The Ca2+ Clock is Not Governed by a Single CaMKII or PKA Phosphorylation Site for Fight or Flight Responses

Both CaMKII and PKA contribute to fight or flight heart rate (HR) increases in response to isoproterenol or activity. CaMKII and PKA promote HR increases, at least in part, by actions on ‘Ca2+ clock’ homeostatic proteins. The Ca2+ clock mechanism for cardiac pacing relies on SR proteins governing SR Ca2+ uptake and release. Inhibition of SR Ca2+ release by ryanodine slows HR but we are unaware of any studies testing the potential for slowing SR Ca2+ uptake to reduce HR. Phospholamban (PLN) is a negative regulator of SERCA that acts to slow SR Ca2+ uptake. PLN phosphorylation is catalyzed by PKA, at serine 16, or CaMKII, at threonine 17. PKA and CaMKII can also promote SR Ca2+ release by catalyzing phosphorylation of the ryanodine receptor (RyR2). CaMKII phosphorylation at Ser 2814 and PKA phosphorylation at Ser 2808 can increase RyR2 Ca2+ leak that drives cell membrane depolarizing inward current through the Na+/Ca2+ exchanger. Despite the mounting evidence that CaMKII and PKA sites on PLN and RyR2 are important for cardiac pacing, the relative importance of these sites is unknown and the potential for any particular site to exert a controlling influence over fight or flight physiology is untested. Here we have taken advantage of genetically modified mouse models where CaMKII and PKA sites are specifically ablated to interrogate the role of each site and determine if any of these SR protein sites exercises a decisive influence on HR responses to isoproterenol or activity. The results from genetically modified mice harboring various PLN mutantions (PLN-/-, N27A, S16A, T17A) and RYR2 mutations (S2808A and S2814A) suggest that established CaMKII and PKA sites do not, by themselves, control fight or flight HR responses.