Regulators and interactomes of CaV1.2 in health and disease

My overall goal is to elucidate the mechanisms underlying maladaptive electrical changes to cardiomyocytes in heart failure (HF). During the fight or flight response, beta-adrenergic activation of protein kinase A (PKA) increases cardiac Ca influx and contractility. The mechanism of adrenergic regulation has remained unclear and the subject of controversy for several decades. In an effort to identify the regulator(s) of CaV1.2 that transduce adrenergic stimulation, we developed an in situ CaV1.2 proximity labeling system. Using quantitative proteomics to identify the interactome of CaV1.2 during stimulation with beta-adrenergic agonists, were able to detect the distancing of a channel inhibitor known Rad from the channel. Further study has confirmed that Rad is the target of PKA during adrenergic stimulation that is required for modulation of CaV1.2. In search of pathogenic modulators of CaV1.2 in HF, I have applied this same proximity proteomics technique to mice with HF. My initial findings show significant changes in 71 of nearly 2000 biotin-labeled proteins, including a two-fold increase in gal-1 and enrichment of the 26s proteasome. Gal-1 has been found to disrupt interaction between the core CaV1.2 subunits, which we have shown is critical to adrenergic modulation of the channel. It has also been shown to increase proteasomal degradation of the channel. I hypothesize that rising gal-1 in HF reduces adrenergic sensitivity by eliminating these interactions and increases degradation of the channel. To explore this theory, I will first obtain mice with gal-1 deletion and produce a gal-1 cardiac overexpression mouse and observe the effects on adrenergic modulation in cardiomyocytes. We have also generated a transgenic mouse with a bungarotoxin-binding site (BBS) on an extracellular loop of a YFP-tagged channel expressed exclusively in the heart. Treating isolated myocytes with fluorophore-conjugated bungarotoxin, we can track total, and surface, expression with flow-cytometry. Crossing these mice with gal-1 deletion or overexpression, I can identify the overall effect of gal-1 on CaV1.2 expression in normal physiology. I can also examine if deletion of gal-1 spares mice this loss of cardiac CaV1.2 and potentially the loss of adrenergic reserve in HF. Using markers and inhibitors of the proteasome and other breakdown pathways, I will explore at a deeper level the underlying mechanisms for loss of CaV1.2 in HF.