Division of Medical Genetics and Genomics, The Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310058, China.
Institute of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China.
Int J Mol Sci. 2023 Jun 28;24(13):10800. doi: 10.3390/ijms241310800.
Autophagy has stabilizing functions for cardiomyocytes. Recent studies indicate that an impairment in the autophagy pathway can seriously affect morphology and function, potentially leading to heart failure. However, the role and the underlying mechanism of the endosomal sorting complex required for transport (ESCRT) family protein, in particular the AAA-ATPase vacuolar protein sorting 4a (Vps4a), in regulating myocardial autophagy remains unclear. In the present study, cardiomyocyte-specific Vps4a knockout mice were generated by crossing () with transgenic mice. As a result, we observed a partially dilated left ventricular (LV) chamber, a significant increase in heart weight to body weight ratio (HW/BW), and heart weight to tibial length ratio (HW/TL), hypertrophic cardiomyopathy and early lethality starting at 3 months of age. Hematoxylin-eosin (HE), immunofluorescence assay (IFA), and Western blot (WB) revealed autophagosome accumulation in cardiomyocytes. A transcriptome-based analysis and autophagic flux tracking by AAV-RFP-GFP-LC3 showed that the autophagic flux was blocked in Vps4a knockout cardiomyocytes. In addition, we provided in vitro evidence demonstrating that Vps4a and LC3 were partially co-localized in cardiomyocytes, and the knockdown of Vps4a led to the accumulation of autophagosomes in cardiomyocytes. Similarly, the transfection of cardiomyocytes with adenovirus (Adv) mCherry-GFP-LC3 further indicated that the autophagic flux was blocked in cells with deficient levels of Vps4a. Finally, an electron microscope (EM) showed that the compromised sealing of autophagosome blocked the autophagic flux in Vps4a-depleted cardiomyocytes. These findings revealed that Vps4a contributed to the sealing of autophagosomes in cardiomyocytes. Therefore, we demonstrated that Vps4a deletion could block the autophagic flux, leading to the accumulation of degradation substances and compromised cardiac function. Overall, this study provides insights into a new theoretical basis for which autophagy may represent a therapeutic target for cardiovascular diseases.
自噬对心肌细胞具有稳定作用。最近的研究表明，自噬途径的损伤会严重影响形态和功能，可能导致心力衰竭。然而，内体分选复合物必需的运输蛋白（ESCRT）家族蛋白，特别是 AAA-ATP 酶液泡蛋白分选 4a（Vps4a），在调节心肌自噬中的作用和潜在机制尚不清楚。在本研究中，通过将（）与转（）基因小鼠杂交，生成了心肌细胞特异性 Vps4a 敲除小鼠。结果，我们观察到左心室（LV）腔部分扩张，心重/体重比（HW/BW）和心重/胫骨长度比（HW/TL）显著增加，肥厚型心肌病和早发性死亡始于 3 月龄。苏木精-伊红（HE）、免疫荧光分析（IFA）和 Western blot（WB）显示心肌细胞中自噬体积累。基于转录组的分析和 AAV-RFP-GFP-LC3 的自噬流追踪显示，Vps4a 敲除心肌细胞中的自噬流被阻断。此外，我们提供了体外证据表明 Vps4a 和 LC3 在心肌细胞中部分共定位，并且 Vps4a 的敲低导致心肌细胞中自噬体的积累。同样，用腺病毒（Adv）mCherry-GFP-LC3 转染心肌细胞进一步表明，Vps4a 水平降低的细胞中自噬流被阻断。最后，电子显微镜（EM）显示，自噬体封闭的缺陷导致 Vps4a 耗尽的心肌细胞中自噬流被阻断。这些发现表明 Vps4a 有助于心肌细胞中自噬体的封闭。因此，我们证明了 Vps4a 的缺失可以阻断自噬流，导致降解物质的积累和心脏功能受损。总的来说，这项研究为自噬可能成为心血管疾病治疗靶点提供了新的理论依据。
