André Renner

Inhibition of Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Improves Contractility in Human Failing Myocardium

Rationale: Heart failure (HF) is known to be associated with increased Ca2+/calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium. Objective: We sought to investigate detailed CaMKII&dgr; expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Methods and Results: Expression analysis revealed that CaMKII&dgr;, both cytosolic &dgr;C and nuclear &dgr;B splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca2+ loading. This was confirmed in isolated myocytes by a reduced SR Ca2+ spark frequency and hence SR Ca2+ leak, resulting in increased SR Ca2+ load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca2+ leak, was found to be markedly reduced in KN-93–treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae. Conclusions: The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKII&dgr; expression is increased. The mechanism proposed consists of a reduced SR Ca2+ leak and consequently increased SR Ca2+ load. Thus, CaMKII inhibition appears to be a possible therapeutic option for patients with HF and merits further investigation.

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase A Differentially Regulate Sarcoplasmic Reticulum Ca2+ Leak in Human Cardiac Pathology

Background —Sarcoplasmatic reticulum (SR) Ca2+-leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF). However, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy (Hy) to HF are still controversial. Methods and Results —We investigated healthy control myocardium (NF, n=5) as well as myocardium from patients with compensated Hy (n=25) and HF (n=32). In Hy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylate RyR2 at levels which are not different from NF. Accordingly, inhibitors of these kinases reduce the SR Ca2+-leak. In HF, however, the SR Ca2+-leak is nearly doubled compared to Hy leading to reduced systolic Ca2+-transients, a depletion of SR Ca2+-storage and elevated diastolic Ca2+-levels. This is accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation is not increased in HF and is independent of previous β-blocker treatment. In HF CaMKII inhibition but not inhibition of PKA yields a reduction of the SR Ca2+-leak. Moreover, PKA inhibition further reduces SR Ca2+-load and systolic Ca2+-transients. Conclusions —In human Hy CaMKII as well as PKA functionally regulate RyR2 and may induce SR Ca2+-leak. In the transition from Hy to HF the diastolic Ca2+-leak increases and disturbed Ca2+-cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2-phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.Background— Sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca2+ leak. In HF, however, the SR Ca2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca2+ transients, a depletion of SR Ca2+ storage and elevated diastolic Ca2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation was not increased in HF and was independent of previous &bgr;-blocker treatment. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca2+ leak. Moreover, PKA inhibition further reduced SR Ca2+ load and systolic Ca2+ transients. Conclusions— In human hypertrophy, both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca2+ leak. In the transition from hypertrophy to HF, the diastolic Ca2+ leak increases and disturbed Ca2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.

CaMKII and PKA Differentially Regulate SR Ca2+-Leak in Human Cardiac Pathology

Background —Sarcoplasmatic reticulum (SR) Ca2+-leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF). However, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy (Hy) to HF are still controversial. Methods and Results —We investigated healthy control myocardium (NF, n=5) as well as myocardium from patients with compensated Hy (n=25) and HF (n=32). In Hy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylate RyR2 at levels which are not different from NF. Accordingly, inhibitors of these kinases reduce the SR Ca2+-leak. In HF, however, the SR Ca2+-leak is nearly doubled compared to Hy leading to reduced systolic Ca2+-transients, a depletion of SR Ca2+-storage and elevated diastolic Ca2+-levels. This is accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation is not increased in HF and is independent of previous β-blocker treatment. In HF CaMKII inhibition but not inhibition of PKA yields a reduction of the SR Ca2+-leak. Moreover, PKA inhibition further reduces SR Ca2+-load and systolic Ca2+-transients. Conclusions —In human Hy CaMKII as well as PKA functionally regulate RyR2 and may induce SR Ca2+-leak. In the transition from Hy to HF the diastolic Ca2+-leak increases and disturbed Ca2+-cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2-phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.Background— Sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca2+ leak. In HF, however, the SR Ca2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca2+ transients, a depletion of SR Ca2+ storage and elevated diastolic Ca2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation was not increased in HF and was independent of previous &bgr;-blocker treatment. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca2+ leak. Moreover, PKA inhibition further reduced SR Ca2+ load and systolic Ca2+ transients. Conclusions— In human hypertrophy, both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca2+ leak. In the transition from hypertrophy to HF, the diastolic Ca2+ leak increases and disturbed Ca2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure

Aims Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

Clampless Off-Pump Versus Conventional Coronary Artery Revascularization A Propensity Score Analysis of 788 Patients

Background— This study aimed to assess if clampless off-pump coronary artery bypass grafting (CABG) decreases risk-adjusted mortality, stroke rate, and morbidity in unselected patients in comparison to conventional CABG. Methods and Results— Between July 2009 and November 2010, data of 1282 consecutive patients undergoing isolated CABG were prospectively recorded. In 30.8% (n=395), clampless off-pump revascularization was used, either with the PAS-Port automated central venous anastomosis system (n=310) or as total arterial revascularization without central anastomoses (n=85). Propensity score (PS) matching was performed based on 15 preoperative risk variables to correct for selection bias. In-hospital mortality and stroke rate as primary end point, as well as major complications and follow-up outcome of clampless off-pump (lessOPCAB) and conventional CABG (cCABG) were compared in 394 matched patient pairs (total: 788 patients). The clampless off-pump technique decreased the in-hospital rate of death (odds ratio, 0.25; 95% confidence interval, 0.05–1.18, P=0.080) and stroke (odds ratio, 0.36; 95% confidence interval, 0.13–0.99, P=0.048) significantly. Complications such as low cardiac output syndrome, prolonged ventilation and rethoracotomy were also reduced by lessOPCAB. Over a 2-year follow-up period overall survival, cerebrovascular and major adverse event rate were significantly lower in the lessOPCAB group, while the repeat revascularization rate was comparable. Conclusions— In a retrospective PS-matched analysis, clampless off-pump CABG lowers mortality, stroke rate and other morbidity in an unselected group of patients with coronary artery disease.

Ca2+/calmodulin‐dependent protein kinase II equally induces sarcoplasmic reticulum Ca2+ leak in human ischaemic and dilated cardiomyopathy

The sarcoplasmic reticulum (SR) Ca2+ leak is an important pathomechanism in heart failure (HF). It has been suggested that Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is only relevant for the induction of the SR Ca2+ leak in non‐ischaemic but not in ischaemic HF. Therefore, we investigated CaMKII and its targets as well as the functional effects of CaMKII inhibition in human ischaemic cardiomyopathy (ICM, n = 37) and dilated cardiomyopathy (DCM, n = 40).

Extra cellular matrix remodelling after heterotopic rat heart transplantation: gene expression profiling and involvement of ED-A+ fibronectin, alpha-smooth muscle actin and B+ tenascin-C in chronic cardiac allograft rejection

Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A+ Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B+ Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A+ Fn, ASMA and B+ Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A+ Fn, ASMA and B+ Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A+ Fn and B+ Tn-C might functionally contribute to the development of chronic cardiac rejection.

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.

Antiarrhythmic effects of dantrolene in human diseased cardiomyocytes

BACKGROUND Cardiac type 2 ryanodine receptors (RyR2s) play a pivotal role in cellular electrophysiology and contractility. Increased RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca2+ release is linked to heart failure (HF) and arrhythmias. Dantrolene, a drug used for the treatment of malignant hyperthermia, is known to stabilize RyRs in skeletal muscle. OBJECTIVE The purpose of this study was to investigate the effects of dantrolene on arrhythmogenic triggers and contractile function in human atrial fibrillation (AF) and HF cardiomyocytes (CM). METHODS Human CM were isolated from either patients with HF (ventricular) or patients with AF (atrial), and Ca2+ imaging, patch-clamp, or muscle strip experiments were performed. RESULTS After exposure to dantrolene, human atrial AF and left ventricular HF CM showed significant reductions in proarrhythmic SR Ca2+ spark frequency and diastolic SR Ca2+ leak. Moreover, dantrolene decreased the frequency of Ca2+ waves and spontaneous Ca2+ transients in HF CM. Patch-clamp experiments revealed that dantrolene significantly suppressed delayed afterdepolarizations in HF and AF CM. Importantly, dantrolene had no effect on action potential duration in AF or in HF CM. In addition, dantrolene had neutral effects on contractile force of human isometrically twitching ventricular HF trabeculae. CONCLUSION Our study showed that dantrolene beneficially influenced disrupted SR Ca2+ homeostasis in human HF and AF CM. Cellular arrhythmogenic triggers were potently suppressed by dantrolene, whereas action potential duration and contractility were not affected. As a clinically approved drug for the treatment of malignant hyperthermia, dantrolene may be a potential antiarrhythmic drug for patients with rhythm disorders and merits further clinical investigation.

Expression of extra domain A containing fibronectin in chronic cardiac allograft rejection

BACKGROUND Cardiac allograft vasculopathy (CAV) and fibrosis are important in chronic cardiac allograft rejection. The aim of our study was to analyze the up-regulation of extra domain A (ED-A) containing fibronectin (ED-A(+) Fn) in cardiac allografts after heterotopic rat heart transplantation using a human recombinant antibody applicable for targeted drug delivery. METHODS Cardiac allografts were subjected to immunofluorescence double labelling procedures combining a human recombinant small immunoprotein (SIP) format antibody recognizing ED-A(+) Fn (F8) with antibodies recognizing CD31, ASMA or CD45. Protein expression levels of ED-A(+) Fn were measured by quantitative confocal laser scanning microscopy and messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction. RESULTS A distinct re-expression of ED-A(+) Fn was detectable with the F8 antibody, especially in vessel structures exhibiting CAV and in fibrotic areas. ED-A(+) Fn protein deposition but not messenger RNA expression levels increased with rising rejection grade (p ≤ 0.001). There were clear co-localizations of ED-A(+) Fn and α-smooth muscle actin in vessels and in fibrotic areas. CONCLUSIONS We could show first that ED-A(+) Fn is expressed in rat cardiac allografts in association with CAV and cardiac fibrosis. The protein is detectable with the human recombinant antibody F8 usable for targeted drug delivery to the side of disease. Second, protein expression levels increase with rising rejection grade. Thus, ED-A(+) Fn might be usable to monitor and target CAV as well as fibrosis after heart transplantation.