Cholinergic nerve regulation of heart regeneration

Background Cardiac nerves regulate many important physiological functions of the heart such as heart rate and contractility. The emerging role of cardiac nerves during tissue homeostasis and regeneration is beginning to be appreciated. We discovered that neonatal mice are capable of regenerating their hearts following injury within a brief period after birth by proliferation of the pre-existing cardiomyocytes. Furthermore, we have demonstrated that cholinergic nerves play an important role in guiding the neonatal heart regenerative response. However, the adult mammalian heart, including the human heart, is incapable of regeneration following injury. Thus, there is great excitement about understanding the evolutionarily conserved mechanisms of endogenous cardiac regeneration, so that we can explore potential avenues to reawaken this process in adult humans. Purpose Our overarching goal is to define the mechanisms by which cholinergic nerves regulate heart regeneration following ischemic injury by using the neonatal mouse heart regeneration model. These studies will uncover novel pathways by which cholinergic signaling promotes cardiomyocyte proliferation and heart regeneration, which holds significant therapeutic potential for treatment of adult heart disease. Methods In this project, we employed genetically engineered mouse models of the critical receptors for cholinergic signaling in the heart to define the mechanisms of cholinergic nerve regulation of heart regeneration. First, we generated a cardiomyocyte-specific deletion of the muscarinic receptor (M2), the most predominant muscarinic receptor subtype present in the heart. In addition, we utilized the α7 nicotinic receptor (Chrna7) knockout mice to study the role of Chrna7 in endogenous immune cells, which is the main mediator of the cholinergic anti-inflammatory pathway. These mouse models will address how cholinergic nerves regulate heart regeneration via the M2 muscarinic receptor signaling and the inflammatory response following injury. Results Our results demonstrate that inhibition of two different cholinergic receptors (muscarinic and nicotinic) results in a reduction in cardiomyocyte proliferation and inhibition of the neonatal cardiac regenerative response following injury. More importantly, we demonstrate that cholinergic signaling mediates the cardiac regenerative response mainly through suppression of pro-inflammatory cytokines via the cholinergic anti-inflammatory pathway. Conclusions Cholinergic nerve signaling plays an important role in mounting a robust cardiac regenerative response following injury. These results have significant therapeutic potential, which will forge new paradigms with respect to the role of cardiac nerves during mammalian cardiac regeneration and reveal potential mechanisms regarding the benefits of nerve stimulation following cardiac injury in humans. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): American Heart Association, Wisconsin Partnership Program

Lack of mucosal cholinergic innervation is associated with increased risk of enterocolitis in Hirschsprung’s disease

Background & AimsHirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric nervous cells (ganglia). The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates extramural innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown.MethodsWe enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal cholinergic innervation using immunohistochemistry. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using quantitative reverse-transcribed real-time polymerase chain reaction (qRT-PCR) of whole colonic tissue and fluorescence-activated cell sorting (FACS) analysis of isolated colonic immune cells. Fiber-associated immune cells were identified using confocal immunofluorescence microscopy and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic patient tissue using 16S rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation.ResultsWe provided evidence that extrinsic mucosal innervation correlated with reduced interleukin (IL)-17 cytokine levels and T-helper-17 (Th17) cell frequencies. Bipolar CD14high macrophages colocalized with neurons and expressed significantly less interleukin-23, a Th17-promoting cytokine. HSCR patients lacking mucosal cholinergic nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis.ConclusionThe mucosal fiber phenotype might serve as a new prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new future therapeutic options. (www.clinicaltrials.gov accessing number NCT03617640)