Christoph Ziskoven

Mechanisms of β3-adrenoceptor-induced eNOS activation in right atrial and left ventricular human myocardium

β‐adrenoceptors are important modulators of cardiac function. The present study investigated β3‐adrenergic eNOS activation in human myocardium. We measured nitric oxide (NO) liberation (diaminofluorescence) and signal transduction (immunohistochemistry, phosphorylation of eNOSSer1177, eNOSThr495, eNOSSer114, Akt/protein kinase B (Akt/PKB), and eNOS translocation) in human right atrial (RA, aortocoronary‐bypass OP) and left ventricular nonfailing (LV, rejected donor hearts) myocardium after application of BRL 37344 (BRL), a preferential β3‐adrenoceptor agonist. In both RA and LV, BRL (10 μl) induced a liberation of NO. An eNOS activation via translocation was only observed in RA after application of BRL (10 μM). Yet, the NO liberation in both LV and RA was accompanied by phosphorylation of eNOSSer1177 and Akt/PKB. BRL‐induced eNOS phosphorylation was abolished by LY292004, a blocker of PI‐3 kinase. eNOS‐Ser114 phosphorylation was unchanged in RA, but decreased in LV after β3‐adrenergic stimulation. BRL did not alter phosphorylation of eNOSThr495. In conclusion, receptor‐dependent eNOS activation is differentially regulated in the human heart. In the left ventricle, eNOS activation via phosphorylation seems to be of major importance, whereas in human atrial myocardium eNOS translocation is the predominant mechanism induced by β3‐adrenergic activation.

Oxidative stress in secondary osteoarthritis: from cartilage destruction to clinical presentation?

Due to an increasing life expectance, osteoarthritis (OA) is one of the most common chronic diseases. Although strong efforts have been made to regenerate degenerated joint cartilage, OA is a progressive and irreversible disease up to date. Among other factors the dysbalance between free radical burden and cellular scavenging mechanisms defined as oxidative stress is a relevant part of OA pathogenesis. Here, only little data are available about the mediation and interaction between different joint compartments. The article provides a review of the current literature regarding the influence of oxidative stress on cellular aging, senescence and apoptosis in different joint compartments (cartilage, synovial tissue and subchondral bone). Free radical exposure is known to promote cellular senescence and apoptosis. Radical oxygen species (ROS) involvement in inflammation, fibrosis control and pain nociception has been proven. The data from literature indicates a link between free radical burden and OA pathogenesis mediating local tissue reactions between the joint compartments. Hence, oxidative stress is likely not only to promote cartilage destruction but also to be involved in inflammative transformation, promoting the transition from clinically silent cartilage destruction to apparent OA. ROS induced by exogenous factors such as overload, trauma, local intraarticular lesion and consecutive synovial inflammation cause cartilage degradation. In the affected joint, free radicals mediate disease progression. The interrelationship between oxidative stress and OA etiology might provide a novel approach to the comprehension and therefore modification of disease progression and symptom control.

Physiology and pathophysiology of nitrosative and oxidative stress in osteoarthritic joint destruction

Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synovial tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.

Effects of the β3-Adrenergic Agonist BRL 37344 on Endothelial Nitric Oxide Synthase Phosphorylation and Force of Contraction in Human Failing Myocardium

BACKGROUND In nonfailing myocardium, beta(3)-adrenergic signaling causes a decrease in contractility via endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. This study investigates the hypothesis that beta(3)-adrenergic signaling undergoes alterations in failing myocardium. METHODS We compared eNOS- and beta(3)-adrenoceptor expression using Western blot analysis in human nonfailing myocardium versus failing myocardium. With the use of immunohistochemistry, we investigated the distribution of the beta(3)-adrenoceptor protein and eNOS translocation and phosphorylation under basal conditions. beta(3)-adrenergic, eNOS activation, and inotropy were measured in failing myocardium using BRL37344 (BRL, a beta(3)-adrenoceptor agonist). RESULTS beta(3)-adrenoceptor expression was increased in failing myocardium. Under basal conditions, Akt- and eNOS(Ser1177) phosphorylation were reduced in failing myocardium. During stimulation with BRL in failing myocardium, a further dephosphorylation of eNOS(Ser1177) and Akt was observed, whereas eNOS(Ser114) phosphorylation was increased. These results suggest a deactivation of eNOS via beta(3)-adrenergic stimulation. Nevertheless, BRL decreased contractility in failing myocardium, but this effect was not observed in the presence of the NO blocker L-NMA. In failing myocardium, the beta(3)-adrenoceptor was predominantly expressed in endothelial cells. In the cardiomyocytes, the beta(3)-adrenoceptor was mainly located at the intercalated disks. CONCLUSION In failing cardiomyocytes, beta(3)-adrenergic stimulation seems to deactivate rather than activate eNOS. At the same time, beta(3)-adrenergic stimulation induced a NO-dependent negative inotropic effect. Because beta(3)-adrenoceptors are expressed mainly in the endothelium in failing myocardium, our observations suggest a paracrine-negative inotropic effect via NO liberation from the cardiac endothelial cells.

Increased Ca2+ sensitivity and protein expression of SERCA 2a in situations of chronic β3-adrenoceptor deficiency

This study investigated the influence of chronic β3-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca2+-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and β3-adrenoceptor knockout mice (β3-KNO, n=7). Morphometric heart analysis showed no difference between WT and β3-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in β3-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in β3-KNO. Alterations in phospholamban phosphorylation were independent of alterations in β1/β2-adrenoceptor distribution and protein expression of G proteins in β3-KNO. Measurement of myofibrillar Ca2+ sensitivity showed no difference in the Ca2+/force relation for WT and β3-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and β3-KNO. The conclusion is that depression of β3-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca2+ uptake. Thus, β3-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.

Surgical and nonsurgical treatment of total rupture of the pectoralis major muscle in athletes: update and critical appraisal.

The complete rupture of the pectoralis major tendon is an uncommon injury but has become increasingly common among athletes in recent years. This may be due to a higher number of individuals taking part in high-impact sports and weightlifting as well as the use of anabolic substances, which can make muscles and tendons vulnerable to injury. In recent literature, there are only few recommendations to rely on conservative treatment alone, but there are a number of reports and case series recommending early surgical intervention. Comparing the results of the two treatment regimens, there is clear evidence for a superior outcome after surgical repair with better cosmesis, better functional results, regaining of muscle power, and return to sports compared with the conservative treatment. In summary, anatomic surgical repair is the treatment of choice for complete acute ruptures of the pectoralis major tendon or muscle in athletes.

Reply to the Letter to the Editor: Is the flexion strength really increased after tenodesis for tendinopathy of the Long Head of the Biceps?

In regard to the other two studies, post-operative strength analysis alone does not allow valid conclusion in regard to improvement of elbow flexion strength. However, the authors agree that pain reduction certainly is a factor that can have an influence on the elbow flexion strength preoperatively. It is not analysed in the study that the increase in flexion strength is solely a result of the tenodesis or can probably be achieved by a biceps tenotomy as well. Whether the elbow flexion strength is partly compensated by intact short head of the biceps is difficult to evaluate but certainly a factor that must be considered. The authors are aware that the present study has some limitations, which are extensively emphasized in the discussion section. In regard to prospective randomized trials comparing tenodesis or tenotomy, the authors agree that high powered studies with a large number of participants are necessary in order to provide further evidence for the selection of the ideal treatment of long head of biceps pathology in our patients. But, as pointed out before, including patients with concomitant rotator cuff repair as in the study initiated by Gurnani et al. does certainly not provide any viable answers. In conclusion, a prospective randomized trial should be conducted on isolated biceps lesion only and must include supination and elbow flexion strength measured preoperatively and post-operatively as well as the evaluation of an objectively defined Popeye sign deformity in order to allow any further conclusion that can influence the choice for either treatment. This type of study has been started in form of a national DVSE multicentre study in 2015, and preliminary results are expected soon. The authors thank Gurnani et al. for their interest in the study and for once again pointing out the limitations and We would like to thank the Gurnani et al. for their interest and comments on our recent article titled “The influence of suprapectoral arthroscopic biceps tenodesis for isolated biceps lesions on elbow flexion force and clinical outcomes” that prospectively evaluates the influence of all-arthroscopic suprapectoral biceps tenodesis for isolated biceps lesions on elbow flexion force, cosmetic and clinical outcome [3]. Gurnani et al. bring up some points of interest. They noted that not tenodesis but pain reduction is the major factor that is responsible for the increase in elbow flexion strength. The writers deduct this argument from their own review [2] in which they analysed five available studies comparing tenodesis or tenotomy in regard to elbow flexion strength. However, in three of the five included studies patients with concomitant rotator cuff repair were included serving as a huge bias because the influence of the rotator cuff tears must not be neglected. In the other two studies, the elbow flexion strength was only measured post-operatively, bringing no further information in terms of the preoperative status [1, 4–7]. None of those does allow any objective conclusions in regard to the elbow flexion strength in the treatment of long head of biceps pathology. Simultaneous rotator cuff repair must be considered as a major bias on the clinical scores and might also have an influence on elbow flexion strength by impairing shoulder function. Furthermore, one must keep in mind that rotator cuff repair was the leading pathology indicating shoulder surgery in those studies analysed.

LBS-Tenotomie oder LBS-Tenodese?

ZusammenfassungLäsionen der langen Bizepssehne (LBS) verursachen glenohumerale Schmerzen, die durch eine proximale LBS-Desinsertion therapiert werden können. Ein Kraftverlust für die Ellenbogenflexion und Unterarmsupination nach Tenotomie ist beschrieben und kann auch mit einer Tenodese nicht immer komplett vermieden werden. Auch konsekutive „Popeye-sign“-Deformitäten und temporäre Bizepskrämpfe sind bekannt. Bis auf das vermehrt nach Tenotomie auftretende „Popeye sign“ kann bisher kein Konsensus über den Vorteil der Tenodese gefunden werden, wobei es an prospektiv randomisierten Studien zum Vergleich der Tenotomie mit suffizienten LBS-Tenodesetechniken mangelt. Hierbei werden neben offener und arthroskopisch subakromialer oder glenohumeraler Technik verschiedene Fixationsimplantate an supra- oder subpektoraler Position unterschieden. Als Goldstandard hat sich die intraossäre Sehnenfixation mit einer Interferenzschraube erwiesen. Vorteile der subpektoralen Technik sind die suffiziente Weichteildeckung und die Entfernung des intrakanalikulären Teils der LBS. Ein Nachteil der suprapektoralen arthroskopischen subakromialen im Vergleich zur glenohumeralen Technik ist die vermehrte subdeltoidale Weichteilsakrifizierung.AbstractLesions of the long head of the biceps tendon (LHB) are painful but can be treated by biceps tenotomy. A decrease in elbow flexion force and lower arm supination force following tenotomy is well known and cannot be totally avoided by biceps tenodesis. A Popeye deformity and temporary cramps of the biceps muscle have been regularly described after LHB tenotomy. There is no consensus about the advantages of LHB tenodesis except the appearance of a Popeye deformity; however, there is a lack of prospective randomized trials comparing tenotomy with sufficient LHB tenodesis techniques. In this respect, in addition to arthroscopic subacromial, glenohumeral and open techniques, various implants for fixation at the suprapectoral and subpectoral positions are distinguished. The evaluation of intraosseous tendon fixation using an interference screw has yielded excellent results and is therefore considered to be the gold standard. Advantages of the subpectoral technique are sufficient soft tissue covering and removal of the intracanalicular part of the LHB. A disadvantage of the suprapectoral arthroscopic technique via the subacromial approach in comparison to the glenohumeral approach is the higher amount of soft tissue sacrificed when using the subacromial approach.

390 DEPENDENCY OF ENDOTHELIAL NITRIC OXIDE SYNTHASE-ACTIVATION MECHANISMS ON INTRACELLULAR CA2+-CONCENTRATION IN HUMAN ATRIAL MYOCARDIUM

We have recently shown two ways of endothelial nitric oxide synthase (eNOS)-activation in human myocardium (Brixius et al. 2004): 1) a translocation and 2) an Akt-dependent phosphorylation of the enzyme at Ser1177. The present study investigates the Ca2+ dependency of these two mechanisms. Methods and Results: Human right atrial tissue was obtained from patients undergoing coronary bypass-operation. In immunohistochemical experiments, the translocated form of eNOS as well as the (Ser1177)-phosphorylated eNOS (peNOS) and the phosphorylated form of Akt (pAkt) were labelled using specific antibodies. eNOS-translocation was measured in the absence and presence of the Ca2+ chelator BAPTA after application of BRL 37344 (BRL), a preferential β3-adrenoceptor agonist, which has been shown to increase eNOS activity in human right atrial myocardium. In the absence of BAPTA, BRL (10 μM) time-dependently increased staining intensity of translocated eNOS (+BRL, t=5 min, +53.52±15.21%; n=6; p≤0.05), whereas in the presence of BAPTA, this effect was blunted (20 μM; +BRL (10 μM; t=5 min): -42.11±11.26%; n=4; p(0.05). In contrast, BRL 37344 significantly increased staining of peNOS (+BRL 37344 (10 μM; t=5 min): +156.6±26.4%; n=3; p≤0.05) and pAkt in the presence of BAPTA. Using the NO-sensitive dye diaminofluorescein (DAF), we could show that BRL induced a strong release of NO (+BRL 37344 (10 μM): +237.1±70.4%). This effect however was completely abolished in the presence of BAPTA. Though Ca2+ dependent, the translocation state of myocardial eNOS was not changed by the adenylate cyclase activator forskolin (0.3 μM) (+forskolin (t=5 min): -20.2±12.3%; n=3; p(0.05) nor was NO-release induced (n=2). Conclusions: 1) In human myocardium, BRL induced eNOS-translocation is dependent on intracellular Ca2+ whereas eNOS-phosphorylation is not. 2) At least in atrial myocardium, eNOS-translocation and not eNOS-phosphorylation generates bulk NO. 3) Though Ca2+ dependent, eNOS-translocation and NO-release could not be mimicked by adenylate cyclase activation as a mediator of β-adrenergic stimulation.