Fig 2: Generation and characterization of cardiac Casq2 tissue-specific mouse models.(A) Each mouse model is shown with its representative Casq2 alleles before and after Cre expression in the Purkinje cells (PCs) and resulting Casq2 protein expression in the VM and PCs. The Casq2 floxed allele contains the promoter (P) and exon 1 (Ex1) in either the forward or reverse gene orientation flanked by loxP sites (triangles). Cre expression (if present) flips the orientation of the promoter and exon 1, resulting in 4 models: WT (global Casq2+/+ expression); Casq2–/– (global Casq2–/–); PC-Casq2–/– (Casq2 knocked out only in PCs); and VM-Casq2–/– (Casq2 knocked out only in the VM). Cntn2 is only expressed in PCs. (B) Western blots from Casq2 tissue-specific mouse hearts. C57BL/6J (BL/6J) was included as a positive control. Casq2 null (KO) is an independent Casq2 germline deletion model (7) and was included as a negative control (the same Bl/6J and KO samples were loaded on both gels). GAPDH was used as a loading control. (C) Representative immunostaining for Cntn2 and Casq2 from sectioned mouse hearts. Scale bar: 50 μm. (D) Representative ECG traces from each mouse model after i.p. administration of 3 mg/kg ISO plus 60 mg/kg caffeine. Arrows denote premature ventricular contractions, and dashed lines denote episodes of VT. Scale bar: 500 ms. Quantification of VEBs (E) and VT (F) incidence in 8–38-week-old mice. WT sample, n = 39; Casq2KO, n = 31; PC-Casq2–/–, n = 16; VM-Casq2–/–, n = 28. (E) Data reported with mean ± SD. ***P < 0.001 versus WT or PC-Casq2–/– by Kruskal-Wallis test followed by Dunn’s multiple comparisons post hoc test (E) or the Fisher exact test (F).

Fig 3: Endocardial ablation reduces arrhythmia burden in Casq2 null hearts.Lugol’s solution was injected either into the LV or RV to ablate the endocardial wall. Optical mapping before (A) and after (B) injection of Lugol’s solution into the LV. Breakthroughs are denoted in the left-most panels by red stars. Activation of the LV epicardium was maintained even after endocardial ablation. (C) Representative QRS waveform morphology before and after injection with Lugol’s solution. (D) Origin of VEBs after injection of vehicle or Lugol’s solution into the LV or RV. Arrhythmias were stimulated by perfusion with 100 nM ISO (n = 8 hearts/group). Data are reported as mean ± SD. ##P < 0.01 by 2-sided Student’s t test when comparing ectopic origin percentage from the RV with that of no ablation; **P < 0.01 by 2-sided Student’s t test when comparing ectopic origin percentage from the LV with that of no ablation.

Fig 4: Expression of protein targets contributing to sarcoplasmic reticulum Ca2+ATPase (SERCA) and Na+/K+ ATPase (NKA) activities before (Pre‐white bars) and after (Post‐black bars) supplementation with olive oil (n = 7) and fish oil (n = 10). No effect of supplementation on the expression of SERCA 1a and 2a, calsequestrin 1 and 2 (CSQ1, CSQ2) (a, b); NKAα1, NKAα2, NKAβ1, NKAβ2, NKAβ3 (c, d). Values are reported as means ± SEM. Data were analyzed by paired t test

Fig 5: Casq2 expression in subendocardial ventricular myocytes juxtaposed to Purkinje cells reduces PVC burden and prevents arrhythmia.(A) Immunostaining for Cntn2 (a Purkinje cell marker) and Casq2 in selected hearts from VM-Casq2–/– mice. Scale bar: 200 μm. A subset of mice expressed Casq2, in addition to the Purkinje cells, also in ventricular myocytes next to Purkinje cells, denoted as “juxta-PC Casq2” (see top right image in A). Other mice co-expressed Casq2 only in Cntn2-positive cells (see lower right-side image in A). Scale bar in right-side images: 50 μm. (B) Ratio of Casq2 to Cntn2-positive immunostaining in hearts categorized as VM-Casq2–/– or juxta-PC Casq2 by a reviewer blinded to the genotype. (C) Percentage of Cntn2-labeled fibers having contiguous Casq2 staining in ventricular myocytes juxtaposed to the fiber. (D) NND distributions for Casq2-positive immunostaining relative to Cntn2-positive immunostaining. Data are displayed in 15 μm bins (individual distributions are shown in Supplemental Figure 2). (E) Median NND for each heart. (F) VEB and (G) VT incidence (>2 consecutive VEBs); n = 10 and 8 hearts/group, respectively. (B, D, and E) Data are reported with mean ± SD and compared using a 2-sided Mann-Whitney test. (F) Data were compared using the Fisher exact test.