperfusion during standard CPB and cardioplegic arrest is associated with reactive oxygen species (ROS)-mediated pulmonary tissue injury and pneumocyte apoptosis induction, and whether ROS scavenging using N-acetylcysteine (NAC) attenuates these alterations. Twelve pigs (41 ± 8 kg) were randomized to receive either NAC (100 mg/kg prior to CPB; n = 7) or placebo (n = 5) and subjected to CPB and 60 min of cold (4°C) crystalloid cardioplegic arrest. We collected lung biopsies prior to CPB, at 60 min CPB, as well as at 30, 60, and 120 min post CPB. Lung specimens were immunocytochemically stained against nitrotyrosine, 8-isoprostaglandin-F2α, and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) as indicators for ROS-mediated tissue injury and active caspase-3, an apoptosis signal pathway key enzyme. Oxidative stress markers were judged using a scale from 1 to 4 (low to intensive staining), and caspase-3-positive pneumocytes were counted per view field. In placebo, the number of caspase-3-positive pneumocytes significantly increased over time to reach a maximum at 120 min post CPB (p =. 03 vs baseline). NAC significantly prevented caspase-3 activation in pneumocytes (p =. 001 vs Placebo). Pneumocyte nitrotyrosine and 8-OH-dG staining significantly increased over time (p =. 003) in the placebo group, but decreased in the NAC group (p =. 004). In both groups staining for 8-isoprostaglandin-F2α showed no significant changes. This yields the conclusion that standard CPB and cardioplegic arrest initiate ROS-mediated tissue injury and apoptosis in pneumocytes that can be reduced by NAC. Thus, ROS scavenging using NAC may represent a novel approach to minimize lung injury associated with CPB.

Regulation of endothelial nitric oxide synthase (eNOS) in myocardium subjected to cardioplegic arrest.

BACKGROUND Nitric oxide (NO) production by both coronary endothelial cells and cardiomyocytes is thought to play a significant role in myocardial pathophysiology following ischemia/reperfusion (I/R). METHODS In thirteen pigs subjected to 1 hour cardioplegic arrest (CA) on CPB, left ventricular (LV) biopsies were collected prior to CPB (baseline), at 60 min CPA, at 15 and 30 min reperfusion on CPB, and at 120 min post CPB. LV specimens were immunocytochemically stained against phospho-eNOS (Ser1177), phospho-eNOS (Thr495), phosphorylated ERK1/2, and AKT/PKB. Four additional pigs without CA served as controls. Cardiomyocytes were quantitatively investigated using TV densitometry (gray units: U). RESULTS After 60 min CA phosphorylation of eNOS (Ser1177) increased significantly and remained elevated until 30 min of reperfusion. In contrast, eNOS (Thr495) phosphorylation remained unchanged during CA and throughout reperfusion. In control animals, eNOS phosphorylation remained unchanged. Akt/PKB activity significantly increased after 60 min CA and decreased thereafter. ERK1/2 activity remained unchanged during ischemia but increased during reperfusion. CONCLUSIONS ENOS activation during ischemia occurs through phosphorylation at Ser1177 mediated by Akt/PKB. ERK1/2 does not seem to be involved in myocardial eNOS regulation especially not via phosphorylation at eNOS (Thr495).

Experimental Impact of Mucopolysaccharidosis on Right Atrial Contractile Capacity of Skinned Fibers

Objective: Mucopolysaccharidosis is a group of inherited lysosomal storage disorders caused by the absence of an enzyme, contributing to the degradation of the Glycosaminoglycans (GAG). To evaluate if storage of GAGs affects the contractile capacity, we compared the contractile performance of patients with and without MPS in a skinned fiber model. Methods: Right auricle tissue from 12 patients (4 MPS, 8 Non-MPS) undergoing cardiac operation for Mitral Valve Stenosis (MS) or Mitral Valve Regurgitation (MR) was harvested prior to implementation of extracorporal Circulation (ECC). The trabeculae are resected from the right auricle; the tissue was dissected down to small bundles of fibers. They were chemically skinned by removal of cell membrane-dependent properties to gain the actin-myosinformation. These bundles are dissected in single fiber stripes. The fibers were exposed to a gradual increase of calcium concentration (pCa) and the corresponding force was measured and recorded. Results: 1.) pCa-force development was significant lower in fibers with MPS (p max 2.2 ± 0.1 mN) than in fibers with MS (3.4 ± 0.7 mN, p 0.03) or MR (4.6 ± 0.9 mN, p 0.04). 2.) The pCa-force values of fibers with mitral valve stenosis and regurgitation did not differ significantly at single steps of calcium concentration among each other (p 0.3). 3.) Fibers with MR developed significant more force at almost all steps of calcium concentration than those with MPS (p 0.02) 4.) Fibers with MS developed only once significant more force compared to MPS fibers: pCa 5.5: 1.6 mN vs. 0.3 mN, p 0.02. 5.) Calcium sensitivity is similar in patients with MPS and MR (pCa 4.5), but different in patients with MS (pCa 5). Conclusion: Patients with MPS develop significantly less force at similar calcium concentrations compared to patients with a mitral valve stenosis or regurgitation. We suggest that impairment of the contractile apparatus due to the accumulation of GAGs leads to early onset left ventricular hypertrophy and diastolic dysfunction limiting the ventricular filling. This stands in opposition to patients with mitral valve regurgitation with volume overload due to incomplete valve closure.

Oxygen Radical Scavengers

The myocardium can tolerate only relatively short periods of total myocardial ischemia without myocardial cell death. Following short ischemic periods, ischemic damage is reversible by reperfusion. However, with increasing duration and severity of ischemia, the damage inflicted to cardiomyocytes following reperfusion becomes irreversible. The combined pathologic events in the myocardium that follow a critical period of ischemia and leading to either reversible or irreversible damage to both cardiomyocytes and cardiac microvasculature is known as ischemia-reperfusion injury (Goldhaber and Weiss 1992).

Auf dem Weg zu einem detaillierteren Verständnis atrialer Rhythmusstörungen: Darstellung biphasischer Kalziumtransienten

ZusammenfassungDurch akute mechanische Alterationen (Muskeldehnung bzw. -verkürzung) können atriale Rhythmusstörungen induziert werden. Die vorliegende, unter exakt kontrollierten experimentellen Bedingungen durchgeführte Analyse leistet einen Beitrag, mögliche Ursachen hierfür aufzuklären.Isolierte menschliche atriale Trabekel (0,3 x 5,0 mm) wurden Patienten vom rechten Herzohr entnommen, die sich einer koronaren Bypassoperation unterzogen (n = 17). Nach Ladung mit dem Fluoreszenzfarbstoff FURA-2 zur Kalziumbestimmung (Messbedingungen: optimale Länge, isometrische Bedingungen, oxygenierte Krebs-Henseleit-Lösung, 37° C, supramaximale Stimulation mit 0,5 Hz) und einer Äquilibrierungsperiode wurden bei den Präparaten rasche Entdehnungsprozeduren bis hin zur “slack-length” (A), auf 90 % (B) bzw. auf 95 % der optimalen Länge (Lmax) (C) durchgeführt. Systolische und diastolische Kraftentwicklung sowie systolisches und diastolisches Kalzium wurden simultan über 2 min nach Auslösung der experimentellen Prozeduren registriert.Die rasche ”unloading procedure” war assoziiert mit Extrakontraktionen bei 13 von 17 Muskelpräparaten bei Muskellängenreduktion auf slack length (A), bei 8 von 17 Präparaten bei Entdehnung auf 90 % Lmax (B) und bei 2 von 17 Präparaten bei Entdehnung auf 95 % Lmax (C). Überraschenderweise zeigte die Form des intrazellulären Kalziumtransienten in einigen Präparaten substanzielle Veränderungen mit einem biphasischen Kalziumtransienten bei monophasischer Kraftantwort, was wiederum auf wenigstens zwei Populationen von Muskelfasern im selben multizellulären Präparat hinweist. Dieses Phänomen wurde dokumentiert bei 8 von 17 in (A), 4 von 17 in (B) und 1 von 17 in (C).Die Befunde zeigen erstmals die Präsenz von mindestens zwei Muskelfaserpopulationen im multizellulären Muskelpräparat, die unterschiedliche Arten der elektromechanischen Kopplung aufweisen. Damit ist nach unserer Einschätzung ein Ausgangspunkt möglicher atrialer Rhythmusstörungen unter experimentellen Bedingungen nachgewiesen worden. Der Entdeckung mechanisch induzierter biphasischer Kalziumtransienten (mit monophasischer Kraftantwort) kommt möglicherweise entscheidende Bedeutung für das Verständnis der Entstehung atrialer Rhythmusstörungen zu.AbstractAcceleration and decelaration procedures are associated with induction atrial arrhythmias. The present study was designed to investigate the underlying pathophysiological mechanisms using exactly controlled experimental conditions.Isolated intact human atrial trabeculae (0.3 x 5.0 mm) obtained from patients undergoing CABG (n=17) were loaded for calcium measurement with FURA 2 (measurement conditions: optimal length, isometric, 37 °C, oxygenated Krebs Henseleit solution, supramaximal stimulation with 0.5 Hz). After an equilibration period the fibres were subjected to rapid unloading procedures to slack length (A), to 90 % Lmax (B) and to 95 % Lmax (C). Systolic and diastolic force, and systolic and diastolic calcium were simultaneously recorded for 2 min after this procedure.The rapid unloading procedure induced extra contractions in 13/17 fibres at slack length (A), in 8/17 fibres at 90 % Lmax (B) and in 2/17 fibres at 95 Lmax (C). Surprisingly the shape of the calcium transient was substantially altered in some preparations showing a biphasic response with a monophasic isometric force response, indicating the presence of at least two populations of muscle cells with different electromechanical coupling within the same multicellular preparation. This phenomenon was documented in 8/17 in (A), 4/17 in (B) and 1/17 in (C).This is the first time that the presence of two populations of muscular cells with different electromechanical properties have been detected within the same preparation. Thus, the source of arrhythmias was directly monitored under experimental conditions. The discovery of mechanically induced biphasic calcium transients within a monophasic contraction may become crucial for the understanding of atrial arrhythmias.