Abstract
The development of sympathetic innervation is crucial for proper cardiac structure and function [1, 2, 3]. Disruptions to this innervation have been implicated in numerous cardiovascular diseases [2, 3, 6, 13, 16, 17]. Previous work suggests that the spontaneously hypertensive rat (SHR), a useful model of human essential hypertension, demonstrates increased sympathetic fiber growth to the heart as compared to its normotensive counterpart, the Wistar Kyoto (WKY) rat [29]. This sympathetic hyperinnervation precedes hypertension onset in SHRs [Kreipke, Huang, and Birren, unpublished], but its molecular mechanisms are unresolved. We probed the interaction between the three fundamental components of the sympatho-cardiac system—satellite glia (SGs), postganglionic sympathetic neurons, and cardiomyocytes—in the establishment of sympathetic fiber growth to the heart. SGs and sympathetic neurons were isolated from the superior cervical ganglia of neonatal WKY and SHR animals and co-cultured with ventricular myocytes for a period of four days in vitro. Fiber density in contact with myocytes was quantified. The sympathetic innervation density of SHR myocytes was significantly greater than that of the WKY, suggesting cardiomyocytes as cellular drivers of sympathetic hyperinnervation. Addition of satellite glia or glial-conditioned media (GCM) to neuron-myocyte co-cultures significantly increased the sympathetic fiber growth onto myocytes in WKY, but not SHR cultures. These data suggest that glial signals, or neuronal responses to these signals are disrupted in SHR animals. To assess this idea, WKY and SHR neurons were grown in the presence of a glial-derived growth inhibitor, insulin-like growth factor binding protein 4 (Igfbp4). We observed a trend towards a decrease in the fiber length of WKY neurons grown with increasing concentrations of Igfbp4, but not SHR neurons. Lastly, we evaluated a potential role for SGs in activity-dependent structural remodeling. We chronically drove neuronal activity using excitatory DREADDs and observed a decrease in the sympathetic innervation density of WKY myocytes in both neuron-myocyte and neuron-glia-myocyte co-cultures. In the presence of glia, there was an additional trend towards a decrease in the fiber density between myocytes under stimulation conditions. By coupling an increase in neuronal activity with a decrease in release sites, this finding implies a homeostatic role for structural plasticity in the maintenance of cardiac sympathetic drive. Overall, our results establish the growth-regulatory potential of satellite glia in the sympathetic system, and suggest disrupted neuronal responses to glial signals as a possible hypertensive mechanism in the SHR.