Atrial fibrillation and rapid acute pacing regulate adipocyte/adipositas-related gene expression in the atria.

PURPOSE: Atrial fibrillation (AF) has been associated with increased volumes of epicardial fat and atrial adipocyte accumulation. Underlying mechanisms are not well understood. This study aims to identify rapid atrial pacing (RAP)/AF-dependent changes in atrial adipocyte/adipositas-related gene expression (AARE). METHODS: Right atrial (RA) and adjacent epicardial adipose tissue (EAT) samples were obtained from 26 patients; 13 with AF, 13 in sinus rhythm (SR). Left atrial (LA) samples were obtained from 9 pigs (5 RAP, 4 sham-operated controls). AARE was analyzed using microarrays and RT-qPCR. The impact of diabetes/obesity on gene expression was additionally determined in RA samples (RAP ex vivo and controls) from 3 vs. 6 months old ZDF rats. RESULTS: RAP in vivo of pigs resulted in substantial changes of AARE, with 66 genes being up- and 53 down-regulated on the mRNA level. Differential expression during adipocyte differentiation was confirmed using 3T3-L1 cells. In patients with AF (compared to SR), a comparable change in RA mRNA levels concerned a fraction of genes only (RETN, IGF1, HK2, PYGM, LOX, and NR4A3). RA and EAT were affected by AF to a different extent. In patients, concomitant disease contributes to AARE changes. CONCLUSIONS: RAP, and to lesser extent AF, provoke significant changes in atrial AARE. In chronic AF, activation of this gene panel is very likely mediated by AF itself, AF risk factors and concomitant diseases. This may facilitate the development of an AF substrate by increasing atrial ectopic fat and fat infiltration of the atrial myocardium.

A calcium-sensitive promoter construct for gene therapy.

Targeting diseased cells is a challenging issue in both pharmacological and biological therapeutics. Gene therapy is emerging as a novel approach for treating rare diseases and for illnesses for which there is no other alternative. An important limitation of gene therapy has been the off-target effects and therefore efforts have been focused on increasing the specificity of gene transfer to the targeted organ. Here, we describe a promoter containing six nuclear factor of activated T cells (NFAT) consensus sequences, which is as efficient as the cytomegalovirus (CMV) promoter to drive expression in vascular smooth muscle cells both in vitro and in vivo. In contrast to the CMV promoter it is activated in a Ca(2+)-dependent manner after endoplasmic reticulum depletion and allows the transgene expression only in proliferative/diseased cells. Overexpression of sarco/endoplasmic reticulum (SR/ER) Ca(2+) ATPase 2a under the control of this NFAT promoter inhibits restenosis after angioplasty in rats. In conclusion, this promoter may be useful for gene therapy in vascular proliferative diseases and other diseases involving upregulation of the NFAT pathway.

How to optimize in vivo gene transfer to cardiac myocytes: mechanical or pharmacological procedures?

An efficient gene delivery system is a prerequisite for myocardial gene therapy. Among the various procedures studied so far, catheter-based percutaneous gene delivery to the myocardium through the coronary vessels seems the most relevant to routine clinical practice; however, the optimal conditions remain to be determined. We selectively infused adenoviral vectors encoding luciferase (1 x 10(9) PFU) or beta-galactosidase (1 x 10(10) PFU) into coronary arteries of adult rabbits in various experimental conditions. Coronary artery occlusion for 30 sec, during and after adenovirus delivery, was required to observe luciferase activity in the target area of the circumflex artery (4.0 +/- 1.0 x 10(5) vs. 1.1 +/- 0.2 x 10(4) RLU/mg with and without coronary occlusion, respectively, p < 0.01, and 1.0 +/- 0.1 x 10(3) RLU/mg using nonselective infusion). When adenoviruses were delivered using high-pressure infusion (82 +/- 12 vs. 415 +/- 25 mmHg before and during infusion, respectively, p < 0.01), luciferase activity increased to 8.5 +/- 2.5 x 10(5) RLU/mg (p < 0.05 vs coronary occlusion alone). Coronary venous sinus occlusion with saline buffer retroinfusion starting before and during anterograde adenovirus delivery resulted in a further 4.7-fold increase in luciferase activity (4.4 +/- 0.8 x 10(6) RLU/mg, p < 0.01) with 5-25% blue-stained myocytes in the target area, compared with 0-5% with the other procedures. Histamine or VEGF-A(165) pretreatment, used to increase vascular permeability, slightly increased gene transfer efficiency (8.5 +/- 2.0 x 10(5) and 9.0 +/- 2.5 x 10(5) RLU/mg respectively, p < 0.05 vs. coronary occlusion alone). We conclude that catheter-mediated adenoviral gene transfer to cardiac myocytes through coronary vessels can be a very efficient procedure for myocardial gene therapy, particularly when the vector residence time and perfusion pressure in the vessels are increased.

Mechanisms of L-type Ca(2+) current downregulation in rat atrial myocytes during heart failure.

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.

Characterization of effects of endothelin-1 on the L-type Ca2+ current in human atrial myocytes.

The effects of endothelin-1 (ET-1) on the L-type Ca2+ current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. Depending on the initial current density, ET-1 (10 nM) increased the amplitude of I(Ca) by 99 +/- 7% or decreased it by 33 +/- 2%. The stimulatory effect predominated on current of low density (2.3 +/- 0.2 pA/pF), whereas I(Ca) of higher density (5.8 +/- 0.3 pA/pF) was inhibited by ET-1. After I(Ca) stimulation by 1 microM isoproterenol, ET-1 always inhibited the current by 32 +/- 7% (P < 0.05), an effect that was suppressed by pretreating myocytes with pertussis toxin. Atrial natriuretic peptide (ANP) inhibited I(Ca) (41 +/- 3%) by reducing intracellular cAMP concentration. In ANP-treated myocytes, the stimulatory effect of ET-1 on I(Ca) predominated (52 +/- 7%). The inhibitory effect of ET-1 on I(Ca) was blocked by the ET(A) antagonist BQ-123, whereas the stimulatory effect was suppressed by the ET(B) agonist BQ-788. We conclude that ET-1 has opposite effects on I(Ca) depending on the baseline amplitude of current, and both subtype ET receptors are implicated in the signal transduction pathways.

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.

Cumulative inactivation of the outward potassium current: a likely mechanism underlying electrical memory in human atrial myocytes.

The influence of the mode of cell stimulation on the outward K+ current (I(o)) was studied in whole-cell patch-clamped human atrial myocytes. Acceleration of the rate of membrane depolarization at 1 Hz or during prolonged 5-s test pulses at 0.1 Hz increased the rate and extent of I(o) inactivation, resulting in enhanced inactivating (4.9+/-0.6 v 6.3+/-0.7 pA/pF) and suppressed maintained (5.9+/-1.2 v 3.2+/-0.3 pA/pF) current components. These alterations were associated with a leftward shift of the voltage-dependency of I(o), and persisted on returning to a control depolarization protocol (750-ms test pulses delivered at 0.1 Hz). The effects of increasing external K+ concentrations (40 m m) on the kinetics of I(o) were more pronounced following both rapid and prolonged depolarization (changes in I(t)/I(o)caused by 40 m m K+: 8.9+/-3.5% v 15.5+/-3.1% before and after prolonged depolarization; and 9.2+/-1.2% v 15.4+/-1.7% before and after rapid depolarization). The phosphatase inhibitor, okadaic acid, enhanced the effect of rapid and prolonged depolarization on I(o)whereas the inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with KN-62 or KN-93, or by intracellular application of the autocamtide-2-related inhibitory peptide, suppressed it. In conclusion, rapid and prolonged membrane depolarization both cause a cumulative increase in the rate and extent of I(o)inactivation. This process involves slow potassium channel inactivation mechanisms, is regulated by CaMK-II, and may contribute to the electrical memory of the atrial myocardium.

Low catecholamine concentrations protect adult rat ventricular myocytes against apoptosis through cAMP-dependent extracellular signal-regulated kinase activation.

Catecholamines have complex effects on cardiac myocyte growth and survival, including the triggering of apoptosis at high concentration. Here, we examined whether at a lower concentration, catecholamine protected adult rat ventricular myocytes from apoptosis in vitro. Myocytes were exposed to staurosporine (ST, 10 microM) for 18 h, with or without epinephrine (0.1 or 10 microM) or fetal calf serum (10%). Apoptosis was assessed after 48 h of culture in terms of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method, DNA gel electrophoresis). Epinephrine (0.1 microM) and serum reduced ST-induced myocyte apoptosis by approximately 50% (n = 12 cultures, P <.001), whereas epinephrine and serum alone did not influence the low apoptotic rate in control cultures. In contrast, 10 microM epinephrine induced marked apoptosis in ST-free conditions. The protective effects of 0.1 microM epinephrine and serum were blunted by the tyrosine kinase inhibitor genistein (n = 12 cultures, P <. 001). Extracellular signal-regulated kinase (ERK) activity was stimulated by 0.1 microM epinephrine but not by 10 microM epinephrine. Furthermore, the protective effect of epinephrine was mimicked by isoproterenol (1 microM) and forskolin (1 microM) but not by phenylephrine (10 microM) and was blunted by propranolol (10 microM) but not by prazozin (10 microM). Finally, isoproterenol and forskolin activated ERK, an effect that was blunted by propranolol. In conclusion, low epinephrine concentrations attenuate ST-induced apoptosis of adult cardiac myocytes in vitro, an effect mediated by coupling between the cAMP pathway and ERK activation. This suggests that a minimal adrenergic tone is essential for myocyte survival in conditions of unusual stress.

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.

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes.

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.

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.

Regulation of the transient outward K(+) current by Ca(2+)/calmodulin-dependent protein kinases II in human atrial myocytes.

Ca(2+)/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K(+) current (I(to)) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 micromol/L), but not its inactive analog, KN-92, accelerated the inactivation of I(to) (tau(fast): 66.9+/-4.4 versus 43.0+/-4.4 ms, n=35; P<0.0001) and inhibited its maintained component (at +60 mV, 4.9+/-0.4 versus 2.8+/-0.4 pA/pF, n = 35; P<0. 0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2-related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed I(to) inactivation, increased I(sus), and inhibited the effects of KN-93. Changes in [Ca(2+)](i) by dialyzing cells with approximately 30 nmol/L Ca(2+) or by using the fast Ca(2+) buffer BAPTA had opposite effects on I(to). In BAPTA-loaded myocytes, I(to) was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent I(sus), KN-93 still increased the extent of inactivation of I(to). Western blot analysis of atrial samples showed that delta-CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of I(to) in human atrial myocytes, a process that could contribute to I(to) alterations observed during chronic atrial fibrillation.

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.

Species differences in the activity of the Na(+)-Ca2+ exchanger in mammalian cardiac myocytes.

1. Species differences in the activity of the exchanger were evaluated in isolated myocytes from rat, guinea-pig, hamster ventricles and human atria. Fluorescence measurements using fura-2 were carried out in conjunction with the whole-cell patch-clamp technique for simultaneous recording of membrane currents and intracellular Ca2+ concentration. 2. Ca2+ release from sarcoplasmic reticulum (SR) induced either by rapid application of caffeine or by Ca2+ current elicited inward Na(+)-Ca2+ exchange currents (INa-Ca). The magnitude of INa-Ca was largest in hamster, smallest in rat, with guinea-pig and human myocytes having intermediate values. The ratio of caffeine-induced exchanger current densities, normalized with respect to the peak Ca2+ release, was 4:2:1.5:1 for hamster > guinea-pig > or = human > or = rat myocytes. 3. The rates of Ca2+ removal in the presence of caffeine, which reflect primarily the Ca2+ extruding activity of the Na(+)-Ca2+ exchanger, followed the same order of hamster > guinea-pig > or = human > or = rat. 4. The kinetics of INa-Ca vs. Ca2+ transients were different among species. In rat myocytes, the kinetics of the INa-Ca and the Ca2+ transients were similar, with INa-Ca linearly proportional to intracellular Ca2+ concentration ([Ca2+]i). In hamster myocytes, the time course of INa-Ca tracked only the declining phase of the Ca2+ transient with INa-Ca having faster kinetics during the Ca2+ release. These findings suggest that the Ca2+ concentrations in the vicinity of the exchanger were significantly higher than those of the cytosol during Ca2+ release in hamster myocytes. 5. We concluded that there are significant species differences in the exchanger activity of cardiac myocytes, arising from differences in exchanger densities, their modulation and/or their spatial distribution with respect to the ryanodine receptors of cardiac myocytes.