Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder of desmosomal dysfunction, and plakophilin-2 (PKP2) has been reported to be the most common disease-causing gene when mutation-positive. In the early “concealed” phase, the ACM heart is at high risk of sudden cardiac death before cardiac remodeling occurs due to mistargeted ion channels and altered Ca2+ handling. However, the results of pathogenic PKP2 variants on myocyte contraction in ACM pathogenesis remain unknown.
We studied the outcomes of a human truncating variant of PKP2 on myocyte contraction using a novel knock-in mouse model with insertion of thymidine in exon 5 of Pkp2, which mimics a familial case of ACM (PKP2-L404fsX5). We used serial echocardiography, electrocardiography, blood pressure measurements, histology, cardiomyocyte contraction, intracellular calcium measurements, and gene and protein expression studies.
Serial echocardiography of Pkp2 heterozygous (Pkp2-Het) mice revealed progressive failure of the right ventricle (RV) in animals older than three months of age. By contrast, left ventricular (LV) function remained normal. Electrocardiograms of six-month-old anesthetized Pkp2-Het mice showed normal baseline heart rates and QRS complexes. Cardiac responses to β-adrenergic agonist isoproterenol (2 mg.kg-1) plus caffeine (120 mg.kg-1) were also normal. However, adrenergic stimulation enhanced the susceptibility of Pkp2-Het hearts to tachyarrhythmia and sudden cardiac death. Histologic staining showed no significant fibrosis or adipocyte infiltration in the RVs and LVs of six- and twelve-month-old Pkp2-Het hearts. Contractility assessment of isolated myocytes demonstrated progressively reduced Pkp2-Het RV cardiomyocyte function consistent with RV failure measured by echocardiography. However, aging Pkp2-Het and control RV myocytes loaded with intracellular Ca2+ indicator Fura-2 showed comparable Ca2+ transients. Western blotting of Pkp2-RV homogenates revealed a 40% decrease in actin, while actin immunoprecipitation followed by a 2, 4-dinitrophenylhydrazine staining showed doubled oxidation level. This correlated with a 39% increase in troponin-I phosphorylation. In contrast, Pkp2-Het LV myocytes had normal contraction, actin expression and oxidation, and troponin-I phosphorylation. Finally, Western blotting of cardiac biopsies revealed actin expression was 40% decreased in RVs of end-stage ACM patients.
During the early “concealed” phase of ACM, reduced actin expression drives loss of RV myocyte contraction, contributing to progressive RV dysfunction.