The antiarrhythmic and cardioprotective effect of increasing gap junction intercellular communication during ischemia/ reperfusion injury has not been studied.

Summary.  Objective: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI‐039), an orally bioavailable antagonist of PAI‐1, in rat models of thrombosis. Methods and results: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI‐039 was administered orally 90 min before injury (1–30 mg kg−1). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI‐039 administration. PAI‐039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg−1, respectively. Time to occlusive thrombosis was increased from 18.2 ± 4.6 min in controls to 32.5 ± 8.7 (P = ns), 46.1 ± 7.0 (P < 0.05), and 41.6 ± 11.3 min (P < 0.05) in the respective PAI‐039 treatment groups. In the vena cava protocol, PAI‐039 pretreatment significantly reduced thrombus weight at PAI‐039 doses of 3, 10 and 30 mg kg−1. When PAI‐039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI‐039 doses of 3, 10 and 30 mg kg−1. PAI‐039 (10, 30 and 100 mg kg−1) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI‐039 had no effect on circulating, low‐levels of PAI‐1 activity. In contrast, circulating PAI‐1 activity increased 5‐fold following the induction of vascular injury, which was completely neutralized by PAI‐039. Conclusions: PAI‐039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p Ͻ 0.01). In the canine sterile pericarditis model, conduction time (CT; n ϭ 5), atrial effective refractory period (AERP; n ϭ 3), and AF/AFL duration/inducibility (n ϭ 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 Ϯ 1.0 versus 62.0 Ϯ 1.0 ms; p Ͻ 0.001) and 200 ms (64.4 Ϯ 0.9 versus 61.0 Ϯ 1.3 ms; p Ͻ 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 Ϯ 0.6 versus 1.6 Ϯ 0.8; p Ͻ 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.

Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.

To further investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue physiology, the production and regulation of PAI-1 was determined in primary cultures of human preadipocytes. When expressed as production per cell and cultured under identical conditions, human preadipocytes from both visceral (omental) and sc depots of lean and obese individuals released significant, yet similar, amounts of PAI-1 protein into the conditioned medium. High steady-state PAI-1 messenger RNA (mRNA) concentrations were observed in visceral and sc preadipocytes, with the relative level of expression equivalent to beta-actin mRNA. Tumor necrosis factor alpha significantly decreased PAI-1 production in a concentration-dependent manner in both visceral and sc cultures, whereas transforming growth factor beta significantly elevated PAI-1 production, but only in sc preadipocytes from obese individuals. Addition of insulin had no effect on antigen levels in conditioned medium of preadipocyte cultures. Stimulation of the preadipocyte cultures with a defined medium resulted in differentiation to the adipocyte phenotype, as determined by flow cytometric analysis, verifying the cultures as human preadipocyte. These studies are the first to observe significant PAI-1 mRNA expression and protein production in primary cultures of a human adipose tissue cellular component, and they suggest that nascent adipocytes contribute significantly to the elevated plasma PAI-1 observed in obesity.