Arrhythmogenic Cardiomyopathy (ACM) is a genetic condition hallmarked by ventricular fibro-fatty replacement and arrhythmias. Cardiac mesenchymal stromal cells (C-MSC) differentiate into adipocytes in ACM hearts, through the activation of PPARγ, caused by ACM mutations (e.g. PKP2). The clinical phenotype of ACM is variable for poorly understood reasons. The only recognized cofactor is physical exercise, which is known to increases oxidative stress. An accepted marker of exercise-induced oxidative stress is 13HODE, a component of oxLDL and direct activator of PPARγ. In macrophages, during foam cell formation, 13HODE creates a feed-forward loop increasing both PPARγ and the oxLDL receptor CD36, resulting in fat accumulation. To investigate oxLDL effects on ACM adipogenesis and to dissect the involved pathways. We analyzed plasmas (n=42) and ventricular tissues (n=4) of ACM patients and matched healthy controls (HC). For in vitro experiments, ACM and HC C-MSC (n=10) have been used, while in vivo experiments have been conducted in heterozygous Pkp2 knock-out mice (Pkp2+/−; n=10). We observed higher plasma oxLDL in ACM patients compared to HC (ACM 246.70±55.89 vs HC 102.5±17.95ng/ml; p=0.019). oxLDL levels also discriminate between ACM patients with overt phenotype and their unaffected relatives carriers of the same causative mutations (p=0.03). We observed higher oxidative stress (MDA intensity 40.87±11.76 fold; p=0.015) and CD36 levels (14.72±2.10 fold; p=0.0007) in ACM ventricular tissue, compared to HC. In basal conditions, ACM C-MSC showed greater oxidative stress (MDA intensity 8.83±2.78 fold p=0.017) and higher expression of PPARγ (1.47±0.14 fold; p=0.009) compared to HC C-MSC. The adipogenic stimulation led to a parallel increase of CD36 and lipid accumulation, mainly in ACM C-MSC (slopes statistically different p=0.016). OxLDL and 13HODE administration increased lipid accumulation in ACM C-MSC (ORO staining ACM vs ACM+oxLDL p=0.01; ACM vs ACM+13HODE p=0.014). On the contrary, the antioxidant N-Acetylcysteine (NAC) prevented lipid accumulation in ACM C-MSC (ORO staining ACM+13HODE vs ACM+13HODE+NAC p=0.0009). Through CD36 silencing of ACM C-MSC, we obtained a significantly lower lipid accumulation than non-silenced cells (ORO staining 0.35±0.10 fold; p=0.003). Pkp2+/− mice do not spontaneously accumulate adipocytes in the heart, however Pkp2+/− C-MSC are more prone to lipid accumulation in vitro than WT cells (p=0.007). Accordingly, mice have low plasma oxLDL and cardiac oxidative stress. By increasing plasma cholesterol and oxidative stress through high fat diet, we observed fibro-fatty substitution in Pkp2+/− hearts (p=0.046). Figure 1 These findings reveal a modulatory role of oxidized lipids in ACM adipogenesis at a cellular, tissue and clinical level, enlightening novel targets for pharmacological strategies to prevent adipogenic substitution and consequent ACM clinical phenotypes. Telethon Foundation; Italian Ministry of Health

Background: Anthracyclines, like doxorubicin (DOX), are among the most potent antitumor drugs.However, their clinical use is hampered by severe cardiotoxicity.We previously demonstrated that inhibition of phosphoinositide 3-kinase c (PI3Kc) protects against anthracyclineinduced cardiomyopathy (manuscript under revision), but the underlying molecular mechanisms are still unexplored.Purpose: Here, we further test the hypothesis that anthracycline-damaged mitochondria activate Toll-like receptor 9 (TLR9)/PI3Kc signaling, which in turn inhibits protective autophagy, thus exacerbating anthracycline cardiotoxicity.Methods: Neonatal cardiomyocytes (NCMs) were isolated from mice expressing a kinase inactive PI3Kc (PI3Kc kinase-dead; KD) and wild-type (WT) controls, and treated with DOX (1 mM) or TLR9 agonist ODN1826 (1 mg/ml) 6 PI3Kc inhibitor AS605240 (500 nM) or TLR9 antagonist ODN2088 (1 mg/ml), for 1 hour before analyzing Akt/mTOR/Ulk-1 signaling.For in vivo studies, WT and KD mice were treated with 4 mg/kg DOX weekly for 3 weeks.Cardiac function was analyzed with echocardiography 6 weeks after the first injection.Electron microscopy (EM) study of morphology and signaling transduction were studied 3 days after the treatment.To investigate the role of protective autophagy in KD hearts, mice were treated with hydroxychloroquine (HCQ) or AAV9-shATG7, which silences the autophagy regulator ATG7 specifically in cardiomyocytes, together with DOX, as described above.Results: In NCMs, DOX significantly increased the phosphorylation of PI3K downstream targets and autophagy inhibitors, Akt, mTOR and Ulk-1.These effects were completely prevented by the TLR9 antagonist ODN2088, the PI3Kc selective inhibitor AS605240 and genetic PI3Kg inactivation (KD NCMs).Notably, the TLR9 agonist ODN1826, mimicking mitochondrial DNA (mitoDNA), similarly upregulated Akt/mTOR/Ulk-1 signaling in WT but not in KD NCMs.These results suggest that DOX activates PI3Kc through mitoDNA/TLR9.In vivo, EM studies showed more abundance of autolysosomes containing injured mitochondria in KD than WT hearts, highlighting the efficient autophagydependent disposal of DOX-damaged mitochondria in KD mice.Enhanced activation of PI3Kgdependent pathway correlated with a significant blunt of autophagy in DOX-treated hearts, whereas inhibition of PI3Kc promoted autophagy and decreased DOX-induced contractile dysfunction.Finally, inhibition of autophagy by HCQ or AAV9-shATG7 erased the protection in KD mice.Conclusion: Overall, this study demonstrates that PI3Kc prevents autophagic disposal of DOXdamaged mitochondria, resulting in cardiomyopathy.We propose PI3Kc inhibition as a novel strategy to reactivate targeted autophagy and limit cancer therapy-related heart disease.

Arrhythmogenic cardiomyopathy (AC) is characterized by progressive fibro-fatty replacement of the myocardium, arrhythmias and sudden death in young athletes. Due to a desmosomal gene mutation, AC patients present an autosomal-dominant trait of inheritance, with incomplete penetrance and variable expressivity, suggesting the involvement of possible co-factors determining the pathological phenotype.

Shear stress (SS), the tangential component of hemodynamic forces, modulates the expression of several genes in endothelial cells. However, no information is available about its effect on chromatin structure, which plays a key role in gene transcription. In this study, a link between SS and chromatin remodeling was established in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to SS of 10 dyne/cm(2) per second, in the presence or absence of the histone deacetylase inhibitor trichostatin A, and assayed for histone H3 and histone H4 modifications. SS induced histone H3 serine phosphorylation at position 10 (S10) and lysine acetylation at position 14 (K14) but required trichostatin A to induce H3 phosphoacetylation and H4 acetylation. The phosphatidylinositol 3-kinase inhibitor wortmannin and the mitogen-activated protein kinase inhibitor PD98059 decreased SS-dependent histone H3 phosphorylation, without affecting its acetylation; the p38 inhibitor SB203580 reduced both H3 phosphorylation and acetylation, whereas the protein kinase A inhibitor PKI-tide reduced histone H3 acetylation. Remarkably, the abrogation of histone acetylation inhibited SS-dependent c-fos expression. SS also activated ribosomal S6 kinase-2 and mitogen- and stress-activated kinase-1 protein kinases and promoted the formation of a cAMP-responsive element-binding protein (CREB)/CREB-binding protein complex, providing the molecular basis for the increase in histone acetyltransferase activity observed in HUVECs exposed to SS. Finally, the effect of SS on chromatin remodeling was examined. In HUVECs exposed to SS, chromatin within c-fos and c-jun promoters was specifically immunoprecipitated by an antibody against acetylated histone H3 on K14. These results indicate that SS induces posttransduction modifications of histones; this is an early step toward the flow-dependent regulation of gene expression.