Hannes Reuter

Knockout mice for pharmacological screening: testing the specificity of Na+-Ca2+ exchange inhibitors.

The role of the Na+-Ca2+ exchanger as a major determinant of cell Ca2+ is well defined in cardiac tissue, and there has been much effort to develop specific inhibitors of the exchanger. We use a novel system to test the specificity of two putative specific inhibitors, KB-R7943 and SEA0400. The drugs are applied to electrically stimulated heart tubes from control mouse embryos or embryos with the Na+-Ca2+ exchanger knocked out. We monitored effects of the drugs on Ca2+ transients. Both drugs depress the Ca2+ transients at low concentrations even in the absence of any Na+-Ca2+ exchanger. KB-R7943 and SEA0400 are not completely specific and should be used with caution as Na+-Ca2+ exchange inhibitors.

The Na+/Ca2+ Exchange Molecule

Abstract: An overview of the molecular physiology of the Na+/Ca2+ exchanger is presented. This includes information on the variety of exchangers that have been described and their regulatory properties. Molecular insight is most detailed for the cardiac Na+/Ca2+ exchanger (NCX1). Parts of the NCS1 molecule involved in regulation and ion transport have been elucidated, and initial information on the topology and structure is available.

Mild therapeutic hypothermia in cardiogenic shock syndrome.

Objective:Mortality in patients with cardiogenic shock after out-of-hospital cardiac arrest remains high despite advances in resuscitation and early revascularization strategies. Recent studies suggest a reduced mortality in survivors of cardiac arrest subjected to mild therapeutic hypothermia, but the underlying mechanisms are not yet clear. Because positive hemodynamic effects of mild therapeutic hypothermia have been suggested, we aimed at testing the hypothesis that patients in cardiogenic shock might benefit from mild therapeutic hypothermia. Methods:Hemodynamic effects of mild therapeutic hypothermia in 20 consecutive patients admitted in cardiogenic shock after successful resuscitation from out-of-hospital cardiac arrest were investigated. A historic normothermic control group was matched (one-to-one) by means of a propensity score. Patients were cooled to 33°C for 24 hrs using an endovascular cooling device and hemodynamic variables were continuously recorded by means of pulse contour analysis. Cardiac performance was determined by echocardiography. Results:Mild therapeutic hypothermia induced a significant decrease in heart rate from 74 to 64 beats per minute. Despite the reduction in heart rate, cardiac index remained unchanged under mild therapeutic hypothermia likely due to an increase in ejection fraction from 43 ± 4% to 55 ± 4%. Mean arterial pressure increased rapidly from 75 ± 2 mm Hg to 84 ± 3 mm Hg (p = .001) upon induction of hypothermia paralleled by an initial increase in systemic vascular resistance. Accordingly, patients with mild therapeutic hypothermia required lower cumulative doses of vasopressors and inotropes. Conclusions:We conclude that in cardiogenic shock mild therapeutic hypothermia provides circulatory support and an increase in systemic vascular resistance that leads to reduced vasopressor use and may result in lower oxygen consumption. These findings suggest that mild therapeutic hypothermia could be a therapeutic option in hemodynamically unstable patients independent of cardiac arrest and further randomized clinical studies are needed.

Cardiac excitation-contraction coupling in the absence of Na(+) - Ca2+ exchange.

We investigate cardiac excitation-contraction coupling in the absence of sarcolemmal Na(+) - Ca(2+) exchange using NCX1 knock out mice. Knock out of NCX1 is embryonic lethal, and we measure Ca(2+) transients and contractions in heart tubes from embryos at day 9.5 post coitum. Immunoblot and electron microscopy both indicate that sarcoplasmic reticular membranes are diminished in the knock out (NCX(-/-)) heart tubes. Both Ni(2+) and nifedipine block excitation-contraction coupling in NCX-containing (NCX+) and NCX(-/-) heart tubes indicating an essential role for the L-type Ca(2+) current. Under basal conditions (1Hz stimulation), the NCX(-/-) heart tubes have normal Ca(2+) transients but are unable to maintain homeostasis when Ca(2+) fluxes are increased by various interventions (increased stimulation frequency, caffeine, isoproterenol). In each case, the NCX(-/-) heart tubes respond to the intervention in a more deleterious manner (increased diastolic Ca(2+), decreased Ca(2+) transient) than the NCX+ heart tubes. Expression of the sarcolemmal Ca(2+) pump was not upregulated. The sarcolemmal Ca(2+) pump, however, was able to compensate surprisingly well for the absence of Na(+) - Ca(2+) exchange under basal conditions.

Differential increase of CD34, KDR/CD34, CD133/CD34 and CD117/CD34 positive cells in peripheral blood of patients with acute myocardial infarction.

ObjectiveCirculating progenitor cells (CPC) may contribute to cardiac regeneration and neovascularization after acute myocardial infarction (AMI). For potential therapeutic use, understanding the endogenous mechanisms after ischemia is inevitable. We investigated the absolute number, but also the subset composition of CD34+ CPC after AMI.MethodsCD34+, KDR+/ CD34+, CD133+/CD34+ and CD117+/CD34+ CPC were analyzed by FACS in peripheral blood of 10 patients with acute MI (59±5 yrs, m/f=8/2) at day of AMI (day 0) and days 1–5. For comparison patients with stable coronary artery disease (CAD, n=12, 66±2 yrs, m/f=10/2) and young healthy volunteers (n=7, 26±2 yrs, m/f=3/4) were studied.ResultsCD34 and KDR/CD34, CD133/CD34, CD117/CD34 were increased day 3 and 4 after AMI. KDR+ fraction within CD34+ population remained unchanged (58.3±7.8% vs 55.3±10.6%), whereas CD133+ (64.9±3.1% vs 43.5±5.9%, P=0.006) and CD117+ fractions (71.7±5.6% vs 50.1±5.5%, P=0.02) were elevated. In CAD, all CPC and fractions were similar as AMI day 0. Healthy volunteers had more CD34+ than CAD and AMI day 0. Double positive CPC were also higher, but fractions were unchanged vs CAD with more KDR/CD34 in trend (72.8±10.6% vs 50.5±5.6%, P=0.058). After AMI both absolute numbers of CD34+ and their subset composition change, suggesting selective mobilization of CPC. Increased CPC after AMI never reach numbers of young healthy volunteers.

Mechanisms of Ca2+-Dependent Calcineurin Activation in Mechanical Stretch-Induced Hypertrophy

Pressure overload is the major stimulus for cardiac hypertrophy. Accumulating evidence suggests an important role for calcium-induced activation of calcineurin in mediating hypertrophic signaling. Hypertrophy is an important risk factor for cardiovascular morbidity and mortality. We therefore employed an in vitro mechanical stretch model of cultured neonatal cardiomyocytes to evaluate proposed mechanisms of calcium-induced calcineurin activation in terms of inhibition of calcineurin activity and hypertrophy. The protein/DNA ratio and ANP gene expression were used as markers for stretch-induced hypertrophy. Stretch increased the calcineurin activity, MCIP1 gene expression and DNA binding of NFATc as well as the protein/DNA ratio and ANP mRNA in a significant manner. The specific inhibitor of calcineurin, cyclosporin A, inhibited the stretch-induced increase in calcineurin activity, MCIP1 gene expression and hypertrophy. The L-type Ca2+ channel blocker nifedipine and a blocker of the Na+/H+ exchanger (cariporide) both suppressed stretch-dependent enhanced calcineurin activity and hypertrophy. Also application of a blocker of the Na+/Ca2+ exchanger (KB-R7943) was effective in preventing calcineurin activation and increases in the protein/DNA ratio. Inhibition of capacitative Ca2+ entry with SKF 96365 was also sufficient to abrogate calcineurin activation and hypertrophy. The blocker of stretch-activated ion channels, streptomycin, was without effect on stretch-induced hypertrophy and calcineurin activity. The present work suggests that of the proposed mechanisms for the calcium-induced activation of calcineurin (L-type Ca2+ channels, capacitative Ca2+ entry, Na+/H+ exchanger, Na+/Ca2+ exchanger and stretch-activated channels) all but stretch-activated channels are possible targets for the inhibition of hypertrophy.

Aldosterone induces electrical remodeling independent of hypertension

BACKGROUND Treatment of heart failure patients with aldosterone antagonists has been shown to reduce the occurrence of sudden cardiac death. Therefore we aimed at determining the consequences of chronic exposure to aldosterone and the aldosterone antagonists eplerenone and spironolactone on the electrophysiological properties of the heart in a rat model. METHODS AND RESULTS Male Wistar rats were chronically treated (4weeks) with aldosterone (ALD) via an osmotic minipump. Spironolactone (SPI) or eplerenone (EPL) was administered with the rat chow. ALD treated animals developed left ventricular hypertrophy, prolonged QT-intervals, a higher rate of ventricular premature beats and non-sustained ventricular tachycardia despite normal blood pressure values. Spironolactone and eplerenone were both able to inhibit the alterations. Left-ventricular mRNA expressions of Kv4.2 and Kv4.3 (Ito), Kv1.5 (IKur), Kir2.1 and Kir2.3 (IK1) and of Cav1.2 (L-type Ca(2+) channel) were significantly down-regulated in ALD. Correspondingly, the protein expressions of subunits Kv1.5, Kir2.3 and Cav1.2 were significantly decreased. A diminished calcineurin activity and mRNA expression of the Aß subunit of calcineurin were found in ALD, which was insensitive to aldosterone antagonists. CONCLUSIONS Chronic aldosterone-overload induces blood pressure independent structural and electrical remodeling of the myocardium resulting in an increased risk for malignant ventricular arrhythmias.

Sarcoplasmic Ca2+ release is prolonged in nonfailing myocardium of diabetic patients

Background Asymptomatic diabetic patients have a high incidence of clinically unrecognized left ventricular dysfunction with an abnormal cardiac response to exercise. We, therefore, examined subclinical defects in the contraction–relaxation cycle and intracellular Ca2+ regulation in myocardium of asymptomatic type 2 diabetic patients. Methods Alterations in the dynamics of the intracellular Ca2+ transient and contractility were recorded in right atrial myocardium of type 2 diabetic patients and non-diabetic control tissue loaded with fura-2. In order to gain an insight into mechanisms underlying the altered Ca2+ handling in diabetic myocardium levels of mRNA, protein expression and phosphorylation of key proteins in sarcoplasmic Ca2+ handling were determined. Results In isolated atrial trabeculae of diabetic myocardium the rise of systolic Ca2+ was significantly prolonged, but relaxation of the Ca2+ transient was unaltered compared to control tissue. Accordingly, the levels of expression of mRNA and protein of the Ca2+ release channel (RyR2) of the sarcoplasmic reticulum were reduced by 68 and 22%, respectively. Endogenous phosphorylation of RyR2 by protein kinases C, however, was increased by 31% in diabetic myocardium, as assessed by the back-phosphorylation technique. Levels of expression of SERCA2 and phospholamban were unaltered between both groups. Conclusions Intracellular Ca2+ release is prolonged in non-failing myocardium of type 2 diabetic patients and this may be primarily due to a decreased expression of RyR2. This defective Ca2+ release may represent an early stage of ventricular dysfunction in type 2 diabetes and would favor the abnormal response to exercise frequently observed in asymptomatic diabetic patients.

The increased angiotensin II (type 1) receptor density in myocardium of type 2 diabetic patients is prevented by blockade of the renin–angiotensin system

Aims/hypothesisThe angiotensin II (type 1) (AT1) receptor mediates many biological effects of the renin–angiotensin system (RAS), leading to remodelling of cardiac tissue. The present study was designed to analyse changes in the function and expression of the AT1 receptor as principal effector of the RAS in myocardium from type 2 diabetic patients compared with non-diabetic myocardium as control. In addition, we determined the effect of treatment with ACE inhibitors or AT1 receptor blockers on expression levels of the receptor in diabetic patients.MethodsGene expression of the AT1 receptor was analysed by quantitative RT-PCR and protein expression was determined by immunoblot analysis in human right atrial myocardium. We investigated functional coupling of the receptors by measuring contractility in isolated trabeculae stimulated with increasing concentrations of angiotensin II.ResultsDiabetic myocardium showed a significant increase in protein expression (170 ± 16% of control) and median mRNA expression (186% of control) of the AT1 receptor. The additional receptors were functionally coupled, resulting in a stronger inotropic response upon stimulation with angiotensin II (89 ± 5.5% vs 29 ± 1.6% in controls), whereas receptor affinity was similar in both groups. However, myocardium from diabetic patients treated with ACE inhibitors or AT1 receptor blockers showed no increase in AT1 receptor expression.Conclusions/interpretationAT1 receptor expression in myocardium of type 2 diabetic patients is dynamic, depending on the level of glycaemic control and the activity of the RAS. These findings could at least in part explain the strong therapeutic benefit of RAS inhibition in diabetic patients.

Mortality in a cardiac intensive care unit

ObjectiveThere are no reliable data on mortality and morbidity of adult patients in modern university-based cardiac intensive care units. Therefore, the present study was aimed to provide complete data in respect to mortality and morbidity of all patients admitted between 1 January 2008 and 31 December 2009 to the newly opened cardiac intensive care unit of the Heart Centre of the Cologne University Hospital.MethodsAll patients admitted to the 6-bed intensive care unit of the Heart Centre of the University of Cologne between January 1 2008 and December 31 2009 were included in this study.ResultsA total of 684 patients were investigated. The majority of patients (71.1%) were male. The overall in-hospital mortality was 32.5%. The most frequent diagnosis was acute coronary syndrome (43.6%). Coronary angiography was performed in 45.5% of all patients. Cardiopulmonary resuscitation was the reason for admission in 30.8%, the in-hospital mortality of those patients (46.0%) was much higher compared to the overall mortality.ConclusionsOur data demonstrate that despite state-of-the-art university-based intensive care medicine with modern equipment the mortality remains high. These findings will help in calculating the resources required to meet the increasing demand for intensive care medicine.