Virginia Via Research Day Book 2026

Medical Student Research Biomedical

10 LOSS OF DESMOPLAKIN DISRUPTS ION CHANNEL EXPRESSION IN AN EARLY MOUSE MODEL OF ARRHYTHMOGENIC CARDIOMYOPATHY

Trushaben Patel; Chandra Bain; Steven Poelzing, PhD Corresponding author: tpatel01@vcom.edu

Trushaben Patel, VCOM-Virginia, Blacksburg, Virginia Chandra Bain and Steven Poelzing, VTC Fralin Biomedical Research Instituation

Background: Arrhythmogenic cardiomyopathy (ACM), including arrhythmogenic right ventricular cardiomyopathy (ARVC), is strongly linked to pathogenic variants in desmosomal proteins such as desmoplakin (DP). Beyond structural disruption, evidence suggests that desmosomal dysfunction alters the expression that contributes to electrical instability. One of the proteins that is thought to be altered by changes in DP expression is Kir2.1, which is encoded by KCNJ2. Kir2.1 is an inwardly rectifying potassium channel important for maintaining resting membrane potential. It helps regulate cellular excitability and influences the shape of action potential. Objective: To determine how desmoplakin knockdown (KD) influences transcriptional and translational expression of cardiac ion channel, Kir2.1, in a mouse model of DP-KD. Methods: Using an in vivo desmoplakin knockdown (DP-KD) mouse model, we quantified left ventricular (LV) gene expression of KCNJ2 and GAPDH using quantitative PCR (qPCR), and assessed Kir2.1 protein expression in LV via Western blotting. Real Time PCR: Using previously prepared

Results: Real Time PCR: Expression of KCNJ2 gene was significantly reduced in both male and female DP-KD mice. Western Blotting: Initial Kir2.1 blot showed detectable bands at ~75 kDa for all samples. Gapdh, as a housekeeping protein, looked fairly even across all samples. Significant decrease in Kir2.1 protein was seen in the DP-KD mice compared to the control. There was no sex difference in expression of Kir2.1. Technical replicates of the same western blots, yielding a larger n still showed a decrease in Kir2.1 in DP-KD conditions and revealed a statistically significant sex difference with Kir2.1 expression between control groups. Conclusions: Desmoplakin knockdown significantly reduced Kir2.1 gene and protein expression, linking desmosomal disruption to ion channel remodeling. These findings suggest that impaired Kir2.1 regulation may contribute to the electrical instability characteristic of arrhythmogenic cardiomyopathy, highlighting a mechanistic bridge between structural defects and pro-arrhythmic electrophysiologic changes.

cDNA, each sample was run in triplicate to ensure technical reproducibility and minimize pipetting variation. Reaction mixtures containing gene specific forward and reverse primers for KCNJ2 and GAPDH, molecular-grade water, and SYBR Green master mix were prepared following manufacturer guidelines. Samples were loaded into a 96-well plate and amplified under optimized cycling conditions. Cycle-threshold values were obtained, and relative gene expression was quantified using the 2-ΔΔCt method. Western Blotting: Using previously prepared total protein lysate, samples were normalized to 30 µg of protein per lane and prepared with a loading buffer. Proteins were separated by SDS–PAGE. After electrophoresis, proteins were transferred onto a PVDF membrane. Membranes were rinsed, blocked, and incubated overnight at 4 °C with primary antibodies against Kir2.1 and Gapdh. The following day, membranes were incubated with species-appropriate HRP-conjugated secondary antibodies. Protein bands were visualized using chemiluminescent detection, and densitometric quantification was performed using imaging analysis software.

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2026 Research Recognition Day