Nfix: a transcription factor with an important functional role in cardiac automaticity

Cardiac automaticity is a fascinating physiological phenomenon, a remarkable challenge driving physiologists and researchers along the centuries.

Already Galen of Pergamon, one of the greatest physicians of antiquity who lived in the 2^nd century, starting from his observations that an excised, denervated heart continued spontaneous beating after removal from animal's body, stated that the vital spirit was based on the heart (De usu partium corporis humani). Nowadays, there is a consensus that the spontaneous cardiac activity, in healthy condition, is generated by a specialized population of cardiomyocytes referred to as pacemaker cells grouped in the sino-atrial node (SAN), a highly heterogeneous thin tissue structure located in the right atrium.¹ However, the mechanism underlying pacemaker cells automaticity is still controversial and not totally elucidated. In this issue of ACTA Physiologica, Landi and coworkers² describe an unexpected role of Nfix, a transcriptional factor belonging to the Nuclear Factor 1 (NFI) family, in automaticity of pacemaker cardiomyocytes.

The sino-atrial node (SAN) pacemaker cells have a unique action potential profile, characterized by a slow spontaneous depolarizing phase of membrane potential known as “diastolic depolarization”.³ Diastolic depolarization slope is a crucial factor in automaticity: the steeper the slope the higher the heart rate and vice versa.

All along the action potential diastolic depolarization phase of SAN pacemaker cells, a net inward ionic current is required to maintain membrane potential depolarization. This ionic current is the resultant of a complex functional interplay among membrane ion channels (mainly HCN4, L- and T-type Ca⁺⁺ channels, and Na⁺ channels) and proteins involved in intracellular calcium dynamic (such as RyR2 receptors, SERCA pump, and type 1 sodium calcium exchanger). The ensemble of these intracellular proteins and plasma membrane ion channels underpins automaticity.^3-5

The embryonic origin of SAN pacemaker cells is distinct from that of myocytes of the working heart chambers and those of the His–Purkinje network.⁵ Recent results have provided new insights into molecular regulators required for pacemaker cell differentiation and function.

These include several transcriptional factors such Tbx3, Nkx2.5, Tbx5, Tbx18, Pitx2, Shox2, Isl1, and BMP4 that have been shown to play a regulatory role in SAN development.^6-10

The paper by Landi and collaborators² describes, for the first time, the effect of the inhibition of Nfix transcription factor in heart physiology.

In vertebrates, the NFI proteins is encoded by four closely related genes, named Nfia, Nfib, Nfic, and Nfix.^{11, 12} Their specific expression pattern along embryonic development¹³ and studies involving NFI-deficient animals¹⁴ suggested the involvement of NFI-related genes in the regulation of developmental processes. In particular, Nfix is essential in several organ systems,¹⁵ but its specific expression and functional role in heart is still unresolved. The paper by Landi and coworkers fills this gap. To that, they employ a Nfix-null mouse model (Nfix^−/−). Their data on Nfix cardiac expression show that mRNA coding for Nfix was absent at E10.5, increased linearly to reach a plateau at first postnatal week and remained constant up to adulthood. These results strengthen the hypothesis of a functional role of Nfix on heart function regulation rather on its embryonic development. Neither morphological differences nor alterations in expression of NFI-related genes (Nfia, Nfib, and Nfic) and myocardium-specific genes (cTn1, Myh6, Myh7, and Mlc2v) were detected in hearts from Nfix^−/− when compared to control hearts, suggesting no compensatory effects associated with Nfix inhibition. Landi and coworkers, using in vivo ECG telemetric recordings, unmasked tachycardia in Nfix^−/− mice that was confirmed in isolated SAN cells demonstrating that tachycardia is an intrinsic characteristic of SAN independently from the status of the autonomic nervous system. They show that the higher firing rate recorded in SAN cells from Nfix^−/− animals was due to an increase in L-type Ca⁺⁺ current. These results were further confirmed in primary culture of spontaneously beating neonatal rat ventricular cardiomyocytes. Data from mRNA expression levels experiments performed on genes related to cardiac pacemaker activity allow to speculate that augmented conductance of L-type Ca⁺⁺ current may be due to upregulation of the L-type Ca_v1.3 channel isoform, which is now widely accepted as one of the crucial components participating to the generation and regulation of cardiac diastolic depolarization phase.^16-19

In conclusion, work by Landi et al. comprehensively establishes a crucial role of Nfix in pacemaker tissue suggesting an unappreciated function of this transcription factor in setting heart rate to physiologic levels by acting as a postnatal modulator. Moreover, mRNA expression data reveal different Nfix expression levels in distinct cardiac regions: high expression in atria and ventricles and significant low expression in SAN tissue. This interesting discovery suggests new role for Nfix as a possible transcriptional trigger toward automatic or non-automatic myocytes in cardiac tissue development. This point deserves, certainly, further investigations because it could be a consistent step forward towards the understanding of the complex molecular pathway underlying SAN tissue development.

The authors have nothing to report.

P. Mesirca: Conceptualization; writing – original draft.

No conflict of interest to declare.