Circulating mitochondria carrying cGAS promote endothelial Secreted group IIA phospholipase A2-mediated neuroinflammation through activating astroglial/microglial Integrin-alphavbeta3 in subfornical organ to augment central sympathetic overdrive in heart failure rats

Abstract

Background

Sympathoexcitation, a manifestation of heart–brain axis dysregulation, contributes to the progression of heart failure (HF). Our recent study revealed that circulating mitochondria (C-Mito), a newly identified mediator of multi-organ communication, promote sympathoexcitation in HF by aggravating endothelial cell (EC)-derived neuroinflammation in the subfornical organ (SFO), the cardiovascular autonomic neural center. The precise molecular mechanism by which C-Mito promotes SFO-induced endothelial neuroinflammation has not been fully elucidated.

Objective

C-Mito carrying cGAS promote sympathoexcitation by targeting PLA2G2A in ECs of the SFO in HF rats.

Methods

Male Sprague–Dawley (SD) rats received a subcutaneous injection of isoprenaline (ISO) at a dosage of 5 mg/kg/day for seven consecutive days to establish a HF model. C-Mito were isolated from HF rats and evaluated. The level of cGAS, a dsDNA sensor recently discovered to be directly localized on the outer membrane of mitochondria, was detected in C-Mito. C-Mito from HF rats (C-Mito^HF) or control rats (C-Mito^Ctrl) were intravenously infused into HF rats. The accumulation of C-Mito in the ECs in the SFO was detected via double immunofluorescence staining. The SFO was processed for RNA sequencing (RNA-Seq) analysis. Secreted group IIA phospholipase A2 (PLA2G2A), the key gene involved in C-Mito^HF-associated SFO dysfunction, was identified via bioinformatics analysis. Upregulation of PLA2G2A in the SFO ECs was assessed via immunofluorescence staining and immunoblotting, and PLA2G2A activity was evaluated. The interaction between cGAS and PLA2G2A was detected via co-immunoprecipitation. The dowstream molecular mechanisms of which PLA2G2A induced astroglial/microglial activation were also investigated. AAV9-TIE-shRNA (PLA2G2A) was introduced into the SFO to specifically knockdown endothelial PLA2G2A. Neuronal activation and glial proinflammatory polarization in the SFO were also evaluated. Renal sympathetic nerve activity (RSNA) was measured to evaluate central sympathetic output. Cardiac sympathetic hyperinnervation, myocardial remodeling, and left ventricular systolic function were assessed in C-Mito-treated HF rats.

Results

Respiratory functional incompetence and oxidative damage were observed in C-Mito^HF compared with C-Mito^Ctrl. Surprisingly, cGAS protein levels in C-Mito^HF were significantly higher than those in C-Mito^Ctrl, while blocking cGAS with its specific inhibitor, RU.521, mitigated respiratory dysfunction and oxidative injury in C-Mito^HF. C-Mito entered the ECs of the SFO in HF rats. RNA sequencing revealed that PLA2G2A is a key molecule for the induction of SFO dysfunction by C-Mito^HF. The immunoblotting and immunofluorescence results confirmed that, compared with C-Mito^Ctrl, C-Mito^HF increased endothelial PLA2G2A expression in the SFO of HF rats, which could be alleviated by attenuating C-Mito^HF-localized cGAS. Furthermore, we found that cGAS directly interacts with PLA2G2A, increased the activity of PLA2AG2, which produced arachidonic acid, and also promoted PLA2G2A secretion in brain ECs. In addition, the inhibition of PLA2G2A in brain ECs significantly mitigated the proinflammatory effect of conditioned cell culture medium from C-Mito^HF-treated ECs on astroglia and microglia. Also, we found that PLA2G2A secreted from ECs insulted by C-Mito induced neuroinflammation through activating astriglial/microglial Integrin-alphavbeta3 in the SFO, which further promote central sympathetic overdrive in HF rats. Specific knockdown of endothelial PLA2G2A in the SFO mitigated C-Mito^HF-induced presympathetic neuronal sensitization, cardiac sympathetic hyperinnervation, RSNA activation, myocardial remodeling, and systolic dysfunction in HF rats.

Conclusion

C-Mito carrying cGAS promoted cardiac sympathoexcitation by directly targeting PLA2G2A in the ECs of the SFO in HF rats. Secreted PLA2G2A derived from ECs insulted by C-Mito induced neuroinflammation through activating astriglial/microglial Integrin-alphavbeta3 in the SFO, which further promote central sympathetic overdrive in HF rats. Our study indicated that inhibiting cGAS in C-Mito might be a potential treatment for central sympathetic overdrive in HF.