Sympathetic neurons densely innervate the myocardium with non‐random topology and establish structured contacts (i.e. neuro‐cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated with sympathetic denervation, supporting the hypothesis that retrograde ‘cardiomyocyte‐to‐sympathetic neuron’ communication is essential for heart cellular homeostasis. We aimed to determine whether cardiomyocytes provide nerve growth factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signalling. Immunofluorescence on murine and human heart slices shows that NGF and its receptor, tropomyosin‐receptor‐kinase‐A, accumulate, respectively, in the pre‐ and post‐junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co‐cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that this mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real‐time imaging of tropomyosin‐receptor‐kinase‐A vesicle movements demonstrate that efficiency of neurotrophic signalling parallels the maturation of such structured intercellular contacts. Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte‐driven heart drivers. Key points CMs are the cell source of nerve growth factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, ever‐changing, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of ‘CM‐to‐SN’ signalling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases.
Background Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Neurotrophins released by cardiac cells control SN viability and myocardial distribution, which are impaired in heart diseases with reduced (e.g. heart failure) or heterogenous sympathetic stimulation (e.g. arrhythmias). We previously demonstrated that SNs interact directly with cardiomyocytes (CMs) at neuro-cardiac junctions (NCJ), and such structured contact sites allow neurons to efficiently activate β-adrenoceptors on the myocyte membrane. Aims We here asked whether NCJs are functional for retrograde (myocyte to neuron) neurotrophic signaling. Methods and results Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed that the NGF receptor, TrkA, is preferentially found in correspondence of the NCJ. Consistently, neurons taking structured contact with CMs showed fast TrkA activation and its retrograde transport to the soma, which was monitored using live confocal imaging in cells expressing TrkA-RFP. In accord with NGF dependent effects, CM-contacted SN showed larger synaptic varicosities and did not require NGF supplementation in the culture medium. In support that NGF locally released at NCJs sustains SN viability, the neurotrophin concentration in the culture medium was 1.61 pg/mL, and did not suffice to maintain neuronal viability, which was also perturbed (66% decrease of neuronal density) by silencing NGF expression in CMs. These results support that the NCJ is essential for intercellular neurotrophin signaling. Consistently, by applying competitive inhibition of TrkA with increasing doses of K252a, we estimated NGF concentration at the contact site to be about 1000-fold higher than that released by CM in the culture medium. To seek for the structural determinants of the NCJ, we focused on dystrophin, based on the finding that the protein accumulates on the CM membrane portion contacted by SNs, as observed in mouse heart slices, and co-cultured CMs. In support of a role of CM-expressed dystrophin in neurotrophic signaling, hearts from dystrophin-KO (mdx) mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In line with the purported role of NCJs, in co-cultures between wild type SNs and mdx CMs, TrkA activation (TrkA movements toward SN soma (%): WTCM-WTSN=18±4; MDXCM-WTSN= 12±3; p<0,05) and neuronal survival were reduced. Conclusions Taken together, our results suggest that NGF-dependent signaling to SNs requires a direct and specialized interaction with myocytes, and that loss of dystrophin at the CM membrane impairs retrograde signaling to the neurons leading to cardiac sympathetic dys-innervation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Padova
Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy interruption, with devastating consequences on patient survival and quality of life. Although the adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure, recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and morphometric analyses revealed alterations in SN innervation density and topology in hearts from DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons in vivo. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients. Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation, unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is involved in both cardiac physiology and pathology, including heart failure and arrhythmias.
Different from skeletal muscle, the heart autonomously generates rhythmic contraction independently from neuronal inputs. However, speed and strength of the heartbeats are continuously modulated by environmental, physical or emotional inputs, delivered by cardiac innervating sympathetic neurons, which tune cardiomyocyte (CM) function, through activation of β-adrenoceptors (β-ARs). Given the centrality of such mechanism in heart regulation, β-AR signaling has been subject of intense research, which has reconciled the molecular details of the transduction pathway and the fine architecture of cAMP signaling in subcellular nanodomains, with its final effects on CM function. The importance of mechanisms keeping the elements of β-AR/cAMP signaling in good order emerges in pathology, when the loss of proper organization of the transduction pathway leads to detuned β-AR/cAMP signaling, with detrimental consequences on CM function. Despite the compelling advancements in decoding cardiac β-AR/cAMP signaling, most discoveries on the subject were obtained in isolated cells, somehow neglecting that complexity may encompass the means in which receptors are activated in the intact heart. Here, we outline a set of data indicating that, in the context of the whole myocardium, the heart orchestra (CMs) is directed by a closely interacting and continuously attentive conductor, represented by SNs. After a roundup of literature on CM cAMP regulation, we focus on the unexpected complexity and roles of cardiac sympathetic innervation, and present the recently discovered Neuro-Cardiac Junction, as the election site of “SN-CM” interaction. We further discuss how neuro-cardiac communication is based on the combination of extra- and intra-cellular signaling micro/nano-domains, implicating neuronal neurotransmitter exocytosis, β-ARs and elements of cAMP homeostasis in CMs, and speculate on how their dysregulation may reflect on dysfunctional neurogenic control of the heart in pathology.
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