From MS/MS library implementation to molecular networks: Exploring oxylipin diversity with NEO-MSMS.

Oxylipins, small polar molecules derived from the peroxidation of polyunsaturated fatty acids (PUFAs), serve as biomarkers for many diseases and play crucial roles in human physiology and inflammation. Despite their significance, many non-enzymatic oxygenated metabolites of PUFAs (NEO-PUFAs) remain poorly reported, resulting in a lack of public datasets of experimental data and limiting their dereplication in further studies. To overcome this limitation, we constructed a high-resolution tandem mass spectrometry (MS/MS) dataset comprising pure NEO-PUFAs (both commercial and self-synthesized) and in vitro free radical-induced oxidation of diverse PUFAs. By employing molecular networking techniques with this dataset and the existent ones in public repositories, we successfully mapped a wide range of NEO-PUFAs, expanding the strategies for annotating oxylipins, and NEO-PUFAs and offering a novel workflow for profiling these molecules in biological samples.

The Effect of Hypothermia and Osmotic Shock on the Electrocardiogram of Adult Zebrafish.

The use of zebrafish to explore cardiac physiology has been widely adopted within the scientific community. Whether this animal model can be used to determine drug cardiac toxicity via electrocardiogram (ECG) analysis is still an ongoing question. Several reports indicate that the recording configuration severely affects the ECG waveforms and its derived-parameters, emphasizing the need for improved characterization. To address this problem, we recorded ECGs from adult zebrafish hearts in three different configurations (unexposed heart, exposed heart, and extracted heart) to identify the most reliable method to explore ECG recordings at baseline and in response to commonly used clinical therapies. We found that the exposed heart configuration provided the most reliable and reproducible ECG recordings of waveforms and intervals. We were unable to determine T wave morphology in unexposed hearts. In extracted hearts, ECG intervals were lengthened and P waves were unstable. However, in the exposed heart configuration, we were able to reliably record ECGs and subsequently establish the QT-RR relationship (Holzgrefe correction) in response to changes in heart rate.

TREK-1 in the heart: Potential physiological and pathophysiological roles.

The TREK-1 channel belongs to the TREK subfamily of two-pore domains channels that are activated by stretch and polyunsaturated fatty acids and inactivated by Protein Kinase A phosphorylation. The activation of this potassium channel must induce a hyperpolarization of the resting membrane potential and a shortening of the action potential duration in neurons and cardiac cells, two phenomena being beneficial for these tissues in pathological situations like ischemia-reperfusion. Surprisingly, the physiological role of TREK-1 in cardiac function has never been thoroughly investigated, very likely because of the lack of a specific inhibitor. However, possible roles have been unraveled in pathological situations such as atrial fibrillation worsened by heart failure, right ventricular outflow tract tachycardia or pulmonary arterial hypertension. The inhomogeneous distribution of TREK-1 channel within the heart reinforces the idea that this stretch-activated potassium channel might play a role in cardiac areas where the mechanical constraints are important and need a particular protection afforded by TREK-1. Consequently, the main purpose of this mini review is to discuss the possible role played by TREK -1 in physiological and pathophysiological conditions and its potential role in mechano-electrical feedback. Improved understanding of the role of TREK-1 in the heart may help the development of promising treatments for challenging cardiac diseases.

New role of TRPM4 channel in the cardiac excitation-contraction coupling in response to physiological and pathological hypertrophy in mouse.

The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including ß-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.

Grape polyphenols and exercise training have distinct molecular effects on cardiac hypertrophy in a model of obese insulin-resistant rats.

Obesity and exercise lead to structural changes in heart such as cardiac hypertrophy. The underlying signaling pathways vary according to the source of the overload, be it physiological (exercise) or pathologic (obesity). The physiological pathway relies more on PI3K-Akt signaling while the pathologic pathway involves calcineurin-Nuclear factor of activated T-cells activation and fibrosis accumulation. Independently, exercise and polyphenols have demonstrated to prevent pathologic cardiac hypertrophy. Therefore, we investigated the molecular adaptations of the combination of exercise training and grape polyphenols supplementation (EXOPP) in obese high-fat fed rats on heart adaptation in comparison to exercise (EXO), polyphenols supplementation (PP) and high-fat fed rats (HF), alone. Exercised and PP rats presented a higher heart weight/body weight ratio compared to HF rats. EXO and EXOPP depicted an increase in cell-surface area, P-Akt/Akt, P-AMPK/AMPK ratios with a decreased fibrosis and calcineurin expression, illustrating an activation of the physiological pathway, but no additional benefit of the combination. In contrast, neither cell-surface area nor Akt signaling increased in PP rats; but markedly decreased fibrosis, calcineurin expression, systolic blood pressure, higher SERCA and P-Phospholamdan/Phospholamdan levels were observed. These data suggest that PP rats have a shift from pathologic toward physiological hypertrophy. Our study demonstrates that polyphenols supplementation has physical-activity-status-specific effects; it appears to be more protective in sedentary obese insulin-resistant rats than in the exercised ones. Exercise training improved metabolic and cardiac alterations without a synergistic effect of polyphenols supplementation. These data highlight a greater effect of exercise than polyphenols supplementation for the treatment of cardiac alterations in obese insulin-resistant rats.

Prolonged elevated levels of c-kit+ progenitor cells after a myocardial infarction by beta 2 adrenergic receptor priming.

Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction (MI). The beta 2 adrenergic receptor (ß2-AR) pathway induces proliferation of c-kit+ cardiac progenitor cells (CPC) in vitro. We investigated if ß2-AR pharmacological stimulation could ameliorate endogenous CPC-mediated regeneration after a MI. C-kit+ CPC ß1-AR and ß2-AR expression was evaluated in vivo and in vitro. A significant increase in the percentage of CPCs expressing ß1-AR and ß2-AR was measured 7 days post-MI. Accordingly, 24 hrs of low serum and hypoxia in vitro significantly increased CPC ß2-AR expression. Cell viability and differentiation assays validated a functional role of CPC ß2-AR. The effect of pharmacological activation of ß2-AR was studied in C57 mice using fenoterol administered in the drinking water 1 week before MI or sham surgery or at the time of the surgery. MI induced a significant increase in the percentage of c-kit+ progenitor cells at 7 days, whereas pretreatment with fenoterol prolonged this response resulting in a significant elevated number of CPC up to 21 days post-MI. This increased number of CPC correlated with a decrease in infarct size. The immunofluorescence analysis of the heart tissue for proliferation, apoptosis, macrophage infiltration, cardiomyocytes surface area, and vessel density showed significant changes on the basis of surgery but no benefit due to fenoterol treatment. Cardiac function was not ameliorated by fenoterol administration when evaluated by echocardiography. Our results suggest that ß2-AR stimulation may improve the cardiac repair process by supporting an endogenous progenitor cell response but is not sufficient to improve the cardiac function.

No Additive Effects of Polyphenol Supplementation and Exercise Training on White Adiposity Determinants of High-Fat Diet-Induced Obese Insulin-Resistant Rats.

One of the major insulin resistance instigators is excessive adiposity and visceral fat depots. Individually, exercise training and polyphenol intake are known to exert health benefits as improving insulin sensitivity. However, their combined curative effects on established obesity and insulin resistance need further investigation particularly on white adipose tissue alterations. Therefore, we compared the effects on different white adipose tissue depot alterations of a combination of exercise and grape polyphenol supplementation in obese insulin-resistant rats fed a high-fat diet to the effects of a high-fat diet alone or a nutritional supplementation of grape polyphenols (50 mg/kg/day) or exercise training (1 hr/day to 5 days/wk consisting of treadmill running at 32 m/min for a 10% slope), for a total duration of 8 weeks. Separately, polyphenol supplementation and exercise decreased the quantity of all adipose tissue depots and mesenteric inflammation. Exercise reduced adipocytes' size in all fat stores. Interestingly, combining exercise to polyphenol intake presents no more cumulative benefit on adipose tissue alterations than exercise alone. Insulin sensitivity was improved at systemic, epididymal, and inguinal adipose tissues levels in trained rats thus indicating that despite their effects on adipocyte morphological/metabolic changes, polyphenols at nutritional doses remain less effective than exercise in fighting insulin resistance.

Biological activities of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) derived from EPA and DHA: New anti-arrhythmic compounds?

ω3 Polyunsaturated fatty acids (ω3 PUFAs) have several biological properties including anti-arrhythmic effects. However, there are some evidences that it is not solely ω3 PUFAs per se that are biologically active but the non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) like isoprostanes and neuroprostanes. Recent question arises how these molecules take part in physiological homeostasis, show biological bioactivities and anti-inflammatory properties. Furthermore, they are involved in the circulations of childbirth, by inducing the closure of the ductus arteriosus. In addition, oxidative stress which can be beneficial for the heart in given environmental conditions such as the presence of ω3 PUFAs on the site of the stress and the signaling pathways involved are also explained in this review.

The TRPM4 channel is functionally important for the beneficial cardiac remodeling induced by endurance training.

Cardiac hypertrophy (CH) is an adaptive process that exists in two distinct forms and allows the heart to adequately respond to an organism's needs. The first form of CH is physiological, adaptive and reversible. The second is pathological, irreversible and associated with fibrosis and cardiomyocyte death. CH involves multiple molecular mechanisms that are still not completely defined but it is now accepted that physiological CH is associated more with the PI3-K/Akt pathway while the main signaling cascade activated in pathological CH involves the Calcineurin-NFAT pathway. It was recently demonstrated that the TRPM4 channel may act as a negative regulator of pathological CH by regulating calcium entry and thus the Cn-NFAT pathway. In this study, we examined if the TRPM4 channel is involved in the physiological CH process. We evaluated the effects of 4 weeks endurance training on the hearts of Trpm4 +/+ and Trpm4 -/- mice. We identified an elevated functional expression of the TRPM4 channel in cardiomyocytes after endurance training suggesting a potential role for the channel in physiological CH. We then observed that Trpm4 +/+ mice displayed left ventricular hypertrophy after endurance training associated with enhanced cardiac function. By contrast, Trpm4 -/- mice did not develop these adaptions. While Trpm4 -/- mice did not develop gross cardiac hypertrophy, the cardiomyocyte surface area was larger and associated with an increase of Tunel positive cells. Endurance training in Trpm4 +/+ mice did not increase DNA fragmentation in the heart. Endurance training in Trpm4 +/+ mice was associated with activation of the classical physiological CH Akt pathway while Trpm4 -/- favored the Calcineurin pathway. Calcium studies demonstrated that TRPM4 channel negatively regulates calcium entry providing support for activation of the Cn-NFAT pathway in Trpm4 -/- mice. In conclusion, we provide evidence for the functional expression of TRPM4 channel in response to endurance training. This expression may help to maintain the balance between physiological and pathological hypertrophy.

Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F4t-neuroprostane protects the heart against reperfusion injury.

Acute myocardial infarction leads to an increase in oxidative stress and lipid peroxidation. 4(RS)-4-F4t-Neuroprostane (4-F4t-NeuroP) is a mediator produced by non-enzymatic free radical peroxidation of the cardioprotective polyunsaturated fatty acid, docosahexaenoic acid (DHA). In this study, we investigated whether intra-cardiac delivery of 4-F4t-NeuroP (0.03mg/kg) prior to occlusion (ischemia) prevents and protects rat myocardium from reperfusion damages. Using a rat model of ischemic-reperfusion (I/R), we showed that intra-cardiac infusion of 4-F4t-NeuroP significantly decreased infarct size following reperfusion (-27%) and also reduced ventricular arrhythmia score considerably during reperfusion (-41%). Most notably, 4-F4t-NeuroP decreased ventricular tachycardia and post-reperfusion lengthening of QT interval. The evaluation of the mitochondrial homeostasis indicates a limitation of mitochondrial swelling in response to Ca2+ by decreasing the mitochondrial permeability transition pore opening and increasing mitochondria membrane potential. On the other hand, mitochondrial respiration measured by oxygraphy, and mitochondrial ROS production measured with MitoSox red® were unchanged. We found decreased cytochrome c release and caspase 3 activity, indicating that 4-F4t-NeuroP prevented reperfusion damages and reduced apoptosis. In conclusion, 4-F4t-NeuroP derived from DHA was able to protect I/R cardiac injuries by regulating the mitochondrial homeostasis.

Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance.

AIMS/HYPOTHESIS: Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice. METHODS: We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action. RESULTS: Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells. CONCLUSIONS/INTERPRETATION: Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

Non-enzymatic cyclic oxygenated metabolites of omega-3 polyunsaturated fatty acid: Bioactive drugs?

Non-enzymatic oxygenated metabolites derived from polyunsaturated fatty acids (PUFA) are formed in vivo through free radical reaction under oxidative stress conditions. It has been over twenty-five years since the discovery of cyclic oxygenated metabolites derived from arachidonic acid (20:4 n-6), the isoprostanes, and since then they have become biomarkers of choice for assessing in vivo OS in humans and animals. Chemical synthesis of n-3 PUFA isoprostanoids such as F3-Isoprostanes from eicosapentaenoic acid (20:5 n-3), and F4-Neuroprostanes from docosahexaenoic acid (22:6 n-6) unravelled novel and unexpected biological properties of such omega-3 non-enzymatic cyclic metabolites as highlighted in this review.

p11 modulates calcium handling through 5-HT4R pathway in rat ventricular cardiomyocytes.

BACKGROUND: The role of the serotonin receptor 4 (5-HT4R) pathway in cardiac excitation-contraction coupling (ECC) remains unclear. In the brain, induction of the calcium (Ca(2+))-binding protein p11 enhances 5-HT4R translocation and signaling and could therefore be considered as a modulator of the 5-HT4R pathway in the myocardium. p11 expression is increased by brain-derived neurotrophic factor (BDNF) or antidepressant drugs (imipramine). Thus, we investigated whether p11 regulates the 5-HT4R pathway in the heart in physiological conditions or under pharmacological induction and the effects on calcium handling. METHODS AND RESULTS: p11 expression was induced in vivo in healthy Wistar rats by imipramine (10 mg/kg/21 days) and in vitro in left ventricular cardiomyocytes exposed to BDNF (50 ng/ml/8h). Cell shortening and real-time Ca(2+) measurements were processed on field-stimulated intact cardiomyocytes with the selective 5-HT4R agonist, prucalopride (1 µM). Both imipramine and BDNF-induced cardiomyocyte p11 expression unmasked a strong response to prucalopride characterized by an increase of both cell shortening and Ca(2+) transient amplitude compared to basal prucalopride associated with a high propensity to trigger diastolic Ca(2+) events. Healthy rats treated with BDNF (180 ng/day/14 days) exhibited a sustained elevated heart rate following a single injection of prucalopride (0.1 mg/kg) which was not observed prior to treatment. CONCLUSIONS: We have identified a novel role for p11 in 5-HT4R signaling in healthy rat ventricular cardiomyocytes. Increased p11 expression by BDNF and imipramine unraveled a 5-HT4R-mediated modulation of cardiac Ca(2+) handling and ECC associated with deleterious Ca(2+) flux disturbances. Such mechanism could partly explain some cardiac adverse effects induced by antidepressant treatments.

Trpm4 gene invalidation leads to cardiac hypertrophy and electrophysiological alterations.

RATIONALE: TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear. OBJECTIVES: We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4-/-) model. METHODS AND RESULTS: Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase. CONCLUSIONS: TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

The IgA1 immune complex-mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy.

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.

The TRPM4 channel controls monocyte and macrophage, but not neutrophil, function for survival in sepsis.

A favorable outcome following acute bacterial infection depends on the ability of phagocytic cells to be recruited and properly activated within injured tissues. Calcium (Ca(2+)) is a ubiquitous second messenger implicated in the functions of many cells, but the mechanisms involved in the regulation of Ca(2+) mobilization in hematopoietic cells are largely unknown. The monovalent cation channel transient receptor potential melastatin (TRPM) 4 is involved in the control of Ca(2+) signaling in some hematopoietic cell types, but the role of this channel in phagocytes and its relevance in the control of inflammation remain unexplored. In this study, we report that the ablation of the Trpm4 gene dramatically increased mouse mortality in a model of sepsis induced by cecal ligation and puncture. The lack of the TRPM4 channel affected macrophage population within bacteria-infected peritoneal cavities and increased the systemic level of Ly6C(+) monocytes and proinflammatory cytokine production. Impaired Ca(2+) mobilization in Trpm4(-/-) macrophages downregulated the AKT signaling pathway and the subsequent phagocytic activity, resulting in bacterial overgrowth and translocation to the bloodstream. In contrast, no alteration in the distribution, function, or Ca(2+) mobilization of Trpm4(-/-) neutrophils was observed, indicating that the mechanism controlling Ca(2+) signaling differs among phagocytes. Our results thus show that the tight control of Ca(2+) influx by the TRPM4 channel is critical for the proper functioning of monocytes/macrophages and the efficiency of the subsequent response to infection.

Physiological roles of the TRPM4 channel extracted from background currents.

Calcium-activated nonselective cationic currents have been known for 30 years, but their physiological implications have remained unresolved until the recent cloning of the TRPM4 ion channel. Since then, TRPM4 has been identified as a key modulator of numerous calcium-dependent mechanisms such as the immune response, insulin secretion, cerebral artery constriction, respiratory rhythm, and cardiac conduction.

The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells.

Dendritic cell (DC) maturation and migration are events critical for the initiation of immune responses. After encountering pathogens, DCs upregulate the expression of costimulatory molecules and subsequently migrate to secondary lymphoid organs. Calcium (Ca(2+)) entry governs the functions of many hematopoietic cell types, but the role of Ca(2+) entry in DC biology remains unclear. Here we report that the Ca(2+)-activated nonselective cation channel TRPM4 was expressed in and controlled the Ca(2+) homeostasis of mouse DCs. The absence of TRPM4, which elicited Ca(2+) overload, did not influence DC maturation but did considerably impair chemokine-dependent DC migration. Our results establish TRPM4-regulated Ca(2+) homeostasis as crucial for DC mobility but not maturation and emphasize that DC maturation and migration are independently regulated.

TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells.

OBJECTIVE: A calcium-activated nonselective cation channel (NSC(Ca)) has been recently described in several cardiac preparations. This channel is over-expressed in models of ventricular hypertrophy showing electrophysiological perturbations of heart activity, including occurrence of spontaneous activity. While these perturbations are currently attributed to a modification of the pacemaker I(f) current activity, arguments are also in favor of participation of an NSC(Ca). Similarly, the NSC(Ca) may be expressed in specialized pacemaker cells, i.e. sino-atrial node (SAN) cells. The aim of the present study was to detect such current in mouse pacemaker cells. METHODS: The inside-out configuration of the patch-clamp technique was used in freshly isolated SAN cells from adult mice. Also, RT-PCR and Western-blotting studies were used to probe for TRPM4 mRNA and protein expression. RESULTS: In these cells, an NSC(Ca) activity was detected. The channel is voltage dependant with a conductance of 20.9+/-0.5 pS (n = 11). It is equally permeable for Na+ and K+ but does not conduct Ca2+. It is activated by rise in intracellular calcium concentrations and blocked by intracellular ATP (0.5 mmol/L). Also, as a new property in cardiac cells, the channel is activated by internal application of phosphatidylinositol 4,5-bisphosphate (10 microM). It is reversibly inhibited by flufenamic acid and glibenclamide. This channel shows the hallmarks of the TRPM4 molecule, a member of the TRP melastatin subfamily. We confirm the expression of this TRP channel on SAN cells by Western blotting and RT-PCR and validate that TRPM4 is glibenclamide sensitive. CONCLUSION: TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.

Calcium-activated nonselective cation channels in mammalian cardiomyocytes.

A number of calcium-activated nonselective cation (NSC(Ca)) currents recorded from cardiac preparations have been described in the literature. These currents are implicated in membrane depolarization and in the modulation of cardiac activity. The discovery of a novel family of cation channels, the "transient receptor potential" (TRP) protein family, has revived interest in the study of nonselective cation channels. In particular, the TRPM4 protein provides the basis for an NSC(Ca) channel detected in heart preparations. The role of this channel should not be neglected in the description of our understanding of heart activity and the development of arrhythmias induced by calcium waves. This review focuses on the electrophysiologic and regulatory properties of this native NSC(Ca) channel in cardiac preparations compared with those of the TRPM4 protein. Physiologic and pathologic implications of the current carried by this channel are discussed.