Eric Raddatz

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

AIMS It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart. METHODS AND RESULTS TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the α1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first- and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity. CONCLUSIONS These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Arrhythmogenesis in the Developing Heart During Anoxia‐Reoxygenation and Hypothermia‐Rewarming: An In Vitro Model

Introduction: The spatio‐temporal pattern of arrhythmias in the embryonic/fetal heart subjected to a transient hypoxic or hypothermic stress remains to be established.

Oxidative and Glycogenolytic Capacities within the Developing Chick Heart

Cardiac morphogenesis and function are known to depend on both aerobic and anaerobic energy-producing pathways. However, the relative contribution of mitochondrial oxidation and glycogenolysis, as well as the determining factors of oxygen demand in the distinct chambers of the embryonic heart, remains to be investigated. Spontaneously beating hearts isolated from stage 11, 20, and 24HH chick embryos were maintained in vitro under controlled metabolic conditions. O2 uptake and glycogenolytic rate were determined in atrium, ventricle, and conotruncus in the absence or presence of glucose. Oxidative capacity ranged from 0.2 to 0.5 nmol O2/(h·μg protein), did not depend on exogenous glucose, and was the highest in atria at stage 20HH. However, the highest reserves of oxidative capacity, assessed by mitochondrial uncoupling, were found at the youngest stage and in conotruncus, representing 75 to 130% of the control values. At stage 24HH, glycogenolysis in glucose-free medium was 0.22, 0.17, and 0.04 nmol glucose U(h·μg protein) in atrium, ventricle, and conotruncus, respectively. Mechanical loading of the ventricle increased its oxidative capacity by 62% without altering glycogenolysis or lactate production. Blockade of glycolysis by iodoacetate suppressed lactate production but modified neither O2 nor glycogen consumption in substrate-free medium. These findings indicate that atrium is the cardiac chamber that best utilizes its oxidative and glycogenolytic capacities and that ventricular wall stretch represents an early and major determinant of the O2 uptake. Moreover, the fact that O2 and glycogen consumptions were not affected by inhibition of glyceraldehyde-3-phosphate dehydrogenase provides indirect evidence for an active glycerol-phosphate shuttle in the embryonic cardiomyocytes.

Mapping of the oxygen consumption in the gastrulating chick embryo

Abstract The spatial variations of the O2 uptake in the area pellucida (AP) and area opaca (AO) of the chick embryo were measured in vitro by scanning microspectrophotometry. In the AP, the O2 fluxes (stage 4: 20–90, stage 6–7: 30–110 nl·h−1·mm−2) as well as the oxidative activity (stage 4: 14–25, stage 6–7: 10–28 nl·h−1· (μg protein)−1) show distinct regional differences. The most active regions are not those corresponding to the axial embryonic structures but those located in the latero-posterior, relatively undifferentiated zone of the AP. The total O2 uptake increases during 6 h of development from 0.3 to 0.6 μl·h−1 but the average oxidative activity and O2 uptake per cell remain constant (18 nl·h−·(μg protein)−1 and 2.5 pl·h−1·cell−1). In the AO, the O2 uptake is rather homogenous and all respiratory parameters increase during the development: total O2 uptake from 1.6 to 5.5 μl·h−1, oxidative activity from 28 to 45 nl·h−1·(μg protein)−1 and cell respiration from 7 to 16 pl·h−1 cell−1. The described metabolic variations are discussed with respect to the known morphogenetic activities taking place in the chick blastodisc during the studied period of development

Activation of transient receptor potential canonical 3 (TRPC3)-mediated Ca2+ entry by A1 adenosine receptor in cardiomyocytes disturbs atrioventricular conduction.

Background: A1-subtype of the adenosine receptors (A1AR) is arrhythmogenic. Results: A1AR activation enhanced Ca2+ entry through TRPC3 channel. Conclusion: TRPC3 is involved in conduction disturbances induced by A1AR. Significance: TRPC3 represents a promising target to prevent conduction disturbances. Although the activation of the A1-subtype of the adenosine receptors (A1AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A1AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A1AR by CCPA induced sarcolemmal Ca2+ entry. However, A1AR stimulation did not induce Ca2+ release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A1AR-enhanced Ca2+ entry. Ca2+ entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A1AR-enhanced Ca2+ entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A1AR-induced conduction disturbances in the embryonic heart. Our data showing that A1AR activation subtly mediates a proarrhythmic Ca2+ entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca2+ entry and cardiac function are altered. Thus, the A1AR-TRPC3 axis may represent a potential therapeutic target.

Mdx myotubes have normal excitability but show reduced contraction–relaxation dynamics

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 ± 0.03 ms and 23 ± 4 V vs. 0.39 ± 0.07 ms and 22 ± 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 ± 2 and 142 ± 13 ms, respectively) than in control myotubes (26 ± 1 and 85 ± 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells.

Store-operated Ca2+ channels (SOCs) are voltage-independent Ca2+ channels activated upon depletion of the endoplasmic reticulum Ca2+ stores. Early studies suggest the contribution of such channels to Ca2+ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca2+ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca2+ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca2+ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.

Oxygen and glucose uptakes in the early chick embryo

The uptake of O2 and glucose and the production of CO2 and lactate were measured using non-invasive techniques in the intact in vitro developing chick blastodisc during gastrulation-neurulation (18 to 24 h of incubation).

Myocardial impairment in chronic hypoxia is abolished by short aeration episodes: involvement of K+ATP channels

In vivo exposure to chronic hypoxia is considered to be a cause of myocardial dysfunction, thereby representing a deleterious condition, but repeated aeration episodes may exert some cardioprotection. We investigated the possible role of ATP-sensitive potassium channels in these mechanisms. First, rats (n = 8/group) were exposed for 14 days to either chronic hypoxia (CH; 10% O2) or chronic hypoxia with one episode/day of 1-hr normoxic aeration (CH+A), with normoxia (N) as the control. Second, isolated hearts were Langendorff perfused under hypoxia (10% O2, 30 min) and reoxygenated (94% O2, 30 min) with or without 3 μM glibenclamide (nonselective K+ATP channel-blocker) or 100 μM diazoxide (selective mitochondrial K+ATP channel-opener). Blood gasses, hemoglobin concentration, and plasma malondialdehyde were similar in CH and CH+A and in both different from normoxic (P < 0.01), body weight gain and plasma nitrate/nitrite were higher in CH+A than CH (P < 0.01), whereas apoptosis (number of TUNEL-positive nuclei) was less in CH+A than CH (P < 0.05). During in vitro hypoxia, the efficiency (ratio of ATP production/pressure x rate product) was the same in all groups and diazoxide had no measurable effects on myocardial performance, whereas glibenclamide increased end-diastolic pressure more in N and CH than in CH+A hearts (P < 0.05). During reoxgenation, efficiency was markedly less in CH with respect to N and CH+A (P < 0.0001), and rate x pressure product remained lower in CH than N and CH+A hearts (P < 0.001), but glibenclamide or diazoxide abolished this difference. Glibenclamide, but not diazoxide, decreased vascular resistance in N and CH (P < 0.005 and < 0.001) without changes in CH+A. We hypothesize that cardioprotection in chronically hypoxic hearts derive from cell depolarization by sarcolemmal K+ATP blockade or from preservation of oxidative phosphorylation efficiency (ATP turnover/myocardial performance) by mitochondrial K+ATP opening. Therefore K+ATP channels are involved in the deleterious effects of chronic hypoxia and in the cardioprotection elicited when chronic hypoxia is interrupted with short normoxic aeration episodes.

Spatio-temporal micromeasurements of the oxygen uptake in the developing chick embryo

A method has been developed for the determination of the oxygen uptake of small areas (0.01 mm2) in an entire chick embryo cultured in vitro under defined metabolic conditions. It is based on the recordings of the spectral changes of the hemoglobin used as oxygen source for the respiring tissue (Barzu and Borza, 1967). Rapid scanning of the hemoglobin absorbance over the preparation allows a comparison of the O2 uptake of various regions. Values of the order of 10(-2) 1 O2 . min-2 are measured in less than 10 sec with a spatial resolution of 100 micron. The differentiation of embryonic tissue is not disturbed by the measurements. The O2 diffusion in the media and in the tissue has been analyzed by digital simulation. The O2 uptake of the Hensens node was measured from embryos starting at the stage of definitive primitive streak (stage 4) up to the stage of 10 somites. It increases from 0.6 to 1.1 nl . h-1 with a marked acceleration between stages 4 and 5. The values corrected for the protein content of the Hensens node at stage 4, 5, 6 and 8 are 32, 30 and 28 microliter . mg-1 . h-1 respectively. The first scanning results show different patterns of the O2 utake at the level of the Hensens node and of the neural plate. At stage 6-7, the corrected O2 uptake is 30 microliter . mg-1 . h-1 for . the former and 43 microliter . mg-1 . h-1 for the latter.