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Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage. [ABSTRACT FROM AUTHOR]

Background and purpose: Uncoupling of cardiac gap junction channels is an important arrhythmogenic mechanism in ischemia/reperfusion. Antiarrhythmic peptide AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH) has been shown to prevent acidosis-induced uncoupling and ischemia-related increase in dispersion. Previous structure-effect investigations and subsequent computer modeling studies indicated that the tricyclic antidepressant desipramine may exert similar effects as AAP10. Methods: We assessed the binding of C-AAP10 to membranes of rabbit cardiac ventricles and its displacement with desipramine in a classical radioligand binding and competition study. Gap junction currents were measured between isolated pairs of human atrial cardiomyocytes under normal and acidotic (pH 6.3) conditions with or without 1 μmol/l desipramine using dual whole-cell voltage clamp. The effect of 1 μmol/l desipramine was assessed in isolated rabbit hearts (Langendorff technique) undergoing local ischemia by coronary occlusion with 256-channel electrophysiological mapping and subsequent analysis of connexin43 (Cx43) expression, phosphorylation (Western blot), and subcellular localization (immunohistology). Results: We found saturable C-AAP10 binding to cardiac membranes ( K, 0.29 ± 0.11 nmol/l; B, 42.5 ± 7.2 pmol/mg) which could be displaced by desipramine with a K = 0.14 μmol/l and a K = 22 μmol/l. Acidosis reduced the gap junction conductance in human cardiomyocyte pairs from 24.1 ± 4.7 to 11.5 ± 2.5 nS, which could be significantly reversed by desipramine (26.6 ± 4.8 nS). In isolated hearts, ischemia resulted in significantly increased dispersion of activation-recovery intervals, loss of membrane Cx43, and dephosphorylation of Cx43, which all could be prevented by desipramine. Conclusion: Desipramine seems to prevent the uncoupling of cardiac gap junctions and ischemia-related increase in dispersion. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF) and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1, R1 =OH, R2 =NH2), a compound currently under clinical evaluation. Selected compounds with the desired in-vitro profile demonstrated positive in vivo results in the mouse CaCl2 arrhythmia model upon oral administration. [Copyright &y& Elsevier]

Previously, it was shown that antiarrhythmic peptides and our lead substance AAP10 enhance electrical intercellular communication via gap junctions. Now, we wanted to elucidate whether AAP10 acts preferably in the ischemic area and the molecular mechanism of this peptide. Seventeen rabbit hearts were isolated, perfused according to Langendorff, and submitted to 30-min local ischemia by LAD occlusion with/without AAP10 (50 nM). Electrophysiology was assessed by 256 channel epicardial mapping. Finally, the ischemic zone, border zone, and non-ischemic zone were excised, and the cardiac gap junction protein connexin43 (Cx43), its phosphorylation state, and the distribution at the polar and lateral membrane of cardiomyocytes were determined by Western blot and immunofluorescence. Ischemia led to a decrease in activation recovery interval (ARI) homogeneity, which could be completely prevented by AAP10. Moreover, ischemia-induced activation wave slowing in the ischemic border zone was antagonized by AAP10. In ischemic center and border zone, but not in the non-ischemic area, (phospho-Cx43/dephospho-Cx43)-ratio decreased. This was also significantly antagonized by AAP10. Serine 368 was identified as one phosphorylation site for the activity of AAP10. In the non-ischemic area, AAP10 had no influence on Cx43 phosphorylation state. Interestingly, ischemia led to a loss of Cx43 from the cell poles and lateral sides in the ischemic area and border zone. AAP10 completely prevented the ischemia-induced decrease in polar Cx43 presence. In the ischemic area, AAP10 prevents from ischemia-induced Cx43 dephosphorylation and loss of Cx43 from the gap junction at cell poles and in parallel prevents the decrease in ARI homogeneity and attenuates ischemia-induced slowing of activation wave propagation. The AAP10 action seems confined to the ischemic area. [ABSTRACT FROM AUTHOR]

In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 ( n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribution of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis revealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercelluar communication by enhancing phosphorylation of Cx43. [ABSTRACT FROM AUTHOR]

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43. [ABSTRACT FROM AUTHOR]

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH 2 ) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo . ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH 2 ). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H 2 O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 ± 6% and 146 ± 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCα inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro , but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides. [ABSTRACT FROM AUTHOR]

To establish a specific and sensitive radioimmunoassay (RIA) for antiarrhythmic peptide (AAP) and study serum AAP levels in normal Sprague-Dawley rats (NR) and spontaneous hypertension rats (SHR), AAP-bovine serum albumin (BSA) conjugate was prepared by ammonium bicarbonate method. The New Zealand rabbits were immunized by administering intradermally the conjugate. Then the rabbit anti-AAP serum was produced and iodinated AAP was made by Bolton-Hunter method. The RIA for serum AAP was set up and serum AAP levels in NR and SHR were determined. The minimal detectable range of the AAP RIA was (0.45±0.06) μg/L. The affinity constant for antiserum was 1.05×109 L/mol, and the rate of cross-reactivity with atrial peptide (AP) and human growth hormone (hGH) were 0.12% and 0.20%, respectively. The mean recovery rate of high, medium and low doses was 97.6%, and the mean coefficients of variation for intra-and interbath-assay were (6.43±0.85)% and (9.62±1.04)%, respectively. The mean levels of AAP in NR with different age (3 months, 8–10 months and 18–20 months) were (1.75±0.13) μg/L, (1.74±0.11) μg/L and (1.79±0.15) μg/L, respectively, while those in SHR with different age (3 months, 8–10 months and 18–20 months) were (2.38±0.35) μg/L, (2.54±0.25) μg/L and (2.83±0.21) μg/L, respectively. The levels of serum AAP showed a positive correlation with blood pressure ( r=0.8667, P<0.05). It was indicated that this AAP RIA had high specificity, high accuracy and good reproducibility. The levels of serum AAP had a close relation with blood pressure. [ABSTRACT FROM AUTHOR]

Gap junction channels are low resistance pathways allowing an action potential to propagate from one cell to the neighboring. Moreover, small molecules (<1000 Da) may pass the channel providing a possibility for metabolic coupling, growth and differentiation control of a cell by its surrounding. Antiarrhythmic peptides can enhance the conductivity of the channels while other peptides, angiotensin or extracellular loop peptides, reduce intercellular communication. On the other hand, peptides like angiotensin II or endothelin-1 can increase expression of certain gap junction channel proteins and, thereby, may affect intercellular coupling chronically. Thus, intercellular communication can be controlled using peptide drugs. [Copyright &y& Elsevier]

Common antiarrhythmic agents affect ionic membrane channels and thereby alter cellular electrical activity. Since this accounts for the proarrhythmic effects as well we tried to find new substances with different profiles of actions. A new antiarrhythmic peptide, HN-Gly-Ala-Gly-4Hyp-Pro Tyr-CONH (AAP 10), was synthetized using the Fmoc-strategy. This peptide was analyzed for its electrophysiological profile of action in normal isolated rabbit hearts perfused according to the Langendorff technique either under control conditions or after induction of a regional ischemia. For this purpose 256 channel epicardial mapping was employed allowing the determination of the timepoints of activation at each electrode thus identifying the origins of epicardial activation (socalled breakthrough-points, BTP). Epicardial spread of activation was then described mathematically by activation vectors which gave direction and velocity of the epicardial activation wave at each electrode. Single heart beats were analyzed under control conditions and under treatment with AAP 10 or under regional ischemia with or without AAP 10-pretreatment (10 mol/l). We calculated the percentage of similar vectors (VEC) with unaltered direction (deviation <-5°) and the percentage of identical breakthroughpoints (deviation ≤ 1 mm) compared to control conditions. In addition, apparent epicardial velocities, total activation time of a given region and activation-recovery interval (ARI) as well as dispersion of ARI (i.e. standard deviation of ARI) and distribution of ARI were analyzed. Under control conditions treatment with AAP 10 (10 to 3*10 mol/l) led to a significant decrease in ARI-dispersion without alteration of any of the other parameters under investigation. Left ventricular regional ischemia resulted in a marked alteration of the activation patterns (a significant decrease in vectorfield-and breakthroughpoint-similarity) which could be significantly inhibited by pretreatment with AAP 10. In addition, we found that AAP 10 depressed the increase in ARI-dispersion during the first minutes of ischemia and accelerated normalization of ARI-dispersion during reperfusion. In additional experiments, it could be shown that AAP 10 did not alter action potential duration, maximum dU/dt, amplitude or resting membrane potential of isolated guinea pig muscles using a common intracellular action potential recording technique. From these results it is concluded that (a) AAP 10 inhibits ischemia induced alterations of the activation pattern (b) that it decreases ARI-dispersion (c) that this effect seems not to be due to an action on ionic channels (d) that the effect of AAP 10 may be due to an improvement of cellular coupling and finally (e) that AAP 10 may be an interesting new approach to the problem of prophylaxis of ischemia-associated ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

Disturbances in gap junction distribution and a decrease in the connexin43 content of the heart were shown to occur after myocardial infarction and in ischemic heart disease, respectively. These changes are now thought to play an important role in the genesis of arrhythmias associated with these diseases. It is thought that agents that can increase cellular coupling might be beneficial in these situations. Recently, we presented data showing that the synthetic peptide AAP10 acts antiarrhythmically in a model of regional ischemia. The data suggested that AAP10 might act via an increase in cellular coupling. The goal of this study was to establish whether AAP10 can interact with cardiac gap junctions. Measurements of the stimulus-response-interval (SRI) in guinea pig papillary muscle showed that high concentrations of AAP10 (1 µM) can decrease the SRI by about 10% under normoxic conditions. At lower concentrations (10 nM) AAP10 had no effect on SRI under normoxic conditions but prevented the increase in the SRI induced by perfusion with hypoxic, glucose-free Tyrode’s solution. Double-cell voltage-clamp experiments confirmed that AAP10 can interact with cardiac gap junctions. 10 nM AAP10 could either diminish or reverse the run-down of gap junction conductance normally observed in pairs of guinea pig ventricular myocytes. During control gap junction conductance decreased with a rate of -2.5 ± 2.0 nS/min. After application of 10 nM AAP10 gap junction conductance increased with a rate of +1.0 ± 0.7 nS/min ( p < 0.01). After washout of AAP10 gap junction conductance decreased again with a rate not significantly different from control. Our results show that AAP10 does interact with gap junctions. Because no other effects of AAP10 on other electrophysiological parameters could be found, this action on gap junctions might be the basis of AAP10’s antiarrhythmic effect seen in previous studies [ABSTRACT FROM AUTHOR]