DIFFERENTIAL EXPRESSION OF KATP CHANNELS IN NEONATAL CARDIAC FIBROBLASTS AND MYOCYTES FROM 4 CHAMBERS OF THE RAT HEART: A ROLE FOR FORKHEAD TRANSCRIPTION FACTORS?

Philip-Couderc P ., Roatti A., Baertschi AJ.

KATP channels are of considerable pathophysiological interest, since they protect the heart during ischemia, but may cause arrhythmia and atrial fibrillation. These channels are heterotetramers composed of 4 pore-forming potassium inwardly rectifying channel (KIR) subunits 6.x, and 4 regulatory sulfonylurea receptors (SURyz). Different subtypes exist having different channel conductance, metabolic sensitivity, and pharmacological profiles. Current concepts hold that the cardiac myocytes express the KIR6.2-SUR2A subtype, while cardiac fibroblasts are not supposed to express KATP channels. We now report that there were significantly higher levels (2-4-times) of KIR6.1 and SUR2B mRNA in fibroblasts than in cardiomyocytes, with particularly high levels in the atria, especially the right atrium. KIR6.1 protein was detected in the fibroblast fraction, with high levels in the right atrium. KIR6.1 is more expressed in atria than in ventricle, and SUR2B is more expressed in the right than left heart. Cardiac fibroblasts display approximately 10-fold higher expression of KIR6.1 protein than skin fibroblasts. KIR6.1 was localized by immunocytochemistry in fibroblasts and myofibroblasts, as characterized by alphaSMA. While a striated pattern appeared in myocytes, KIR6.1 immunostaining was distributed along stress fibres in fibroblasts. Patch-clamp techniques showed a 40pA current with diazoxide in right atrial fibroblasts, and a significant difference in sensitivity to diazoxide between right and left ventricular myocytes. In order to better understand these differences in KATP gene expression, we performed a bioinformatic analysis (Rvista2.0) of the KIR6.1 (KCNJ8) and 6.2 (KCNJ11) gene promoters. We detected a set of 15 potential transcription factors which could bind to both promoters within a library of 412 sequence families for transcription factor binding sites. A cross-analysis with ANP or BNP permitted to distinguish between an atrial and ventricular pattern of transcription factors for the Kir6.x promoters. Because of the redundancy in binding sites (10 to 70%), the Forkhead family of transcription factors is a strong candidate in KATP gene regulation. Real time PCR identified much higher mRNA levels of FoxA2, F2 and C2 in ventricles than atria. An inverse correlation exists between FoxF2 mRNA level and KIR6.1 mRNA (r=-0.96; n=3), suggesting a repressor role for FoxF2. In conclusion, since fibroblasts bridge gaps and transmit current between myocytes, these results suggest that KATP channels in fibroblasts - and possibly myofibroblasts - should be considered in the generation of atrial conduction and fibrillation. For the first time, a relationship is shown between Forkhead transcription factors, KIR6.1 and K+ current in different chambers of the heart.