THE TRANSCRIPTION FACTOR NFIX AS A NOVEL MODULATOR OF HEART RATE

NFIX is a transcription factor involved in the development of various organs among which the brain and the skeletal muscle. In the skeletal muscle, NFIX induces the switch from embryonic to fetal myogenesis and it regulates the regeneration process in the adult mouse. Considering that skeletal and cardiac muscle share several signaling pathways beyond the same mesodermal origin, here we investigated for the first time the expression profile and role of NFIX in the heart. We discovered that Nfix gene is barely expressed at early stages of heart development but its expression increases after birth until postnatal day 7. Histological analysis revealed no apparent morphological alterations Nfix-null hearts when compared to WT littermates. In agreement with these data, no differences in the expression of the sarcomeric genes such as cardiac myosins (Myh6, Myh7, Mlc2v), troponin (cTnI) and of the transcription factor Nkx2.5 were found between WT and Nfix-null mice. In the adult heart, Nfix gene is highly expressed in both atria and ventricles but significantly less expressed in the sinoatrial node (SAN) where the heart rhythm originates. Electrocardiograms recordings in freely moving mice show that Nfix-null mice (523.10±13.25 bpm, n=4) present tachycardia compared to WT mice (466.98±8.42 bpm, n=5). Isolated SAN cardiomyocytes from Nfix-null mice display a higher intrinsic heart rate (577.80±52.80 bpm, n=7) than WT cells (403.80±45.00 bpm, n=7) suggesting that in vivo tachycardia is not caused by a dysregulation of autonomic nervous system. Using RNA interference, we silenced Nfix gene in neonatal rat ventricular cardiomyocytes (NRVCs) and confirmed a +62.2% increase in action potential rate in Nfix-silenced (87.60±7.80 bpm, n=20) compared to scrambled NRVCs (54.00±9.00 bpm, n=19). It is well established the role of pacemaker currents (funny current and calcium currents) in setting action potential rate. We did not find differences in funny current between Nfix-silenced versus control NRVCs and in Nfix-null versus WT SAN cells, but we demonstrated an increase of L-type calcium current in Nfix-silenced NRVCs compared with the control. In line with these functional data, we found a higher expression of Cacna1d-mRNA in Nfix-null SAN than in WT SAN according with a possible increase of CaV1.3 L-type calcium channel density. In conclusion, our data suggest that NFIX acts as negative modulator of the heart rate probably affecting L-type calcium current; however, further analysis are necessary to properly clarify the molecular pathway in which NFIX acts.