Oxidized LDL‐dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy

Abstract Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro‐adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low‐density lipoprotein (oxLDL)‐dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient‐derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock‐out mice through high‐fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.


Introduction
Arrhythmogenic cardiomyopathy (ACM) is a heart condition with genetic traits. It is hallmarked by a gradual fibro-adipose replacement of the ventricular myocardium, heart failure, malignant arrhythmias, and sudden death (Basso et al, 2009). ACM inheritance has mainly an autosomal dominant pattern and most causative mutations reside in desmosomal genes, especially PKP2 (Lazzarini et al, 2015). The reasons underpinning the characteristic low penetrance and variable expressivity (Pinamonti et al, 2014) are still not properly understood. Genetic determinants and environmental factors could play a role as phenotypic modulators (Xu et al, 2010;König et al, 2017). For instance, physical exercise worsens both ACM arrhythmic burden (La Gerche, 2015) and myocardial dysfunction (Saberniak et al, 2014).
Disease severity markers must be improved, and only few pharmacological approaches to counteract disease progression have been proposed (Chelko et al, 2016;Chelko et al, 2019).
ACM electrical dysfunction is determined by genetically impaired cardiomyocytes and worsened by adipose tissue presence (Samanta et al, 2016;De Coster et al, 2018), which we showed to derive, at least partly, from differentiation of cardiac mesenchymal stromal cells (C-MSCs; (Lombardi et al, 2016;Sommariva et al, 2016;Stadiotti et al, 2017)). C-MSC, being primary human cells directly obtained from patients ventricular biopsy, easy to isolate and to amplify , carrying the whole patient's genetic setting and recapitulating ACM adipogenesis in vitro (Sommariva et al, 2016), represent a valid cell model to study ACM.
A key regulator of ACM adipogenesis is the proliferator-activated receptor gamma (PPARc), whose activation is dependent on Wnt/ bcatenin- (Garcia-Gras et al, 2006) and Hippo-pathway (Chen et al, 2014) impairment. Accordingly, PPARc modulators rosiglitazone or 13-hydroxy-octadecadienoic acid (13HODE) have been used to shift glycolysis to fatty acid metabolism to model ACM lipogenesis in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CM) (Kim et al, 2013). In other conditions such as atherosclerosis, the involvement of oxidized low-density lipoproteins (oxLDL) and their component 13HODE in regulating cell lipid accumulation is well known. In particular, after oxLDL cellular internalization by scavenger receptors, such as CD36 (Sun et al, 2007), 13HODE directly induces PPARc expression , provoking CD36 expression increase, thus facilitating additional oxLDL uptake and further PPARc activation in a feed-forward circle (Jostarndt et al, 2004). Notably, 13HODE blood concentrations have been used as an exercise-induced oxidative stress marker in athletes (Powers & Jackson, 2008;Nieman et al, 2014;Sugama et al, 2015).
An adjunctive clue hinting to a link between LDL levels and cardiac adipocyte accumulation comes from available ACMtransgenic mice, which fail to accumulate considerable amount of myocardial fat (McCauley & Wehrens, 2009). In fact, mice, unlike humans, show low circulating total cholesterol, which mostly resides in HDL fractions (Kashyap et al, 1995).
We thus hypothesized that oxLDL/CD36/PPARc circuitry may be a trigger of adipogenesis, and related clinical phenotypes, in ACM. Through the investigation of ACM patient features, in vitro experiments on patient-derived cardiac cells (both C-MSC and hiPSC-CM) and in vivo studies with Pkp2 heterozygous knock-out mice (Pkp2+/À), we consistently showed for the first time that oxLDL and elevated oxidative status contribute to ACM phenotype severity.

ACM patients show high plasma and cardiac lipid peroxidation indices
We evaluated oxLDL and 13HODE plasma concentrations in ACM patients and HC, matched for sex, age, and cardiovascular risk factors (Appendix Table S1). As reported in Fig 1A, ACM patients showed higher plasma oxLDL compared to HC (n = 36; ACM 137.90 AE 20.85 vs. HC 66.74 AE 5.79 ng/ml; P = 0.015). Accordingly, plasma 13HODE was found significantly higher in patients than controls (Appendix Table S1).
Interestingly, we observed a difference in oxLDL mean levels between patients with overt ACM phenotype carrying ACM-related causative mutations and their relatives, carriers of the same mutations but not clinically affected by the pathology (n = 7 vs. n = 9; ACM 384.50 AE 139.1 vs. unaffected relatives 66.99 AE 17.09 ng/ml; P = 0.02; Fig Table S2). This evidence points to an association between a fully penetrant disease and high oxLDL plasma concentration.
In addition, we did not observe differences in oxLDL plasma concentrations between patients with PKP2 mutations and patients with mutations in other ACM-associated genes or gene elusive (n = 12 vs. n = 22; Fig 1C; Appendix Table S3; Appendix Fig S2;  Appendix Table S5), indicating that oxLDL elevation is not linked only to PKP2 forms.
To understand whether high plasma oxLDL and 13HODE have a correspondence at cardiac tissue levels, we quantified the lipid peroxidation marker MDA on ACM and control RV sections, finding higher oxidative stress in ACM hearts (n = 4; MDA relative densitometric analysis (d.a.)/nuclei numbers ACM 36.25 AE 10.43 vs. HC 1.00 AE 0.72; P = 0.015; Fig 1D).
CD36 receptor has a central role in oxLDL uptake . CD36 immunostaining on RV tissue from ACM and HC donors revealed higher CD36 expression in ACM samples (n = 4; CD36 relative d.a./nuclei numbers ACM 14.72 AE 2.10 vs. HC 1.00 AE 0.40; P = 0.0007; Fig 1E), which was mainly distributed in replacement tissue areas.

Elevated oxLDL plasma concentrations are associated with structural and functional impairment and arrhythmic burden in ACM patients
Since Fig 1B suggested a role of oxLDL in ACM phenotype worsening, we retrospectively investigated the association between plasma oxLDL levels and structural, functional, and arrhythmic features in our whole ACM patient cohort. A ROC curve analysis identified the cut-off value of 86 ng/ml which best discriminates ACM patients vs. HC (n = 36; 63.41% sensitivity and 65.85% specificity; Fig 2A). Based on this cut-off value, we subdivided our ACM patient cohort in two groups (n = 26 < 86 ng/ml oxLDL and n = 41 > 86 ng/ml oxLDL). In the sub-cohort of ACM patients for which MRI was performed in our hospital, we quantified the mass of ventricular fat infiltration (Aquaro et al, 2014). Strikingly, patients with oxLDL plasma concentrations above the cut-off showed significantly higher myocardial fat infiltration (n = 14 vs. n = 25; fat infiltration mass < oxLDL 2.27 AE 1.35 vs. > oxLDL 15.32 AE 4.62 grams; P = 0.04; Fig 2B). Beside greater structural impairment, the group with higher oxLDL also showed a higher frequency of RV dysfunction defined as in (Marcus et al, 2010) (n = 26 vs. n = 41; % of patients with RV dysfunction < oxLDL 26.9% (7/26) vs. > oxLDL 53.7% (22/41); P = 0.04; Fig 2C), biventricular dysfunction (n = 26 vs. n = 41; % of patients with biventricular dysfunction < oxLDL 0% (0/26) vs.

ACM C-MSC show elevated oxidative stress and PPARc and CD36 expression
To model adipogenesis in vitro, we obtained ACM and HC C-MSC from RV endomyocardial biopsies (Casella et al, 2020), as previously described . To evaluate ACM C-MSC reactive oxygen species (ROS), we performed a dichlorofluorescein (DCF) test. As shown in Fig 3A, oxidative stress is significantly higher in ACM C-MSC compared to HC C-MSC (n = 5; relative mean DCF emission ACM 1.63 AE 0.26 vs. HC 1.00 AE 0.06; P = 0.049). At least part of the ACM C-MSC oxidative stress resulted in lipid peroxidation, as shown by MDA cell immunofluorescence (n = 4 vs. n = 5; MDA relative d.a./nuclei number ACM 8.83 AE 2.78 vs. HC 1.00 AE 0.51; P = 0.017; Fig 3B). To understand whether excess oxidative stress is due to a defect in antioxidant capacity, we measured the ratio between reduced (GSH) and oxidized glutathione levels in HC and ACM C-MSC, obtaining no differences between the two groups (n = 8; Fig 3C).

Lipid accumulation and CD36 levels increase in ACM C-MSC upon adipogenic stimulus
Since corresponding transcription levels of CD36 and PPARc, as well as lipid accumulation, are described for other cell types , we evaluated whether ACM C-MSC, already predisposed to PPARc activation (Sommariva et al, 2016), were more prone to expose CD36 on the plasma membrane during lipid accumulation. We performed a double staining with Nile Red, marking neutral lipids, and anti-CD36 antibody in ACM and HC C-MSC, cultured in adipogenic medium (AM) for different time-points (0, 3, 6, and 10 days). Figure 3E shows that, during adipogenic differentiation, ACM C-MSC simultaneously increase CD36 and lipid content significantly more than HC cells. In both ACM and HC cells, a linear correlation is present between these two parameters (P = 0.008 and 0.03, respectively); however, a higher slope in ACM C-MSC was evident (n = 4 vs. n = 5; ACM slope 1.33; R 2 = 0.99 vs. HC slope 0.69; R 2 = 0.93; slopes statistically different P = 0.016). To explore CD36 activity during adipogenic differentiation, we evaluated C-MSC oxLDL internalization, by assessing the intracellular fluorescence after 10 lg/ml DiI dye-conjugated oxLDL treatment (DiI-oxLDL), either in GM or in AM. ACM C-MSC internalized more DiI-oxLDL in AM compared to GM (n = 3; DiI internalization ACM GM 2.22 AE 0.16 vs. ACM AM 6.29 AE 0.65; P = 0.002) and 3-fold more than controls in AM (P = 0.01; Fig 3F). Furthermore, lipidomic assays confirmed ACM C-MSC transition toward an adipogenic lineage (Appendix Fig S3).
By mimicking PKP2 haploinsufficiency through a silencing approach, we demonstrated that oxLDL susceptibility is dependent on desmosomal protein defect (Appendix Fig S4).
A oxLDL plasma concentration in ACM patients and HC (n = 36; Mann-Whitney test). B oxLDL plasma concentration in mutated ACM (n = 7) and non-ACM relatives, carriers of the same causative mutation (n = 9; Mann-Whitney test). C oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n = 10 vs. n = 26; one-way ANOVA). D Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n = 4 biological replicates; two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E Representative images of CD36 immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n = 4 biological replicates; two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue).
The antioxidant compound NAC reduces lipid accumulation in ACM C-MSC In an attempt to prevent the effects of oxidized agents on lipid droplet accumulation in ACM cells, we tested the effect of the antioxidant NAC. 5 mmol/l NAC treatment was able to contain 13HODE-dependent lipid accumulation in ACM cells ( Fig 4C).
Moreover, NAC addition to AM led to lipid accumulation reduction in ACM C-MSC compared to AM alone, suggesting that oxidative stress plays a role in the lipogenic process per se (n = 12; ORO quantification ACM AM 3.60 AE 0.82 vs. ACM AM+NAC 2.38 AE 0.61; P = 0.03; n = 7; PPARc/GAPDH d.a. ACM AM 1.67 AE 0.16 vs. ACM AM+NAC 1.03 AE 0.08; P = 0.007; Fig 4B and C). The effect is possibly due to a direct action of NAC on gene expression as previously described in macrophages during foam cell formation (Sung et al, 2012). A ROC curve shows the capability of plasma oxLDL concentration in discriminating ACM vs. HC subjects (data as in Fig 1A). The red dot shows the point where the difference between sensitivity and specificity is minimized; this value, corresponding to the oxLDL cut-off = 86 ng/ml, was used to part the whole ACM cohort in two subpopulations. B Left panels: representative images of three cases of ACM in steady-state free procession sequences at cardiac MRI. On the left, a case with RV dilation, regional bulging of the RV wall without fat infiltration, and oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass of the two ACM subpopulations (above or below the cut-off) whose MRI was available for re-analysis (n = 14 oxLDL < 86 ng/ml, n = 25 oxLDL > 86 ng/ml; two-tailed Student's t-test). C Frequency of patients showing RV dysfunction (defined as in (31)) in the two ACM cohort subgroups (n = 26 oxLDL < 86 ng/ml vs. n = 41 oxLDL > 86 ng/ml; twotailed Student's t-test). D Frequency of patients showing biventricular dysfunction in the two ACM cohort subgroups (n = 26 oxLDL < 86 ng/ml vs. n = 41 oxLDL > 86 ng/ml; two-tailed Student's t-test). E Frequency of patients showing RV wall motion abnormalities in the two ACM cohort subgroups (n = 26 oxLDL < 86 ng/ml vs. n = 41 oxLDL > 86 ng/ml; Fisher's exact test). F RV ejection fraction % of the patients classified in the two ACM cohort subgroups (n = 23 oxLDL < 86 ng/ml vs. n = 39 oxLDL > 86 ng/ml; two-tailed Student's t-test). G Kaplan-Meier analysis of actual MAE-free survival of patient belonging to the two ACM cohort subgroups in the first 5-year follow-up (n = 26 oxLDL < 86 ng/ml vs. n = 41 oxLDL > 86 ng/ml; log-rank P < 0.0001; HR = 0.223[0.116-0.428]).
Data information: mean AE SEM. *P < 0.05. Source data are available online for this figure.
ª 2021 The Authors EMBO Molecular Medicine 13: e14365 | 2021 CD36 silencing and PPARc inhibition in ACM C-MSC impair the lipid accumulation mechanism To directly assess CD36 causal role in ACM cell adipogenic process, we performed CD36 siRNA-mediated silencing in AM supplemented with oxLDL. 32% mean reduction in CD36 levels (n = 7; P = 0.014; Fig 5A) in ACM C-MSC was enough for inducing significantly less lipid accumulation if compared to the non-silenced counterparts (n = 7; ORO relative lipid accumulation scramble 1.00 AE 0.19 vs. siRNA 0.35 AE 0.10; P = 0.003; Fig 5B), along with PPARc reduced levels (n = 7; PPARc/GAPDH relative d.a. scramble 1.00 AE 0.18 vs. siRNA 0.76 AE 0.15; P = 0.05; Fig 5A). In addition, CD36 levels (both with and without silencing) correlated with PPARc (n = 14; slope=1.05; R 2 = 0.76; P = 0.0002) and to a lesser extent with ORO staining (n = 14; slope=0.52; R 2 = 0.26; P = 0.07; Fig 5C). As expected, reduced CD36 levels determined lower oxLDL image quantification (n = 4 biological replicates ACM, n = 5 biological replicates HC; two-tailed Student's t-test). C GSH/GSSG ratio quantification in HC and ACM C-MSC cultured in GM (n = 8 biological replicates). D Top panel: representative images of Western blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARc antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARc (n = 18 biological replicates) and CD36 (n = 17 vs. n = 16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test). E Results of a FACS analysis of ACM and HC C-MSC, cultured in GM (time-point 0) or AM for 3, 6 or 10 days (time-points 3, 6,and 10, respectively) and marked with anti-CD36 antibody and Nile red. The mean (n = 4 biological replicates ACM, n = 5 biological replicates HC) fluorescence of CD36 and Nile Red is shown for each condition, the relative regression line, its equation, R 2 , and P-value (X-Y correlation). F Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10 lg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n = 3 biological replicates; two-way ANOVA).
Data information: mean AE SEM. *P < 0.05 and **P < 0.01. Source data are available online for this figure.
In addition, we confirmed that lipid accumulation and CD36 levels and functionality increased also in human ACM cardiomyocytes (using hiPSC-CM derived from ACM patients vs. unaffected relatives) upon PPARc agonism (Appendix Fig S5).
HFD administration leads to cardiac lipid accumulation and dysfunction in Pkp2+/À mice To validate our hypothesis on an ACM in vivo model, we took advantage of the well-established Pkp2+/À mouse (Grossmann et al, 2004;Sato et al, 2009). Despite adult Pkp2+/À mice showed no cardiac adipogenesis, low oxidative stress and CD36 levels, and no cardiac dysfunction, C-MSC obtained from the mutant mice accumulated lipids in vitro (Appendix Figs S6 and S7).
We thus fed Pkp2+/À mice a HFD for 3 months to test the hypothesis that increasing cholesterol and oxidative stress levels A Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150 µg/ml oxLDL. Middle panel: image quantification (n = 11 biological replicates; two-way ANOVA). Right panels: representative images of Western blot of CD36, PPARc, and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150 µg/ml oxLDL (n = 5 biological replicates) and d.a. normalized on the housekeeping GAPDH (two-way ANOVA). B Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20 lg/ml 13HODE, 5 mmol/l NAC, or both. Right panel: image quantification (n = 13 biological replicates; two-way ANOVA). C Left panel: representative images of Western blot of CD36, PPARc, and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20 lg/ml 13HODE, 5 mmol/l NAC, or both (n = 8 biological replicates). Right panels: d.a. normalized on the housekeeping GAPDH (two-way ANOVA).

Discussion
Current information about ACM genetic basis does not fully explain reduced penetrance and phenotypic variability (Pinamonti et al, 2014). Among ACM pathogenic pathways, metabolic dysfunctions are thought to play a relevant role, given the well-established involvement of PPARc and recent discoveries on the topic (van Opbergen et al, 2019; Song et al, 2020).
In the present study, we defined the contribution of oxLDL/ CD36/PPARc circuitry as ACM penetrance cofactor, demonstrating, for the first time, that oxidative stress and oxidized lipid metabolism modulate ACM adipogenic phenotype, both in vitro and in vivo. Interestingly, such biological axis is pharmacologically targetable in order to reduce the adipogenic phenotype and consequent disease severity in ACM.
This hypothesis-driven study was prompted by the observations in ACM patient plasma of higher oxLDL and 13HODE plasma levels as compared to matched HC. Our cohort was composed of 81% male and 38% athletic patients. Of note, strenuous exercise and male sex are the only accepted cofactors precipitating ACM phenotype. The association of both exercise and male gender with oxidative stress is well known (Miller et al, 2007). Oxidative stress can induce oxidation of different proteins and complexes, including LDL (Sanchez-Quesada et al, 1995).
Our data suggest that a higher oxLDL concentration in ACM patient plasma is not a mere consequence of the causative genetic defect. Indeed, we observed that oxLDL plasma levels are higher in ACM patients than in their unaffected relatives, carriers of the same ACM causative genetic mutation. Further, oxLDL elevation is not linked to a specific genetic form of ACM, even if, given the low prevalence in our cohort of carriers of pathogenic mutations in desmosomal genes other than PKP2, testing each single gene association was not possible. On the other hand, we identified background variants in genes associated with oxidative stress or dyslipidemia co-segregating with the ACM phenotype (Appendix Fig S1; Appendix Table S4; Dataset EV1). These results will need confirmation in larger cohorts. Other factors, including lifestyle and diet, or unidentified protective genetic variants, may play a role in modulating oxLDL concentrations.
Notably, we unraveled in our ACM patients a strong association between oxLDL plasma levels above the cut-off of 86 ng/ml and pathognomonic ACM structural and clinical features. Such circulating oxLDL cut-off allowed to segregate ACM patient population with a severe clinical phenotype in terms of fat infiltration, ventricular dysfunction, and risk of major arrhythmic events in the long term. In addition, these data are relevant to confirm oxLDL pathogenic role at a clinical level and to introduce oxLDL plasma levels as new ◀ Figure 6. HFD increases plasma oxLDL, cardiac adipose substitution, PPARc and CD36 expression, and lipid peroxidation, and impairs RV function in ACM mice.
A Left panel: plasma total cholesterol in the different lipoprotein fractions of WT and Pkp2+/À mice fed a CD (green line) or a 3-month HFD (blue line; n = 7). Right panel: plasma total cholesterol quantity in fractions 29-32, corresponding to low-, very-low-, and intermediate-density lipoproteins, of WT and Pkp2+/À mice fed a CD (green line) or a 3-month HFD (blue line; n = 7; Mann-Whitney test). B Plasma concentration of oxLDL in WT and Pkp2+/À mice samples, fed a CD and a 3-month HFD (n = 6; two-tailed Student's t-test). C Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/À cardiac sections. Right panel: quantification of ORO positive area percentage (n = 10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/À mice (n = 9; two-way ANOVA) is shown (Appendix Fig S3). D Representative images of PPARc (green) immunostaining on HFD-fed WT and Pkp2+/À mice cardiac sections (n = 10; two-way ANOVA). Quantification of the PPARc staining in HFD is shown relative to the values of CD (n = 9). E Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/À mice cardiac sections (n = 10; two-way ANOVA). Quantification of the MDA staining in HFD is shown relative to the values of CD (n = 9). F Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/À mice cardiac sections (n = 10; two-way ANOVA). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n = 9). G RV EF and FS percentages, RVID in systole and diastole of WT and Pkp2+/À mice after (right) 3-month HFD feeding (n = 10; two-tailed Student's t-test).
Data information: mean AE SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. We used human C-MSC obtained from ACM heart biopsies and HC as in vitro model, as they subsidize ACM-driven cardiac adipogenesis and represent a reliable cell model to study pathogenic mechanisms (Sommariva et al, 2016;Sommariva et al, 2017). As previously demonstrated in other cell models (Garcia-Gras et al, 2006;Chen et al, 2014;Sommariva et al, 2017), in ACM C-MSC we found a higher expression of PPARc. In agreement with the oxLDL receptor CD36 interdependence with PPARc, described in cardiovascular atherogenic conditions (Nicholson et al, 1995), we found also in ACM C-MSC a linear correlation between the activation of PPARc, with consequent lipid accumulation, and CD36 expression and function. The proposed mechanism was confirmed by CD36 silencing, which entailed a significant reduction of lipid accumulation in ACM C-MSC, as well as by PPARc inhibition, which caused a reduction of CD36 expression and function. Moreover, we described, for the first time, that increased oxidative stress is present in ACM C-MSC, without changes in their antioxidant capability. Thus, an analysis of mitochondrial dysfunction as a source of oxidative stress is awaited (van Opbergen et al, 2019).
We further demonstrated that oxLDL enhances C-MSC adipogenic differentiation. Accordingly, Parhami et al (1999) have previously shown that oxLDL promotes adipogenic differentiation of bone marrow-derived MSC. We showed that 13HODE prompts, in ACM patient C-MSC, a PPARc/CD36-dependent adipogenesis as an intrinsic ACM pathogenic mechanism. 13HODE effect was previously used in ACM hiPSC-CM as a mere in vitro supplement to activate PPARc (Kim et al, 2013). Our results do not exclude that the Wnt/bcatenin pathway may exert a modulatory role in the balance of PPARc activation. In addition, we showed that PPARc-agonism induces CD36 increased expression and function in ACM hiPSC-CM.
Importantly, cell exposure to the antioxidant NAC not only prevented 13HODE-dependent lipogenesis and CD36/PPARc expression, but ameliorated C-MSC phenotype, possibly by reducing the effects of basal oxidized lipids in their ability to activate PPARc, as well as directly acting on the transcriptional profile of the cells, including CD36 and PPARc expression (Ji et al, 2011;Sung et al, 2012).
We confirmed in vitro findings with in vivo experiments by means of the Pkp2+/À mouse model (Grossmann et al, 2004), since PKP2 heterozygous mutations are the most frequent in ACM patients. Notably, available ACM in vivo models, including the Pkp2+/À mouse, do not fully recapitulate the disease phenotype, showing from absence to small amount of ventricular fibro-fatty substitution, possibly due to an intrinsic protective mechanism. Since it is known that mice show low cholesterol plasma levels, we demonstrated that a consistent increase of plasma LDL cholesterol, including the oxidized form, achieved by HFD feeding, provoked fat accumulation in ACM mouse hearts. This phenomenon, borne by C-MSC, as previously proven in humans (Sommariva et al, 2016), was prevalently located in sub-epicardial regions, in line with the epicardial-endocardial gradient observed in ACM human hearts. In accordance with in vitro and patient ex vivo results, cardiac oxidative stress and CD36 significantly increased in ACM mouse hearts following HFD. Importantly, HFD treatment provoked an initial impairment of RV function, in line with the concept that myocardial substrate alterations lead to functional impairment (Pabon et al, 2018;te Riele et al, 2014) and adipocyte-secreted factors negatively influence CM contractility (Lamounier-Zepter et al, 2009;Gastaldelli et al, 2012;Lamounier-Zepter et al, 2014;Pabon et al, 2018). In addition, proarrhythmic (Peters et al, 2012;Canpolat et al, 2013) ECG changes were detected in the Pkp2+/À mice fed HFD. These results confirmed that ACM causative mutations are necessary but not sufficient to generate an overt ACM phenotype in mice, which was instead obtained when increasing oxLDL levels, thus recognizing in oxLDL a likely contributory pathogenic cause.
We cannot exclude that disease progression itself may, in turn, further increase oxLDL levels in patients, e.g., by boosting ROS production, inflammation, or other mechanisms. However, our in vitro, in vivo, and clinical data clearly showed that maintaining the levels of oxLDL low is beneficial in terms of disease progression.
A Left panel: plasma total cholesterol in the different lipoprotein fractions of WT and Pkp2+/À mice fed a 3-month HFD (blue line) or a 3-month HFD+atorvastatin (red line). Right panel: plasma total cholesterol quantity in fractions 29-32, corresponding to low-, very-low-, and intermediate-density lipoproteins, of WT and Pkp2+/À mice fed a 3-month HFD (blue line; n = 7) or a 3-month HFD+atorvastatin (red line; n = 9; two-tailed Student's t-test). B oxLDL plasma concentration in WT and Pkp2+/À mouse samples, fed a 3-month HFD plus atorvastatin (n = 8), compared to HFD (as in Fig 6B; two-tailed Student's t-test). C Left panel: representative images of ORO staining of cardiac sections of Pkp2+/À mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n = 9) is compared to that in HFD (as in Fig 6C; two-tailed Student's t-test). D Representative images of PPARc immunostaining (green) on cardiac sections of Pkp2+/À mice fed a 3-month HFD plus atorvastatin (n = 9). Quantification is compared to the values of Pkp2+/À in HFD and relative to WT in CD (as in Fig 6D; two-tailed Student's t-test). E Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/À mice fed a 3-month HFD plus atorvastatin (n = 9). Quantification is compared to the values of Pkp2+/À in HFD and relative to WT in CD (as in Fig 6E; two-tailed Student's t-test). F Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/À mice fed a 3-month HFD plus atorvastatin (n = 9). Quantification is compared to the values of Pkp2+/À HFD and relative to WT in CD (as in Fig 6F; two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). G RV EF and FS percentages, RVID in systole and diastole of Pkp2+/À mice during (left panel) and after (right panel) 3-month HFD plus atorvastatin feeding (n = 9). For comparison, RV EF of Pkp2+/À mice fed a HFD is shown (as in Fig 6G; two-tailed Student's t-test).
12 of 23 An interesting side-product of this project was the generation of a novel diet-driven ACM mouse model showing fat accumulation and RV dysfunction, thus better recapitulating, beyond arrhythmias (Cerrone et al, 2012), human ACM substrate myocardial phenotype.
In conclusion, by means of a multilayer approach, we demonstrated, for the first time, that oxidative stress increases oxLDL bioavailability, which are internalized in C-MSC by CD36 receptor, thus acting, through 13HODE-mediated PPARc activation, as a cofactor of cardiac adipogenic differentiation and with dependent phenotypes (Synopsis figure), which can be targeted by available therapies such as antioxidants (Shafiei et al, 2018) or statins (Ky et al, 2008). According to our findings, these strategies have the potential to reduce the penetrance of the disease in ACM mutation carriers, by attenuating cardiac adipose substitution, ventricular dysfunction, and arrhythmic phenotypes.

Ethics statement
This study complies with the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. It was approved by "IEO-CCM IRCCS" (12/06/2012) and by "South Tyrol Azienda Sanitaria" (13/03/2014, No. 1/2014) Ethics Committees. Written informed consent was obtained from all participants. HC cardiac samples were obtained from donors (accidental death), from "Treviso Tissue Bank Foundation".

Study patient population
A total of 67 ACM patients were enrolled for this study. ACM diagnosis was reached according to the 2010 International Task Force criteria (Marcus et al, 2010). Thirty-six patients out of the total cohort were matched for age, sex, and cardiovascular risk factors to 36 HC without a previous history of heart disease for plasma analysis (Appendix Table S1 summarizes baseline characteristics of 36 ACM patients and 36 HC, and Appendix Table S3 describes the genetic profile of ACM patients). We further enrolled for the plasma analysis nine ACM patients' relatives with PKP2 mutations but no clinical signs of the disease.
We obtained blood samples from all the recruited ACM patients and HC. ACM patients and HC taking statins or other lipid-lowering drugs were excluded from the analysis. Clinical data were collected for all ACM patients, as available: RV EF % was determined by MRI; RV dysfunction was defined as in Li and Durbin (2010); and MAEs are defined as sustained ventricular tachycardia, ventricular fibrillation, appropriate implantable cardioverter device intervention, and aborted sudden cardiac death. Genetic data on ACMlinked genes were obtained for 65 out of 67 patients (Appendix Table S5).
RV samples were obtained by biopsy procedures from 19 ACM patients.

Plasma preparation
Blood samples (5 ml) were collected in EDTA-coated tubes and centrifuged at 1,500 g for 15 min. Supernatants were collected, centrifuged again at 16,000 g for 15 min to obtain cell-and plateletfree plasma, and stored at À80°C as 400 µl aliquots until usage.

oxLDL in human plasma samples
Plasma samples from ACM patients and HC were used for the determination of oxLDL quantity, using a specific ELISA kit (Immundiagnostik, Bensheim, Germany). The test recognizes MDA-modified apolipoprotein B100. The protocol recommended by manufacturers was observed, and the absorption was determined with a spectrophotometer (Berthold Technologies, Bad Wildbad, Germany) at 450 nm. Results were interpreted by constructing a dose/response curve according to the standards provided in the kit.

Genetic analysis
ACM-associated genes DNA was extracted from blood or C-MSC using QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Next-generation sequencing was performed with the TruSight TM Cardio Sequencing Kit (Illumina NextSeq, San Diego, CA, USA). The alignment of sequence reads to reference human genome (GRCh37/hg19) was performed using GATK software. Variants in DSC2, DSG2, DSP, PKP2, JUP, TMEM43, DES, RYR2, PLN, SCN5A, and LMNA were filtered with Wannovar and pathogenicity classified according to Richards et al (2015).
Exome sequencing NA samples from blood were prepared following the Nextera â Rapid Capture Exome Enrichment kit protocol. Libraries for all samples, except Fam3.I.3, Fam3.I.4, and Fam3.II.3 (Appendix Fig  S1), were sequenced on two lanes of an Illumina HiSeq in pairedend mode and a read length of 100 bp. The remaining three samples were sequenced subsequently on four lanes of an Illumina MiSeq in paired-end mode and a read length of 75 bp. Nextera adapters were trimmed off using SeqPrep (https://github.com/jstjohn/SeqPrep), and read quality was controlled with FastQC (http://www.bioinf ormatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to reference genome GRCh37 with BWA version 0.7.15 (DePristo et al, 2011). Duplicate marking was performed with picard tools version 2.8.1 (https://broadinstitute.github.io/picard/). Indels were realigned and base quality scores recalibrated using GATK 3.7 (Van der Auwera et al, 2013), following the GATK best practice guidelines (Van der Auwera et al, 2013). Quality of bam files was evaluated with QualiMap version 2.2.1 (Garcia-Alcalde et al, 2012), sample contamination estimated with verifyBamId (Jun et al, 2012), and sex validated by inspection of the X chromosome coverage. Intermediate per sample gvcfs were generated with the GATK HaplotypeCaller, followed by joint genotyping on all individuals with GATK GenotypeGVCFs and additional samples not related to this study. Variants were called on the exonic target regions as defined by the Nextera protocol with a padding of 100 bp around the exons. Variants were annotated with Ensembl gene and variant consequence data using the Dintor gcoords2cons tool (Weichenberger et al, 2015). For each variant, the annotation of the transcript with the worst consequence type as according to Ensembl was selected.
First, using the Dintor MendelianFiltration tool, variants were selected that were present either in homo-or heterozygous form in all affected individuals of all three families, requiring a coverage of at least 10X at variant sites in the respective individuals. Variants were restricted to those mapped to a gene of the oxidative stress and dyslipidemia gene panel (Dataset EV1). Further, variants were removed, if their ExAC EUR allele frequency (AF) (Lek et al, 2016), their gnomAD NEF AF (Lek et al, 2016), or their 1,000 Genomes phase 3 EUR AF (Auton et al, 2015) was greater than 0.3. Variants were not required to be in protein-coding genes (Appendix Table S4A).
Next, each family was analyzed individually. Using the Dintor MendelianFiltration tool, variants were selected that were present in a families' affected PKP2 carriers, but absent in the same families' healthy PKP2 carriers, requiring a coverage of at least 10X at the variant sites in the relevant individuals. For the selection, variants segregating in the affected PKP2 carriers in either a dominant or a recessive mode of inheritance were accepted. Variants were restricted to those mapped to a gene of the oxidative stress and dyslipidemia gene panel (Dataset EV1). Further, variants were removed, if their ExAC EUR allele frequency (AF) (Lek et al, 2016), their gnomAD NEF AF (Lek et al, 2016), or their 1,000 Genomes phase 3 EUR AF (Auton et al, 2015) was greater than 0.05. Variants were not required to be in protein-coding genes (Appendix Table S4B).
Per sample, an average of 55 millionAE17 million reads mapped to reference genome, resulting in a mean coverage of 57XAE21X, and 87%AE4% of the exon target region covered at ≥ 10X. The sex of all samples was confirmed by inspection of the coverage of the X chromosome, and sample contamination was below 1%.

Biopsy sampling
Endomyocardial biopsy sampling was performed in ACM-suspected patients for diagnostic purposes, guided by CARTO mapping, as previously described (Casella et al, 2015;Pilato et al, 2018). A biopsy sample from the RV acquired in the area adjacent to the electroanatomical scar was obtained from ACM patients and was processed to obtain heart sections, total tissue protein extracts, and C-MSC (Sommariva et al, 2016). HC RV autoptic samples were treated with the same protocols.

Heart tissue section preparation and immunofluorescence analysis
Human ventricular samples were fixed in 4% paraformaldehyde (Santa-Cruz, Dallas, Texas, USA) in phosphate-buffered saline (PBS; Lonza, Basel, Switzerland) and processed for paraffin embedding. Paraffin-embedded sections (6 lm thick) were de-waxed in xylene and rehydrated in ascending alcohols. The immunofluorescence analysis was performed following antigen retrieval with incubation with target retrieval solution citrate pH6/microwave (Dako, Santa Clara, California, USA). Sections were incubated with primary antibody anti-MDA (1:2,500; Abcam, Cambridge, UK) and anti-CD36 (1:200; BD, Franklin Lakes, New Jersey, USA) at 4°C overnight (see Appendix Table S7). After washing, sections were incubated with the fluorochrome-conjugated antibody goat anti-rabbit IgG Alexa 488 1:200 (Alexa Fluor, Waltham, Massachusetts, USA) for 1 h at room temperature (RT) in the dark. Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1,000; Life Technologies, Carlsbad, California, USA). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each explanted heart subject, three consecutive slices and at least five fields for each slice were examined, excluding autofluorescence and aspecific signals. For ACM biopsy samples, all the samples were sliced and examined.

C-MSC immunofluorescence analysis
C-MSC were plated on 1.8 cm 2 chamber slides (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a density of 20,000 cells/cm 2 . After 24 h of culture in basal conditions, C-MSC were washed with PBS and fixed in 4% paraformaldehyde in PBS. After the blocking step in 10% goat serum (Sigma-Aldrich, St. Louis, Missouri, USA), cells were incubated with primary antibodies anti-MDA (1:2,500; Abcam, Cambridge, UK) and anti-CD36 (1:200; BD, Franklin Lakes, New Jersey, USA) at 4°C overnight. After washing, sections were incubated with the fluorochromeconjugated antibody goat anti-rabbit IgG Alexa 488 1:200 (Alexa Fluor, Waltham, Massachusetts, USA) for 1 h at RT in the dark. Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1,000; Life Technologies, Carlsbad, California, USA). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each dish, 15 fields were examined.

C-MSC lipid staining
C-MSC were plated at a concentration of 20,000 cells/cm 2 and cultured in AM. Fat accumulation was tested by ORO (Sigma-Aldrich, St. Louis, Missouri, USA) intracellular lipid staining. In detail, C-MSC were stained with 1% ORO (Sigma-Aldrich, St. Louis, Missouri, USA) solution in 60% isopropanol for 1 h after 5-min fixation with 4% paraformaldehyde in PBS. After five washes in PBS to ensure the removal of unbound dye, quantitative results were obtained by evaluating luminance in the 255 red channel with the ImageJ program (at least 15 fields were evaluated per condition per patient).

OxLDL preparation
Fresh plasma purchased from healthy donors (Niguarda Ca' Granda Hospital, Milano) was brought to a density of 1.019 g/ml with KBr and subsequently centrifuged at 40,000 rpm for 16 h to remove VLDL particles. After this operation, the gradient was adjusted to a density of 1.063 g/ml and samples centrifuged for further 24 h at the same speed. LDL were then isolated, dialyzed for 48 h at 4°C, sterilized by filtration (0.22 lm; Millipore, Burlington, Massachusetts, USA) and then characterized for their protein and cholesterol content. Aliquots of 2-3 ml were oxidized by addition of an equal volume of CuSO 4 (final concentration 2.7 mM) for 18 h, under stirring. Oxidation was documented by agarose gel electrophoresis and by gas-liquid chromatography analysis, by which we monitored the specific disappearance of polyunsaturated fatty acids, namely linoleic, arachidonic, and eicosapentaenoic ones. OxLDL were then utilized for cell culture experiments, under sterile conditions.

OxLDL internalization assay in C-MSC
C-MSC were plated on 1.8 cm 2 chamber slides (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a density of 20,000 cells/cm 2 either in GM or in AM for 3 days. 10 lg/ml DiI-oxLDL (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added. After 3 h, the cells were fixed for 5 min in 4% paraformaldehyde in PBS, stained with Hoechst 33342 (1:1,000; Life Technologies, Carlsbad, California, USA), and the slides mounted. Pictures were acquired with Zeiss Axio Observer.Z1, with Apotome technology. For the quantification of intracellular DiI, other cells from the same cultures were treated as described above and acquired with FACS Gallios (Beckman Coulter, Brea, California, USA).
PKP2 silencing HC C-MSC were plated at a density of 12,500 cell/cm 2 in growth medium and transduced with pooled lentiviral particles containing shRNAs targeting both variants of human PKP2 (Gene ID 5381) in psi-LVRU6GP (with U6 promoter, eGFP reporter, puromycin resistance; Genecopoeia; Rockville, Maryland) or with the correspondent scrambled control lentiviral particles (Genecopoeia; Rockville, Maryland) for 24 h. After checking the transduction efficiency by detection of the GFP signal, 2 lg/ml puromycin was added to select transduced cells.
After cell amplification, PKP2 reduction was assayed by Western blot. Scrambled control and PKP2 shRNA C-MSC were plated in AM at a concentration of 20,000 cells/cm 2 and treated with 150 lg/ml oxLDL. After 72 h, treatment effects were evaluated by Oil Red O (ORO; Sigma-Aldrich St. Louis, Missouri, USA) staining. (Life Technologies, Carlsbad, California, USA) were added to 300 µl of Opti-MEM medium (Life Technologies, Carlsbad, California, USA). After 15 min at RT, the transfection reactions were added to the cells in 1.5 ml low-serum/no antibiotics medium. After 24 h, the medium was changed to AM supplemented with 150 µg/ml oxLDL for the following 72 h. CD36 reduction was confirmed by Western blot at the end of the experiment.
PPARc antagonism in C-MSC C-MSC were cultured for 72 h in AM with 5 lM GW9662 (Sigma-Aldrich, St. Louis, Missouri, USA). The treatment was added to the medium every 8 h. To check oxLDL internalization, 10 lg/ml DiI-oxLDL (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added 3 h before the end of the experiment, following the protocol described above (see "OxLDL internalization assay"). Pictures were acquired with Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each biological sample, 15 fields were examined.

Flow cytometry in C-MSC
To evaluate C-MSC oxidative status, cells cultured in basal medium were incubated for 30 min with 10 lM dichlorofluorescein (Sigma-Aldrich, St. Louis, Missouri, USA) and detached with TrypLE Select (Life Technology, Carlsbad, California, USA), and the conversion into the fluorescent dye 2 0 ,7 0 -DCF by cell ROS was measured by flow cytometry (Gallios, Beckman Coulter, Brea, California, USA). The mean FITC fluorescence was measured.
To determine the correlation between CD36 expression and lipid accumulation, cells were stained using 12.5 ng/ml Nile Red (Invitrogen, Carlsbad, California, USA), to mark intracellular neutral lipids, and 2.5 ll of anti-CD36 antibody (Life Technologies, Carlsbad, California, USA). The mean of the fluorescence was determined for Nile Red and CD36 for each sample.
To quantify the DiI-oxLDL internalization, cells were treated with 10 lg/ml DiI-oxLDL for 3 h, detached with TrypLE Select (Life Technologies, Carlsbad, California, USA), and acquired with FACS Gallios (Beckman Coulter, Brea, California, USA). The mean APC fluorescence was determined for each sample.

Western blot in C-MSC
Total proteins from C-MSC were obtained by Laemmli lysis buffer. After quantification with DC protein assay (Bio-Rad, Hercules, California, USA), proteins were run on SDS-PAGE gel (NUpage precast 4-12%; Invitrogen, Carlsbad, California, USA) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membrane was blocked in 5% skimmed milk-TBS for 1 h at RT and incubated overnight at 4°C with primary antibodies against GAPDH, PPARc, and CD36 (see Appendix Table S7). After washes, the membrane was incubated for 1 h at RT with the appropriate HRPconjugated secondary antibody goat anti-rabbit or goat anti-mouse (GE Healthcare, Chicago, Illinois, USA). Blots were washed and developed with the ECL system (Bio-Rad, Hercules, California, USA). Images were acquired with the Alliance Mini 2 M System (UVITEC, Cambridge, UK), and densitometric analysis was performed using Alliance Mini4 16.07 software (UVITEC, Cambridge, UK). Data are normalized expressing as 1 the comparison group in order to highlight the fold differences between different groups or treatment.

Glutathione quantification in C-MSC
Levels of GSH and GSSG were determined by a previously described and validated LC-MS/MS method (Squellerio et al, 2012). Briefly, cells cultured in GM have been washed twice with PBS, detached with trypsin, and then collected and centrifuged at 400 g for 10 min. The supernatant was removed, the pellet was resuspended in 50 µl of PBS, and proteins were precipitated with 50 µl of 10% trichloroacetic acid with the addition of 1 mmol/l EDTA and stored at À80°C until the analysis. Thawed samples were further diluted 1:10 with formic acid 0.1% before the LC-MS/MS analysis. The LC-MS/MS analysis was performed using an Accela HPLC System (Thermo Fisher Scientific, Waltham, Massachusetts, USA) coupled to a triple quadrupole mass spectrometer TSQ Quantum Access (Thermo Fisher Scientific, Waltham, Massachusetts, USA) outfitted with electrospray ionization source operating in positive mode. The chromatographic separation was conducted on a Luna PFP column (2.0 mm × 100 mm, particle size 3.0 µm, Phenomenex, Torrance, California, USA) maintained at 35°C. Analytes were eluted under isocratic conditions at 200 µl/min by 1% methanol in 0.75 mM ammonium formate adjusted to pH3.5 with formic acid. The analytes were detected by multiple reaction monitoring, and the transitions monitored (precursor ion > product-fragment ions) were m/z 308.1 ? m/z 76.2, 84.2, 161.9 (GSH) and m/z 613.2 ? m/z 230. 5,234.6,354.8 (GSSG). A linear 6-point calibration curve (range 0.25-8 µM for GSH and 0.008-0.25 µM for GSSG) was used for the quantification.

Analysis of C-MSC lipids
Cell lipids were extracted by hexane/isopropanol 3:2, plus butylated hydroxytoluene 0.005% as antioxidant. Known amounts of proper internal standards (stigmasterol, cholesteryl heptadecanoate, triheptanoin, nonadecanoic acid; Sigma-Aldrich, St. Louis, Missouri, USA) were added for the analysis of free cholesterol (FC) and esterified cholesterol (CE), triglycerides (TG), and free fatty acids (FFA), respectively. Lipid extracts were dried in a steam of nitrogen, aliquoted, and conserved at À80°C in the dark, until use.
An aliquot was loaded onto pre-run and activated channeled Silica TLC plates (BioMap) and run in hexane-diethyl ether-acetic acid (80:20:1 vol/vol/vol). The plates were then sprayed with dichlorofluorescein (0.15% in ethanol), and the spots corresponding to those of FC, FFA, TG, and CE standards were identified by UV light and scraped out of the TLC.
Samples were analyzed by a gas-liquid chromatographer (GLC 1000; DANI Instruments, Cologno Monzese, Italy) equipped with an autosampler HT300A (HTA, Brescia, Italy), a fused silica column (MEGA-5 30 m length, 0.3 mm diameter, 0.15 µm film thickness; Mega Columns, Legnano, Italy) and a flame ionization detector. Hydrogen flow was at a constant pressure of 1.2 bar. The oven temperature was constant (260°C, 8 min run) for FC, while ranged from 120°C to 300°C for FFA, TG, and CE (total run 45 min).
FC was resuspended in hexane/isopropanol and analyzed without derivatization. The other lipid classes were processed with methanolic acid 3N at 30-80°C for 30-120 min and analyzed for their fatty acid content. Peaks were identified by comparing their retention times with those of standard and their area determined by a dedicated software (Clarity). To calculate the total mass of each lipid class, the areas of all the peaks corresponding to the fatty acids were summed and the real mass determined by comparison with the area of the internal standards. Results were normalized by the number of cells in each dish (µg lipid/10 6 cells).

Generation of ACM and HC hiPSC
hiPSC from one ACM patient, carrying the deletion of the whole PKP2 exon 4 leading to a predicted truncated protein (p.N346Lfs*12), and one HC of the same family were obtained and characterized as previously described (Ermon et al, 2018;Meraviglia et al, 2018). Briefly, every hiPSC line has been generated using episomal vectors carrying OCT3/4, SOX2, KLF4, and L-MYC and has been subsequently grown on a mouse embryonic fibroblast (MEF) feeder layer. Clones that passed the quality check were adapted at passage 10-15 to grow in feeder-free condition (without MEF) directly onto six-well plates coated with Matrigel â matrix (Corning, Corning, New York, USA) and cultured in the commercially available xeno-free medium StemMACSTM iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany).

hiPSC-CM immunofluorescence analysis
Cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized (PBS with 0,1% Triton X100) for 10 min at RT. Cells were then blocked in PBS with 5% Goat serum for 1 h at RT and incubated with anti-aSARC overnight at 4°C. After washing, cells were incubated with the proper secondary antibody for 1 h at 37°C. Intracellular lipid droplet accumulation was evaluated using BODIPY 493/503 assay (dilution 0.1 µg/ml in PBS; Thermo Fisher Scientific, Waltham, Massachusetts, USA) incubated for 20 min at RT and nuclei were stained with DAPI (Invitrogen, Carlsbad, California, USA). The images were acquired using confocal microscopy (Leica Microsystem CMs GmBH Type: TCSSP8X) and analyzed with ImageJ software. Intensity fluorescence was normalized on nuclei number.

hiPSC-CM flow cytometry analysis
Cells were dissociated at single cell using Multi Tissue Dissociation Kit 3 (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions and blocked in FACS buffer (PBS containing 0.5% FBS and 2 mM EDTA). Cells were incubated with anti-CD36 for 15 min at 4°C avoiding direct light and then washed and resuspended with FACS buffer. Cardiomyocytes were identified and gated based on their forward and side scatter using the S3Cell Sorter (Bio-Rad, Hercules, California, USA); median fluorescence intensity (MFI) of PE was calculated on the gated cells. Data were analyzed using FlowJo software. Median intensity fluorescence is presented as MFI(PE) sample -MFI(PE) isotype.
ACM murine model: Pkp2+/À mice C57Bl/6 Pkp2 heterozygous knock-out mice (Pkp2+/À) were produced by Prof. Birchmeier, as described (Grossmann et al, 2004). The homozygous mice were embryonic-lethal, while heterozygous mice were healthy and fertile. For our experiments, we used 28 C57Bl/6 Pkp2+/À mice and 28 siblings C57Bl/6 wild type (WT), as control. This number is the result of a power analysis by means of G*Power 3.1.9.2 software. We used a two-way ANOVA test, taking into account the interaction between the differences due to the different strains to be used, and that due to the diets. Thanks to previous results with the same murine model, given the expected differences in cardiac lipid accumulation and the expected intergroup standard deviation, we obtained an effect size f of 0.52. The calculation of sample size showed that˜9 animals/group/treatment are required for an 85% probability of demonstrating differences from the mean, with a P value of 0.05.

Murine C-MSC isolation and culture
Explanted hearts from 5 WT and 5 Pkp2+/À mice (age: 10 weeks) were washed with PBS, cut into 2-3 mm pieces, and incubated at 37°C for 1 h under continuous agitation in IMDM containing 3 mg/ ml collagenase NB4. After a PBS wash, the pellet was resuspended in GM and seeded. Non-adherent cells were removed after 24 h. AM medium was used to prompt the adipogenic differentiation of murine C-MSC for 6 days.
Heart volumes and functionality were assessed before the beginning of the diet and every month by echocardiography. Bodyweight was monitored every month. Blood samples were taken before and after the diet. At sacrifice, hearts were explanted after perfusion with saline solution. Sections of heart specimens were used for lipid accumulation analysis, PPARc, MDA, and CD36 immunofluorescence staining. Protein extracts from total tissue lysates were used to evaluate PPARc, MDA, and CD36 expression through Western blot analysis.

High-fat diet plus atorvastatin
Nine WT and 9 Pkp2+/À mice (age: 10 weeks) were fed for 3 months with a HFD (D12492, Open Source, New Brunswick, New Jersey, USA) to which 20 mg/kg atorvastatin (Khan et al, 2018;Peng et al, 2018) was added. Heart volumes and functionality were assessed before the beginning of the diet and every month by echocardiography. Bodyweight was monitored every month. Blood samples were taken before and after the diet. At sacrifice, hearts were explanted after perfusion with saline solution. Sections of heart specimens were used for lipid accumulation analysis, PPARc, MDA, and CD36 immunofluorescence staining. Protein extracts from total heart tissue lysates were used to evaluate PPARc, MDA, and CD36 expression through Western blot analysis.

Echocardiographic and electrocardiographic analyses
Transthoracic echocardiography was performed using the Vevo2100 high-resolution imaging system (VisualSonics, Toronto, Canada) and a 40-MHz linear transducer with simultaneous electrocardiographic recording, as previously reported (Milano et al, 2014). Analyses were performed on mice lightly anesthetized with 0.5% to 1% isoflurane (heart rate: 480-550 beats/min) at the following timepoints: 1 day before starting the diet (pre-diet) and on 1, 2, and 3 months of CD, HFD or HFD+atorva.
RV parameters in systole and diastole were acquired from a parasternal long-axis view and measured from images acquired in M mode, using the depth interval (in mm) generic measurements tool (Urboniene et al, 2010;Seta et al, 2011;Hansmann et al, 2012).
Two-dimensional short-axis M-mode echocardiography was performed at the level of the midpapillary muscle to measure LV parameters, in systole and diastole.
All measurements were averaged from a minimum of three cycles during diastole and systole corresponding to the electrocardiogram. Data and imaging were analyzed using the VisualSonics Cardiac Measurements Package by a blinded investigator. Parameters were normalized on mice heart weight.
Surface electrocardiographic signal (lead II via limb electrodes) were acquired in all mice during echocardiography (lightly anesthetized mice). CSV files of each mouse physiological data were exported, and analyzed in Excel. Specifically, QRSp (as defined in (Merentie et al, 2015)), terminal activation duration (TAD) and QRS amplitude were measured in at least 10 consecutive beats for each animal by a blinded investigator.

Murine blood sampling
Mice were anesthetized with 4% isoflurane and maintained asleep with 1% isoflurane. The blood sampling was performed through tail vein using 25G needle, after tail pre-heating and local application of anesthetic.
Whole blood was collected into EDTA-coated tubes (Fisher Scientific, Waltham, Massachusetts, USA). Separated plasma was obtained after centrifugation for 15 min at 2,000 g at 4°C and stored at À80°C until the analysis.
We divided each sample into 50 fractions (one every minute), and we collected those from #22 to #45, corresponding to VLDL, IDL, LDL, and HDL. Each obtained fraction (1 ml) was frozen at À80°C, lyophilized, and then reconstituted with 200 ll of water. On portions of these samples, total cholesterol was measured by commercial colorimetric kits (ABX Pentra, Roma, Italy) at 490 nm by a spectrophotometer (Bio-Rad, Hercules, California, USA).

oxLDL in murine plasma samples
For the quantitative determination of oxLDL in murine plasma samples before and after the HFD or HFD+atorva, Mouse Oxidized Low Density Lipoprotein ELISA kit was used (CUSABIO, Houston, Texas, USA), following the manufacturer's instructions. Absorbance was determined with a spectrophotometer (Berthold Technologies, Bad Wildbad, Germany) at 450 nm. Results were inferred according to a calibration curve constructed using the standards provided in the kit.
Histological characterization of Pkp2+/À hearts Explanted hearts from WT and Pkp2+/À mice, in CD, HFD, and HFD+atorva conditions, were fixed in 4% paraformaldehyde (Santa-Cruz, Dallas, Texas, USA) overnight at 4°C. The following day, after three washes in PBS, the hearts were transferred in 15% sucrose in distilled water overday at RT and then in 30% sucrose in distilled water overnight at 4°C. The explanted hearts were embedded in OCT (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and stored at À80°C until use. After sectioning with a cryostat (Thermo Fisher Scientific, Waltham, Massachusetts, USA), OCT embedded sections (6 lm thick) were de-frosted and washed twice in PBS. The immunofluorescence analysis was performed following blocking in 2% goat serum (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 min. Sections were incubated with primary antibodies (see Appendix Table S7)  . Sections were observed with Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each explanted heart, at least 10 fields for five consecutive transversal slices were quantified.
For the lipid accumulation analysis, the sections were stained with ORO (Sigma-Aldrich, St. Louis, Missouri, USA) for 1 h at RT and then washed five times in PBS to ensure the removal of the aspecific dye. Quantitative results were obtained by evaluating red area vs. total tissue area (all the section surface of five consecutive slices for each sample was quantified).
Western blot in murine total heart tissue Total proteins from murine total heart tissue lysates were obtained by Laemmli lysis buffer. After quantification with DC protein assay (Bio-Rad, Hercules, California, USA), proteins were run on SDS-PAGE gel (NUpage precast 4-12%; Invitrogen, Carlsbad, California, USA) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membrane was blocked in 5% skimmed milk-TBS for 1 h at RT and incubated overnight at 4°C with primary antibodies against GAPDH, PPARc, MDA, and CD36 (see Appendix Table S7). After washes, the membrane was incubated for 1 h at RT with HRP-conjugated secondary antibody goat anti-rabbit (GE Healthcare, Chicago, Illinois, USA). Blots were washed and developed with the ECL system (Bio-Rad, Hercules, California, USA). Images were acquired with the Alliance Mini 2 M System (UVITEC, Cambridge, UK), and densitometric analysis was performed using Alliance Mini4 16.07 software (UVITEC, Cambridge, UK). Data are normalized expressing as 1 the comparison group in order to highlight the fold differences between different groups or treatment.

Patient fat infiltration analysis at MRI
Intramyocardial fat mass was evaluated as previously described (Aquaro et al, 2012;Aquaro et al, 2014). Briefly, steady-state free procession (SSFP) were used and the following acquisition parameters were applied: 30 phases, 10-25 views per segment, NEX 1, FOV 40 cm, a matrix of 224 x 224, a 60°flip angle, TR 3.6-4.2 and TE = TR/2. Images were acquired using a 1.5-T unit (Discovery MR450, GE Healthcare, Milwaukee, Minneapolis, USA). Fat in the ventricular myocardium appears as a hyperintense area surrounded by a hypointense band, the so-called chemical shift artifact (Aquaro et al, 2014). Manual contouring of the chemical shift artifact (including the black contour) was drawn, and its extent was measured.

Statistical analysis
Discrete variables were analyzed with Fisher's exact test. Continuous variables were reported as meanAEstandard error. Comparisons between normally distributed groups were performed using either paired or unpaired two-tailed Student's t-tests, whereas populations without a Gaussian distribution were compared using Mann-Whitney tests. The n indicated in each figure legend corresponds to biological replicates. Comparisons among three or more groups were performed with one-way or two-way ANOVA test, in association with Bonferroni multiple comparison post-tests. When both intragroup and intergroup values are tested, red lines and asterisks identify test differences within the same group, while intergroup differences are in black. X-Y correlation analyses have been determined. Comparison of slopes of linear regressions was performed with the following method: t = (b1-b2) / sb1,b2 where b1 and b2 are the two slope coefficients and sb1,b2 the pooled standard error of the slope. Receiver operating characteristic (ROC) plot was used to determine the oxLDL level cutoff value minimizing the difference between sensitivity and specificity in discriminating ACM patients vs. HC individuals. Kaplan-Meier curves were performed to determine the actual risk of MAE and analyzed through log-rank (Mantel-Cox) test. Statistics were performed using GraphPad Prism software. Results were considered statistically significant for P values < 0.05 (see Appendix Table S8 for the details). Power analyses, for both in vitro and in vivo experiments, were performed using G*Power 3.1.9.2 software.

The Paper Explained
Problem Arrhythmogenic cardiomyopathy (ACM) is a genetically determined heart condition. It is hallmarked by a gradual fibro-adipose replacement of the ventricular myocardium, heart failure, malignant arrhythmias, and sudden death. Few information is available regarding the mechanisms mediating the phenotypic variability among carriers of the same ACM mutation. In addition, no pharmacological approaches are available in the clinical practice to counteract cardiac adipogenic substitution.

Results
We demonstrated that oxLDL increase ACM adipogenesis through a mechanism implying over-activation of PPARc, the main effector of lipid accumulation. This novel pathogenic mechanism was investigated in patients and verified with in vitro experiments on two cardiac cell types and in vivo studies. Importantly, antioxidants and atorvastatin treatments counteracted ACM phenotypes.

Impact
This study adds a piece to the puzzle of phenotypic variability among carriers of the same ACM mutation. Increased oxLDL plasma levels may represent both a precision medicine tool to identify patients at high disease burden and a target of therapeutic approaches. A proof of principle is given of the efficacy of a new treatment with atorvastatin and antioxidants in counteracting oxidized lipid-dependent cardiac adipogenesis and related dysfunction. Future drug repositioning studies will compare the efficacy of such treatments in mitigating ACM phenotypes.

Data availability
This study includes no datasets deposited in external repositories.
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