Carsten Zobel

Regulation of cardiac excitation–contraction coupling by action potential repolarization: role of the transient outward potassium current (Ito)

The cardiac action potential (AP) is critical for initiating and coordinating myocyte contraction. In particular, the early repolarization period of the AP (phase 1) strongly influences the time course and magnitude of the whole‐cell intracellular Ca2+ transient by modulating trans‐sarcolemmal Ca2+ influx through L‐type Ca2+ channels (ICa,L) and Na‐Ca exchangers (ICa,NCX). The transient outward potassium current (Ito) has kinetic properties that make it especially effective in modulating the trajectory of phase 1 repolarization and thereby cardiac excitation‐contraction coupling (ECC). The magnitude of Ito varies greatly during cardiac development, between different regions of the heart, and is invariably reduced as a result of heart disease, leading to corresponding variations in ECC. In this article, we review evidence supporting a modulatory role of Ito in ECC through its influence on ICa,L, and possibly ICa,NCX. We also discuss differential effects of Ito on ECC between different species, between different regions of the heart and in heart disease.

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs: Effects of β-Blockade and Angiotensin-Converting Enzyme Inhibition

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by 86Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5±0.5 to 86.0±27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3±1.7 to 129.8±51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1±1.2 nmol/L and at 30±1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7±0.8 to 166.9±91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the &bgr;1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

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.

Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes

Prolonged action potential duration (APD) and decreased transient outward K+ current (Ito) as a result of decreased expression of Kv4.2 and Kv4.3 genes are commonly observed in heart disease. We found that treatment of cultured neonatal rat ventricular myocytes with Heteropoda Toxin3, a blocker of cardiac Ito, induced hypertrophy as measured using cell membrane capacitance and 3H-leucine uptake. To dissect the role of specific Ito-encoding genes in hypertrophy, Ito was selectively reduced by overexpressing mutant dominant-negative (DN) transgenes. Ito amplitude was reduced equally (by about 50%) by overexpression of DN Kv1.4 (Kv1.4N) or DN Kv4.2 (either Kv4.2N or Kv4.2W362F), but only DN Kv4.2 prolonged APD duration (at 1 Hz) and induced myocyte hypertrophy. This hypertrophy was prevented by coexpressing wild-type Kv4.2 channels (Kv4.2F) with the DN Kv4.2 genes, suggesting the hypertrophy is due to Ito reduction and not nonspecific effects of transgene overexpression. The hypertrophy caused by reductions of Kv4.x-based Ito was associated with increased activity of the calcium-dependent phosphatase, calcineurin, and could be prevented by coinfection with Ad-CAIN, a specific calcineurin inhibitor. The hypertrophy and calcineurin activation induced by Kv4.2N infection were prevented by blocking Ca2+ entry and excitability with verapamil or high [K+]o. Our studies suggest that reductions of Kv4.2/3-based Ito play a role in hypertrophy signaling by activation of calcineurin.

Selective Inhibition of Inward Rectifier K+ Channels (Kir2.1 or Kir2.2) Abolishes Protection by Ischemic Preconditioning in Rabbit Ventricular Cardiomyocytes

Volume regulatory Cl− channels are key regulators of ischemic preconditioning (IPC). Because Cl− efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that IK1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPCCM 18.0±2.1% versus control (CCM) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPCCM+AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPCCM+AdEGFPKir2.2DN 47.9±1.4% versus IPCCM; P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both CCM+ AdEGFPKir2.1DN 45.8±0.7% and CCM+AdEGFPKir2.2DN 46.2±1.3% versus CCM; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after IK1 blockade with 20 &mgr;mol/L BaCl2 plus 1 &mgr;mol/L nifedipine (to prevent Ba2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that IK1 channels play an important role in IPC protection.

Calcineurin Increases Cardiac Transient Outward K+ Currents via Transcriptional Up-regulation of Kv4.2 Channel Subunits

Fast transient outward potassium currents (Ito,f) are critical determinants of regional heterogeneity of cardiomyocyte repolarization as well as cardiomyocyte contractility. Additionally, Ito,f densities are markedly down-regulated in cardiac hypertrophy and heart disease, conditions associated with activation of the serine/threonine phosphatase calcineurin (Cn). In this study, we investigated the regulation of Ito,f expression by Cn in cultured neonatal rat ventricular myocytes (NRVMs) with and without α1-adrenoreceptor stimulation with phenylephrine (PE). Overexpression of constitutively active Cn in NRVMs induced hypertrophy and caused profound increases in Ito,f density as well as Kv4.2 mRNA and protein expression and promoter activity, without affecting Kv4.3 or KChIP2 levels. The effects of Cn on hypertrophy, Ito,f, and Kv4.2 transcription were associated with NFAT activation and were abrogated by NFAT inhibition. Despite activating Cn and inducing hypertrophy in NRVMs, PE resulted in profound down-regulation of Ito,f densities as well as Kv4.2, Kv4.3, and KChIP2 expression. Although hypertrophy and NFAT activation were inhibited by the Cn inhibitory peptide CAIN, Ito,f and Kv4.2 expression were further reduced by CAIN, whereas Cn overexpression eliminated PE-induced reductions in Ito,f and Kv4.2 expression without affecting Kv4.3 or KChIP2 levels. We conclude that Cn increases cardiac Ito,f densities by positively regulating Kv4.2 gene transcription. Consistent with this conclusion, we found that Ito,f was increased in myocytes isolated from young mice overexpressing Cn prior to the development of heart disease. This positive regulation of Kv4.2 transcription by Cn activation is expected to minimize the reductions in Ito,f and Kv4.2 expression observed in hypertrophic cardiomyocytes.

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.