Adult Guinea Pig Cardiomyocytes Research Articles

Transfer RNA-mediated restoration of potassium current and electrical correction in premature termination long-QT syndrome hERG mutants

Disease-causing premature termination codons (PTCs) individually disrupt the functional expression of hundreds of genes and represent a pernicious clinical challenge. In the heart, loss-of-function mutations in the hERG potassium channel account for approximately 30% of long-QT syndrome arrhythmia, a lethal cardiac disorder with limited treatment options. Premature termination of ribosomal translation produces a truncated and, for potassium channels, a potentially dominant-negative protein that impairs the functional assembly of the wild-type homotetrameric hERG channel complex. We used high-throughput flow cytometry and patch-clamp electrophysiology to assess the trafficking and voltage-dependent activity of hERG channels carrying patient PTC variants that have been corrected by anticodon engineered tRNA. Adenoviral-mediated expression of mutant hERG channels in cultured adult guinea pig cardiomyocytes prolonged action potential durations, and this deleterious effect was corrected upon adenoviral delivery of a human ArgUGA tRNA to restore full-length hERG protein. The results demonstrate mutation-specific, context-agnostic PTC correction and elevate the therapeutic potential of this approach for rare genetic diseases caused by stop codons.

Aldosterone down-regulates the slowly activated delayed rectifier potassium current in adult guinea pig cardiomyocytes.

There is emerging evidence that the mineralocorticoid hormone aldosterone is associated with arrhythmias in cardiovascular disease. However, the effect of aldosterone on the slowly activated delayed rectifier potassium current (IK s ) remains poorly understood. The present study was designed to investigate the modulation of IK s by aldosterone. Adult guinea pigs were treated with aldosterone for 28 days via osmotic pumps. Standard glass microelectrode recordings and whole-cell patch-clamp techniques were used to record action potentials in papillary muscles and IK s in ventricular cardiomyocytes. The aldosterone-treated animals exhibited a prolongation of the QT interval and action potential duration with a higher incidence of early afterdepolarizations. Patch-clamp recordings showed a significant down-regulation of IK s density in the ventricular myocytes of these treated animals. These aldosterone-induced electrophysiological changes were fully prevented by a combined treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist. In addition, in in vitro cultured ventricular cardiomyocytes, treatment with aldosterone (sustained exposure for 24 h) decreased the IK s density in a concentration-dependent manner. Furthermore, a significant corresponding reduction in the mRNA/protein expression of IKs channel pore and auxiliary subunits, KCNQ1 and KCNE1 was detected in ventricular tissue from the aldosterone-treated animals. Aldosterone down-regulates IK s by inhibiting the expression of KCNQ1 and KCNE1, thus delaying the ventricular repolarization. These results provide new insights into the mechanism underlying K(+) channel remodelling in heart disease and may explain the highly beneficial effects of MR antagonists in HF.

Cardiomyopathy-Causing Mutations in Thin Filament Regulatory Proteins Acutely Affeect Ca2+-Buffering and Ca2+-Dependent Signalling In Situ

Hypertrophic cardiomyopathy (HCM) is caused by mutations in thin filament regulatory proteins that confer distinct primary alterations of cardiac contractility. We believe that altered Ca2+-buffering by mutant thin filaments leads to altered Ca2+ handling and, via Ca2+-dependent signalling pathways, contributes to disease pathogenesis. We are studying the in situ effect on Ca2+-flux of a HCM causing mutations in human cardiac troponin T (R92Q), troponin I (R145G) and α-tropomyosin (D175N) by adenoviral expression in adult guinea pig cardiomyocytes. Western blot and immunolocalisation analysis showed that recombinant protein was found at the I band and comprised ∼50% of the total protein 48 hours after infection. Simultaneous measurement of unloaded sarcomere-shortening and Ca2+-transients, showed the HCM mutations had a significant decrease in the basal sarcomere length coupled with an increase in the diastolic Ca2+ concentration. Caffeine challenging indicated that the SR load was reduced and the sodium calcium exchanger (NCX) dependant tau decay time was increased in the HCM mutant cells. Ca2+-dependent signalling cascades were assessed by observing, the nuclear translocation of the transcription factor NFAT by immunofluorescence. Chronically paced HCM mutant cells had a ∼2.5 fold increase in nuclear NFAT compared to unpaced or wild type infected cells. A potential mechanism for this was ryanodine receptor leak which was increased ∼7 fold in cTnI R145G mutant cells. The same mutation reduced the ability of the SR to respond to PKA dependant contractile sensitization, upon Isoproterinol treatment. These data strongly implicate Ca2+-buffering as a driving mechanism of the macroscopic remodelling observed in the pathological disease state of HCM. Ca2+ Buffering is now being directly measured by simultaneously integrating the NCX current of patch clamped mutant cardiomyocytes, as the cells are challenged with caffeine.

Abstract 19413: Regulation of I Ks Current Amplitude by Differential Trafficking Patterns and Interactions Of KCNQ1 and KCNE Subunits

KCNQ1 (Q1, pore-forming subunit) and KCNE1 (E1, modulatory subunit) associate to form the slow delayed rectifier (I Ks ) channel that functions as ‘repolarization reserve’ in human heart. We have shown that in adult guinea pig (GP) cardiomyocytes Q1 & E1 cluster to different subcellular compartments, and the degree of Q1/E1 colocalization affects the I Ks amplitude. We have also shown that KCNE2 (E2) is expressed in human heart, and can associate with Q1 or Q1/E1 to suppress the I Ks amplitude. Objectives: To study the patterns of trafficking and interactions of Q1, E1 and E2. Methods: Q1, E1 and E2 are labeled with eGFP (Q1-G) or mono-dsR (Ex-R, x = 1 or 2) and expressed in COS-7 cells, neonatal rat (NRVM) and adult GP (AGPV) ventricular myocytes; the latter 2 by adenoviral (Adv) gene transfer. Protein trafficking/interactions are monitored by live cell imaging, in conjunction with patch clamping, immunocytochemistry & immunoblotting. Results: Time courses of Q1-G & Ex-R distribution in COS-7 cells suggest that they are translated, packaged and shipped to the cell surface independent of each other. They meet on the cell surface and form Q1-G/Ex-R complexes. Q1-G/Ex-R complexes are endocytosed and colocalized in a cytosolic tubulo-vesicular system. We identify 2 main effects of Ex-R on Q1-G trafficking: (1) Ex-R accelerates Q1-G recovery after photobleaching, (2) 24-48 hr after transfection, E2-R brings Q1-G with it to lysosomes for increased degradation. The distribution patterns of Q1-G and Ex-R in NRVM are similar to those in COS-7 cells. However, under the same Adv incubation conditions, Q1-G and Ex-R are expressed at a much lower level in AGPV than NRVM. In AGPV, Q1-G is distributed mainly as cytosolic striations while Ex-R mainly on cell surface, similar to the native Q1 and Ex immunofluorescence patterns seen in control myocytes. Conclusions: Assembly of Q1 & E1 into I Ks does not occur early during biogenesis. Q1 & E1 meet after they reach the plasma membrane and travel together thereafter. E1 increases the mobility of Q1, while E2 increases Q1 degradation in lysosomes. In adult cardiomyocytes the expression levels and subcellular distribution patterns of Q1 & Ex are tightly regulated, with the sarcomere structure playing a key role in Q1 localization.

Investigating the Effect of Cardiomyopathy-Causing Mutations in Cardiac Troponin-T on Calcium Buffering In Situ

In vitro investigation of the effects of cardiomyopathy-causing mutations in thin filament regulatory proteins has demonstrated that hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by distinct primary alterations of cardiac contractility and myofilament calcium affinity. We hypothesise that chronically altered calcium-buffering by mutant thin filaments leads to altered calcium handling and, via calcium-dependent signalling pathways, contributes to disease pathogenesis. We aim to study the in situ effect on calcium flux of a HCM and a DCM causing mutation in human cardiac troponin T (cTnT) (R92Q, R131W respectively), by adenoviral mediated expression of mutant protein in adult guinea pig cardiomyocytes. The adenoviral vectors co-express GFP and western blot analysis of FACS-sorted, GFP-expressing cells showed that recombinant cTnT comprised 45-50% of the total cTnT in these cardiomyocytes, 48 hours after infection. Analysis of unloaded sarcomere shortening showed that at an excitation frequency of 2 Hertz, cardiomyocytes infected with R131W cTnT elongated the time to 50% relaxation and reduced the magnitude of contraction, whilst R92Q cTnT reduced the time to 50% relaxation and increased the contractile magnitude compared to wild type. Analysis of calcium transients of the same cells using fura-2 loading, indicates that the R92Q mutation reduces calcium transient amplitude, whilst the R131W mutation increases the time to complete calcium reuptake, with no change to the transient amplitude, despite the observed decrease in contraction. We are currently assessing the caffeine transients of these cells to investigate alterations to overall SR load and measuring alterations to components of calcium-dependent signalling cascades which may link the acute effects of cTnT mutations to macroscopic remodelling observed in the pathological disease states of HCM and DCM.

Demonstration of functional dipeptide transport with expression of PEPT2 in guinea pig cardiomyocytes

The transporters PEPT1 and PEPT2 accept a broad spectrum of substrates including small, naturally occurring peptides and peptidomimetic drugs. This study aimed to investigate for the first time whether these transporters are expressed and active in isolated cardiomyocytes. PEPT1/PEPT2 expression in rat kidney (positive control), guinea pig kidney and cardiomyocytes were investigated by reverse transcription polymerase chain reaction. L-Glycyl-L-[(14)C]sarcosine (Gly-sar) uptake was characterised using freshly isolated suspensions of adult male guinea pig cardiomyocytes. PEPT2-specific primers recognised mRNA of appropriate size and sequence in cardiomyocytes and kidney, whilst PEPT1 was expressed in the kidney only. The initial uptake (30 s) of 200 microM Gly-sar was dependent on extracellular pH with a maximum at pH 6.0 (237.8 +/- 12.2 pmol/microl) and a minimum at pH 8.0 (72.1 +/- 13.4 pmol/microl, n = 6 +/- SE, p < 0.01, T test). The K (m) and V (max) of Gly-sar uptake at pH 6.0 were 495.5 +/- 69.6 microM and 1470.5 +/- 69.6 pmol microl(-1) min(-1). The addition of 10 mM fosinopril, cefadroxil, carnosine, cyclacillin or a variety of L-amino acid containing dipeptides/tripeptides significantly reduced Gly-sar uptake. Gly-sar uptake was not affected by 10 mM D-ala-D-ala, glycine or sarcosine. These results support the presence of a functional dipeptide transporter in isolated cardiomyocytes, with accompanying expression of PEPT2.

Effects of sustained beta-adrenergic stimulation on ionic currents of cultured adult guinea pig cardiomyocytes.

Short-term stimulation of beta-receptors is known to affect cardiac ion channels; however, the impact of longer-term stimulation on intrinsic channel function is poorly understood. To evaluate this, cultured guinea pig ventricular myocytes were exposed to isoproterenol (10 nM), vehicle, or isoproterenol plus propranolol (1 microM) for 48 h. Sustained exposure to isoproterenol decreased the density of the inward rectifier (I(K1)), slow delayed rectifier (I(Ks)), and L-type Ca2+ (I(Ca L)) currents, effects that were fully prevented by propranolol. Changes in K+ currents were prevented by the beta1-selective antagonist CGP-20712A, unaffected by the beta2-antagonist ICI-118,551, and mimicked by the membrane-permeable cAMP analog 8-bromo-cAMP. Isoproterenol did not alter the current-voltage relationship of the K+ currents but increased the density of T-type Ca2+ current (I(Ca T)) and thereby increased the proportion of the total Ca2+ current at more negative potentials. We conclude that sustained exposure to isoproterenol reduces I(K1), I(Ks), and I(Ca L) density and increases the density of I(Ca T). The direct ionic current remodeling effects of sustained beta-adrenoceptor stimulation resemble changes reported with heart failure and may be important in arrhythmogenic ionic remodeling.

Protein Kinase Cα Mediates the Effect of Antiarrhythmic Peptide on Gap Junction Conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH2, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (Gj) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced Gj (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCgamma and -alpha) and CGP 54345 (PKCalpha). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP10. For further analysis, we incubated HeLa-Cx43 cells with [32P]orthophosphate (0.05 mCi/ml) for 4 h at 37 degrees C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of 32P into Cx43 was significantly enhanced in the presence of AAP10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [14C]-AAP10 as radioligand was performed. We found a saturable binding of [14C]-AAP10 with a KD of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP10. In conclusion; AAP10 increases Gj in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP10 leads to enhanced phosphorylation of Cx43 via activation of PKCalpha. A membrane receptor exists for antiarrhythmic peptides.

Maintenance of Ca i2+transients during prolonged cardiac arrest aids rapid contractile recovery

We explored the effects of contractile arrest maintained for 24–72 h in the presence of 2,3-butanedione monoxime or a Ca 2+ channel blocker (nifedipine or verapamil) on contractile activity, Ca i 2+ transients, and myofibrillar protein content and ultrastructure in long-term cultures of spontaneously beating adult guinea-pig cardiomyocytes. The contractions were not affected by 5 mM 2,3-butanedione monoxime, but they were strongly or fully suppressed by 10 and 18 mM 2,3-butanedione monoxime, respectively, while the Ca i 2+transients triggered by the maintained spontaneous electrical activity were either not changed at all (5 and 10 mM 2,3-butanedione monoxime) or decreased only slightly (18 mM 2,3-butanedione monoxime). The uncoupling of excitation from contraction by 10–18 mM for 24–72 h did not affect the content of the myofibrillar proteins. Confocal laser microscopy showed that these exposures affected the assembly of myofilaments, giving an overall deranged appearance to the myofibrils. In spite of this effect, the cells' contractile activity was readily regained within 15–60 min upon the washout of 2,3-butanedione monoxime. The 24–72-h exposures to 5 μM nifedipine or 10 μM verapamil, which blocked fully both the Ca i 2+ transients and contractility, did not affect the myofibrillar protein content nor their assembly. However, the recovery of contractile activity after exposure to a Ca 2+-channel blocker was significantly slower (several days) than after 2,3-butanedione monoxime exposure. Furthermore, cultures exposed to Ca 2+-channel blockers also had significantly decreased sensitivity to β-adrenergic stimulation. Altogether, these data indicate the importance of regular Ca 2+ influx for the maintenance of the functional integrity of adult cardiomyocytes during prolonged periods of contractile arrest.

Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes.

The present study was designed to investigate how prolonged (24-72 h) exposure to modifiers of Ca transarcolemmal transport affects the myofibrillar structure, protein turnover and content of myofibrillar proteins in adult guinea pig cardiomyocytes maintained beating synchronously in long-term cultures. First we established the functional responses (the contractile activity and [Ca]i transients) of the cultured myocytes to acute exposures to several drugs used in this study. The ultrastructural characteristics of these cultures under the various treatments were determined using immunohistochemistry and confocal scanning laser microscopy, and their biochemical properties were evaluated using analysis of total cellular protein content, myofibrillar protein content and SDS-PAGE electrophoretic examination. We compared the effects of 24, 36 and 72 h-long exposures to the various specific Ca-flux modifiers. Increased Ca influx via CaL-channel agonist (Bay K 8644) or via the reversed-mode of the Na/Ca exchanger (veratrine) did not alter the myofibrillar structure or the specific protein profiles or proteosynthesis. However, when cytosolic Ca was increased by three different types of inhibitors of Ca extrusion from the cells via Na/Ca exchange, (Na-free solution, 5 mM NiCl2 and 10(-6) M ouabain), very significant changes in all investigated parameters occurred almost immediately. Twenty-four h-long exposure to Na-free did not affect significantly the total cellular protein (TCP), but the protein synthesis was decreased by 87% and the total myofibrillar protein (TMP) content was decreased by 38%. The myofibrils were heavily fragmented. Similarly, 24 h-long exposure to 5 mM NiCl2 did not affect the TCP, but it reduced protein synthesis by about 90% and decreased the total myofibrillar protein content by 30%. These effects were even more pronounced at 72 h of exposure and they were accompanied with a complete disassembly of myofilaments. Exposure to 10(-6) M ouabain over 72 h resulted in > 80% inhibition of protein synthesis, a 45% decrease in TCP content and a 53% in TMP content. In contrast, 10(-7) M ouabain did not produce any such changes. The changes produced by the Na/Ca-exchange inhibitors were accompanied by only minor changes in DNA content, indicating that the myocytes remained viable. Moreover, these effects were not due to the associated contractile arrest, since exposure to CaL-channel antagonists (5-20 microM nifedipine or 10 microM verapamil) produced only very minor changes in the myofibrillar structure and in protein profiles. Our data demonstrate that short-term (up to 72 h) increased Ca influx or contractile arrest of well-interconnected, spontaneously beating adult cardiomyocytes does not affect their ultrastructural characteristics or their myofibrillar protein turnover greatly, while any situations leading to Ca accumulation (via inhibition of Na/Ca exchange) affect cardiomyocyte function and ultrastructure almost immediately. These data are in sharp contrast to those previously reported from immature, neonatal myocytes.

Adenylyl cyclase in the heart: an enzymocytochemical and immunocytochemical approach.

This review provides a discussion of the localization of adenylyl cyclase (AC) in normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes. This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin. With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes. Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction. Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes demonstrated by enzymocytochemistry.

Differences in the Structural Characteristics of Adult Guinea Pig and Rat Cardiomyocytes during Their Adaptation and Maintenance in Long-Term Cultures: Confocal Microscopy Study

In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term (1–5 week) cultures of adult guinea pig cardiomyocytes and adult rat cardiomyocytes. Providing that the preparation of freshly isolated guinea pig cardiomyocytes consists mostly (>80%) of rod-shaped, Ca 2+-tolerant, and quiescent cells and these are plated under optimal conditions and density (10 5cells/cm 2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for 1 week; (2) within 10–14 days, they reestablish intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layer; (3) their myofibrillar striations remain regular all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) they form adherence junctions (as indicated by their immunoreactivity to an antibody against N-cadherin) over the entire cellular surface; (5) they appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate. In contrast, rat cardiomyocytes cultured under very similar and optimal conditions exhibit very different characteristics during their adaptation in long-term cultures: (1) although 85–90% of freshly isolated cells are also rod-shaped and Ca 2+tolerant they exhibit slow spontaneous contractions; (2) they round up during the first few days, and during the first week they dedifferentiate, losing their regular striated appearance; (3) they spread, becoming irregularly shaped, and, unlike the guinea pig cardiomyocytes, they do not form confluent layers, no matter what the plating density is; (4) they atrophy at a very early stage in the cultures, so that by the fourth week, they have lost most of their myofibrils; (5) the initial rounding up is largely eliminated by exposure for 24 h to 1 μM ryanodine or 20 mM butanedione monoxime, compounds that suppress the spontaneous contractions. In conclusion, our studies demonstrate that adult guinea pig cardiomyocytes adapt and survive in long-term (1–5 week) cultures much better than do adult rat cardiomyocytes, indicating that the long-term cultures of adult guinea pig ventricular myocytes provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under the acute or chronic influence of various intrinsic and/or extrinsic factors.

ANG II modifies cardiomyocyte function via extracardiac and intracardiac neurons: in situ and in vitro studies.

To determine whether angiotensin II (ANG II) affects cardiac performance via neurons in intrathoracic cardiac ganglia, studies were performed on anesthetized dogs. To exclude possible vascular regulatory effects of ANG II, experiments were also performed using long-term cultures of adult guinea pig ventricular cardiomyocytes with or without intrathoracic neurons. 1) In in situ experiments in 10 anesthetized dogs, cardiac augmentation occurred when ANG II (10 microl or 0.1 ml; 10-100 microM) was administered into limited loci within acutely decentralized stellate or middle cervical ganglia that were neurally connected to, but not those disconnected from, the heart. In another 18 dogs, ANG II increased intrinsic cardiac neuronal activity when administered adjacent to such neurons or into their local arterial blood supply. Ventricular ionotropic effects elicited by ANG II were eliminated by timolol, whereas increases in intrinsic cardiac neuronal activity were not affected. Effects elicited by ANG II were eliminated by administration of a selective AT1 receptor antagonist (losartan) but not by a selective AT2 receptor antagonist (PD-123319). 2) In in vitro experiments, ANG II (100 nM) induced positive chronotropic effects on cultured adult guinea pig cardiomyocytes innervated with adult extrinsic or intrinsic cardiac neurons, but not those cultured without neurons. The frequency of calcium inward current (Ca(i)) transients (recorded by fura 2 fluorescence) increased in innervated cocultures but not in the noninnervated cardiomyocyte cultures; however, the amplitude of Ca(i) transients was not affected by ANG II in cultures or in freshly isolated adult guinea pig cardiomyocytes. ANG II-induced effects in cocultures were blocked by losartan but not PD-123319 or timolol. Thus 1) ANG II-sensitive neurons exist in intrathoracic extracardiac and intrinsic cardiac ganglia; 2) these neurons possess AT1 receptors; and 3) these neurons appear to act directly and indirectly via adrenergic neurons to enhance cardiomyocyte function.

Anti-peptide antibodies sensitive to the ‘active’ state of the β2-adrenergic receptor

Antibodies directed against a peptide corresponding to the second loop of the human β 2-adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freund's adjuvant. The resulting antibodies were affinity-purified and shown to be monospecific for the target receptor. They were able to stimulate the L-type Ca 2+ channels in whole-cell patch-clamp experiments on isolated adult guineapig cardiomyocytes. This effect was similar to that obtained by the specific β 2-adrenergic agonist zinterol. The antibody effects could be blocked with the specific β 2-adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the β 2-adrenergic receptor.

Changes in the Expression and Distribution of Connexin 43 in Isolated Cultured Adult Guinea Pig Cardiomyocytes

In the present study, we have investigated the changes in the expression and distribution of the principal gap-junction channel protein in ventricular muscle, connexin 43 (Cx43), during the first 2 weeks of culturing adult guinea pig cardiomyocytes at low density to prevent formation of cellular contacts. In freshly isolated cardiomyocytes, immunoreactive Cx43 occupied 6.5 ± 0.4% of the pixel area of the cell, with 85% being localized to dense particles at the step-like end projections of the myocytes (intercalated disk regions) and 15% being within the sarcoplasm or along the lateral surface of the myocytes (“nondisk” distribution). During the myocytes’ first 48 h in culture, immunoreactive Cx43 decreased by 27.5% from control values, to 4.7 ± 0.5% of the cells’ pixel area (P< 0.01). Cx43 particles also redistributed: after 48 h in culture ∼90% of the immunoreactive Cx43 was localized in the sarcoplasm and nondisk regions of the myocyte. After 7 days, immunoreactive Cx43 only occupied 50% of the cells’ control pixel area (P< 0.01) and was nearly uniform in its punctate pattern throughout the sarcoplasm. This distribution remained the same during the 2nd week in culture. Changes in myosin light chain staining during 8 days in culture largely paralleled those in Cx43 staining. Laser confocal microscopic analysis of double-immunolabeled myocytes that had been in culture for 24–48 h showed colocalization of Cx43 with clathrin in ∼30% of the sarcoplasmic Cx43 particles. Thus it is demonstrated that the expression of Cx43 decreases significantly during the first 48 h in culture after myocyte isolation and that Cx43 also undergoes substantial redistribution but for the next 2 weeks remains more or less unchanged and at relatively high levels (∼50%). These data indicate that cardiomyocytes in isolation maintain their ability to reconnect with each other for up to at least 2 weeks. This is the first time that this property has been investigated in cultured adult ventricular cardiomyocytes.

Modulation of hypertensive heart disease by estrogen

Left ventricular hypertrophy is an independent risk factor for morbidity and mortality in patients with hypertensive heart disease. Cardiac hypertrophy, associated with increased cardiac fibrosis and myocardial relaxation impairment, shows gender-based differences with significantly higher mortality in men. The role of estrogen in the pathogenesis of this process is poorly understood. After our previous demonstration that cardiac myocytes and fibroblasts contain functional estrogen receptors, we therefore investigated: 1) the influence of different estrogen metabolites on cardiac fibroblast growth; 2) the influence of different estrogen metabolites on the expression of the immediate early gene c- Fos; 3) the influence of estrogen on the L-type calcium channel in cardiomyocytes. Methods: 1) Neonatal rat cardiac fibroblasts were incubated with 17β-estradiol, estrone, 2-hydroxyestrone, and 2-methoxyestradiol (all 10 −9 M). Bromodeoxyuridine incorporation was measured after 24 h. 2) c- Fos expression was demonstrated by immunoblotting. 3) L-type (Ca 2+) current with and without 17β-estradiol was assessed in adult guinea pig cardiomyocytes by whole cell patch clamp. Results: Cardiac fibroblast growth was stimulated by estrogen metabolites with 2-hydroxyestrone as the most potent activator; in addition, 10 −5 M 17β-estradiol reduced the L-type Ca 2+ current by about 20% in cardiomyocytes. Conclusions: Estrogen induces both short term effects (non-genomic) and long term effects (genomic) on the heart and may therefore account for gender- and age-based differences in hypertensive heart disease.

Purinergic modulation of adult guinea pig cardiomyocytes in long term cultures and co-cultures with extracardiac or intrinsic cardiac neurones.

To determine the capacity of ATP to modify cardiomyocytes directly or indirectly via peripheral autonomic neurones, the effects of various purinergic agents were studied on long term cultures of adult guinea pig ventricular myocytes and their co-cultures with extracardiac (stellate ganglion) or intrinsic cardiac neurones. Ventricular myocytes and cardiac neurones were enzymatically dissociated and plated together or alone (myocytes only). Myocyte cultures were used for experiments after three to six weeks. The electrical and contractile properties of cultured myocytes and myocyte-neuronal networks were investigated. The spontaneous beating frequency of ventricular myocytes co-cultured with stellate ganglion neurones increased by approximately 140% (p < 0.001) following superfusion with 10(-5) M ATP. This effect was not modified significantly by tetrodotoxin or by beta adrenoceptor blockade (10(-5) M timolol), but was eliminated following application of the P2 antagonist suramin (10(-5) M). Basal spontaneous contractile rate was reduced by approximately 86% (p < 0.001) in the presence of suramin, indicating the existence of tonically active purinergic synaptic mechanisms in stellate ganglion neurone-myocyte cocultures. Suramin did not significantly affect non-innervated myocyte cultures. ATP increased myocyte contractile rate in intrinsic cardiac neurone-myocyte co-cultures by approximately 40% (p < 0.01) under control conditions, but when beta adrenergic receptors of tetrodotoxin sensitive neural responses were blocked, ATP induced greater augmentation (> 100%). In contrast, ATP induced much smaller effects in non-innervated myocyte cultures (approximately 26%, p < 0.01). Analogues of AT) showed the following order of potency: ATP > UTP > MSATP > beta gamma ATP > alpha beta ATP. Adenosine (10(-4) M) attenuated the beating frequency of myocytes in both types of co-culture, while not significantly affecting non-innervated myocyte cultures. The experimental model used in this study showed that extrinsic and intrinsic cardiac neurones which possess P2 receptors can greatly enhance cardiac myocyte contractile rate when activated by ATP. Since adenosine reduced contractile rate in both types of co-cultures while not affecting non-innervated myocytes, it is concluded that some of these neurones possess P1 receptors.

A cardiodepressant factor isolated from blood blocks Ca2+ current in cardiomyocytes

A cardiodepressant factor (CDF) was isolated (salt free) from the plasma of dogs after hypovolemic-traumatic shock by column chromatography. CDF was found to exert a concentration-dependent negative inotropic effect in guinea pig papillary muscle; it reduced the amplitude of cell wall motion, the contraction and relaxation velocity, and the frequency of spontaneously beating rat cardiomyocytes in culture, effects that were rapidly reversible upon washout. By means of electrophysiological methods (whole cell recording with patch electrodes and voltage clamp) we tested the effect of CDF on adult guinea pig cardiomyocytes: application of CDF initially decreased the plateau by 7 mV (150 ms after peak of action potential) and reduced the action potential duration by 93 ms (76% of control action potential duration) at 50% and 88 ms (79%) at 90% of repolarization. The plateau was further reduced by 13 mV and the action potential duration was prolonged by 25 ms (106%) at 50% and was prolonged drastically by 156 ms (137%) at 90% of repolarization compared with control. Voltage-clamp experiments have shown that the most prominent effect of CDF is a strong reduction of ICa accompanied by inhibition of IK and subsequent repolarization. Similar results have been obtained with neonatal rat cardiomyocytes. Blockage of the calcium inward current can explain the negative inotropic and chronotropic effect of CDF in cardiomyocytes.

Stretch-Activated Channels in Heart Cells

Stretch-activated channels have been proposed as the transduction mechanism between load and protein synthesis in cardiac hypertrophy. Under this hypothesis, cardiac deformation is linked to an increased sodium (Na) influx, which, in turn, increases protein synthesis. We have tested whether stretch actually increases Na influx by applying patch-clamp techniques to cultured chick embryo cardiac myocytes and to freshly isolated adult guinea pig cardiomyocytes. Our experiments, in excised and cell-attached patches, revealed the existence of ionic channels that opened, or increased their frequency of opening, upon the application of negative pressures to the lumen of the patch-clamp pipettes. These stretch-sensitive channels allowed the passage of the major monovalent physiological cations, Na and potassium (K), and, to a much lesser extent, the major divalent cations calcium (Ca) and magnesium (Mg). Under normal conditions, the channels had a high open channel noise that prevented the customary, straightforward statistical analysis of single channel data. However, when one of the major monovalent cations was iso-osmotically replaced by sucrose, the open channel noise decreased significantly and permitted a good delineation of the open and closed channel states and, therefore, application of standard patch-clamp, statistical analysis techniques. Under these "sucrose," "monoionic" conditions, the reversal potential was, as one should expect, close to the equilibrium potential for the major monovalent cation present. When high extracellular K solution was used to minimize the cell resting potential, the reversal potential for these stretch-activated currents was estimated to be around -40 mV. Therefore, under normal conditions, stretch should induce an inward, depolarizing current, carried mostly by Na ions.(ABSTRACT TRUNCATED AT 250 WORDS)