Perfluorooctanoic acid-induced toxicities in chicken embryo primary cardiomyocytes: Roles of PPAR alpha and Wnt5a/Frizzled2

Perfluorooctanoic acid (PFOA) is a widespread persistent organic pollutant and may induce developmental toxicities, including developmental cardiotoxicity. To explore the potential mechanism of developmental cardiotoxicity induced by PFOA exposure, chicken embryo primary cardiomyocytes were extracted either from chicken embryos pretreated with PFOA (2 mg/kg), or from untreated embryos and then directly exposed cells to PFOA (1, 10, 30 or 100 mu g/ml) in culture. Additionally, peroxisome proliferator activated receptor alpha (PPAR alpha) silencing lentivirus was applied to the embryos on embryonic day (ED2). Cell viability was measured with CCK-8 kit, morphology was assessed with hematoxylin and eosin staining, and intracellular Ca2+ concentrations were determined with Fluo-4 AM probe. Western blotting was utilized to confirm PPAR alpha silencing efficiency and the protein abundance of Wnt5a and Frizzled2. The results indicated that both PFOA pretreatment and direct exposure decreased primary cardiomyocyte viability, altered cell morphology and increased intracellular Ca2+ concentrations. While L-carnitine co-treatment effectively abolished such changes, PPAR alpha silencing only abolished most of the changes in PFOA pretreatment group, but not in cells directly exposed to relatively high doses of PFOA. The protein abundance of Wnt5a and Frizzled2 was increased by PFOA pretreatment, while direct exposure to PFOA increased Frizzled2 abundance but decreased Wnt5a abundance. PPAR alpha silencing resulted in over 50% decrease of PPAR alpha expression level, which abolished the Wnt5a/Frizzled2 expression alterations following PFOA exposure. In conclusion, PFOA-induced primary cardiomyocyte toxicity is associated with PPAR alpha and Wnt5a/Frizzled2, in which PPAR alpha seems to play regulatory roles towards Wnt5a/Frizzled2.

全氟辛酸（PFOA）是一种广泛存在的持久性有机污染物，可能诱发发育毒性，包括发育性心脏毒性。为探究PFOA暴露诱发发育性心脏毒性的潜在机制，从经PFOA（2mg/kg）预处理的鸡胚或未经处理的鸡胚中提取鸡胚原代心肌细胞，然后在培养过程中将细胞直接暴露于PFOA（1、10、30或100μg/ml）。此外，在胚胎期第2天（ED2）向胚胎施用过氧化物酶体增殖物激活受体α（PPARα）沉默慢病毒。用CCK - 8试剂盒测定细胞活力，用苏木精 - 伊红染色评估细胞形态，用Fluo - 4 AM探针测定细胞内Ca²⁺浓度。采用蛋白质印迹法确认PPARα沉默效率以及Wnt5a和Frizzled2的蛋白丰度。结果表明，PFOA预处理和直接暴露均降低了原代心肌细胞活力，改变了细胞形态并增加了细胞内Ca²⁺浓度。虽然左旋肉碱共同处理有效地消除了这些变化，但PPARα沉默仅消除了PFOA预处理组中的大部分变化，而在直接暴露于相对高剂量PFOA的细胞中则未消除。PFOA预处理使Wnt5a和Frizzled2的蛋白丰度增加，而直接暴露于PFOA使Frizzled2丰度增加但使Wnt5a丰度降低。PPARα沉默导致PPARα表达水平降低50%以上，这消除了PFOA暴露后Wnt5a/Frizzled2的表达变化。总之，PFOA诱导的原代心肌细胞毒性与PPARα和Wnt5a/Frizzled2有关，其中PPARα似乎对Wnt5a/Frizzled2起调节作用。