Intrinsic and extrinsic factors affect cardiac sympathetic innervation

Project #1. Impairment of TrkA/NGF signalling affects cardiac sympathetic innervation in ADOA patients. Rationale: Heart activity is finely tuned by cardiac sympathetic neurons (cSNs) whose function and viability strictly depend on mitochondrial function. OPA1 affects mitochondrial dynamics, ATP production and apoptosis, and its haploinsufficiency causes Autosomal Dominant Optic Atrophy (DOA), a neurodegenerative disease characterized by retinal ganglion cell death and visual loss. Interestingly, DOA patients also display peripheral neuropathy and cardiac rhythm abnormalities, prompting to test the hypothesis that OPA1-deficient mitochondria may also affect cSNs. Methods/Results: SNs are affected in DOA, as shown by significant decrease in SN density in skin biopsies from DOA patients, compared to controls. To isolate the effect of DOA genetics on SNs, we generated a mouse model with SNs selective OPA1 haploinsufficiency (TH-OPA1+/- mice). These mice had, as expected, intact heart morphology and contractile function, but SN mitochondria were altered in dynamics/structure. In support of SN abnormalities, ECG-telemetry showed decreased HRV and increased arrhythmia incidence, which were exacerbated during ageing. Consistently, confocal IF and multiphoton imaging of clarified hearts revealed reduced cSN density in adult TH-OPA1+/- mice, which worsened during ageing, and was accompanied to arrhythmogenic alterations in SN distribution and morphology. In vitro assays in PC12-derived SNs, and cSNs from TH-OPA1+/- mice, demonstrated that reduction in OPA1 content was sufficient to affect SNs. Molecular and functional assays attributed these effects to impairment of NGF/TrkA signaling, which increased neuronal susceptibility to NGF withdrawal, a condition mimicking the reduction of cardiac neurotrophin, occurring during ageing. Conclusions: OPA1 reduction impacts on cSN homeostasis, by impinging on neurotrophin signaling. Our data indicates that OPA1 haplo-insufficiency also affects peripheral innervation, thus explaining additional symptoms experienced by DOA patients. Project #2. Circulating muscle-derived miR-206 links skeletal muscle dysfunction to heart sympathetic denervation. Purpose: Skeletal muscle-specific ablation of the autophagy-related protein Atg7, in MLC-Atg7-/- mice, causes block of autophagy, sarcopenia and destabilization of the neuromuscular junction (NMJ). Loss of Atg7 also increases the release of extracellular vesicles (ECVs), enriched in the muscle-specific microRNA, miR206, which was found elevated in the plasma and heart of MLC-Atg7-/- mice. Since miR-206 it not expressed in the heart, in physiologic conditions, we assessed its effect on cardiac homeostasis. Methods/Results: Standard histology in heart sections from MLC-Atg7-/- mice did not reveal morphological alterations. However, IF staining and 3-D reconstruction of the neuronal network in heart blocks showed SN fragmentation and reduced SN density. Consistent with SN denervation, heart rate increase, upon atropine administration, was reduced in MLC-Atg7-/- mice, which also showed arrhythmias. Similar results were obtained in normal mice injected with ECVs carrying miR-206, supporting the primary role of this miR in affecting heart neurons. Consistently, ECV-carried miR-206 directly targets cultured cardiac SNs, leading to morphological signs of neurodegeneration. Bioinformatics, luciferase and molecular/biochemical assays indicate that the neuronal effects are due to miR-206 downregulating the low-affinity NGF receptor, p75. This impairs formation of p75/TrkA heterodimers, which alters pro-survival NGF transport, resulting in SN degeneration. Notably, these results were confirmed in vivo.Conclusions: Our results indicate that a primary muscle defect leads to release, into the bloodstream, of miR-206 which may affect long-distance tissues, such as the myocardium. In addition, we uncover a novel target of miR-206, affecting cardiac innervation.