Bridging vascular physiology to vascular medicine: an integrative laboratory class.

Vascular diseases of the legs are highly prevalent and constitute an important part of medical curricula. The understanding of these diseases relies on strongly interwoven aspects of vascular physiology and vascular medicine. We aimed to connect these within a horizontally integrated laboratory class on vascular physiology of the leg that was designed in cooperation between the departments of physiology and vascular surgery. Conceptually, we applied examination techniques of vascular medicine to visualize physiological parameters that are altered by the most frequent diseases. This facilitates integrative discussions on malfunctions, trains diagnostic skills, and bridges to vascular medicine. In four experiments, we use oscillometry and impedance venous occlusion plethysmography to address key aspects of the arterial and venous system of the legs: 1) arterial pulse wave, 2) arterial systolic blood pressure, 3) venous capacitance and venous outflow, and 4) reactive hyperemia. After the experiments, physiological vascular function, the associated diseases, their impact on the recorded parameters, and diagnostic options are discussed. To allow reproduction, we describe the course structure and the experimental setup in detail. We present the experimental data of a cohort of medical students and document learning success and student satisfaction. All experiments were feasible and provided robust data on physiologically and clinically relevant vascular functions. The activity was perceived positively by the students and led to a substantial improvement of knowledge. With this work, we offer a template for reproduction or variation of a proven concept of horizontally integrated teaching of vascular physiology of the leg.NEW & NOTEWORTHY This article presents an integrative laboratory class on vascular physiology bridging to vascular medicine. The four experiments rely on oscillometry and venous occlusion plethysmography. We describe in detail this new class regarding structure, experimental setup, and experimental procedure, and we give insight into the applied materials. Moreover, we present the experimental data of 74 students and a quantitative evaluation of the students' learning success and acceptance.

A Comparative Analysis of Bupivacaine and Ropivacaine Effects on Human Cardiac SCN5A Channels.

BACKGROUND: Intoxication with local anesthetics may induce cardiac arrhythmias by interaction with ion channels. Ropivacaine has been introduced into clinical anesthesia as a safer alternative to bupivacaine, which is associated with a relatively high risk of cardiac arrhythmias. Diverging safety profiles may result from differences in the mode of interaction with cardiac Na(+) channels. We conducted this study to test this hypothesis and to provide experimental basis for the ongoing discussion regarding the cardiotoxic profiles of these local anesthetics. METHODS: The influence of bupivacaine and ropivacaine on the electrophysiological properties of Na(+) channels was investigated in human embryonic kidney-293 cells stably transfected with SCN5A channels cloned from the human heart using the patch-clamp technique in the outside-out configuration. RESULTS: Open-channel block of SCN5A channels was concentration dependent, with bupivacaine being approximately 4.5-fold more potent than ropivacaine (IC50 = 69.5 ± 8.2 µM vs IC50 = 322.2 ± 29.9 µM). Both drugs influenced the voltage dependency of channel activation and steady-state inactivation by shifting the membrane potential of half-maximal activation/inactivation toward somewhat more negative membrane potentials. In their inactivated state, SCN5A channels were slightly more sensitive toward bupivacaine than toward ropivacaine (IC50 = 2.18 ± 0.16 µM vs IC50 = 2.73 ± 0.27 µM). Blockade of inactivated channels developed in a concentration-dependent manner, with comparable time constants for both drugs, whereas recovery from block was approximately 2-fold faster for ropivacaine than for bupivacaine. CONCLUSIONS: Human cardiac Na(+) channels show state-dependent inhibition by ropivacaine, and the mode of interaction is comparable to that of bupivacaine. Therefore, modest differences in cardiotoxicity between these local anesthetic drugs are compatible with subtle differences in their interaction with human cardiac Na(+) channels.

Nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated calcium signaling and arrhythmias in the heart evoked by ß-adrenergic stimulation.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca(2+) release in cardiac myocytes evoked by ß-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca(2+) signals sensitive to inhibitors of both acidic Ca(2+) stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca(2+) transients caused by high concentrations of the ß-adrenergic agonist isoproterenol. Ca(2+) transients were recorded both as increases of the free cytosolic Ca(2+) concentration and as decreases of the sarcoplasmic luminal Ca(2+) concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca(2+) transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.

Development of a drug screening platform based on engineered heart tissue.

RATIONALE: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. OBJECTIVE: Here, we present a new miniaturized and automated method based on engineered heart tissue (EHT). METHODS AND RESULTS: Neonatal rat heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 microL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12x3x3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. CONCLUSIONS: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.

Bupivacaine destabilizes action potential duration in cellular and computational models of long QT syndrome 1.

BACKGROUND: The effects of the local anesthetic bupivacaine on cardiac action potentials (APs) are mainly attributed to inhibition of cardiac Na(+) channels. The relevance of its ability to also induce high-affinity blockade of human ether-à-gogo-related gene (hERG) channels is unclear. We investigated whether this interaction may functionally become more significant in cellular and computational models of long (L)QT syndromes. METHODS: Left ventricular cardiomyocytes were isolated from adult guinea pig hearts, and bupivacaine-induced effects on APs were investigated using the patch-clamp technique. LQT-like states were pharmacologically induced by either blocking I(Ks) (LQT1-like, 10 µmol/L chromanol 293B), or I(Kr) (LQT2-like, 10 µmol/L E4031). Computational analysis of bupivacaine's effects was based on the Luo-Rudy dynamic model. RESULTS: Bupivacaine induced dose-dependent AP shortening in control myocytes. However, in the presence of 1 to 30 µmol/L bupivacaine, a high variability in AP duration with AP prolongations of up to 40% was observed. This destabilizing effect on AP duration was significantly increased in LQT1-like but not in LQT2-like myocytes. Similarly, the incidence of AP prolongations in the presence of 3 µmol/L bupivacaine was significantly increased from 6% in control myocytes to 24% in LQT1-like but not in LQT2-like myocytes. Computational modeling supported the concept that this bupivacaine-induced AP instability and the AP prolongations in the control and LQT1-like myocytes were caused by inhibition of hERG channels. CONCLUSIONS: This study provides evidence that bupivacaine induces inhibition of hERG channels, which is functionally silent under normal conditions but will become more relevant in LQT1-like states in which repolarization relies to a larger degree on hERG channels. Interactions with ion channels other than cardiac Na(+) channels may, therefore, determine the net cardiac effects of bupivacaine when the normal balance of ionic currents is altered.

The subtype-specific effects of droperidol on action potential duration in cellular and computational models of long QT syndrome.

BACKGROUND: Droperidol is a highly potent butyrophenone used for the therapy of postoperative nausea and vomiting. Its cardiac safety in cardiovascular-healthy patients and those with long QT (LQT) syndrome is a matter of debate. In this study, we investigated whether droperidol has subtype-specific effects in cellular and computational models of LQT syndrome. METHODS: Left ventricular cardiac myocytes were isolated from adult guinea pig hearts. LQT1-like behavior was pharmacologically induced by chromanol 293B (10 micromol/L) and LQT2-like states by E4031 (10 micromol/L). Computational analysis was performed using the Luo-Rudy dynamic model. Data are given as mean + or - SEM. RESULTS: In control myocytes, droperidol lengthened action potentials in a concentration-dependent manner with a maximal prolongation of 37% + or - 13% (n = 4) at a concentration of 0.6 micromol/L. In LQT1-like myocytes, droperidol (0.6 micromol/L) further prolonged action potentials by 31% + or - 6% (n = 6) but shortened action potentials of LQT2-like myocytes by 11% + or - 2% (n = 8). Computational modeling supported the concept that droperidol, in addition to the rapid component of the delayed K(+) current, blocks depolarizing targets, such as the L-type Ca(2+) current, the Na(+)-Ca(2+) exchanger, and the Na(+)-K(+) adenosine triphosphatase. CONCLUSIONS: Droperidol has more detrimental effects on cardiac repolarization of LQT1-like than of LQT2-like myocytes suggesting subtype-specific cardiotoxic effects in patients with LQT syndrome. The subtype specificity of droperidol seems to be caused by a complex interaction of droperidol with several different molecular targets. This interaction deserves further investigation to establish the feasibility of a subtype-directed approach in the perioperative management of patients with LQT syndrome.

Nitro-fatty acids suppress ischemic ventricular arrhythmias by preserving calcium homeostasis.

Nitro-fatty acids are electrophilic anti-inflammatory mediators which are generated during myocardial ischemic injury. Whether these species exert anti-arrhythmic effects in the acute phase of myocardial ischemia has not been investigated so far. Herein, we demonstrate that pretreatment of mice with 9- and 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA) significantly reduced the susceptibility to develop acute ventricular tachycardia (VT). Accordingly, epicardial mapping revealed a markedly enhanced homogeneity in ventricular conduction. NO2-OA treatment of isolated cardiomyocytes lowered the number of spontaneous contractions upon adrenergic isoproterenol stimulation and nearly abolished ryanodine receptor type 2 (RyR2)-dependent sarcoplasmic Ca2+ leak. NO2-OA also significantly reduced RyR2-phosphorylation by inhibition of increased CaMKII activity. Thus, NO2-OA might be a novel pharmacological option for the prevention of VT development.

Augmentation of myocardial If dysregulates calcium homeostasis and causes adverse cardiac remodeling.

HCN channels underlie the depolarizing funny current (If) that contributes importantly to cardiac pacemaking. If is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice (HCN4tg/wt) to assess functional consequences of HCN4 overexpression-mediated If increase in cardiomyocytes to levels observed in human heart failure. HCN4tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. If augmentation induces a diastolic Na+ influx shifting the Na+/Ca2+ exchanger equilibrium towards 'reverse mode' leading to increased [Ca2+]i. Changed Ca2+ homeostasis results in significantly higher systolic [Ca2+]i transients and stimulates apoptosis. Pharmacological inhibition of If prevents the rise of [Ca2+]i and protects from ventricular remodeling. Here we report that augmented myocardial If alters intracellular Ca2+ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial If per se may constitute a therapeutic mechanism to prevent cardiomyopathy.

Analysis of fibrosis in control or pressure overloaded rat hearts after mechanical unloading by heterotopic heart transplantation.

Mechanical unloading (MU) by implantation of left ventricular assist devices (LVAD) has become clinical routine. This procedure has been shown to reverse cardiac pathological remodeling, with the underlying molecular mechanisms incompletely understood. Most studies thus far were performed in non-standardized human specimens or MU of healthy animal hearts. Our study investigates cardiac remodeling processes in sham-operated healthy rat hearts and in hearts subjected to standardized pathological pressure overload by transverse aortic constriction (TAC) prior to MU by heterotopic heart transplantation (hHTx/MU). Rats underwent sham or TAC surgery. Disease progression was monitored by echocardiography prior to MU by hHTx/MU. Hearts after TAC or TAC combined with hHTx/MU were removed and analyzed by histology, western immunoblot and gene expression analysis. TAC surgery resulted in cardiac hypertrophy and impaired cardiac function. TAC hearts revealed significantly increased cardiac myocyte diameter and mild fibrosis. Expression of hypertrophy associated genes after TAC was higher compared to hearts after hHTx/MU. While cardiac myocyte cell diameter regressed to the level of sham-operated controls in all hearts subjected to hHTx/MU, fibrotic remodeling was significantly exacerbated. Transcription of pro-fibrotic and apoptosis-related genes was markedly augmented in all hearts after hHTx/MU. Sarcomeric proteins involved in excitation-contraction coupling displayed significantly lower phosphorylation levels after TAC and significantly reduced total protein levels after hHTx/MU. Development of myocardial fibrosis, cardiac myocyte atrophy and loss of sarcomeric proteins was observed in all hearts that underwent hHTX/MU regardless of the disease state. These results may help to explain the clinical experience with low rates of LVAD removal due to lack of myocardial recovery.

Mechanical unloading of the rat heart involves marked changes in the protein kinase-phosphatase balance.

Mechanical unloading of failing hearts by left ventricular (LV) assist devices is regularly used as a bridge to transplantation and may lead to symptomatic improvement. The latter has been associated with altered phosphorylation of cardiac regulatory proteins, but the underlying mechanisms remained unknown. Here, we tested whether cardiac unloading alters protein phosphorylation by affecting the corresponding kinase-phosphatase balance. Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation in rats for two weeks (n=8). Native in situ hearts from the recipient animals were used as controls (n=8). The steady-state protein kinase A (PKA) and/or Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) phosphorylation levels of phospholamban (PLB, Ser(16) and Thr(17)) and troponin I (TnI, Ser(23/24)) were decreased by 40-60% in unloaded hearts. Consistently, in these hearts PKA activity was decreased by approximately 80% and the activity of protein phosphatase 1 and 2A was increased by 50% and 90%, respectively. In contrast, CaMKII activity was approximately 60% higher, which may serve as a partial compensation. These data indicate that unloading shifts the kinase-phosphatase balance towards net dephosphorylation of PLB and TnI. This shift may also contribute to the reduction in phosphorylation levels of cardiac phosphoproteins observed in diseased human hearts after LVAD.

A New Animal Model for Investigation of Mechanical Unloading in Hypertrophic and Failing Hearts: Combination of Transverse Aortic Constriction and Heterotopic Heart Transplantation.

OBJECTIVES: Previous small animal models for simulation of mechanical unloading are solely performed in healthy or infarcted hearts, not representing the pathophysiology of hypertrophic and dilated hearts emerging in heart failure patients. In this article, we present a new and economic small animal model to investigate mechanical unloading in hypertrophic and failing hearts: the combination of transverse aortic constriction (TAC) and heterotopic heart transplantation (hHTx) in rats. METHODS: To induce cardiac hypertrophy and failure in rat hearts, three-week old rats underwent TAC procedure. Three and six weeks after TAC, hHTx with hypertrophic and failing hearts in Lewis rats was performed to induce mechanical unloading. After 14 days of mechanical unloading animals were euthanatized and grafts were explanted for further investigations. RESULTS: 50 TAC procedures were performed with a survival of 92% (46/50). When compared to healthy rats left ventricular surface decreased to 5.8±1.0 mm² (vs. 9.6± 2.4 mm²) (p = 0.001) after three weeks with a fractional shortening (FS) of 23.7± 4.3% vs. 28.2± 1.5% (p = 0.01). Six weeks later, systolic function decreased to 17.1± 3.2% vs. 28.2± 1.5% (p = 0.0001) and left ventricular inner surface increased to 19.9±1.1 mm² (p = 0.0001). Intraoperative graft survival during hHTx was 80% with 46 performed procedures (37/46). All transplanted organs survived two weeks of mechanical unloading. DISCUSSION: Combination of TAC and hHTx in rats offers an economic and reproducible small animal model enabling serial examination of mechanical unloading in a truly hypertrophic and failing heart, representing the typical pressure overloaded and dilated LV, occurring in patients with moderate to severe heart failure.

Parthenogenetic stem cells for tissue-engineered heart repair.

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation.

Observational clinical and ex vivo studies have established a strong association between atrial fibrillation and inflammation. However, whether inflammation is the cause or the consequence of atrial fibrillation and which specific inflammatory mediators may increase the atria's susceptibility to fibrillation remain elusive. Here we provide experimental and clinical evidence for the mechanistic involvement of myeloperoxidase (MPO), a heme enzyme abundantly expressed by neutrophils, in the pathophysiology of atrial fibrillation. MPO-deficient mice pretreated with angiotensin II (AngII) to provoke leukocyte activation showed lower atrial tissue abundance of the MPO product 3-chlorotyrosine, reduced activity of matrix metalloproteinases and blunted atrial fibrosis as compared to wild-type mice. Upon right atrial electrophysiological stimulation, MPO-deficient mice were protected from atrial fibrillation, which was reversed when MPO was restored. Humans with atrial fibrillation had higher plasma concentrations of MPO and a larger MPO burden in right atrial tissue as compared to individuals devoid of atrial fibrillation. In the atria, MPO colocalized with markedly increased formation of 3-chlorotyrosine. Our data demonstrate that MPO is a crucial prerequisite for structural remodeling of the myocardium, leading to an increased vulnerability to atrial fibrillation.

Treatment with atorvastatin partially protects the rat heart from harmful catecholamine effects.

AIMS: Atorvastatin blunts the response of cardiomyocytes to catecholamines by reducing isoprenylation of G gamma subunits. We examined whether atorvastatin exerts similar effects in vivo and protects the rat heart from harmful effects of catecholamines. METHODS AND RESULTS: Rats were treated with atorvastatin (1 or 10 mg/kg x day) or H(2)O for 14 days per gavage. All three animal groups were subjected to restraint stress on day 10 and to infusions of isoprenaline (ISO; 1 mg/kg x day) or NaCl via minipumps for the last 4 days. Heart rate was measured by telemetry, left ventricular atrial natriuretic peptide (ANP) transcript levels by RT-PCR, and left atrial contractile function in organ baths. Heart rate was similar in all six study groups. In animals pre-treated with water, infusion of ISO induced an increase in heart-to-body weight ratio (HW/BW) by approximately 20%, an increase in ANP mRNA by approximately 350%, and a reduction in the inotropic effect of isoprenaline in left atrium by approximately 50%. In animals pre-treated with high-dose atorvastatin, the effects of ISO on HW/BW, ANP, and left atrial force were approximately 40, 50, and 40% smaller, respectively. Low dose atorvastatin had similar, albeit smaller effects. Atorvastatin treatment of NaCl-infused rats had only marginal effects. In cardiac homogenates from atorvastatin-treated rats (both NaCl- and ISO-infused), G gamma and G alpha(s) were partially translocated from the membrane to the cytosol. CONCLUSION: In the rat heart, treatment with atorvastatin results in translocation of cardiac membrane G gamma and G alpha(s) to the cytosol. This mechanism might contribute to protecting the heart from harm induced by chronic isoprenaline infusion without affecting heart rate.

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization.

BACKGROUND: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. METHODS: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. RESULTS: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. CONCLUSIONS: Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.