Daniel Potreau

Functional characterization of a Ca2+‐activated non‐selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i‐sensitive currents: the Na+–Ca2+ exchange current, a Ca2+‐activated chloride current and a Ca2+‐activated non‐selective cationic current. Using the cell‐free configuration of the patch‐clamp technique, we have characterized the properties of a Ca2+‐activated non‐selective cation channel (NSCCa) in freshly dissociated human atrial cardiomyocytes. In excised inside‐out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channels open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 μm. Channel activity was reduced in the presence of 0.5 mm ATP or 10 μm glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 mm flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the ‘TRP melastatin’ subfamily. In conclusion, the NSCCa channel is a serious candidate to support the delayed after‐depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation.

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes (CMs) and progressive fibrosis are known to occur simultaneously. To investigate the nature of interactions between ventricular CMs and cardiac fibroblasts (CFs) in these conditions, we have established a “dedifferentiated model” of adult murine CMs in coculture with CFs. In such a model, which is recognized to study cardiac cell hypertrophy in vitro, dedifferentiated CMs in culture and in coculture were characterized by immunopositive staining to ANP (atrial natriuretic peptide) and β‐myosin heavy chain (β‐MHC). The results confirm that ANP secretion by CMs was significantly increased during the cultures. The increase size of cultured CMs was significantly higher in CM/CF cocultures than in CM cultures which was also observed when CMs were cultured with fibroblast conditioned medium (FCM). In addition, fibroblast proliferation studies showed that CMs favored fibroblast adhesion and/or growth at the beginning of the coculture and fibroblast proliferation throughout the time course of the coculture. Furthermore, a significant level of interleukin‐6 (IL‐6) production was detected by ELISA in CM/CF cocultures. A similar higher increase was observed when CMs were cultured in the presence of FCM. These results demonstrate that CFs enhance myocyte hypertrophy and that CMs regulate fibroblast adhesion and/or proliferation, suggesting a paracrine interaction between CMs and CFs which could involve IL‐6.

Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation.

The process of cardiac hypertrophy is considered to involve two components: that of cardiac myocyte (CM) enlargement and cardiac fibroblast (CF) proliferation. The interleukin‐6 (IL‐6) family cytokines have been implicated in a variety of cellular and molecular interactions between myocytes and non‐myocytes (NCMs), which in turn have important roles in the development of cardiac hypertrophy. In the study of these interactions, we previously detected very high levels of IL‐6 in supernatants of a “dedifferentiated model” of adult ventricular CMs cultured with CFs. In the present study, we have used this in vitro coculture system to examine how IL‐6 is involved in the interactions between CMs and CFs during CM hypertrophy and CF proliferation. IL‐6 and its signal transducer, 130‐kDa glycoprotein (gp130), were detected by immunostaining cultured CMs and CFs with anti‐IL‐6 or anti‐gp130 antibodies. Addition of anti‐IL‐6 or anti‐gp130 antagonist antibodies into CM/CF cocultures induced a significant decrease in expression of atrial natriuretic peptide (ANP) and β‐myosin heavy chain (β‐MHC) in CMs. The presence of IL‐6 antagonist also resulted in a decrease in the surface area of 12‐day‐old CMs cultured with CFs or in the presence of fibroblast conditioned medium (FCM), and decreased fibroblast proliferation in CM/CF cocultures, particularly in the presence of a gp130 antagonist. The results also show that angiotensin II (AngII) is mainly secreted by CFs and induces IL‐6 secretion in CMs cultured with CFs or with FCM. In addition, the effects of IL‐6 on cardiomyocyte hypertrophy and fibroblast proliferation were inhibited by addition of the AT‐1 receptor antagonist, losartan. These results suggest that IL‐6 contributes significantly to CM hypertrophy by an autocrine pathway and to fibroblast proliferation by a paracrine pathway and that these effects could be mediated by AngII.

Functional Expression of the TRPM4 Cationic Current in Ventricular Cardiomyocytes From Spontaneously Hypertensive Rats

Cardiac hypertrophy is associated with electrophysiological modifications, including modification of action potential shape that can give rise to arrhythmias. We report here a higher detection of a calcium-activated nonselective cation current in cardiomyocytes of spontaneously hypertensive rats (SHRs), a model of hypertension and heart hypertrophy when compared with Wistar-Kyoto (WKY) rat, its normotensive equivalent. Freshly isolated cells from the left ventricles of 3- to 6-month-old WKY rats or SHRs were used for patch-clamp recordings. In inside-out patches, the channel presented a linear conductance of 25±0.5 pS, did not discriminate Na+ over K+, and was not permeable to Ca2+. Open probability was increased by depolarization and a rise in [Ca2+]i (dissociation constant=10±5.4 &mgr;mol/L) but reduced by 0.5 mmol/L [ATP]i, 10 &mgr;mol/L glibenclamide, or flufenamic acid (IC50=5.5±1.7 &mgr;mol/L). Thus, it owns the fingerprint of the TRPM4 current. Although rarely detected in WKY cardiomyocytes, the current was present in >50% of patches from SHR cardiomyocytes. Moreover, by performing RT-PCR from ventricular samples, we observed that TRPM4 mRNA detection was higher in SHRs than in WKY rats. We propose that a TRPM4 current is expressed in ventricular cardiomyocytes from SHRs. According to its properties, this channel may contribute to the transient inward current implicated in delayed-after-depolarizations observed during [Ca2+] overload of cardiomyocytes.

Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture

Electrophysiological characteristics of neonatal rat ventricular cardiomyocytes in primary culture were studied using the whole-cell patch-clamp recording technique. Cell size, estimated by measurement of membrane capacitance, was significantly increased throughout the culture from 22.4±5.4 pF at day 2 to 55.0±16.1 pF at day 7, reflecting the hypertrophic process which characterises postnatal cell development. The Ca2+ current was investigated at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. At 2 days of culture, two types of Ca2+ current could be distinguished, as also observed in freshly dissociated newborn ventricular cells. From their potential dependence and pharmacological characteristics, they could be attributed to the T- (ICa-T) and L-type (ICa-L) Ca2+ current components. After 7 days of culture, only the latterICa-L was present and its density was significantly increased when compared to the density in 2-day-old cells, but lower than that obtained in freshly dissociated adult cells. As the age of the culture progressed, the steady-state inactivation curve was shifted toward negative potentials, in the direction of the inactivation curve obtained for adult cells. Compared to the serum-free control conditions, the density ofICa-L was significantly increased in the presence of fetal calf serum throughout the culture. Consequently, the density ofICa-L obtained in 7-day-old cells was similar to the density ofICa-L obtained in freshly dissociated adult cardiac cells. These results show that in rat neonatal ventricular cardiomyocytes, the changes in Ca2+ current during development in primary culture can be compared to that observed in vivo during the first weeks of the postnatal period. The data suggest that the composition of the culture medium is a conditioning factor in the development of cardiac cells in culture. However, the determination and the role of specific factors contained in the serum need to be investigated. The data are also discussed in terms of a possible correlation between the expression and maturation of the Ca2+ current components and the capabilities of the neonatal cardiac cells to proliferate and/or to hypertrophy. For these reasons primary cultures of neonatal rat cardiac cells could constitute a valuable in vitro model for studies of postnatal development.

Human cardiomyocyte hypertrophy induced in vitro by gp130 stimulation

OBJECTIVES Recent in vivo and in vitro studies in animals have demonstrated that cytokines of the IL-6 family are involved in cardiac hypertrophy and in protection of cardiomyocytes against apoptosis. The present study aims to analyse the capacity of human atrial cardiac cells (i.e., cardiomyocytes and fibroblasts) to display the gp130 receptor subunit, and to evaluate its functionality. METHODS Twenty human atrial biopsies were used for immunohistochemistry, in situ hybridisation, and western blot analysis or dissociated for isolation and primary culture of cardiac cells. RESULTS Fibroblasts present in tissue or maintained in primary culture clearly express gp130 whereas the signal in cardiomyocytes is weaker. Culture of cardiac cells with a gp130 agonist antibody enhances atrial natriuretic peptide (ANP), beta myosin heavy chain (beta-MHC) expression in cardiomyocytes, and significantly increases the cell surface area microm(2)). This process could involve STAT3 (signal transducer and activator of transcription 3) phosphorylation. CONCLUSIONS These results demonstrate that gp130 activation in human cardiac cells leads to cardiomyocyte hypertrophy. We discuss several hypotheses on the role of IL-6-type cytokines on cardiomyocyte functions.

Circulating soluble gp130, soluble IL-6R, and IL-6 in patients undergoing cardiac surgery, with or without extracorporeal circulation

OBJECTIVE Soluble forms of interleukin-6 (IL-6) receptors are known to modulate biological activities of IL-6. The purpose of the study was to measure circulating levels of IL-6, sIL-6R and sgp130 in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (CPB group) or without CPB (non-CPB group). METHODS The CPB group included 19 patients and the non-CPB group 12 patients. Sera levels of IL-6, sIL-6R and sgp130 were measured by specific ELISA at the beginning of the operation (T0, 15 min before skin incision) and 6 h later (T1). RESULTS IL-6 sera levels were respectively 9+/-20 pg/ml (mean+/-SD) and 13+/-19 pg/ml at T0 and reached 340+/-250 pg/ml and 965+/-1060 pg/ml at T1 in CPB and non-CPB groups, indicating a significant increase from T0 to T1, but no differences between the two groups. When compared to T0 values, sgp130 levels decreased in both groups (respectively 105+/-37 and 115+/-35 ng/ml at T0 for CPB and non-CPB groups, and 72+/-25 and 84+/-29 ng/ml at T1) while we are not able to detect differences between the groups. Whatever the group or the time, sIL-6R concentrations remained unchanged. CONCLUSIONS We showed that the increase of IL-6 after artery bypass grafting was similar between patients operated with CPB or without CPB. We conclude that the main inductor of IL-6 release is linked to surgical trauma rather than a reaction to CPB. Since it is known that gp130 inhibits IL-6-biological activities, we suggest that the decrease of sgp130 sera levels could further enhance the inflammatory effects of IL-6 in cardiac surgery.

Depressed transient outward current in single hypertrophied cardiomyocytes isolated from the right ventricle of ferret heart

OBJECTIVE The aim of this study was to investigate transient outward potassium current (Ito) changes as a basis for the prolongation of the action potential associated with cardiac hypertrophy. METHODS Right ventricular hypertrophy was induced by chronic pulmonary artery constriction in adult male ferrets. After 4-6 weeks, hearts were excised and single myocytes were isolated from the right ventricles of banded and sham-operated (control) animals by enzymatic dissociation. Ito was recorded by means of the whole cell patch clamp technique. RESULTS Heart weight:body weight ratio and cell membrane capacitance, as indications of hypertrophy, were increased by 17% (P < 0.05) and 32% (P < 0.01) respectively in the banded group. Analysis of Ito showed that in hypertrophied myocytes compared to normal controls: (1) the density of current was significantly reduced; (2) both the time to peak and the time constant of inactivation were increased; (3) the voltage-dependent steady-state availability was not changed, with similar potentials for half activation (30.4 +/- 6.8 mV in control and 33.9 +/- 8.5 mV in hypertrophied cells) and half inactivation (-12.3 +/- 3.3 mV in control and -11.4 +/- 2.7 mV in hypertrophied cells); (4) the time constant for recovery from inactivation was significantly increased regardless of the holding potentials (-50 mV, -70 mV or -90 mV). CONCLUSIONS Alterations of the transient outward potassium current with respect to its density, kinetics and recovery from inactivation can explain the prolongation of the action potential in myocytes isolated from pressure-overload hypertrophied heart and may thus be an important step in such cardiac adaptation.

Characterization of a hyperpolarization-activated current in dedifferentiated adult rat ventricular cells in primary culture

1 The presence of a hyperpolarization‐activated pacemaker (If)‐like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole‐cell patch clamp technique. 2 An If‐like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from ‐92.0 ± 2.5 and ‐63.0 ± 1.0 mV at the beginning (4‐day‐cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at ‐170 mV progressively increased from ‐2.34 ± 0.36 to ‐6.12 ± 0.64 pA pF−1 between the two farthest stages of cellular remodelling. In freshly isolated cells the current was activated at ‐108.0 ± 1.5 mV and its current density measured at ‐170 mV was ‐1.97 ± 0.56 pA pF−1. 3 The current was blocked by extracellular CsCl (3 mM) in a voltage‐dependent manner. Modification of reversal potentials obtained at various values of [K+]o (5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions. 4 It is concluded that the hyperpolarization‐activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of If. In adult rat ventricular cells it is activated in a non‐physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture.

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066+/-0.005 min(-1) (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.