Lars Maier - Academia.edu (original) (raw)

Papers by Lars Maier

Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem ce... more Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into

Journal of molecular and cellular cardiology, 2002

The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been ... more The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been related to a number of subcellular alterations including desensitization of the beta -adrenergic system. However, it is unknown whether alterations in SR Ca(2+)-handling contribute to blunted inotropic responsiveness of failing myocardium. We tested the hypothesis that the reduced effectiveness of Ca(2+)-dependent inotropic interventions results from the inability of the SR to sufficiently increase its Ca(2+)-content in failing human myocardium. Experiments were performed in ventricular muscle preparations from a total of four non-failing and 18 end-stage failing hearts. Isometric twitch force and SR Ca(2+)-content (using rapid cooling contractures; RCCs) were assessed under basal experimental conditions (1 Hz, 37 degrees C, [Ca(2+)](o) 2.5 mmol/l), and at increasing [Ca(2+)](o) (1.25-15 mmol/l), increasing concentrations of the beta -adrenergic agonist isoproterenol (ISO; 0.01-10 microm...

Cardiovascular research, 2005

Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SER... more Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) but increased function of sarcolemmal Na+/Ca2+ exchanger (NCX), leading to decreased SR Ca2+ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca2+ handling. Therefore, we characterized TG (n=35) vs. wild-type (WT) control rats (n=39) under physiological conditions (37 degrees C, stimulation rate <8 Hz). Twitch force, intracellular Ca2+ transients ([Ca2+]i), and SR Ca2+ content were measured in isolated muscles. The contribution of transsarcolemmal Ca2+ influx (I(Ca)) through L-type Ca2+ channels (LTCC) and reverse mode NCX (I(Na/Ca)) to Ca2+ cycling were studied in isolated myocytes. With increasing frequency, force increased in TG muscles by 168+/-35% (8 Hz...

The Annals of thoracic surgery, Jan 5, 2015

Transcatheter valve-in-valve (VIV) implantation evolved as a therapeutic alternative, despite an ... more Transcatheter valve-in-valve (VIV) implantation evolved as a therapeutic alternative, despite an increased risk of coronary obstruction in comparison with a regular transcatheter aortic valve implantation (TAVI). We report a comprehensive single-institution experience emphasizing strategies to reduce the risk of myocardial ischemia. Since 2009, 639 patients underwent a TAVI procedure in our institution. All patients are prospectively collected into an institutional registry. In total 31 patients underwent a VIV procedure at our institution (age 77.8 ± 6.3 years; The Society or Thoracic Surgeons predicted risk of mortality 20.9% ± 8.8%; New York Heart Association (NYHA) 3.0 ± 0.6). Degenerated bioprostheses included 24 Mitroflow, 6 Edwards Perimount, and Cryo-Valve O' Brien with label sizes from 21 to 27 mm. The type of failure was mostly regurgitation with or without concomitant stenosis (78%). Patients were provided with 5 Medtronic CoreValves, 15 Edwards SapienXT, 1 Edwards Sa...

Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology, Jan 10, 2015

The isolation of the pulmonary veins (PVs) is the mainstay of atrial fibrillation (AF) ablation, ... more The isolation of the pulmonary veins (PVs) is the mainstay of atrial fibrillation (AF) ablation, which with current ablation techniques can be achieved in almost all cases. Reconnection of PVs constitutes the most frequent cause of AF recurrence. Visually guided laser balloon ablation (VGLA) is a novel system with very high rate of persistence of pulmonary vein isolation (PVI) three months after the first procedure shown in preclinical and clinical studies. We aimed to determine the acute efficiency of the laser energy during PVI with the help of adenosine provocation. Twenty-six patients (19 male; mean age 64 ± 9 years) with symptomatic paroxysmal AF were included in the study. Pulmonary vein isolation was performed using the VGLA system. After successful PVI, we studied the effects of intravenous adenosine (18 mg) on activation of each PV at least 20 min after PVI. A total of 104 PVs were targeted. The balloon catheter could not be placed in two PVs. Of the remaining 102 PVs 99 (9...

but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featur... more but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted~250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca 2þ channel Ca V b subunits that enhance L-type channels in heterologous systems. Ca V b expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca 2þ current, we obtained single-channel recordings of native L-type channels. Ca V b subunits strikingly increased open probability, while decreasing inactivation (b, control; c, Ca V b expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration. A moderate amount of exercise has been reported to improve the symptom of heart failure (HF). Dilated cardiomyopathy (DCM) is one of major causes of HF characterized by ventricular dilatation and contractile dysfunction, and often associated with sudden death (SD) by lethal arrhythmia. Applicability of exercise to DCM patients is not established yet. In this study, we evaluated effects of voluntary exercise on arrhythmogenic changes in a mouse model of inherited DCM that closely mimics human phenotype. Mice with a deletion mutation of K210 (DK210) in cardiac troponin T, which decreases Ca 2þ sensitivity in myofilaments, were used as DCM model. Wild type (WT) and DCM mice at various ages were housed with free access to a running wheel and their voluntary running activity was measured. Ventricular muscles were excised and gene expressions of ion channels were determined by real-time PCR analysis.Membrane potential signals were optically determined to detect functional changes in myocardium. Homozygous DK210 mice developed cardiac enlargement and showed frequent SD with t1/2 of 70 days. Down regulation of multiple types of Kþ channels was detected in DCM mice. In parallel, prolongation of action potential duration and frequent spontaneous activity were observed. At 2-month of age, DCM mice showed similar wheel-running activity to WT. Some of DCM mice at 3 months or later decreased running activity with lung edema. In DCM mice with high activity, down-regulation of K þ channels was less marked. The DCM mice that started running at a young age showed significantly improved survival rate. These results indicate that (1) running wheel was useful to detect sign of HF, and that (2) voluntary exercise is beneficial to DCM mice. Potential mechanisms contributing to the improved survival rate will be discussed. Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB 1 and CB 2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB 1 and CB 2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 mM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 mM, anandamide reduced APD 90 by 38 5 8 % (n = 7, p < 0.001). In addition, 10 mM R-(þ)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB 2 receptor antagonist AM 630, but not the CB 1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 mM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca 2þ handling. Since these effects appear only to be partially inhibited by CB 2 receptor blockade, it is likely that anandamide has actions which are independent of CB 1 and CB 2 receptors. , Cesare M. Terracciano. Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice. Vibratome-cut slices (350 mm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.3250.61 & 1.3750.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values. Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 46754 ms (n=42/10), similar to previously reported data using ventricular wedges. Longitudinal conduction velocity (CV) was faster than transversal CV (5153 & 2051 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1. Ikr blocker E4031 (1 mM) and Iks blocker Chromanol 293B (20 mM) prolonged FPD by 6453% (n=20/7, p<0.001) and 2252% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The nonspecific K þ channel blocker 4-aminopyridine (1mM) prolonged FPD by 4752% (n=6/2, p<0.001). Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy. Optical imaging of cardiac electrical activity is of considerable interest, especially in cardiac pathophysiology and pharmacology studies. Voltage sensitive fluorescent dyes are widely used in this context, but have a limited applicability in long-term studies. We hypothesized that novel genetically encoded voltage sensitive fluorescent proteins (VSFPs) can be stably expressed in mouse hearts to (1) specifically label sarcolemmal membranes and (2) monitor membrane voltage transients. Methods: cDNA encoding for the VSFP2.3 voltage probe (see W. Akemann et al., 2010 Nat Methods) was placed under the control of the cardiomyocyte-specific alpha myosin heavy chain (aMHC) promoter. Transgenic mice were generated by pronuclear injection. Myocardial structure and performance were assessed by echocardiography (in 16-week old mice). Myocyte-and sarcolemma-restricted transgene activity was studied in isolated cardiomyocytes using confocal laser scanning microscopy and FRET-imaging. Results: We established 4 independent aMHC-VSFP2.3 mouse lines (TG#97, #107, #108, #123). Heart morphology and function did not differ in transgenic and wildtype mice with an average heart-tobody weight ratio of 4.350.5 (n=26) and 4.250.2 (n=8), respectively. Fluorescent imaging of intact hearts showed homogeneous pattern of CFP and YFP expression throughout the ventricles. Fluorescence intensity varied between the established lines (#123>#97>#108>#107). On a single cell basis, prominent sarcolemmal targeting was observed. Interestingly, the T-tubular system was clearly labeled with a predicted periodicity of 1.8850.02 and 1.8250.02 mm (from 7/11 cells) in wildtype and transgenic mice, respectively. Voltage transients could be readily detected using optical imaging at the level of intact hearts and isolated myocytes. Conclusion: We have established the first mouse model with cardiac-restricted VSFP-expression. aMHC-VSFP2.3 mice demonstrated unimpaired myocardial structure and function. We

Cardiovascular Research, 2015

To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. E... more To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.

Journal of molecular and cellular cardiology, 2006

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic chang... more Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca-CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca-CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca-CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca-CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca-CaM sensing probe for 24-48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca-CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca-CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon beta-adrenergic stimulati...

Heart Rhythm, 2015

Currently available antiarrhythmic agents for the treatment of atrial fibrillation (AF) have impo... more Currently available antiarrhythmic agents for the treatment of atrial fibrillation (AF) have important limitations, leaving an unmet need for safe and effective therapy. Ranolazine is an approved antianginal agent with a favorable safety profile and electrophysiologic properties suggesting a potential role in the treatment of AF. The purpose of this study was to assess the safety and efficacy of ranolazine in the prevention of AF recurrence after successful electrical cardioversion and to ascertain the most appropriate dose of this agent. This prospective, multicenter, randomized, double-blind, placebo-control parallel group phase II dose-ranging trial randomized patients with persistent AF (7 days to 6 months) 2 hours after successful electrical cardioversion to placebo, or ranolazine 375 mg, 500 mg, or 750 mg bid. Patients were monitored daily by transtelephonic ECG. The primary end-point was the time to first AF recurrence. Of 241 patients randomized, 238 took at least 1 drug dose. Ranolazine proved to be safe and tolerable. No dose of the drug significantly prolonged time to AF recurrence. AF recurred in 56.4%, 56.9%, 41.7%, and 39.7% of patients in the placebo, ranolazine 375 mg, ranolazine 500 mg, and ranolazine 750 mg groups, respectively. The reduction in overall AF recurrence in the combined 500-mg and 750-mg groups was of borderline significance compared to the placebo group (P = .053) and significant compared to 375-mg group (P = .035). No dose of ranolazine significantly prolonged time to AF recurrence. However, the 500-mg and 750 mg-groups combined reduced AF recurrences, suggesting a possible role for this agent in the treatment of AF.

American journal of physiology. Heart and circulatory physiology, 2014

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing ... more Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A ...

Journal of the American College of Cardiology, 2012

Journal of Molecular and Cellular Cardiology, 2002

34, 919À939. Calcium (Ca) is the key regulator of cardiac contraction during excitationÀcontracti... more 34, 919À939. Calcium (Ca) is the key regulator of cardiac contraction during excitationÀcontraction (EÀC) coupling. However, differences exist between the amount of Ca being transported into the myocytes upon electrical stimulation as compared to Ca released from the sarcoplasmic reticulum (SR). Moreover, alterations in EÀC coupling occur in cardiac hypertrophy and heart failure. In addition to the direct effects of Ca on the myo®laments, Ca plays a pivotal role in activation of a number of Ca-dependent proteins or second messengers, which can modulate EÀC coupling. Of these proteins, calmodulin (CaM) and Ca-CaM-dependent kinase II (CaMKII) are of special interest in the heart because of their role of modulating Ca in¯ux, SR Ca release, and SR Ca uptake during EÀC coupling. Indeed, CaM and CaMKII may be associated with some ion channels and Ca transporters and both can modulate acute cellular Ca handling. In addition to the changes in Ca, CaM and CaMKII signals from beat-to-beat, changes may occur on a longer time scale. These may occur over seconds to minutes involving phosphorylation/dephosphorylation reactions, and even a longer time frame in altering gene transcription (excitationÀtranscription (EÀT) coupling) in hypertrophic signaling and heart failure. Here we review the classical role of Ca in EÀC coupling and extend this view to the role of the Ca-dependent proteins CaM and CaMKII in modulating EÀC coupling and their contribution to EÀT coupling.

Frontiers in Pharmacology, 2014

Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failin... more Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failing myocardium, alterations in the expression and activity of voltage-gated K channels are frequently observed and contribute to the increased propensity for life-threatening arrhythmias. Thus, understanding the mechanisms of disturbed K channel regulation in heart failure (HF) is of critical importance. Amongst others, Ca/calmodulin-dependent protein kinase II (CaMKII) has been identified as an important regulator of K channel activity. In human HF but also various animal models, increased CaMKII expression and activity has been linked to deteriorated contractile function and arrhythmias. This review will discuss the current knowledge about CaMKII regulation of several K channels, its influence on action potential properties, dispersion of repolarization, and arrhythmias with special focus on HF.

Circulation …, 2003

Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) ␦ is the predominant cardiac isoform, and ... more Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) ␦ is the predominant cardiac isoform, and the ␦ C splice variant is cytoplasmic. We overexpressed CaMKII␦ C in mouse heart and observed dilated heart failure and altered myocyte Ca 2ϩ regulation in 3-month-old CaMKII␦ C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca 2ϩ ] i transient amplitude, and diastolic [Ca 2ϩ ] i were all reduced by Ϸ50% in TG versus WT. This is explained by Ͼ50% reduction in SR Ca 2ϩ content in TG versus WT. Peak Ca 2ϩ current (I Ca ) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca 2ϩ load and diastolic [Ca 2ϩ ] i , fractional SR Ca 2ϩ release was increased and resting spontaneous SR Ca 2ϩ release events (Ca 2ϩ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca 2ϩ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca 2ϩ spark frequency and I Ca , consistent with direct CaMKII activation of ryanodine receptors (and I Ca ) in TG. The rate of [Ca 2ϩ ] i decline during caffeine exposure was faster in TG, indicating enhanced Na ϩ -Ca 2ϩ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca 2ϩ leak (via sparks), reduced SR Ca 2ϩ -ATPase expression, and increased Na ϩ -Ca 2ϩ exchanger explain the reduced diastolic [Ca 2ϩ ] i and SR Ca 2ϩ content in TG. We conclude that CaMKII␦ C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure. (Circ Res. 2003;92:904-911.) Key Words: calcium Ⅲ Ca 2ϩ /calmodulin-dependent protein kinase II Ⅲ sarcoplasmic reticulum Ⅲ ryanodine receptor Ⅲ heart Original

Biophysical …, 2011

but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featur... more but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted~250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca 2þ channel Ca V b subunits that enhance L-type channels in heterologous systems. Ca V b expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca 2þ current, we obtained single-channel recordings of native L-type channels. Ca V b subunits strikingly increased open probability, while decreasing inactivation (b, control; c, Ca V b expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration. A moderate amount of exercise has been reported to improve the symptom of heart failure (HF). Dilated cardiomyopathy (DCM) is one of major causes of HF characterized by ventricular dilatation and contractile dysfunction, and often associated with sudden death (SD) by lethal arrhythmia. Applicability of exercise to DCM patients is not established yet. In this study, we evaluated effects of voluntary exercise on arrhythmogenic changes in a mouse model of inherited DCM that closely mimics human phenotype. Mice with a deletion mutation of K210 (DK210) in cardiac troponin T, which decreases Ca 2þ sensitivity in myofilaments, were used as DCM model. Wild type (WT) and DCM mice at various ages were housed with free access to a running wheel and their voluntary running activity was measured. Ventricular muscles were excised and gene expressions of ion channels were determined by real-time PCR analysis.Membrane potential signals were optically determined to detect functional changes in myocardium. Homozygous DK210 mice developed cardiac enlargement and showed frequent SD with t1/2 of 70 days. Down regulation of multiple types of Kþ channels was detected in DCM mice. In parallel, prolongation of action potential duration and frequent spontaneous activity were observed. At 2-month of age, DCM mice showed similar wheel-running activity to WT. Some of DCM mice at 3 months or later decreased running activity with lung edema. In DCM mice with high activity, down-regulation of K þ channels was less marked. The DCM mice that started running at a young age showed significantly improved survival rate. These results indicate that (1) running wheel was useful to detect sign of HF, and that (2) voluntary exercise is beneficial to DCM mice. Potential mechanisms contributing to the improved survival rate will be discussed. Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB 1 and CB 2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB 1 and CB 2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 mM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 mM, anandamide reduced APD 90 by 38 5 8 % (n = 7, p < 0.001). In addition, 10 mM R-(þ)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB 2 receptor antagonist AM 630, but not the CB 1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 mM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca 2þ handling. Since these effects appear only to be partially inhibited by CB 2 receptor blockade, it is likely that anandamide has actions which are independent of CB 1 and CB 2 receptors. , Cesare M. Terracciano. Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice. Vibratome-cut slices (350 mm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.3250.61 & 1.3750.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values. Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 46754 ms (n=42/10), similar to previously reported data using ventricular wedges. Longitudinal conduction velocity (CV) was faster than transversal CV (5153 & 2051 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1. Ikr blocker E4031 (1 mM) and Iks blocker Chromanol 293B (20 mM) prolonged FPD by 6453% (n=20/7, p<0.001) and 2252% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The nonspecific K þ channel blocker 4-aminopyridine (1mM) prolonged FPD by 4752% (n=6/2, p<0.001). Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy. Optical imaging of cardiac electrical activity is of considerable interest, especially in cardiac pathophysiology and pharmacology studies. Voltage sensitive fluorescent dyes are widely used in this context, but have a limited applicability in long-term studies. We hypothesized that novel genetically encoded voltage sensitive fluorescent proteins (VSFPs) can be stably expressed in mouse hearts to (1) specifically label sarcolemmal membranes and (2) monitor membrane voltage transients. Methods: cDNA encoding for the VSFP2.3 voltage probe (see W. Akemann et al., 2010 Nat Methods) was placed under the control of the cardiomyocyte-specific alpha myosin heavy chain (aMHC) promoter. Transgenic mice were generated by pronuclear injection. Myocardial structure and performance were assessed by echocardiography (in 16-week old mice). Myocyte-and sarcolemma-restricted transgene activity was studied in isolated cardiomyocytes using confocal laser scanning microscopy and FRET-imaging. Results: We established 4 independent aMHC-VSFP2.3 mouse lines (TG#97, #107, #108, #123). Heart morphology and function did not differ in transgenic and wildtype mice with an average heart-tobody weight ratio of 4.350.5 (n=26) and 4.250.2 (n=8), respectively. Fluorescent imaging of intact hearts showed homogeneous pattern of CFP and YFP expression throughout the ventricles. Fluorescence intensity varied between the established lines (#123>#97>#108>#107). On a single cell basis, prominent sarcolemmal targeting was observed. Interestingly, the T-tubular system was clearly labeled with a predicted periodicity of 1.8850.02 and 1.8250.02 mm (from 7/11 cells) in wildtype and transgenic mice, respectively. Voltage transients could be readily detected using optical imaging at the level of intact hearts and isolated myocytes. Conclusion: We have established the first mouse model with cardiac-restricted VSFP-expression. aMHC-VSFP2.3 mice demonstrated unimpaired myocardial structure and function. We

Cardiovascular Research, 2015

Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene... more Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis.

Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem ce... more Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into

Journal of molecular and cellular cardiology, 2002

The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been ... more The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been related to a number of subcellular alterations including desensitization of the beta -adrenergic system. However, it is unknown whether alterations in SR Ca(2+)-handling contribute to blunted inotropic responsiveness of failing myocardium. We tested the hypothesis that the reduced effectiveness of Ca(2+)-dependent inotropic interventions results from the inability of the SR to sufficiently increase its Ca(2+)-content in failing human myocardium. Experiments were performed in ventricular muscle preparations from a total of four non-failing and 18 end-stage failing hearts. Isometric twitch force and SR Ca(2+)-content (using rapid cooling contractures; RCCs) were assessed under basal experimental conditions (1 Hz, 37 degrees C, [Ca(2+)](o) 2.5 mmol/l), and at increasing [Ca(2+)](o) (1.25-15 mmol/l), increasing concentrations of the beta -adrenergic agonist isoproterenol (ISO; 0.01-10 microm...

Cardiovascular research, 2005

Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SER... more Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) but increased function of sarcolemmal Na+/Ca2+ exchanger (NCX), leading to decreased SR Ca2+ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca2+ handling. Therefore, we characterized TG (n=35) vs. wild-type (WT) control rats (n=39) under physiological conditions (37 degrees C, stimulation rate <8 Hz). Twitch force, intracellular Ca2+ transients ([Ca2+]i), and SR Ca2+ content were measured in isolated muscles. The contribution of transsarcolemmal Ca2+ influx (I(Ca)) through L-type Ca2+ channels (LTCC) and reverse mode NCX (I(Na/Ca)) to Ca2+ cycling were studied in isolated myocytes. With increasing frequency, force increased in TG muscles by 168+/-35% (8 Hz...

The Annals of thoracic surgery, Jan 5, 2015

Transcatheter valve-in-valve (VIV) implantation evolved as a therapeutic alternative, despite an ... more Transcatheter valve-in-valve (VIV) implantation evolved as a therapeutic alternative, despite an increased risk of coronary obstruction in comparison with a regular transcatheter aortic valve implantation (TAVI). We report a comprehensive single-institution experience emphasizing strategies to reduce the risk of myocardial ischemia. Since 2009, 639 patients underwent a TAVI procedure in our institution. All patients are prospectively collected into an institutional registry. In total 31 patients underwent a VIV procedure at our institution (age 77.8 ± 6.3 years; The Society or Thoracic Surgeons predicted risk of mortality 20.9% ± 8.8%; New York Heart Association (NYHA) 3.0 ± 0.6). Degenerated bioprostheses included 24 Mitroflow, 6 Edwards Perimount, and Cryo-Valve O' Brien with label sizes from 21 to 27 mm. The type of failure was mostly regurgitation with or without concomitant stenosis (78%). Patients were provided with 5 Medtronic CoreValves, 15 Edwards SapienXT, 1 Edwards Sa...

Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology, Jan 10, 2015

The isolation of the pulmonary veins (PVs) is the mainstay of atrial fibrillation (AF) ablation, ... more The isolation of the pulmonary veins (PVs) is the mainstay of atrial fibrillation (AF) ablation, which with current ablation techniques can be achieved in almost all cases. Reconnection of PVs constitutes the most frequent cause of AF recurrence. Visually guided laser balloon ablation (VGLA) is a novel system with very high rate of persistence of pulmonary vein isolation (PVI) three months after the first procedure shown in preclinical and clinical studies. We aimed to determine the acute efficiency of the laser energy during PVI with the help of adenosine provocation. Twenty-six patients (19 male; mean age 64 ± 9 years) with symptomatic paroxysmal AF were included in the study. Pulmonary vein isolation was performed using the VGLA system. After successful PVI, we studied the effects of intravenous adenosine (18 mg) on activation of each PV at least 20 min after PVI. A total of 104 PVs were targeted. The balloon catheter could not be placed in two PVs. Of the remaining 102 PVs 99 (9...

but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featur... more but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted~250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca 2þ channel Ca V b subunits that enhance L-type channels in heterologous systems. Ca V b expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca 2þ current, we obtained single-channel recordings of native L-type channels. Ca V b subunits strikingly increased open probability, while decreasing inactivation (b, control; c, Ca V b expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration. A moderate amount of exercise has been reported to improve the symptom of heart failure (HF). Dilated cardiomyopathy (DCM) is one of major causes of HF characterized by ventricular dilatation and contractile dysfunction, and often associated with sudden death (SD) by lethal arrhythmia. Applicability of exercise to DCM patients is not established yet. In this study, we evaluated effects of voluntary exercise on arrhythmogenic changes in a mouse model of inherited DCM that closely mimics human phenotype. Mice with a deletion mutation of K210 (DK210) in cardiac troponin T, which decreases Ca 2þ sensitivity in myofilaments, were used as DCM model. Wild type (WT) and DCM mice at various ages were housed with free access to a running wheel and their voluntary running activity was measured. Ventricular muscles were excised and gene expressions of ion channels were determined by real-time PCR analysis.Membrane potential signals were optically determined to detect functional changes in myocardium. Homozygous DK210 mice developed cardiac enlargement and showed frequent SD with t1/2 of 70 days. Down regulation of multiple types of Kþ channels was detected in DCM mice. In parallel, prolongation of action potential duration and frequent spontaneous activity were observed. At 2-month of age, DCM mice showed similar wheel-running activity to WT. Some of DCM mice at 3 months or later decreased running activity with lung edema. In DCM mice with high activity, down-regulation of K þ channels was less marked. The DCM mice that started running at a young age showed significantly improved survival rate. These results indicate that (1) running wheel was useful to detect sign of HF, and that (2) voluntary exercise is beneficial to DCM mice. Potential mechanisms contributing to the improved survival rate will be discussed. Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB 1 and CB 2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB 1 and CB 2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 mM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 mM, anandamide reduced APD 90 by 38 5 8 % (n = 7, p < 0.001). In addition, 10 mM R-(þ)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB 2 receptor antagonist AM 630, but not the CB 1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 mM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca 2þ handling. Since these effects appear only to be partially inhibited by CB 2 receptor blockade, it is likely that anandamide has actions which are independent of CB 1 and CB 2 receptors. , Cesare M. Terracciano. Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice. Vibratome-cut slices (350 mm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.3250.61 & 1.3750.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values. Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 46754 ms (n=42/10), similar to previously reported data using ventricular wedges. Longitudinal conduction velocity (CV) was faster than transversal CV (5153 & 2051 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1. Ikr blocker E4031 (1 mM) and Iks blocker Chromanol 293B (20 mM) prolonged FPD by 6453% (n=20/7, p<0.001) and 2252% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The nonspecific K þ channel blocker 4-aminopyridine (1mM) prolonged FPD by 4752% (n=6/2, p<0.001). Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy. Optical imaging of cardiac electrical activity is of considerable interest, especially in cardiac pathophysiology and pharmacology studies. Voltage sensitive fluorescent dyes are widely used in this context, but have a limited applicability in long-term studies. We hypothesized that novel genetically encoded voltage sensitive fluorescent proteins (VSFPs) can be stably expressed in mouse hearts to (1) specifically label sarcolemmal membranes and (2) monitor membrane voltage transients. Methods: cDNA encoding for the VSFP2.3 voltage probe (see W. Akemann et al., 2010 Nat Methods) was placed under the control of the cardiomyocyte-specific alpha myosin heavy chain (aMHC) promoter. Transgenic mice were generated by pronuclear injection. Myocardial structure and performance were assessed by echocardiography (in 16-week old mice). Myocyte-and sarcolemma-restricted transgene activity was studied in isolated cardiomyocytes using confocal laser scanning microscopy and FRET-imaging. Results: We established 4 independent aMHC-VSFP2.3 mouse lines (TG#97, #107, #108, #123). Heart morphology and function did not differ in transgenic and wildtype mice with an average heart-tobody weight ratio of 4.350.5 (n=26) and 4.250.2 (n=8), respectively. Fluorescent imaging of intact hearts showed homogeneous pattern of CFP and YFP expression throughout the ventricles. Fluorescence intensity varied between the established lines (#123>#97>#108>#107). On a single cell basis, prominent sarcolemmal targeting was observed. Interestingly, the T-tubular system was clearly labeled with a predicted periodicity of 1.8850.02 and 1.8250.02 mm (from 7/11 cells) in wildtype and transgenic mice, respectively. Voltage transients could be readily detected using optical imaging at the level of intact hearts and isolated myocytes. Conclusion: We have established the first mouse model with cardiac-restricted VSFP-expression. aMHC-VSFP2.3 mice demonstrated unimpaired myocardial structure and function. We

Cardiovascular Research, 2015

To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. E... more To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.

Journal of molecular and cellular cardiology, 2006

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic chang... more Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca-CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca-CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca-CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca-CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca-CaM sensing probe for 24-48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca-CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca-CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon beta-adrenergic stimulati...

Heart Rhythm, 2015

Currently available antiarrhythmic agents for the treatment of atrial fibrillation (AF) have impo... more Currently available antiarrhythmic agents for the treatment of atrial fibrillation (AF) have important limitations, leaving an unmet need for safe and effective therapy. Ranolazine is an approved antianginal agent with a favorable safety profile and electrophysiologic properties suggesting a potential role in the treatment of AF. The purpose of this study was to assess the safety and efficacy of ranolazine in the prevention of AF recurrence after successful electrical cardioversion and to ascertain the most appropriate dose of this agent. This prospective, multicenter, randomized, double-blind, placebo-control parallel group phase II dose-ranging trial randomized patients with persistent AF (7 days to 6 months) 2 hours after successful electrical cardioversion to placebo, or ranolazine 375 mg, 500 mg, or 750 mg bid. Patients were monitored daily by transtelephonic ECG. The primary end-point was the time to first AF recurrence. Of 241 patients randomized, 238 took at least 1 drug dose. Ranolazine proved to be safe and tolerable. No dose of the drug significantly prolonged time to AF recurrence. AF recurred in 56.4%, 56.9%, 41.7%, and 39.7% of patients in the placebo, ranolazine 375 mg, ranolazine 500 mg, and ranolazine 750 mg groups, respectively. The reduction in overall AF recurrence in the combined 500-mg and 750-mg groups was of borderline significance compared to the placebo group (P = .053) and significant compared to 375-mg group (P = .035). No dose of ranolazine significantly prolonged time to AF recurrence. However, the 500-mg and 750 mg-groups combined reduced AF recurrences, suggesting a possible role for this agent in the treatment of AF.

American journal of physiology. Heart and circulatory physiology, 2014

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing ... more Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A ...

Journal of the American College of Cardiology, 2012

Journal of Molecular and Cellular Cardiology, 2002

34, 919À939. Calcium (Ca) is the key regulator of cardiac contraction during excitationÀcontracti... more 34, 919À939. Calcium (Ca) is the key regulator of cardiac contraction during excitationÀcontraction (EÀC) coupling. However, differences exist between the amount of Ca being transported into the myocytes upon electrical stimulation as compared to Ca released from the sarcoplasmic reticulum (SR). Moreover, alterations in EÀC coupling occur in cardiac hypertrophy and heart failure. In addition to the direct effects of Ca on the myo®laments, Ca plays a pivotal role in activation of a number of Ca-dependent proteins or second messengers, which can modulate EÀC coupling. Of these proteins, calmodulin (CaM) and Ca-CaM-dependent kinase II (CaMKII) are of special interest in the heart because of their role of modulating Ca in¯ux, SR Ca release, and SR Ca uptake during EÀC coupling. Indeed, CaM and CaMKII may be associated with some ion channels and Ca transporters and both can modulate acute cellular Ca handling. In addition to the changes in Ca, CaM and CaMKII signals from beat-to-beat, changes may occur on a longer time scale. These may occur over seconds to minutes involving phosphorylation/dephosphorylation reactions, and even a longer time frame in altering gene transcription (excitationÀtranscription (EÀT) coupling) in hypertrophic signaling and heart failure. Here we review the classical role of Ca in EÀC coupling and extend this view to the role of the Ca-dependent proteins CaM and CaMKII in modulating EÀC coupling and their contribution to EÀT coupling.

Frontiers in Pharmacology, 2014

Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failin... more Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failing myocardium, alterations in the expression and activity of voltage-gated K channels are frequently observed and contribute to the increased propensity for life-threatening arrhythmias. Thus, understanding the mechanisms of disturbed K channel regulation in heart failure (HF) is of critical importance. Amongst others, Ca/calmodulin-dependent protein kinase II (CaMKII) has been identified as an important regulator of K channel activity. In human HF but also various animal models, increased CaMKII expression and activity has been linked to deteriorated contractile function and arrhythmias. This review will discuss the current knowledge about CaMKII regulation of several K channels, its influence on action potential properties, dispersion of repolarization, and arrhythmias with special focus on HF.

Circulation …, 2003

Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) ␦ is the predominant cardiac isoform, and ... more Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) ␦ is the predominant cardiac isoform, and the ␦ C splice variant is cytoplasmic. We overexpressed CaMKII␦ C in mouse heart and observed dilated heart failure and altered myocyte Ca 2ϩ regulation in 3-month-old CaMKII␦ C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca 2ϩ ] i transient amplitude, and diastolic [Ca 2ϩ ] i were all reduced by Ϸ50% in TG versus WT. This is explained by Ͼ50% reduction in SR Ca 2ϩ content in TG versus WT. Peak Ca 2ϩ current (I Ca ) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca 2ϩ load and diastolic [Ca 2ϩ ] i , fractional SR Ca 2ϩ release was increased and resting spontaneous SR Ca 2ϩ release events (Ca 2ϩ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca 2ϩ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca 2ϩ spark frequency and I Ca , consistent with direct CaMKII activation of ryanodine receptors (and I Ca ) in TG. The rate of [Ca 2ϩ ] i decline during caffeine exposure was faster in TG, indicating enhanced Na ϩ -Ca 2ϩ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca 2ϩ leak (via sparks), reduced SR Ca 2ϩ -ATPase expression, and increased Na ϩ -Ca 2ϩ exchanger explain the reduced diastolic [Ca 2ϩ ] i and SR Ca 2ϩ content in TG. We conclude that CaMKII␦ C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure. (Circ Res. 2003;92:904-911.) Key Words: calcium Ⅲ Ca 2ϩ /calmodulin-dependent protein kinase II Ⅲ sarcoplasmic reticulum Ⅲ ryanodine receptor Ⅲ heart Original

Biophysical …, 2011

but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featur... more but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted~250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca 2þ channel Ca V b subunits that enhance L-type channels in heterologous systems. Ca V b expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca 2þ current, we obtained single-channel recordings of native L-type channels. Ca V b subunits strikingly increased open probability, while decreasing inactivation (b, control; c, Ca V b expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration. A moderate amount of exercise has been reported to improve the symptom of heart failure (HF). Dilated cardiomyopathy (DCM) is one of major causes of HF characterized by ventricular dilatation and contractile dysfunction, and often associated with sudden death (SD) by lethal arrhythmia. Applicability of exercise to DCM patients is not established yet. In this study, we evaluated effects of voluntary exercise on arrhythmogenic changes in a mouse model of inherited DCM that closely mimics human phenotype. Mice with a deletion mutation of K210 (DK210) in cardiac troponin T, which decreases Ca 2þ sensitivity in myofilaments, were used as DCM model. Wild type (WT) and DCM mice at various ages were housed with free access to a running wheel and their voluntary running activity was measured. Ventricular muscles were excised and gene expressions of ion channels were determined by real-time PCR analysis.Membrane potential signals were optically determined to detect functional changes in myocardium. Homozygous DK210 mice developed cardiac enlargement and showed frequent SD with t1/2 of 70 days. Down regulation of multiple types of Kþ channels was detected in DCM mice. In parallel, prolongation of action potential duration and frequent spontaneous activity were observed. At 2-month of age, DCM mice showed similar wheel-running activity to WT. Some of DCM mice at 3 months or later decreased running activity with lung edema. In DCM mice with high activity, down-regulation of K þ channels was less marked. The DCM mice that started running at a young age showed significantly improved survival rate. These results indicate that (1) running wheel was useful to detect sign of HF, and that (2) voluntary exercise is beneficial to DCM mice. Potential mechanisms contributing to the improved survival rate will be discussed. Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB 1 and CB 2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB 1 and CB 2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 mM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 mM, anandamide reduced APD 90 by 38 5 8 % (n = 7, p < 0.001). In addition, 10 mM R-(þ)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB 2 receptor antagonist AM 630, but not the CB 1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 mM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca 2þ handling. Since these effects appear only to be partially inhibited by CB 2 receptor blockade, it is likely that anandamide has actions which are independent of CB 1 and CB 2 receptors. , Cesare M. Terracciano. Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice. Vibratome-cut slices (350 mm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.3250.61 & 1.3750.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values. Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 46754 ms (n=42/10), similar to previously reported data using ventricular wedges. Longitudinal conduction velocity (CV) was faster than transversal CV (5153 & 2051 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1. Ikr blocker E4031 (1 mM) and Iks blocker Chromanol 293B (20 mM) prolonged FPD by 6453% (n=20/7, p<0.001) and 2252% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The nonspecific K þ channel blocker 4-aminopyridine (1mM) prolonged FPD by 4752% (n=6/2, p<0.001). Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy. Optical imaging of cardiac electrical activity is of considerable interest, especially in cardiac pathophysiology and pharmacology studies. Voltage sensitive fluorescent dyes are widely used in this context, but have a limited applicability in long-term studies. We hypothesized that novel genetically encoded voltage sensitive fluorescent proteins (VSFPs) can be stably expressed in mouse hearts to (1) specifically label sarcolemmal membranes and (2) monitor membrane voltage transients. Methods: cDNA encoding for the VSFP2.3 voltage probe (see W. Akemann et al., 2010 Nat Methods) was placed under the control of the cardiomyocyte-specific alpha myosin heavy chain (aMHC) promoter. Transgenic mice were generated by pronuclear injection. Myocardial structure and performance were assessed by echocardiography (in 16-week old mice). Myocyte-and sarcolemma-restricted transgene activity was studied in isolated cardiomyocytes using confocal laser scanning microscopy and FRET-imaging. Results: We established 4 independent aMHC-VSFP2.3 mouse lines (TG#97, #107, #108, #123). Heart morphology and function did not differ in transgenic and wildtype mice with an average heart-tobody weight ratio of 4.350.5 (n=26) and 4.250.2 (n=8), respectively. Fluorescent imaging of intact hearts showed homogeneous pattern of CFP and YFP expression throughout the ventricles. Fluorescence intensity varied between the established lines (#123>#97>#108>#107). On a single cell basis, prominent sarcolemmal targeting was observed. Interestingly, the T-tubular system was clearly labeled with a predicted periodicity of 1.8850.02 and 1.8250.02 mm (from 7/11 cells) in wildtype and transgenic mice, respectively. Voltage transients could be readily detected using optical imaging at the level of intact hearts and isolated myocytes. Conclusion: We have established the first mouse model with cardiac-restricted VSFP-expression. aMHC-VSFP2.3 mice demonstrated unimpaired myocardial structure and function. We

Cardiovascular Research, 2015

Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene... more Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis.