Nerve growth factor transfer from cardiomyocytes to innervating sympathetic neurons activates TrkA receptors at the neuro-cardiac junction.

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.

Tuning the Consonance of Microscopic Neuro-Cardiac Interactions Allows the Heart Beats to Play Countless Genres.

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 ß-adrenoceptors (ß-ARs). Given the centrality of such mechanism in heart regulation, ß-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 ß-AR/cAMP signaling in good order emerges in pathology, when the loss of proper organization of the transduction pathway leads to detuned ß-AR/cAMP signaling, with detrimental consequences on CM function. Despite the compelling advancements in decoding cardiac ß-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, ß-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.

Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.

KEY POINTS: The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions. ABSTRACT: It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro-cardiac communication to occur locally, with a 'quasi-synaptic' mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co-cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro-cardiac communication, CMs, directly contacted by SNs in co-cultures, were larger than the non-targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro-cardiac junctions.

Dynamics of neuroeffector coupling at cardiac sympathetic synapses.

KEY POINTS: The present study demonstrates, by in vitro and in vivo analyses, the novel concept that signal transmission between sympathetic neurons and the heart, underlying the physiological regulation of cardiac function, operates in a quasi-synaptic fashion. This is a result of the direct coupling between neurotransmitter releasing sites and effector cardiomyocyte membranes. ABSTRACT: Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer-based imaging of cAMP in co-cultures, as a readout of cardiac ß-adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino-atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20-50 mg kg-1 ), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site.

Putting together the clues of the everlasting neuro-cardiac liaison.

Starting from the late embryonic development, the sympathetic nervous system extensively innervates the heart and modulates its activity during the entire lifespan. The distribution of myocardial sympathetic processes is finely regulated by the secretion of limiting amounts of pro-survival neurotrophic factors by cardiac cells. Norepinephrine release by the neurons rapidly modulates myocardial electrophysiology, and increases the rate and force of cardiomyocyte contractions. Sympathetic processes establish direct interaction with cardiomyocytes, characterized by the presence of neurotransmitter vesicles and reduced cell-cell distance. Whether such contacts have a functional role in both neurotrophin- and catecholamine-dependent communication between the two cell types, is poorly understood. In this review we will address the effects of the sympathetic neuron activity on the myocardium and the hypothesis that the direct neuro-cardiac contact might have a key role both in norepinephrine and neurotrophin mediated signaling. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy.

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Cardiac sympathetic neurons provide trophic signal to the heart via ß2-adrenoceptor-dependent regulation of proteolysis.

AIMS: Increased cardiac sympathetic neuron (SN) activity has been associated with pathologies such as heart failure and hypertrophy, suggesting that cardiac innervation regulates cardiomyocyte trophism. Whether continuous input from the SNs is required for the maintenance of the cardiomyocyte size has not been determined thus far. METHODS AND RESULTS: To address the role of cardiac innervation in cardiomyocyte size regulation, we monitored the effect of pharmacological sympathetic denervation in mice on cardiac structure, function, and signalling from 24 h to 30 days in the absence of other pathological stimuli. SN ablation caused an immediate reduction in the cardiomyocyte size with minimal consequences on the resting contractile function. Atrophic remodelling was mediated by the ubiquitin-proteasome system through FOXO-dependent early induction of the muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1, which was followed by activation of the autophagy-lysosome system. MuRF1 was found to be determinant in denervation atrophy as remodelling did not develop in denervated MuRF1 knock-out (KO) hearts. These effects were caused by decreased basal stimulation of cardiomyocyte ß2-adrenoceptor (AR), as atrophy was prevented by treatment of denervated mice with the ß2-AR agonist clenbuterol. Consistent with these data, we also observed that ß2-AR KO mice showed cardiac atrophy at rest. CONCLUSION: Cardiac SNs are strong regulators of the cardiomyocyte size via ß2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of ß-AR in cardiovascular diseases and their modulation in therapy.

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.