Deciphering Cellular Signals in Adult Mouse Sinoatrial Node Cells

Abstract

The flexibility of manipulating the mouse genome makes cells from this species an ideal model to study cellular signals that control organ function. Genetically-modified mice provide opportunities to decipher how pacemaking sinoatrial node (SAN) cells are regulated by several second messengers and signaling molecules. However, detailed information on the spatiotemporal dynamics of these signaling events in adult mouse SAN cells has been limited due to the challenge of visualizing them in live cells. Here, a method is described for culturing and immediate infection of adult mouse SAN cells with genetically-encoded FRET-based biosensors to examine cellular dynamics of signaling events. SAN cells maintained in culture media containing blebbistatin or (S)-nitro-blebbistatin retain their elongated morphology and action potential (AP) waveform for up to 40 hours. The culturing condition does not change β-adrenergic-mediated cAMP signal as determined in freshly dissociated and cultured SAN cells from a cardiac-specific cAMP reporter mouse. SAN cells expressing cAMP sensors in different cellular compartments show distinct β-adrenergic-mediated cAMP pools. Examination of cyclic GMP (cGMP), protein kinase A (PKA), Ca2+/CaM kinase II (CaMKII) and protein kinase D (PKD) activity with specific FRET biosensors also show unique responses to different stimuli in SAN cells. Isolation and culture of SAN cells from heart failure mice reveal a decrease in cAMP and cGMP signaling. Thus, a reliable and efficient method for culturing adult mouse SAN cells has been developed to facilitate studies of signaling network dynamics during physiological and pathological conditions.