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.

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.

Increased susceptibility to Ca(2+)-induced permeability transition and to cytochrome c release in rat heart mitochondria with aging: effect of melatonin.

Aging is associated with a decline of cardiac function. The mitochondrial permeability transition (MPT) may be a factor in cardiac dysfunction associated with aging. We investigated the effect of aging and long-term treatment with melatonin (approximately 10 mg/kg b.w./day for 2 months), a known natural antioxidant, on the susceptibility to Ca(2+)-induced MPT opening and cytochrome c release in rat heart mitochondria. The mitochondrial content of normal and oxidized cardiolipin as a function of aging and melatonin treatment was also analyzed. Mitochondria from aged rats (24 month old) displayed an increased susceptibility to Ca(2+)-induced MPT opening, associated with an elevated release of cytochrome c, when compared with young control animals (5 month old). Melatonin treatment counteracted both these processes. Aging was also associated with an oxidation/depletion of cardiolipin which could be counteracted as well by melatonin. It is proposed that the increased level of oxidized cardiolipin could be responsible, at least in part, for the increased susceptibility to Ca(2+)-induced MPT opening and cytochrome c release in rat heart mitochondria with aging. Melatonin treatment counteracts both these processes, most likely, by preventing the oxidation/depletion of cardiolipin. Our results might have implications in the necrotic and apoptotic myocytes cell death in aged myocardium, particularly in ischemia/reperfusion injury.

Melatonin protects against heart ischemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening.

Melatonin, a well-known antioxidant, has been shown to protect against ischemia-reperfusion myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during ischemia-reperfusion and therefore a possible target for cardioprotection. In the present study, we tested the hypothesis that melatonin could protect heart against ischemia-reperfusion injury by inhibiting MPTP opening. Isolated perfused rat hearts were subjected to global ischemia and reperfusion in the presence or absence of melatonin in a Langerdoff apparatus. Melatonin treatment significantly improves the functional recovery of Langerdoff hearts on reperfusion, reduces the infarct size, and decreases necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria isolated from melatonin-treated hearts are less sensitive than mitochondria from reperfused hearts to MPTP opening as demonstrated by their higher resistance to Ca(2+). Similar results were obtained following treatment of ischemic-reperfused rat heart with cyclosporine A, a known inhibitor of MPTP opening. In addition, melatonin prevents mitochondrial NAD(+) release and mitochondrial cytochrome c release and, as previously shown, cardiolipin oxidation associated with ischemia-reperfusion. Together, these results demonstrate that melatonin protects heart from reperfusion injury by inhibiting MPTP opening, probably via prevention of cardiolipin peroxidation.

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release.

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca(2+)-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca(2+)-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca(2+)-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca(2+) overload.

Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin.

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging.