Genetic manipulation of key proteins involved in the process is impossible in rabbit f-SANC. So, we have developed a method to stable culture adult rabbit SANC (c-SANC), have characterized their properties, and have successfully overexpressed proteins in c-SANC via adenovirus directed acute gene-transfer technique. ? On the first day of primary SANC culture, most of the cells tend to spread out and could stay alive for up to 8 days, a reasonable period which would allow us to introduce exogenous proteins into c-SANC. The cultured SANC can beat spontaneously at 34 0.5 oC, and the action potential (AP) firing rate stabilizes at a level of 1.35 0.02 Hz (n=803, 2 to 8 days into culture), which is significantly slower than f-SANC (2.79 0.04 Hz, n=203, p<0.001). c-SANC generate regular AP, global Ca2+ release transients and local Ca2+ releases just prior to the Ca2+ transients triggered by spontaneous AP, and the major characteristics of them are similar to f-SANC. By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a and Sodium-Calcium exchanger.? It is well documented that the peptide PKA inhibitor, PKI can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the relatively low beating rate of c-SANC compared to f-SANC, is possibly due to the down-regulated protein kinase A (PKA) signaling in the cultured cells. Acute stimulation of -adrenergic receptors with 1M isoproterenol accelerates the AP firing rate to a similar maximum in c-SANC (3.34 0.05 Hz, n=150) and in f-SANC (3.55 0.06 Hz, n=126). In additionally, we observed that the phosphorylation level of RyR2, which is indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by the cells total RyR2 fluorescence density, is substantially lower in c-SANC (1.45 0.10, n=43) than that in f-SANC (3.57 0.13, n=56, p<0.001). Preliminary data of the immuno-staining of phosphorylated PLB at Ser16 suggests that this PKA-specific phosphorylation level is also lower in cultured vs. freshly isolated SANC.? Inducing the green fluorescent protein (GFP) into c-SANC via adenovirus directed acute gene-transfer technique, does not affect the cell beating rate, and there is no correlation between AP firing rate and GFP expression level. We also successfully overexpressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIC (a multifunctional Ca2+ dependent kinase), Ht31 (a peptide that binds the PKA regulatory subunit type II (RII) and competes with endogenous A-kinase anchoring protein (AKAP) for RII binding, thus interrupts AKAP mediated PKA anchoring) and its inactive form Ht31p.? We conclude that adult c-SANC provide a reliable model to study the autonomic regulation by acute genetic manipulation of key proteins.
