IDDF2024-ABS-0411 The exploration of the mechanism about calmodulin-3 mediated degeneration of nitrergic neurons in achalasia

<h3>Background</h3> The pathogenesis of achalasia may be related to the degeneration of nitriergic neurons in the lower esophageal sphincter (LES) region. Herpesvirus, ryanodine receptor 2 (RYR2), and calmodulin-like protein 3 (CALML3) were hypothesized to be the upstream factors of the degeneration of nitrergic neurons in our previous 4D label-free proteomic study. We aimed to clarify the role of CALML3-related pathways in the pathogenesis of achalasia. <h3>Methods</h3> Patients with confirmed achalasia were included, and normal serum from healthy volunteers and normal LES tissue from patients with esophageal cancer were included as negative controls in this study. The serum level of CALML3 was detected by enzyme-linked immunosorbent assay (ELISA), while the infection rate of herpes simplex virus type 1 (HSV-1), the expression of RYR2, CALML3, and neuronal nitric oxide synthase (nNOS) were detected by immunohistochemistry. Then, the primary human LES myenteric nerve plexus model was constructed. HSV-1 or CALML3 was regulated by recombinant proteins or siRNA, after which the expression levels of CALML3, RYR2 and nNOS were detected. <h3>Results</h3> A total of 65 achalasia patients, 20 normal LES controls, and serum were included. ELISA and immunohistochemical data have confirmed that the positive rate of HSV-1, the expression of RYR2 and CALML3 were significantly increased, while the number of nitrergic neurons was significantly decreased in patients with achalasia (<i>p</i> all &lt; 0.05, IDDF2024-ABS-0411 Figure 1). In the LES myenteric plexus model (IDDF2024-ABS-0411 Figure 2), the mRNA and protein expression of RYR2 and CALML3 were significantly upregulated, and the expression of nNOS was down-regulated after stimulation with HSV-1 (<i>p</i> all &lt; 0.05, IDDF2024-ABS-0411 Figure 3). After stimulation with CALML3 recombinant protein, the expression of nNOS was also significantly decreased (<i>p</i> &lt; 0.05, IDDF2024-ABS-0411 Figure 4). Knockdown of CALML3 restored the reduction of nNOS induced by HSV-1 stimulation (<i>p</i> &lt; 0.05, IDDF2024-ABS-0411 Figure 5). <h3>Conclusions</h3> The infection with HSV-1 may increase CALML3 expression through RYR2. CALML3 could bind to nNOS in LES and promote the degeneration of nitrergic neurons, ultimately leading to achalasia. Inhibition of ectopic expression of CALML3 may be a new therapeutic strategy for achalasia.