A simple and accurate method to quantify real-time contraction of vascular smooth muscle cell in vitro.

Vascular smooth muscle cells (VSMCs) constitute the medial layer of the blood vessel wall. Their contractile state regulates blood flow in physiological and pathological conditions. Current methods for assessing the contractility of VSMCs are not amenable to the high-throughput screening of pharmaceutical compounds. This study aimed to develop a method to address this shortcoming in the field. Real-time contraction was visualized in living VSMCs using the exogenous expression of green fluorescent protein (GFP). Image-Pro Plus software (IPPS) was used to measure various morphological cell indices. In phenylephrine-treated VSMCs, GFP fluorescence imaging was more accurate than brightfield imaging or phalloidin staining in representing VSMC morphology, as measured using IPPS. Among the multiple indices of VSMC shape, area and mean-diameter were more sensitive than length in reflecting the morphological changes in VSMC. We developed a new index, compound length, by combining the mean-diameter and length to differentiate contracted and uncontracted VSMCs. Based on the compound length, we further generated a contraction index to define a single-VSMC contractile status as single-VSMC contraction-index (SVCI). Finally, compound length and SVCI were validated to effectively assess cell contraction in VSMCs challenged with U46619 and KCl. In conclusion, GFP-based indices of compound length and SVCI can accurately quantify the real-time contraction of VSMCs. In future, the new method will be applied to high-throughput drug screening or basic cardiovascular research.

ATP6AP2 knockdown in cardiomyocyte deteriorates heart function via compromising autophagic flux and NLRP3 inflammasome activation.

Moderate autophagy can remove damaged proteins and organelles. In some inflammatory diseases, autophagy plays a protective role by inhibiting the NOD-like receptor family pyrin domain containing 3(NLRP3). (Pro)renin receptor (PRR, or ATP6AP2) is a critical component of the V-ATPase required for autophagy. It remains controversial about ATP6AP2 in the pathological process. The impact of ATP6AP2 on NLRP3 inflammasome and autophagic flux remains unknown under pressure overload stress. This research explores the potential link between ATP6AP2, autophagic flux, and NLRP3. There was upregulation of ATP6AP2 from 5-day post-TAC, and this expression remained at a high level until 8-weeks post-TAC in wild mice. Meanwhile, autophagic flux switched from early compensatory activation to blocking in the heart failure phase. NLRP3 activation can be seen at 8-week post-TAC. Adenovirus-mediated knockdown of ATP6AP2(shR-ATP6AP2) accelerated the progress of heart failure. After TAC was induced, shR-ATP6AP2 significantly deteriorated heart function and fibrosis compared with the shR-Scr group. Meanwhile, there was an elevated expression of NLRP3 and autophagic flux blockage. A transgenic mouse(Tg) with cardio-restricted ATP6AP2/(P)RR overexpression was constructed. Although high expression in cardiac tissue, there were no spontaneous functional abnormalities under the basal state. Cardiac function, fibrosis, hypertrophy remained identical to the control TAC group. However, SQSTM1/P62 was reduced, which indicated the relief of autophagic flux blockage. Further, Neonatal rat ventricular myocyte (NRVMs) transfected with shR-ATP6AP2 showed more susceptibility than sh-Scr NRVMs to phenylephrine-induced cell death. More reactive oxygen species (ROS) or mito-ROS accumulated in the shR-ATP6AP2 group when phenylephrine stimulation. Blocking NLRP3 activation in vivo partly rescued cardiac dysfunction and fibrosis. In conclusion, ATP6AP2 upregulation is a compensatory response to pressure overload. If not effectively compensated, it compromises autophagic flux, leads to dysfunctional mitochondria accumulation, further produces ROS to activate NLRP3, eventually accelerates heart failure.