Abstract 1091: Phosphatidylinositol-Bisphosphate Regulates Intercellular Coupling in Cardiac Myocytes
Introduction: Phosphatidylinositol-(4,5)bisphosphate (PIP2) levels in the plasma membrane have been shown to regulate a number of channels and transporters relevant to cardiac tissue. Stimulation of Gαq receptors activates phospholipase C which cleaves PIP2 into IP3 and diacylglycerol, thereby reducing PIP2 in the plasma membrane. Whether PIP2 regulates gap junctional intercellular coupling (GJIC) in cardiac tissue is presently unknown, but if so it could add to the risk of arrhythmia during stimulation of Gαq coupled receptors such as angiotensin II or noradrenaline.
Hypothesis: We tested the hypothesis that PIP2 regulates GJIC and that stimulation with Gαq coupled agonists reduces GJIC in part due to a reduction of PIP2.
Methods: Cardiac myocytes were prepared from neonate rats and grown in primary culture. GJIC was measured by dye transfer after localized electroporation of Lucifer Yellow. PIP2 synthesis was inhibited by wortmanin (20 μM), which at this concentration inhibits PI- and PIP-kinase. To stimulate α-adrenergic receptors, noradrenaline was given in combination with propranolol (0.5 μM).
Results: Exposure of myocytes to wortmanin for 1 hour resulted in a significant reduction of GJIC (48 ± 9 % of control, n=10) indicating that PIP2 depletion inhibits GJIC. Likewise, GJIC could be inhibited to similar levels by stimulation with angiotensin II (1 μM, 57 ± 8 % of control, n=10) and noradrenaline (0.5 μM, 52 ± 9 % of control, n=9). To test to what extent the reduction in GJIC was caused by PIP2 depletion; myocytes were stimulated by angiotensin II and then allowed to recover for 10 minutes in control medium with or without wortmanin to inhibit resynthesis of PIP2. GJIC returned to normal when cells were allowed to recover in control medium (109 ± 12 % of control, n=8) whereas no recovery was seen in the presence of wortmanin (61 ± 10 % of control, n=8). Incubation with wortmanin alone for 10 minutes did not affect GJIC (89 ± 12 % of control, n=7).
Conclusion: Reductions in PIP2 inhibits GJIC, and stimulation by physiologically relevant agonists such as angiotensin II and noradrenaline can reduce GJIC by this mechanism.