Cardiac sympathetic neurons are additional cells affected in genetically determined arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes, which accounts for most cases of stress-related arrhythmic sudden death, in young and athletes. AC hearts display fibro-fatty lesions that generate the arrhythmic substrate and cause contractile dysfunction. A correlation between physical/emotional stresses and arrhythmias supports the involvement of sympathetic neurons (SNs) in the disease, but this has not been confirmed previously. Here, we combined molecular, in vitro and ex vivo analyses to determine the role of AC-linked DSG2 downregulation on SN biology and assess cardiac sympathetic innervation in desmoglein-2 mutant (Dsg2mut/mut) mice. Molecular assays showed that SNs express DSG2, implying that DSG2-mutation carriers would harbour the mutant protein in SNs. Confocal immunofluorescence of heart sections and 3-D reconstruction of SN network in clarified heart blocks revealed significant changes in the physiologialc SN topology, with massive hyperinnervation of the intact subepicardial layers and heterogeneous distribution of neurons in fibrotic areas. Cardiac SNs isolated from Dsg2mut/mut neonatal mice, prior to the establishment of cardiac innervation, show alterations in axonal sprouting, process development and distribution of varicosities. Consistently, virus-assisted DSG2 downregulation replicated, in PC12-derived SNs, the phenotypic alterations displayed by Dsg2mut/mut primary neurons, corroborating that AC-linked Dsg2 variants may affect SNs. Our results reveal that altered sympathetic innervation is an unrecognized feature of AC hearts, which may result from the combination of cell-autonomous and context-dependent factors implicated in myocardial remodelling. Our results favour the concept that AC is a disease of multiple cell types also hitting cardiac SNs. KEY POINTS: Arrhythmogenic cardiomyopathy is a genetically determined cardiac disease, which accounts for most cases of stress-related arrhythmic sudden death. Arrhythmogenic cardiomyopathy linked to mutations in desmoglein-2 (DSG2) is frequent and leads to a left-dominant form of the disease. Arrhythmogenic cardiomyopathy has been approached thus far as a disease of cardiomyocytes, but we here unveil that DSG2 is expressed, in addition to cardiomyocytes, by cardiac and extracardiac sympathetic neurons, although not organized into desmosomes. AC-linked DSG2 downregulation primarily affect sympathetic neurons, resulting in the significant increase in cardiac innervation density, accompanied by alterations in sympathetic neuron distribution. Our data supports the notion that AC develops with the contribution of several 'desmosomal protein-carrying' cell types and systems.

Sympathetic neuropathology is revealed in muscles affected by amyotrophic lateral sclerosis.

Rationale: The anatomical substrate of skeletal muscle autonomic innervation has remained underappreciated since it was described many decades ago. As such, the structural and functional features of muscle sympathetic innervation are largely undetermined in both physiology and pathology, mainly due to methodological limitations in the histopathological analysis of small neuronal fibers in tissue samples. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease which mainly targets motor neurons, and despite autonomic symptoms occurring in a significant fraction of patients, peripheral sympathetic neurons (SNs) are generally considered unaffected and, as such, poorly studied. Purpose: In this research, we compared sympathetic innervation of normal and ALS muscles, through structural analysis of the sympathetic network in human and murine tissue samples. Methods and Results: We first refined tissue processing to circumvent methodological limitations interfering with the detection of muscle sympathetic innervation. The optimized "Neuro Detection Protocol" (NDP) was validated in human muscle biopsies, demonstrating that SNs innervate, at high density, both blood vessels and skeletal myofibers, independent of the fiber metabolic type. Subsequently, NDP was exploited to analyze sympathetic innervation in muscles of SOD1G93A mice, a preclinical ALS model. Our data show that ALS murine muscles display SN denervation, which has already initiated at the early disease stage and worsened during aging. SN degeneration was also observed in muscles of MLC/SOD1G93A mice, with muscle specific expression of the SOD1G93A mutant gene. Notably, similar alterations in SNs were observed in muscle biopsies from an ALS patient, carrying the SOD1G93A mutation. Conclusion: We set up a protocol for the analysis of murine and, more importantly, human muscle sympathetic innervation. Our results indicate that SNs are additional cell types compromised in ALS and suggest that dysfunctional SOD1G93A muscles affect their sympathetic innervation.