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High-density lipoprotein (HDL) is known for its cardioprotective properties independent from its cholesterol transport activity. These properties are mediated by activation of protein kinases, such as PKC. Connexin43 (Cx43) is a gap junction protein present in ventricular cardiomyocytes. PKC-dependent phosphorylation modifies Cx43 gap junction channel properties and is involved in cardioprotection. We hypothesized that cardioprotective properties of HDL may be mediated in part by affecting Cx43 gap junction channels.Neonatal rat cardiomyocytes were treated with HDL and Cx43 phosphorylation was evaluated by Western blotting and immunofluorescence. We found that HDL promoted phosphorylation of Cx43 with a maximal induction at 5 min, which was inhibited by pre-treatment with various PKC inhibitors. Sphingosine-1-phosphate (S1P), a component of HDL, induced similar effects as HDL. The effect of S1P on Cx43 phosphorylation was mediated by S1P2 and S1P3 receptors. HDL or S1P significantly reduced diffusion of fluorescent dye between cardiomyocytes (∼50%), which could be prevented by PKC inhibition. As observed during optical recordings of transmembrane voltage, HDL and S1P depressed impulse conduction only minimally ( < 5%). Moreover, 5 min of HDL and S1P treatment at the onset of reperfusion significantly reduced infarct size in response to 30 min of ischemia in ex vivo experiments (Control: 23 ± 3 %, HDL: 13 ± 2 % and S1P: 11 ± 2 %, p < 0.05). Moreover, Left Ventricular Developed Pressure was also significantly improved by 5 min treatment with HDL or S1P at the onset of reperfusion (23 ± 6 mmHg, 32 ± 4 mmHg, respectively, vs. controls: 8 ± 2 mmHg, p < 0.05).Short-term treatment with HDL or S1P induces phosphorylation of Cx43 by a PKC-dependent pathway. HDL-induced phosphorylation of Cx43 reduced the diffusion of large tracer molecules between cells, whereas impulse conduction was maintained. Moreover, 5 min treatment with HDL confers cardioprotection against ischemia/reperfusion injury. These results link Cx43 for the first time to the short-term cardioprotective effects of HDL.