Progression of excitation-contraction coupling defects in doxorubicin cardiotoxicity.

Cardiac failure is a common complication in cancer survivors treated with anthracyclines. Here we followed up cardiac function and excitation-contraction (EC) coupling in an in vivo doxorubicin (Dox) treated mice model (iv, total dose of 10 mg/Kg divided once every three days). Cardiac function was evaluated by echocardiography at 2, 6 and 15 weeks after the last injection. While normal at 2 and 6 weeks, ejection fraction was significantly reduced at 15 weeks. In order to evaluate the underlying mechanisms, we measured [Ca2+]i transients by confocal microscopy and action potentials (AP) by patch-clamp technique in cardiomyocytes isolated at these times. Three phases were observed: 1/depression and slowing of the [Ca2+]i transients at 2 weeks after treatment, with occurrence of proarrhythmogenic Ca2+ waves, 2/compensatory state at 6 weeks, and 3/depression on [Ca2+]i transients and cell contraction at 15 weeks, concomitant with in-vivo defects. These [Ca2+]i transient alterations were observed without cellular hypertrophy or AP prolongation and mirrored the sarcoplasmic reticulum (SR) Ca2+ load variations. At the molecular level, this was associated with a decrease in the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression and enhanced RyR2 phosphorylation at the protein kinase A (PKA, pS2808) site (2 and 15 weeks). RyR2 phosphorylation at the Ca2+/calmodulin dependent protein kinase II (CaMKII, pS2814) site was enhanced only at 2 weeks, coinciding with the higher incidence of proarrhythmogenic Ca2+ waves. Our study highlighted, for the first time, the progression of Dox treatment-induced alterations in Ca2+ handling and identified key components of the underlying Dox cardiotoxicity. These findings should be helpful to understand the early-, intermediate-, and late- cardiotoxicity already recorded in clinic in order to prevent or treat at the subclinical level.

Enhanced activity of the myocardial Na+/H+ exchanger contributes to left ventricular hypertrophy in the Goto-Kakizaki rat model of type 2 diabetes: critical role of Akt.

AIMS/HYPOTHESIS: Diabetes mellitus is a strong risk factor for the development of heart failure, and left ventricular (LV) hypertrophy has been detected in a significant proportion of diabetic patients. Because several studies have suggested that the Na(+)/H(+) exchanger (NHE1) plays a part in the molecular mechanisms involved in cardiac hypertrophy, we investigated its activity and its role in LV myocytes from the Goto-Kakizaki (GK) rat model of type 2 diabetes. MATERIALS AND METHODS: Fluorometric measurements were used to assess sarcolemmal NHE1 activity in isolated myocytes. NHE1 levels and the possible molecular pathways driving and/or related to NHE1 activity were investigated in relation to the diabetic LV phenotype. RESULTS: Enhanced NHE1 activity was associated with LV myocyte hypertrophy. This occurred in the absence of any change in NHE1 protein levels; however, activation of several molecular pathways related to NHE1 activity was demonstrated. Thus, phosphorylation of the extracellular signal-regulated protein kinase (Erk), of the protein kinase Akt (also known as protein kinase B) and of the Ca(2+)/calmodulin-dependent kinase II was increased in GK LV myocytes. Intracellular Ca(2+) levels were also increased. Chronic treatment (10-12 weeks) with the NHE1 inhibitor cariporide normalised NHE1 activity, decreased [Formula: see text] levels and reduced LV myocyte hypertrophy. Moreover, among the various activated pathways, cariporide treatment markedly reduced Akt activity only. CONCLUSIONS/INTERPRETATION: These findings indicate that activation of the Akt pathway represents a likely mechanism mediating the hypertrophic effect of increased NHE1 activity in the GK model of type 2 diabetes.

[Role of extracellular matrix in angiotensin II signalling in aortic smooth muscle cells: relationship with arterial hypertension]. / Rôle de la matrice extracellulaire dans la signalisation de l'angiotensine II dans les cellules musculaires lisses aortiques: relation avec l'HTA.

Angiotensin II (Ang II) is involved in hypertension-related arterial wall hypertrophy [1]. Regulation of AT II transduction pathway in vascular smooth muscle cells (VSMC) may involve cytoskeleton and extracellular matrix (ECM) [2]. We assessed the role of components of ECM on Cai2+ increase induced by Ang II in Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) aortic VSMC. The effect of Ang II (1 mumol) on Ca2+ mobilization was studied in cultured VSMC isolated from the aorta of 6-wk old WKY (MAP (m +/- SE) = 98 +/- 4 mmHg) and SHR (136 +/- 5 mmHg; p < 0.05), using fluorescent imaging microscopy (Fura-2 AM). Cai2+ release from internal stores and Ca2+ influx were assessed in the absence and upon reintroduction of external Ca2+ respectively. Cells were cultured on uncoated glass coverslips (control) or coated with either collagen I (10 micrograms/mL), collagen IV (7 micrograms/mL), vitronectin (0.1 microgram/mL), fibronectin (3 micrograms/mL) and extracellular matrix extract (matrigel, 1/10) and studied at confluence. Paxillin was located in cells by indirect immunofluorescence micrography. Results are expressed in % of Control. Statistical significance (p < 0.05) was assessed with Student's t-test for unpaired data. The effects on Ang II-induced Ca2+ mobilization of growing cells on ECM are in Table. Paxillin in Control cells appeared as dots at the cell boundaries. Density increased in cells grown on collagen I with a diffuse distribution in the WKY cells. On matrigel, paxillin was located in a belt-like fashion at the periphery of the cell. These effects were not linked to differences in cell cycle (flux cytometry).

Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes.

1. The regulation of the L-type Ca(2+) current (I(Ca)) by intracellular cGMP was investigated in human atrial myocytes using the whole-cell patch-clamp technique. 2. Intracellular application of 0.5 microM cGMP produced a strong stimulation of basal I(Ca) (+64 +/- 5 %, n = 60), whereas a 10-fold higher cGMP concentration induced a 2-fold smaller increase (+36 +/- 8 %, n = 35). 3. The biphasic response of I(Ca) to cGMP was not mimicked by the cGMP-dependent protein kinase (PKG) activator 8-bromoguanosine 3',5' cyclic monophosphate (8-bromo-cGMP, 0.5 or 5 microM), and was not affected by the PKG inhibitor KT 5823 (100 nM). 4. In contrast, cGMP stimulation of I(Ca) was abolished by intracellular perfusion with PKI (10 microM), a selective inhibitor of the cAMP-dependent protein kinase (PKA). 5. Selective inhibition of the cGMP-inhibited phosphodiesterase (PDE3) by extracellular cilostamide (100 nM) strongly enhanced basal I(Ca) in control conditions (+78 +/- 13 %, n = 7) but had only a marginal effect in the presence of intracellular cGMP (+22 +/- 7 % in addition to 0.5 microM cGMP, n = 11; +20 +/- 22 % in addition to 5 microM cGMP, n = 7). 6. Application of erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, 30 microM), a selective inhibitor of the cGMP-stimulated phosphodiesterase (PDE2), fully reversed the secondary inhibitory effect of 5 microM cGMP on I(Ca) (+99 +/- 16 % stimulation, n = 7). 7. Altogether, these data indicate that intracellular cGMP regulates basal I(Ca) in human atrial myocytes in a similar manner to NO donors. The effect of cGMP involves modulation of the cAMP level and PKA activity via opposite actions of the nucleotide on PDE2 and PDE3.

Effect of extracellular matrix elements on angiotensin II-induced calcium release in vascular smooth muscle cells from normotensive and hypertensive rats.

The interaction of the vascular smooth muscle cells (VSMCs) with the components of the matrix determines several functions of the cell, such as growth and differentiation. In contrast, an alteration in angiotensin (Ang) II-induced Ca(2+) mechanisms in VSMCs was reported in genetic hypertension. In this study, we wished to assess the effect of different components of the extracellular matrix on the increase of [Ca(2+)](i) induced by Ang II in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto rats (WKY). Results demonstrate for the first time that elements of the extracellular matrix modulate the Ang II-induced Ca(2+) transport mechanisms. This modulation is different in cells from WKY compared with those from SHR. Thus, growing cells from SHR on collagen I, collagen IV, fibronectin, vitronectin, or Matrigel induced a significant decrease in Ang II-induced Ca(2+) release from internal stores, whereas in cells from WKY, no effect could be observed except for those grown on collagen I, which increased Ca(2+) release. Fibronectin and vitronectin, however, induced a decrease in Ang II-induced Ca(2+) influx in WKY, whereas no effect could be observed in SHR. Conversely, collagen I and collagen IV induced an increase in this influx in SHR but not in WKY, whereas Matrigel increased the influx in both strains. These results suggest a modulation of the Ang II-associated signaling events by the matrix elements via the focal adhesion points. The understanding of these synergies should provide insight into issues such as development of hypertrophy of large vessels in hypertension.

Adult cardiac myocytes survive and remain excitable during long-term culture on synthetic supports.

OBJECTIVE: Cardiomyocytes can be transplanted successfully into skeletal and cardiac muscle. Our goal was to determine the feasibility of grafting cardiomyocytes onto various synthetic supports to create an excitable and viable tissue for implantation. METHODS: Adult rat cardiomyocytes were cultured over an 8-week period onto different substitutes, including human glutaraldehyde-treated pericardium (n = 3), equine glutaraldehyde-treated pericardium (n = 3), polytetrafluoroethylene (n = 8), Dacron polyester (n = 16), and Vicryl polyglactin (n = 8). RESULTS: Only the cells seeded on the Dacron survived, with the synthetic fibers colonized at 8 weeks. On the other supports, the number of myocytes progressively decreased from the first week, with their density (number of cells per square millimeter) being, after 20 days, 17 +/- 2 on the polytetrafluoroethylene and 5 +/- 1 on the human or equine pericardium compared with 45 +/- 3 on the Dacron. After 8 weeks of culture on Dacron, the sarcomeric protein (sarcomeric alpha-actinin) was detected in all cells. In addition, the staining was regularly arranged and well aligned in a striated pattern. Spontaneous beating activity was obtained. Moreover, electrical stimulation of the cell preparation resulted in the generation of calcium transients, the frequency of which followed the frequency of the electrical stimulation. CONCLUSIONS: These results suggest that adult cardiac myocytes remain viable and excitable during long-term culture on a 3-dimensional Dacron support, which might constitute a new synthetic cardiac tissue.

Cardiac capillary cells release biologically active nitric oxide at an early stage of in vitro development.

OBJECTIVE: Coronary microvascular endothelial cells (EC) may regulate the myocardial contractile function by releasing cardioactive agents such as nitric oxide (NO). However, understanding of these regulatory mechanisms is complicated by the fact that EC exhibit marked phenotypic changes, such as the loss of endothelial NO synthase (eNOS), when they are placed into culture. Recently, it has been shown that eNOS gene expression is regulated by specific cell-cell interactions with mural cells depending on vascular beds. Since EC and pericytes (PL) are closely associated in capillaries, we have enzymatically isolated these cells from rat hearts to develop a primary culture of capillary cells favoring the re-establishment of cell interactions in vitro. METHODS: Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase chain reaction and Southern blot analysis. Expression of NOS proteins was detected with specific rhodamine-labeled antibodies. Production of NO was assessed (i) from nitrite measurements in culture supernatants by the Griess reaction, and (ii) from its antiproliferative action on cardiac fibroblasts (FIB) in non-contacted cocultures (reporter-cell bioassay) compared to that of sodium nitroprusside in homotypic FIB cultures. Fura-2 fluorescence was used to measure agonist-induced changes in cytosolic free calcium levels. RESULTS: In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the following days (i.e. second phase). The data from RT-PCR analysis demonstrate the presence of mRNAs of both eNOS and iNOS at all developmental stages of the culture. However, eNOS protein was only detected and restricted to EC. During the first phase of cell growth (5-8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferation in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15-20 days), were prevented by hemoglobin (50 microM) and by N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM). At the two periods of culture, EC increased rapidly their cytosolic Ca(2+) concentration in response to bradykinin (10 nM). However, this calcium response was associated with an increase in nitrite production only in older cultures. CONCLUSIONS: Our data indicate that heterotypic cultures of native capillary cells preserve the eNOS expression by EC. This enzyme is basally active at an early stage of in vitro development, and then becomes activatable by a Ca(2+)-mobilizing agonist. NO released by growing EC downregulates the proliferation of cardiac FIB, an effect which could be important in the cardiovascular plasticity.

Left ventricular alterations in a model of fetal left ventricular overload.

Congenital aortic coarctation is well tolerated by the fetus because the foramen ovale and ductus arteriosus equalize intracardiac and great arteries pressures and shunts. The pathologic consequences only emerge after birth with closure of the foramen ovale and ductus arteriosus. There is, however, no documentation of myocardial effects in utero of the left ventricular (LV) pressure overload induced by aortic banding. We investigated whether prenatal aortic banding could be detrimental at the structural and/or functional level. The goal of the present study was to investigate the cardiac effects of LV pressure overload in a fetal lamb model. Nine fetal lambs underwent preductal banding of the aortic arch in utero at midgestation (CoA group), whereas their twins underwent sham surgery. All fetuses were studied between 27 and 37 d after surgery for LV pressure, anatomic and histologic anomalies, and steady state sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA and protein levels and pump activity. Surgery resulted in severe aortic coarctation in all the animals in the CoA group and was associated with a 65% increase in the LV weight to body weight ratio relative to the sham-operated group (p < 0.001). Hemodynamic and histologic studies showed an evolutionary pattern depending on duration of the experimental coarctation with a shift occurring at 30 d of coarctation. The initial response of cardiomyocytes to ventricular overload was hypertrophy of the myocytes, followed by myocyte hyperplasia. Compared with sham, there was an apparent decrease in the percentage of binucleated cells in the CoA group after 30 d of coarctation. The earliest response to LV pressure overload appears to occur at the molecular level. Indeed, sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA levels fell significantly to only 28.6% of the sham group value (p = 0.023), independently of the duration of coarctation. In the fetal lamb, the pressure overload-induced hypertrophy resulting from progressive aortic coarctation leads to hemodynamic and lesional abnormalities and slows ontogenic maturation.

Highly efficient adenovirus-mediated gene transfer to cardiac myocytes after single-pass coronary delivery.

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

Myocardial cell death in fibrillating and dilated human right atria.

OBJECTIVES: The aim of the present study was to determine if myocytes can die by apoptosis in fibrillating and dilated human atria. BACKGROUND: The cellular remodeling that occurs during atrial fibrillation (AF) may reflect a degree of dedifferentiation of the atrial myocardium, a process that may be reversible. METHODS: We examined human right atrial myocardium specimens (n = 50) for the presence of apoptotic myocytes. We used immunohistochemical and Western blotting analysis to examine the expression of a final effector of programmed cell death, caspase-3 (CASP-3) and of regulatory proteins from the BCL-2 family. RESULTS: Sections from atria in AF contained a high percentage of large myocytes with a disrupted sarcomeric apparatus replaced by glycogen granules (64.4 +/- 6.3% vs. 12.2 +/- 5.8%). These abnormal myocytes, which also predominated in atria from hearts with decreased left ventricular ejection fraction (42.3 +/- 10.1%), contained large nuclei, most of which were TUNEL positive, indicating a degree of DNA breakage. None of these abnormal myocytes expressed the proliferative antigen Ki-67. A small percentage of the enlarged nuclei (4.2 +/- 0.8%) contained condensed chromatin and were strongly TUNEL positive. Both the pro- and activated forms of CASP-3 were detected in diseased myocardial samples, which also showed stronger CASP-3 expression than controls. Expression of the antiapoptotic BCL-2 protein was decreased in diseased atria, whereas that of the proapoptotic BAX protein remained unchanged. CONCLUSIONS: In fibrillating and dilated atria, apoptotic death of myocytes with myolysis contributes to cellular remodeling, which may not be entirely reversible.

Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes.

OBJECTIVES: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation. METHODS: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 microM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C2-ceramide (C2-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay. RESULTS: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82 +/- 3 vs. 12 +/- 1%, n = 7; p < 0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715 +/- 243 vs. 785 +/- 99 pmol/mg prot, n = 5; p < 0.01), a phenomenon also detected on day 3 (731 +/- 59 vs. 259 +/- 37 pmol/mg prot, n = 5; p < 0.001). Incubation of myocytes at day 0 with 50 microM C2-cer induced rapid cell shrinkage and DNA fragmentation (45 +/- 3 vs. 10 +/- 1% TUNEL-positive myocytes on day 1 in C2-cer-treated and Cl cultures, respectively; n = 6, p < 0.001). Myocyte exposure to 10 microM PDMP for 7 days (n = 5), caused ceramide accumulation (1.7-fold increase vs. Cl, p < 0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62 +/- 6 vs. 11 +/- 3% in Cl cultures, p < 0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01 +/- 0.64 vs. 5.24 +/- 0.88 pmol/mg prot, n = 6; p < 0.001). CONCLUSION: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.

Early redistribution of plasma membrane phosphatidylserine during apoptosis of adult rat ventricular myocytes in vitro.

In many cell types, DNA fragmentation is a late event of apoptosis which may be lacking. This contrasts with the early translocation of phosphatidylserine (PS) from the internal to the external leaflet of the cell membrane. We examined whether an early PS translocation also occurs during apoptosis induced in adult rat ventricular myocytes grown in the presence of 10% fetal calf serum (FCS), by the protein kinase inhibitor staurosporine. Apoptosis was assessed by the observation of: (i) typical alterations in cell morphology; (ii) nuclear alterations visualized using the permeant intercalating agent Hoechst 33258; (iii) DNA fragmentation detected by the TUNEL method. PS translocation was detected using annexin V binding. Data are expressed as means +/- SEM. Prolonged exposure of myocytes to 10 microM staurosporine from day 3 to day 7 of culture resulted in cell shrinkage, typical nuclear alterations, membrane protrusions and fragmentation of the sarcomeric apparatus in the vast majority of myocytes. At this time, 52.4 +/- 5.7% of staurosporine-treated myocytes were TUNEL positive (vs 6.1 +/- 2.0% in control cultures (CC), p < 0.001) and 69.7 +/- 1.7% were annexin V positive (vs 21.1 +/- 1.0% in CC, p < 0.001). Importantly, PS translocation was detected as early as 35 minutes following staurosporine addition, the percentage of annexin V positive myocytes reaching 10 times the control value (19.2 +/- 2.7 vs 1.8 +/- 0.8%, p < 0.001) after 3 hours. A 18-hour staurosporine exposure of freshly isolated myocytes resulted, at the end of exposure, in 24.3 +/- 1.7% annexin V positive myocytes (vs 9.6 +/- 0.5% in CC, p < 0.05), whereas a marked increase in the percentage of TUNEL positive myocytes was observed only from day 5. Finally, myocyte exposure to the membrane-permeant ceramide analog, C2-ceramide (50 microM), resulted in 63.2 +/- 3.5% annexin V positive myocytes 4 hours later (vs 17.8 +/- 4.4% in CC, p < 0.001), whereas a significant increase in the percentage of TUNEL positive myocytes was detected only the next day (43.7 +/- 3.4 vs 9.9 +/- 1.3%, p < 0.001). Taken together, these results strongly suggest that the loss of PS asymmetry is an early event of cardiac myocyte apoptosis which precedes DNA fragmentation.

The antiarrhythmic agent bertosamil induces inactivation of the sustained outward K+ current in human atrial myocytes.

1. In whole-cell patch-clamped human atrial myocytes, the antiarrhythmic agent bertosamil (10 microM) inhibited the sustained component, Isus (38.6 +/- 3.1%), and enhanced the inactivating component, I(t) (9.1 +/- 6.1%), of the outward K+ current elicited by 750 ms test pulses from -60 mV to +50 mV. Higher concentrations of bertosamil (> 10 microM) inhibited both I(t) and Isus. 2. Suppression of Isus and stimulation of I(t) by 10 microM bertosamil was observed on renewed stimulation following a 2 min rest period during which the drug was applied and persisted after washout, indicating a rest-dependent effect of bertosamil on the outward K+ current. 3. Cell dialysis with an internal solution containing 10 microM bertosamil increased both I(t) (78.0 +/- 14.7%) and Itotal (26.7 +/- 8.4%) and inhibited Isus (28.9 +/- 6.3%, n = 6). In the presence of intracellular bertosamil, external application of the drug inhibited I(t) and Isus in a concentration-dependent and use-dependent manner. 4. Following the suppression of Isus by 200 microM 4-aminopyridine (4-AP), bertosamil (10 microM) inhibited I(t). Washout of 4-AP was associated with a larger I(t) amplitude than that observed in control conditions. In myocytes characterized by a prominent Isus and lack of I(t), bertosamil (10 microM) induced a rapid and partial inactivation of the current, together with inward rectification of the current measured at the end of the test pulse. 5. In the presence of bertosamil the activation/voltage relationships, steady-state inactivation and recovery from inactivation of I(t) were markedly modified, pointing to changes in the conductance underlying I(t). 6. We conclude that bertosamil induces rapid inactivation of sustained outward current which leads to an apparent increase in I(t) and decrease in Isus. This effect, which was distinct from the use-dependent inhibition of the outward K+ current, could represent a new antiarrhythmic mechanism.

Primary culture of human atrial myocytes is associated with the appearance of structural and functional characteristics of immature myocardium.

We examined changes in the structural and physiological characteristics of human atrial myocytes during primary culture in the presence of serum. Action potentials and ionic currents were recorded in freshly dissociated (FM) and cultured (CM) whole-cell patch-clamped myocytes, alpha-smooth muscle actin, sarcomeric alpha-actinin and beta-myosin heavy chains (beta-MHC) were stained with monoclonal antibodies. From day 5 to day 21, myocytes lost their rod shape, spread and exhibited reorganized sarcomeres. These morphological changes were associated with a marked increase in membrane capacitance (+266%). Both beta-MHC and alpha-smooth muscle actin were expressed in CM but not in FM, indicating a dedifferentiation process. CM were characterized by a lower resting potential (-30 +/- 2 v -60 +/- 4 mV, P < 0.05) and, when repolarized, by a shorter action potential duration (APD) than FM (APD-60: 126.9 v 159.6 ms, P < 0.05). The inward rectifier K+ current was absent in CM, thus explaining the low resting potential. The density of the transient component of the voltage-activated K+ current Ito1 was not modified during culture, while that of the sustained component Isus was increased fourfold. The amplitude of ICa was increased, but its density was unchanged, indicating that CM maintained a normal density of functional calcium channels. Neither the voltage dependence nor the inactivation of ICa was modified in CM. The time constants of inactivation of ICa were unchanged, although the amplitude of the rapidly inactivating component of ICa was increased in CM compared to FM. Moreover, ICa was increased by the beta-adrenergic agonist isoproterenol (1 microM) throughout the culture period. Our results demonstrate that in long-term serum-supplemented culture, adaptation of human atrial myocytes to their new environment is associated with differential alterations of the main ionic currents and phenotypic changes characteristic of immature myocardium.

Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.

The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca2+ transients (Cai transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric alpha-actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca2+ current (Ica) elicited a prolonged Cai transient, with an initial rapidly activating phase (slope 1, 23.6 +/- 1.2 s-1) followed by a slowly activating phase (slope 2, 5.8 +/- 0.4 s-1; P < .001 versus slope 1), resulting in a dome-shaped Cai transient. Ryanodine (100 mumol/L) inhibited 79 +/- 6% of the Cai transient, indicating that it was due essentially to sarcoplasmic reticulum Ca2+ release. During step depolarizations, maximal activation of the Cai transient or tail current (Itail) (in cells dialyzed with Ca2+ buffer-free internal solution) preceded that of Ica and did not follow its voltage dependence (n = 12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Cai transient and Itail (n = 5). In a given cell, the two components of the Cai transient could be dissociated by altering the intracellular Ca2+ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of Ica. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, Ica only partially controls the activation of RyRs, with the prolonged and dome-shaped Cai transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca2+ channels.

Differential regulation of voltage-activated potassium currents in cultured human atrial myocytes.

To examine whether the two components of the voltage-activated outward K+ current, an initially rapidly inactivating component (Ito,1) and a slowly inactivating sustained component (Isus), in human atrial myocytes are distinct currents differentially regulated, we studied their behavior during serum-induced growth of cultured myocytes. Currents were recorded in whole cell patch clamped myocytes. After 1 wk of culture (day 8), membrane capacitance was twice the value in freshly dissociated myocytes (178.7 +/- 23 vs. 83.1 +/- 5.5 pF; P < 0.001). Ito,1 density did not differ from that in freshly dissociated myocytes (at +40 mV: 4.38 +/- 0.8 vs. 3.71 +/- 0.6 pA/pF), whereas that of Isus was markedly increased (at +40 mV: 9.76 +/- 2 vs. 2.21 +/- 0.29 pA/pF; P < 0.001). After inactivation of Ito,1 by a prepulse, sustained depolarization elicited in cultured myocytes an Isus with a density of 10.22 +/- 1.18 pA/pF and an apparent tail current reversal potential of -73.5 +/- 3.2 mV, indicating high K+ selectivity. Isus was highly sensitive to 4-aminopyridine (55.4 +/- 4.4% inhibition in 50 microM) and to D-600 (with a concentration inhibiting 50% of maximal response of 34.2 x 10(-6) M). Addition of 5-10 nM staurosporine at day 3 prevented cell growth and reduced Ito,1 density but not the increase in Isus density, which was inhibited by 10 microM staurosporine. Our results indicate that Ito,1 and Isus are regulated independently during in vitro myocyte growth in human atrial myocytes and that the increase in Isus density is not mediated by a protein kinase C-dependent pathway.

Contribution of Na+/Ca2+ exchange to action potential of human atrial myocytes.

The Ca2+ dye indo 1 was used to record internal Ca2+ (Cai) transients in order to investigate the role of the Na+/Ca2+ exchange current (INa/Ca) in whole cell patch-clamped human atrial myocytes After the activation of the L-type Ca2+ current by test pulses (20 ms) at +20 mV, a tail current (I(tail)) was activated at a holding potential of -80 mV with a density of -1.29 +/- 0.06 pA/pF. The time course of I(tail) followed that of Cai transients I(tail) was suppressed by dialyzing cells with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, applying 5 mM caffeine, or substituting external Na+ with Li+, indicating that this current was mainly generated by INa/Ca. Two types of action potential were recorded: type A, which is characterized by a narrow early plateau followed by a late low plateau phase, and type B, which is characterized by a small initial peak followed by a prolonged high plateau phase. Type B action potentials were found in larger cells than type A action potentials (membrane capacitance 81.8 +/- 4.5 and 122.4 +/- 7.0 pF in types A and B, respectively, P < 0.001). Substitution of external Na+ with Li+ shortened the late plateau of the type A action potential and the prolonged plateau of the type B action potential. Suppression of Cai transients by caffeine shortens the late part of both types of action potentials, whereas its lengthening effect on the initial phase of action potentials can result from several different mechanisms. The beat-to-beat dependent relationship between Cai transients and action potentials could be mediated by Ina/Ca- Delayed afterdepolarizations were present in a significant proportion of atrial myocytes in our experimental conditions. They were reversibly suppressed by Li+ substitution for Na+, suggesting that they are generated by INa/Ca. We conclude that INa/Ca plays a major role in the development of action potentials and delayed afterdepolarizations in isolated human atrial myocytes.

Behaviour of human atrial myocytes in culture is donor age dependent.

The characteristics of cultured myocardial cells isolated from small mammals are well documented, but there is a dearth of data on cultured human cardiocytes. The aim of this study was to determine the main features of myocytes isolated from human atria and maintained in culture in the presence of 10% fetal calf serum (FCS), according to the age of the donor. The following characteristics were analysed: (1) yield and viability; (2) adhesive properties; and (3) changes in cell morphology. Myocytes preferentially adhered to laminin-coated dishes and could be maintained in culture for at least 2 weeks, whatever the age of the donor (which was from 6 days to 85 yr). Maintenance in culture induced morphologic changes characterized by myocyte spreading and changes in myofibrillar organization. Interestingly, the time of onset of these changes depended on the age of the donor: they occurred earlier in young atrial myocytes (< 1 yr) than in older cells (> 13 yr).