Abstract
Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.
Highlights
Sympathetic nervous system (SNS) plays a critical role in the maintenance of cardiovascular homeostasis by regulating intrinsic heart functions
To compare neurotrophin gene expression profiling in cardiomyocytes and fibroblasts, Nerve growth factor (NGF), NT3, NT4, CNTF and BDNF gene expression were measured by real time qPCR using mRNA extracted from adult cardiomyocytes or cardiac fibroblasts isolated from healthy rat hearts
In agreement with qPCR gene profiling, western-blot experiments confirmed the overexpression of immature proNGF and mature NGF proteins in cardiac fibroblasts isolated from control hearts by opposition to adult cardiomyocytes in which the two proteins were barely detectable (Figure 1B)
Summary
Sympathetic nervous system (SNS) plays a critical role in the maintenance of cardiovascular homeostasis by regulating intrinsic heart functions. A number of human pathologies associated with either cardiac sympathetic hyperinnervation such as myocardial infarction (MI) [5] or hypoinnervation such as diabetic or asynuclein-associated postganglionic autonomic neuropathies [6,7] are associated with increased cardiac morbi-mortality [8,9]. All these data strongly argue for a crucial role of SNS innervation in the heart, reinforcing the need to improve our knowledge on molecular and cellular mechanisms contributing to the regulation of cardiac SNS innervation
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