Title: Elucidating the mechanism of beta-adrenergic regulation of calcium channels in the heart
Description: Physiologic β-adrenergic activation of PKA during the sympathetic “fight-or-flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. The second chapter of this dissertation describes our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Recent data confirm that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites on α1C or β2B subunits. Proximity proteomic labeling methods led us to other potential PKA targets near the CaV1.2 complex, including Rad, a calcium channel inhibitor that changes its position within the calcium channel neighborhood after β-adrenergic stimulation. With expression of α1C, β2B, and Rad in a heterologous expression system, we reconstituted forskolin-PKA regulation of CaV1.2.

By mutating potential PKA phosphorylation sites on Rad, we identified specific residues that are critical for this mechanism to occur and validated Rad as the functional PKA target for regulation of CaV1.2. In the third chapter, we probe the contribution of both CaV1.2 α1C and β subunits in β-adrenergic regulation. Previous results have shown that binding between α1C and β subunits is required for adrenergic stimulation of the calcium channel in the heart. Using transgenic mouse models, we demonstrate that this phenomenon requires a rigid IS6-AID linker in the α1C subunit, as introduction of glycines in this region increased flexibility of the linker and abolished a response to adrenergic agonists even though α1C was able to bind to β. The fourth chapter examines the role of the auxiliary β subunit in β-adrenergic regulation of CaV1.2. Binding of Rad to the β subunit is also necessary for this mechanism to occur. Although the β2B isoform is the predominant subunit in the heart, we show that transgenic mice with β3 or β4 replacing β2B in the heart are viable and still have normal β-adrenergic regulation of CaV1.2, indicating that this mechanism is universal to other voltage gated calcium channels that bind to β subunits and RGK proteins.

The fifth chapter verifies that Rad is the PKA target in the heart. Using a knock-in mouse model with four PKA phosphorylation sites mutated to alanine, we definitively show that phosphorylation of Rad is necessary for β-adrenergic regulation of CaV1.2 in the heart. We investigate the importance of Rad phosphorylation on many levels. First, we study Rad’s role in regulating the calcium channel. Second, we observe the effect phosphorylation of Rad has on the calcium transient using isolated cardiomyocytes. Third, we examine cardiovascular function in vivo using radiotelemetry and echocardiograms. Finally, we assess the “fight-or-flight” response in an animal model with exercise capacity testing. Together, these findings conclusively show that in the heart, phosphorylation of Rad is the essential mechanism for the sympathetic nervous system control of calcium influx in both atrial and ventricular cardiomyocytes. Additionally, Rad modulates both heart rate and contractility in vivo. In the sixth chapter, we explore the mechanism of Rad modulation of CaV1.2 in depth using a flow-cytometry Förster resonance energy transfer (FRET) two-hybrid assay.

We closely examine the roles of phosphorylation sites on both Rad’s N-terminus and C-terminus. By creating phosphomimetic mutations on Rad, we uncover the importance of phosphorylating the C-terminus for release of Rad from both the membrane and the β subunit. Taken together, these findings elucidate the mechanism behind β-adrenergic regulation of CaV1.2 in the heart – a longstanding query for over forty years in the cardiovascular ion channel field. At baseline, Rad “tunes” the amount of calcium influx into the cell by inhibiting a population of channels as a functional reserve. Upon adrenergic stimulation, Rad is phosphorylated, lessening its interaction with the membrane, and releasing inhibition of the calcium channel. The enhanced local calcium influx allows for additional calcium release into the cytoplasm through ryanodine receptors leading to increased contractility of the heart, a notable characteristic of the evolutionary survival mechanism— “fight-or-flight.”.