Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

A New Bioassay for the Detection of Paralytic and Amnesic Biotoxins Based on Motor Behavior Impairments of Zebrafish Larvae.

The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1-0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

Lipid-Encapsuled Grape Tannins Prevent Oxidative-Stress-Induced Neuronal Cell Death, Intracellular ROS Accumulation and Inflammation.

The central nervous system (CNS) is particularly vulnerable to oxidative stress and inflammation, which affect neuronal function and survival. Nowadays, there is great interest in the development of antioxidant and anti-inflammatory compounds extracted from natural products, as potential strategies to reduce the oxidative/inflammatory environment within the CNS and then preserve neuronal integrity and brain function. However, an important limitation of natural antioxidant formulations (mainly polyphenols) is their reduced in vivo bioavailability. The biological compatible delivery system containing polyphenols may serve as a novel compound for these antioxidant formulations. Accordingly, in the present study, we used liposomes as carriers for grape tannins, and we tested their ability to prevent neuronal oxidative stress and inflammation. Cultured catecholaminergic neurons (CAD) were used to establish the potential of lipid-encapsulated grape tannins (TLS) to prevent neuronal oxidative stress and inflammation following an oxidative insult. TLS rescued cell survival after H2O2 treatment (59.4 ± 8.8% vs. 90.4 ± 5.6% H2O2 vs. TLS+ H2O2; p < 0.05) and reduced intracellular ROS levels by ~38% (p < 0.05), despite displaying negligible antioxidant activity in solution. Additionally, TLS treatment dramatically reduced proinflammatory cytokines' mRNA expression after H2O2 treatment (TNF-α: 400.3 ± 1.7 vs. 7.9 ± 1.9-fold; IL-1ß: 423.4 ± 1.3 vs. 12.7 ± 2.6-fold; p < 0.05; H2O2 vs. TLS+ H2O2, respectively), without affecting pro/antioxidant biomarker expression, suggesting that liposomes efficiently delivered tannins inside neurons and promoted cell survival. In conclusion, we propose that lipid-encapsulated grape tannins could be an efficient tool to promote antioxidant/inflammatory cell defense.

Cardiorespiratory alterations following intermittent photostimulation of RVLM C1 neurons: Implications for long-term blood pressure, breathing and sleep regulation in freely moving rats.

AIM: Sympathoexcitation and sleep-disordered breathing are common contributors for disease progression. Catecholaminergic neurons from the rostral ventrolateral medulla (RVLM-C1) modulate sympathetic outflow and have anatomical projections to respiratory neurons; however, the contribution of highly selective activation of RVLM-C1 neurons on long-term autonomic and breathing (dys)regulation remains to be understood. METHODS: To explore this relationship, a lentiviral vector carrying the light-sensitive cation channel channelrhodopsin-2 (LVV-PRSX8-ChR2-YFP) was unilaterally injected into the RVLM of healthy rats. On the contralateral side, LVV-PRSX8-ChR2-YFP was co-injected with a specific immunotoxin (DßH-SAP) targeted to eliminate C1 neurons. RESULTS: Intermittent photostimulation of RVLM-C1 in vivo, in unrestrained freely moving rats, elicited long-term facilitation of the sympathetic drive, a rise in blood pressure and sympatho-respiratory coupling. In addition, photoactivation of RVLM-C1 induced long-lasting ventilatory instability, characterized by oscillations in tidal volume and increased breathing variability, but only during non-rapid eye movement sleep. These effects were not observed when photostimulation of the RVLM was performed in the presence of DßH-SAP toxin. CONCLUSIONS: The finding that intermittent activation of RVLM-C1 neurons induces autonomic and breathing dysfunction suggest that episodic stimulation of RVLM-C1 may serve as a pathological substrate for the long-term development of cardiorespiratory disorders.

Sleep dysregulation in sympathetic-mediated diseases: implications for disease progression.

The autonomic nervous system (ANS) plays an important role in the coordination of several physiological functions including sleep/wake process. Significant changes in ANS activity occur during wake-to-sleep transition maintaining the adequate cardiorespiratory regulation and brain activity. Since sleep is a complex homeostatic function, partly regulated by the ANS, it is not surprising that sleep disruption trigger and/or evidence symptoms of ANS impairment. Indeed, several studies suggest a bidirectional relationship between impaired ANS function (i.e. enhanced sympathetic drive), and the emergence/development of sleep disorders. Furthermore, several epidemiological studies described a strong association between sympathetic-mediated diseases and the development and maintenance of sleep disorders resulting in a vicious cycle with adverse outcomes and increased mortality risk. However, which and how the sleep/wake control and ANS circuitry becomes affected during the progression of ANS-related diseases remains poorly understood. Thus, understanding the physiological mechanisms underpinning sleep/wake-dependent sympathetic modulation could provide insights into diseases involving autonomic dysfunction. The purpose of this review is to explore potential neural mechanisms involved in both the onset/maintenance of sympathetic-mediated diseases (Rett syndrome, congenital central hypoventilation syndrome, obstructive sleep apnoea, type 2 diabetes, obesity, heart failure, hypertension, and neurodegenerative diseases) and their plausible contribution to the generation of sleep disorders in order to review evidence that may serve to establish a causal link between sleep disorders and heightened sympathetic activity.

Neurodegeneration in Multiple Sclerosis: The Role of Nrf2-Dependent Pathways.

Multiple sclerosis (MS) encompasses a chronic, irreversible, and predominantly immune-mediated disease of the central nervous system that leads to axonal degeneration, neuronal death, and several neurological symptoms. Although various immune therapies have reduced relapse rates and the severity of symptoms in relapsing-remitting MS, there is still no cure for this devastating disease. In this brief review, we discuss the role of mitochondria dysfunction in the progression of MS, focused on the possible role of Nrf2 signaling in orchestrating the impairment of critical cellular and molecular aspects such as reactive oxygen species (ROS) management, under neuroinflammation and neurodegeneration in MS. In this scenario, we propose a new potential downstream signaling of Nrf2 pathway, namely the opening of hemichannels and pannexons. These large-pore channels are known to modulate glial/neuronal function and ROS production as they are permeable to extracellular Ca2+ and release potentially harmful transmitters to the synaptic cleft. In this way, the Nrf2 dysfunction impairs not only the bioenergetics and metabolic properties of glial cells but also the proper antioxidant defense and energy supply that they provide to neurons.

Potential Role of the Retrotrapezoid Nucleus in Mediating Cardio-Respiratory Dysfunction in Heart Failure With Preserved Ejection Fraction.

A strong association between chemoreflex hypersensitivity, disordered breathing, and elevated sympathetic activity has been shown in experimental and human heart failure (HF). The contribution of chemoreflex hypersensitivity in HF pathophysiology is incompletely understood. There is ample evidence that increased peripheral chemoreflex drive in HF with reduced ejection fraction (HFrEF; EF<40%) leads to pathophysiological changes in autonomic and cardio-respiratory control, but less is known about the neural mechanisms mediating cardio-respiratory disturbances in HF with preserved EF (HFpEF; EF>50%). Importantly, it has been shown that activation of the central chemoreflex worsens autonomic dysfunction in experimental HFpEF, an effect mediated in part by the activation of C1 catecholaminergic neurons neighboring the retrotrapezoid nucleus (RTN), an important region for central chemoreflex control of respiratory and autonomic function. Accordingly, the main purpose of this brief review is to discuss the possible role played by activation of central chemoreflex pathways on autonomic function and its potential role in precipitating disordered breathing in HFpEF. Improving understanding of the contribution of the central chemoreflex to the pathophysiology of HFpEF may help in development of novel interventions intended to improve cardio-respiratory outcomes in HFpEF.

Cardiovascular and autonomic dysfunction in long-COVID syndrome and the potential role of non-invasive therapeutic strategies on cardiovascular outcomes.

A significant percentage of COVID-19 survivors develop long-lasting cardiovascular sequelae linked to autonomic nervous system dysfunction, including fatigue, arrhythmias, and hypertension. This post-COVID-19 cardiovascular syndrome is one facet of "long-COVID," generally defined as long-term health problems persisting/appearing after the typical recovery period of COVID-19. Despite the fact that this syndrome is not fully understood, it is urgent to develop strategies for diagnosing/managing long-COVID due to the immense potential for future disease burden. New diagnostic/therapeutic tools should provide health personnel with the ability to manage the consequences of long-COVID and preserve/improve patient quality of life. It has been shown that cardiovascular rehabilitation programs (CRPs) stimulate the parasympathetic nervous system, improve cardiorespiratory fitness (CRF), and reduce cardiovascular risk factors, hospitalization rates, and cognitive impairment in patients suffering from cardiovascular diseases. Given their efficacy in improving patient outcomes, CRPs may have salutary potential for the treatment of cardiovascular sequelae of long-COVID. Indeed, there are several public and private initiatives testing the potential of CRPs in treating fatigue and dysautonomia in long-COVID subjects. The application of these established rehabilitation techniques to COVID-19 cardiovascular syndrome represents a promising approach to improving functional capacity and quality of life. In this brief review, we will focus on the long-lasting cardiovascular and autonomic sequelae occurring after COVID-19 infection, as well as exploring the potential of classic and novel CRPs for managing COVID-19 cardiovascular syndrome. Finally, we expect this review will encourage health care professionals and private/public health organizations to evaluate/implement non-invasive techniques for the management of COVID-19 cardiovascular sequalae.

Effects of enriched-potassium diet on cardiorespiratory outcomes in experimental non-ischemic chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) is a global health problem. Increased sympathetic outflow, cardiac arrhythmogenesis and irregular breathing patterns have all been associated with poor outcomes in CHF. Several studies showed that activation of the renin-angiotensin system (RAS) play a key role in CHF pathophysiology. Interestingly, potassium (K+) supplemented diets showed promising results in normalizing RAS axis and autonomic dysfunction in vascular diseases, lowering cardiovascular risk. Whether subtle increases in dietary K+ consumption may exert similar effects in CHF has not been previously tested. Accordingly, we aimed to evaluate the effects of dietary K+ supplementation on cardiorespiratory alterations in rats with CHF. METHODS: Adult male Sprague-Dawley rats underwent volume overload to induce non-ischemic CHF. Animals were randomly allocated to normal chow diet (CHF group) or supplemented K+ diet (CHF+K+ group) for 6 weeks. Cardiac arrhythmogenesis, sympathetic outflow, baroreflex sensitivity, breathing disorders, chemoreflex function, respiratory-cardiovascular coupling and cardiac function were evaluated. RESULTS: Compared to normal chow diet, K+ supplemented diet in CHF significantly reduced arrhythmia incidence (67.8 ± 15.1 vs. 31.0 ± 3.7 events/hour, CHF vs. CHF+K+), decreased cardiac sympathetic tone (ΔHR to propranolol: - 97.4 ± 9.4 vs. - 60.8 ± 8.3 bpm, CHF vs. CHF+K+), restored baroreflex function and attenuated irregular breathing patterns. Additionally, supplementation of the diet with K+ restores normal central respiratory chemoreflex drive and abrogates pathological cardio-respiratory coupling in CHF rats being the outcome an improved cardiac function. CONCLUSION: Our findings support that dietary K+ supplementation in non-ischemic CHF alleviate cardiorespiratory dysfunction.

Heart rate and cardiac autonomic responses to concomitant deep breathing, hand grip exercise, and circulatory occlusion in healthy young adult men and women.

BACKGROUND: Deep breathing (DB) and handgrip (HG) exercise -with and without circulatory occlusion (OC) in muscle-, have been shown to have beneficial effects on cardiovascular function; however, the combination of these maneuvers on heart rate (HR) and cardiac sympathovagal balance have not been previously investigated. Therefore, the aim of the present study was to evaluate the effect of simultaneous DB, HG, and OC maneuvers on the sympathovagal balance in healthy women and men subjects. METHODS AND RESULTS: Electrocardiogram and ventilation were measured in 20 healthy subjects (Women: n = 10; age = 27 ± 4 years; weight = 67.1 ± 8.4 kg; and height = 1.6 ± 0.1 m. Men: n = 10; age = 27 ± 3 years; weight = 77.5 ± 10.1 kg; and height = 1.7 ± 0.1 m) at baseline and during DB, DB + HG, or DB + HG + OC protocols. Heart rate (HR) and respiratory rate were continuously recorded, and spectral analysis of heart rate variability (HRV) were calculated to indirectly estimate cardiac autonomic function. Men and women showed similar HR responses to DB, DB + HG and DB + HG + OC. Men exhibited a significant HR decrease following DB + HG + OC protocol which was accompanied by an improvement in cardiac autonomic control evidenced by spectral changes in HRV towards parasympathetic predominance (HRV High frequency: 83.95 ± 1.45 vs. 81.87 ± 1.50 n.u., DB + HG + OC vs. baseline; p < 0.05). In women, there was a marked decrease in HR after completion of both DB + HG and DB + HG + OC tests which was accompanied by a significant increase in cardiac vagal tone (HRV High frequency: 85.29 ± 1.19 vs. 77.93 ± 0.92 n.u., DB + HG vs. baseline; p < 0.05). No adverse effects or discomfort were reported by men or women during experimental procedures. Independent of sex, combination of DB, HG, and OC was tolerable and resulted in decreases in resting HR and elevations in cardiac parasympathetic tone. CONCLUSIONS: These data indicate that combined DB, HG and OC are effective in altering cardiac sympathovagal balance and reducing resting HR in healthy men and women.

Exercise intolerance in volume overload heart failure is associated with low carotid body mediated chemoreflex drive.

Mounting an appropriate ventilatory response to exercise is crucial to meeting metabolic demands, and abnormal ventilatory responses may contribute to exercise-intolerance (EX-inT) in heart failure (HF) patients. We sought to determine if abnormal ventilatory chemoreflex control contributes to EX-inT in volume-overload HF rats. Cardiac function, hypercapnic (HCVR) and hypoxic (HVR) ventilatory responses, and exercise tolerance were assessed at the end of a 6 week exercise training program. At the conclusion of the training program, exercise tolerant HF rats (HF + EX-T) exhibited improvements in cardiac systolic function and reductions in HCVR, sympathetic tone, and arrhythmias. In contrast, HF rats that were exercise intolerant (HF + EX-inT) exhibited worse diastolic dysfunction, and showed no improvements in cardiac systolic function, HCVR, sympathetic tone, or arrhythmias at the conclusion of the training program. In addition, HF + EX-inT rats had impaired HVR which was associated with increased arrhythmia susceptibility and mortality during hypoxic challenges (~ 60% survival). Finally, we observed that exercise tolerance in HF rats was related to carotid body (CB) function as CB ablation resulted in impaired exercise capacity in HF + EX-T rats. Our results indicate that: (i) exercise may have detrimental effects on cardiac function in HF-EX-inT, and (ii) loss of CB chemoreflex sensitivity contributes to EX-inT in HF.

Exercise training reduces brainstem oxidative stress and restores normal breathing function in heart failure.

Enhanced central chemoreflex drive and irregular breathing are both hallmarks in heart failure (HF) and closely related to disease progression. Central chemoreceptor neurons located within the retrotrapezoid nucleus (RTN) are known to play a role in breathing alterations in HF. It has been shown that exercise (EX) effectively reduced reactive oxygen species (ROS) in HF rats. However, the link between EX and ROS, particularly at the RTN, with breathing alterations in HF has not been previously addressed. Accordingly, we aimed to determine: i) ROS levels in the RTN in HF and its association with chemoreflex drive, ii) whether EX improves chemoreflex/breathing function by reducing ROS levels, and iii) determine molecular alterations associated with ROS generation within the RTN of HF rats and study EX effects on these pathways. Adult male Sprague-Dawley rats were allocated into 3 experimental groups: Sham (n = 5), volume overloaded HF (n = 6) and HF (n = 8) rats that underwent EX training for 6 weeks (60 min/day, 25 m/min, 10% inclination). At 8 weeks post-HF induction, breathing patterns and chemoreflex function were analyzed by unrestrained plethysmography. ROS levels and anti/pro-oxidant enzymes gene expression were analyzed in the RTN. Our results showed that HF rats have high ROS levels in the RTN which were closely linked to the enhanced central chemoreflex and breathing disorders. Also, HF rats displayed decreased expression of antioxidant genes in the RTN compared with control rats. EX training increases antioxidant defense in the RTN, reduces ROS formation and restores normal central chemoreflex drive and breathing regularity in HF rats. This study provides evidence for a role of ROS in central chemoreception in the setting of HF and support the use of EX to reduce ROS in the brainstem of HF animals and reveal its potential as an effective mean to normalize chemoreflex and breathing function in HF.

Paraquat herbicide diminishes chemoreflex sensitivity, induces cardiac autonomic imbalance and impair cardiac function in rats.

Paraquat (PQT) herbicide is widely used in agricultural practices despite being highly toxic to humans. It has been proposed that PQT exposure may promote cardiorespiratory impairment. However, the physiological mechanisms involved in cardiorespiratory dysfunction following PQT exposure are poorly known. We aimed to determine the effects of PQT on ventilatory chemoreflex control, cardiac autonomic control, and cardiac function in rats. Male Sprague-Dawley rats received two injections/week of PQT (5 mg·kg-1 ip) for 4 wk. Cardiac function was assessed through echocardiography and pressure-volume loops. Ventilatory function was evaluated using whole body plethysmography. Autonomic control was indirectly evaluated by heart rate variability (HRV). Cardiac electrophysiology (EKG) and exercise capacity were also measured. Four weeks of PQT administration markedly enlarged the heart as evidenced by increases in ventricular volumes and induced cardiac diastolic dysfunction. Indeed, end-diastolic pressure was significantly higher in PQT rats compared with control (2.42 ± 0.90 vs. 4.01 ± 0.92 mmHg, PQT vs. control, P < 0.05). In addition, PQT significantly reduced both the hypercapnic and hypoxic ventilatory chemoreflex response and induced irregular breathing. Also, PQT induced autonomic imbalance and reductions in the amplitude of EKG waves. Finally, PQT administration impaired exercise capacity in rats as evidenced by a ∼2-fold decrease in times-to-fatigue compared with control rats. Our results showed that 4 wk of PQT treatment induces cardiorespiratory dysfunction in rats and suggests that repetitive exposure to PQT may induce harmful mid/long-term cardiovascular, respiratory, and cardiac consequences.NEW & NOREWORTHY Paraquat herbicide is still employed in agricultural practices in several countries. Here, we showed for the first time that 1 mo paraquat administration results in cardiac adverse remodeling, blunts ventilatory chemoreflex drive, and promotes irregular breathing at rest in previously healthy rats. In addition, paraquat exposure induced cardiac autonomic imbalance and cardiac electrophysiology alterations. Lastly, cardiac diastolic dysfunction was overt in rats following 1 mo of paraquat treatment.

Optogenetics to Interrogate Neuron-Glia Interactions in Pups and Adults.

In just over 10 years, the use of optogenetic technologies in neuroscience has become widespread, having today a tremendous impact on our understanding of brain function. An extensive number of studies have implemented a variety of tools allowing for the manipulation of neurons with light, including light-activated ion channels or G protein-coupled receptors, among other innovations. In this context, the proper calibration of photostimulation in vivo remains crucial to dissect brain circuitry or investigate the effect of neuronal activity on specific subpopulations of neurons and glia. Depending on the scientific question, the design of specific stimulation protocols must consider from the choice of the animal model to the light stimulation pattern to be delivered. In this chapter, we describe a detailed framework to investigate neuron-glia interactions in both mouse pups and adults using an optogenetic approach.

Heart rate and cardiac autonomic responses to concomitant deep breathing, hand grip exercise, and circulatory occlusion in healthy young adult men and women

BACKGROUND: Deep breathing (DB) and handgrip (HG) exercise -with and without circulatory occlusion (OC) in muscle-, have been shown to have beneficial effects on cardiovascular function; however, the combination of these maneuvers on heart rate (HR) and cardiac sympathovagal balance have not been previously investigated. Therefore, the aim of the present study was to evaluate the effect of simultaneous DB, HG, and OC maneuvers on the sympathovagal balance in healthy women and men subjects. METHODS AND RESULTS: Electrocardiogram and ventilation were measured in 20 healthy subjects (Women: n = 10; age = 27 ± 4 years; weight = 67.1 ± 8.4 kg; and height = 1.6 ± 0.1 m. Men: n = 10; age = 27 ± 3 years; weight = 77.5 ± 10.1 kg; and height = 1.7 ± 0.1 m) at baseline and during DB, DB + HG, or DB + HG + OC protocols. Heart rate (HR) and respiratory rate were continuously recorded, and spectral analysis of heart rate variability (HRV) were calculated to indirectly estimate cardiac autonomic function. Men and women showed similar HR responses to DB, DB + HG and DB + HG + OC. Men exhibited a significant HR decrease following DB + HG + OC protocol which was accompanied by an improvement in cardiac autonomic control evidenced by spectral changes in HRV towards parasympathetic predominance (HRV High frequency: 83.95 ± 1.45 vs. 81.87 ± 1.50 n.u., DB + HG + OC vs. base-line; p < 0.05). In women, there was a marked decrease in HR after completion of both DB + HG and DB + HG + OC tests which was accompanied by a significant increase in cardiac vagal tone (HRV High frequency: 85.29 ± 1.19 vs. 77.93 ± 0.92 n.u., DB + HG vs. baseline; p < 0.05). No adverse effects or discomfort were reported by men or women during experimental procedures. Independent of sex, combination of DB, HG, and OC was tolerable and resulted in decreases in resting HR and elevations in cardiac parasympathetic tone. CONCLUSIONS: These data indicate that combined DB, HG and OC are effective in altering cardiac sympathovagal balance and reducing resting HR in healthy men and women.