Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Sympathetic neuropathology is revealed in muscles affected by amyotrophic lateral sclerosis.

Rationale: The anatomical substrate of skeletal muscle autonomic innervation has remained underappreciated since it was described many decades ago. As such, the structural and functional features of muscle sympathetic innervation are largely undetermined in both physiology and pathology, mainly due to methodological limitations in the histopathological analysis of small neuronal fibers in tissue samples. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease which mainly targets motor neurons, and despite autonomic symptoms occurring in a significant fraction of patients, peripheral sympathetic neurons (SNs) are generally considered unaffected and, as such, poorly studied. Purpose: In this research, we compared sympathetic innervation of normal and ALS muscles, through structural analysis of the sympathetic network in human and murine tissue samples. Methods and Results: We first refined tissue processing to circumvent methodological limitations interfering with the detection of muscle sympathetic innervation. The optimized "Neuro Detection Protocol" (NDP) was validated in human muscle biopsies, demonstrating that SNs innervate, at high density, both blood vessels and skeletal myofibers, independent of the fiber metabolic type. Subsequently, NDP was exploited to analyze sympathetic innervation in muscles of SOD1G93A mice, a preclinical ALS model. Our data show that ALS murine muscles display SN denervation, which has already initiated at the early disease stage and worsened during aging. SN degeneration was also observed in muscles of MLC/SOD1G93A mice, with muscle specific expression of the SOD1G93A mutant gene. Notably, similar alterations in SNs were observed in muscle biopsies from an ALS patient, carrying the SOD1G93A mutation. Conclusion: We set up a protocol for the analysis of murine and, more importantly, human muscle sympathetic innervation. Our results indicate that SNs are additional cell types compromised in ALS and suggest that dysfunctional SOD1G93A muscles affect their sympathetic innervation.

Toll-like receptor 9 signaling after myocardial infarction: Role of p66ShcA adaptor protein.

During myocardial infarction, cellular debris is released, causing a sterile inflammation via pattern recognition receptors. These reactions amplify damage and promotes secondary heart failure. The pattern recognition receptor, Toll-like receptor 9 (TLR9) detects immunogenic fragments of endogenous DNA, inducing inflammation by NFκB. The p66ShcA adaptor protein plays an important role in both ischemic myocardial damage and immune responses. We hypothesized that p66ShcA adaptor protein promotes DNA-sensing signaling via the TLR9 pathway after myocardial infarction. TLR9 protein expression increased in cardiac tissue from patients with end-stage heart failure due to ischemic heart disease. Myocardial ischemia in mice in vivo induced gene expression of key TLR9 pathway proteins (MyD88 and Unc93b1). In this model, a functional link between TLR9 and p66ShcA was revealed as; (i) ischemia-induced upregulation of TLR9 protein was abrogated in myocardium of p66ShcA knockout mice; (ii) when p66ShcA was overexpressed in NFkB reporter cells stably expressing TLR9, NFkB-activation increased during stimulation with the TLR9 agonist CpG B; (iii) in cardiac fibroblasts, p66ShcA overexpression caused TLR9 upregulation. Co-immunoprecipitation showed that ShcA proteins and TLR9 may be found in the same protein complex, which was dissipated upon TLR9 stimulation in vivo. A proximity assay confirmed the co-localization of TLR9 and ShcA proteins. The systemic immune response after myocardial ischemia was dampened in p66ShcA knockout mice as interleukin-4, -17 and -22 expression in mononuclear cells isolated from spleens was reduced. In conclusion, p66ShcA adaptor may be an interaction partner and a regulator of the TLR9 pathway post-infarction.

Neurotoxic Effect of Doxorubicin Treatment on Cardiac Sympathetic Neurons.

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.

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.

Optogenetic Control of Heart Rhythm: Lightly Guiding the Cardiac Pace.

It is well appreciated that, differently from skeletal muscles, the heart contracts independently from neurogenic inputs. However, the speed and force of heartbeats are finely modulated during stresses, emotions, and daily activities, by the autonomic neurons (both parasympathetic and sympathetic) which highly innervate the myocardium. Despite this aspect of cardiac physiology has been known for long, research has only recently shed light on the biophysical mechanisms underlying the meticulous adaptation of heart activity to the needs of the organism. A conceptual advancement in this regard has come from the use of optogenetics, a revolutionary methodology which allows to control the activity of a given excitable cell type, with high specificity, temporal and spatial resolution, within intact tissues and organisms. The method, widely affirmed in the field of neuroscience, has more recently been exploited also in research on heart physiology and pathology, including the study of the mechanisms regulating heart rhythm. The last point is the object of this book chapter which, starting from the description of the physiology of heart rhythm automaticity and the neurogenic modulation of heart rate, makes an excursus on the theoretical basis of such biotechnology (with its advantages and limitations), and presents a series of examples in cardiac and neuro-cardiac optogenetics.

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Neurohumoral Cardiac Regulation: Optogenetics Gets Into the Groove.

The cardiac autonomic nervous system (ANS) is the main modulator of heart function, adapting contraction force, and rate to the continuous variations of intrinsic and extrinsic environmental conditions. While the parasympathetic branch dominates during rest-and-digest sympathetic neuron (SN) activation ensures the rapid, efficient, and repeatable increase of heart performance, e.g., during the "fight-or-flight response." Although the key role of the nervous system in cardiac homeostasis was evident to the eyes of physiologists and cardiologists, the degree of cardiac innervation, and the complexity of its circuits has remained underestimated for too long. In addition, the mechanisms allowing elevated efficiency and precision of neurogenic control of heart function have somehow lingered in the dark. This can be ascribed to the absence of methods adequate to study complex cardiac electric circuits in the unceasingly moving heart. An increasing number of studies adds to the scenario the evidence of an intracardiac neuron system, which, together with the autonomic components, define a little brain inside the heart, in fervent dialogue with the central nervous system (CNS). The advent of optogenetics, allowing control the activity of excitable cells with cell specificity, spatial selectivity, and temporal resolution, has allowed to shed light on basic neuro-cardiology. This review describes how optogenetics, which has extensively been used to interrogate the circuits of the CNS, has been applied to untangle the knots of heart innervation, unveiling the cellular mechanisms of neurogenic control of heart function, in physiology and pathology, as well as those participating to brain-heart communication, back and forth. We discuss existing literature, providing a comprehensive view of the advancement in the understanding of the mechanisms of neurogenic heart control. In addition, we weigh the limits and potential of optogenetics in basic and applied research in neuro-cardiology.

Neuropeptide Y promotes adipogenesis of human cardiac mesenchymal stromal cells in arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic Cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes. AC hearts show fibro-fatty myocardial replacement, which favors stress-related life-threatening arrhythmias, predominantly in the young and athletes. AC lacks effective therapies, as its pathogenesis is poorly understood. Recently, we showed that cardiac Mesenchymal Stromal Cells (cMSCs) contribute to adipose tissue in human AC hearts, although the underlying mechanisms are still unclear. PURPOSE: We hypothesize that the sympathetic neurotransmitter, Neuropeptide Y (NPY), participates to cMSC adipogenesis in human AC. METHODS: For translation of our findings, we combined in vitro cytochemical, molecular and pharmacologic assays on human cMSCs, from myocardial biopsies of healthy controls and AC patients, with the use of existing drugs to interfere with the predicted AC mechanisms. Sympathetic innervation was inspected in human autoptic heart samples, and NPY plasma levels measured in healthy and AC subjects. RESULTS: AC cMSCs expressed higher levels of pro-adipogenic isotypes of NPY-receptors (i.e. Y1-R, Y5-R). Consistently, NPY enhanced adipogenesis in AC cMSCs, which was blocked by FDA-approved Y1-R and Y5-R antagonists. AC-associated PKP2 reduction directly caused NPY-dependent adipogenesis in cMSCs. In support of the involvement of sympathetic neurons (SNs) and NPY in AC myocardial remodeling, patients had elevated NPY plasma levels and, in human AC hearts, SNs accumulated in fatty areas and were close to cMSCs. CONCLUSIONS: Independently from the disease origin, AC causes in cMSCs a targetable gain of responsiveness to NPY, which leads to increased adipogenesis, thus playing a role in AC myocardial remodeling.

Multiphoton Imaging of Ca2+ Instability in Acute Myocardial Slices from a RyR2R2474S Murine Model of Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a familial stress-induced arrhythmia syndrome, mostly caused by mutations in Ryanodine receptor 2 (RyR2), the sarcoplasmic reticulum (SR) Ca2+ release channel in cardiomyocytes. Pathogenetic mutations lead to gain of function in the channel, causing arrhythmias by promoting diastolic spontaneous Ca2+ release (SCR) from the SR and delayed afterdepolarizations. While the study of Ca2+ dynamics in single cells from murine CPVT models has increased our understanding of the disease pathogenesis, questions remain on the mechanisms triggering the lethal arrhythmias at tissue level. Here, we combined subcellular analysis of Ca2+ signals in isolated cardiomyocytes and in acute thick ventricular slices of RyR2R2474S knock-in mice, electrically paced at different rates (1-5 Hz), to identify arrhythmogenic Ca2+ dynamics, from the sub- to the multicellular perspective. In both models, RyR2R2474S cardiomyocytes had increased propensity to develop SCR upon adrenergic stimulation, which manifested, in the slices, with Ca2+ alternans and synchronous Ca2+ release events in neighboring cardiomyocytes. Analysis of Ca2+ dynamics in multiple cells in the tissue suggests that SCRs beget SCRs in contiguous cells, overcoming the protective electrotonic myocardial coupling, and potentially generating arrhythmia triggering foci. We suggest that intercellular interactions may underscore arrhythmic propensity in CPVT hearts with 'leaky' RyR2.

Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disorder at high risk of arrhythmic sudden death in the young and athletes. AC is hallmarked by myocardial replacement with fibro-fatty tissue, favoring life-threatening cardiac arrhythmias and contractile dysfunction. The AC pathogenesis is unclear, and the disease urgently needs mechanism-driven therapies. Current AC research is mainly focused on 'desmosome-carrying' cardiomyocytes, but desmosomal proteins are also expressed by non-myocyte cells, which also harbor AC variants, including mesenchymal stromal cells (MSCs). Consistently, cardiac-MSCs contribute to adipose tissue in human AC hearts. We thus approached AC as a multicellular disorder, hypothesizing that it also affects extra-cardiac bone marrow (BM)-MSCs. Our results show changes in the desmosomal protein profile of both cardiac- and BM- MSCs, from desmoglein-2 (Dsg2)-mutant mice, accompanied with profound alterations in cytoskeletal organization, which are directly caused by AC-linked DSG2 downregulation. In addition, AC BM-MSCs display increased proliferation rate, both in vitro and in vivo, and, by using the principle of the competition homing assay, we demonstrated that mutant circulating BM-MSCs have increased propensity to migrate to the AC heart. Taken altogether, our results indicate that cardiac- and BM- MSCs are additional cell types affected in Dsg2-linked AC, warranting the novel classification of AC as a multicellular and multiorgan disease.

Psychosocial Stress Hastens Disease Progression and Sudden Death in Mice with Arrhythmogenic Cardiomyopathy.

Physiological stressors, such as exercise, can precipitate sudden cardiac death or heart failure progression in patients with arrhythmogenic cardiomyopathy (ACM). Yet, whether and to what extent a highly prevalent and more elusive environmental factor, such as psychosocial stress (PSS), can also increase ACM disease progression is unexplored. Here, we first quantified perceived stress levels in patients with ACM and found these levels correlated with the extent of arrhythmias and cardiac dysfunction. To determine whether the observed correlation is due to causation, we inflicted PSS-via the resident-intruder (RI) paradigm-upon Desmoglein-2 mutant mice, a vigorously used mammalian model of ACM. We found that ACM mice succumbed to abnormally high in-trial, PSS mortality. Conversely, no sudden deaths occurred in wildtype (WT) counterparts. Desmoglein-2 mice that survived RI challenge manifested markedly worse cardiac dysfunction and remodeling, namely apoptosis and fibrosis. Furthermore, WT and ACM mice displayed similar behavior at baseline, but Desmoglein-2 mice exhibited heightened anxiety following RI-induced PSS. This outcome correlated with the worsening of cardiac phenotypes. Our mouse model demonstrates that in ACM-like subjects, PSS is incisive enough to deteriorate cardiac structure and function per se, i.e., in the absence of any pre-existing anxious behavior. Hence, PSS may represent a previously underappreciated risk factor in ACM disease penetrance.

Towards a clearer view of sympathetic innervation of cardiac and skeletal muscles.

It is well appreciated that autonomic neurons have a central role in the homeostatic regulation of organs and systems and participate to the pathogenesis of several disease conditions. As such, the function and signalling pathways activated by sympathetic neurons (SNs) in different cell types and organs have become a matter of intense investigation throughout the years of modern biomedical research. This review is focused on the methods used to address sympathetic innervation of cardiac and skeletal muscles which, quite surprisingly, has remained incompletely understood, mainly due to the technical limitations of the traditional methodologies. The current review provides a summary of the existing literature and, putting together the results obtained with different methodological approaches, provides a comprehensive view of the complexity of the SN network in striated muscles.

A Light Wand to Untangle the Myocardial Cell Network.

The discovery of optogenetics has revolutionized research in neuroscience by providing the tools for noninvasive, cell-type selective modulation of membrane potential and cellular function in vitro and in vivo. Rhodopsin-based optogenetics has later been introduced in experimental cardiology studies and used as a tool to photoactivate cardiac contractions or to identify the sites, timing, and location most effective for defibrillating impulses to interrupt cardiac arrhythmias. The exploitation of cell-selectivity of optogenetics, and the generation of model organisms with myocardial cell type targeted expression of opsins has started to yield novel and sometimes unexpected notions on myocardial biology. This review summarizes the main results, the different uses, and the prospective developments of cardiac optogenetics.