Am J Physiol Regul Integr Comp Physiol 306: R814–R822, 2014. First published April 2, 2014; doi:10.1152/ajpregu.00529.2013.

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats Eugen Brailoiu,1,2 Elena Deliu,1 Romeo A. Sporici,3 Khalid Benamar,4 and G. Cristina Brailoiu5 1

Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania; 2Center for Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania; 3Department of Internal Medicine, Brandywine Hospital, Coatesville, Pennsylvania; 4Center for Substance Abuse, Temple University School of Medicine, Philadelphia, Pennsylvania; and 5Department of Pharmaceutical Sciences, Thomas Jefferson University, Jefferson School of Pharmacy, Philadelphia, Pennsylvania Submitted 2 December 2013; accepted in final form 26 March 2014

Brailoiu E, Deliu E, Sporici RA, Benamar K, Brailoiu GC. HIV1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats. Am J Physiol Regul Integr Comp Physiol 306: R814 –R822, 2014. First published April 2, 2014; doi:10.1152/ajpregu.00529.2013.—The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1– 86) produced a concentration-dependent increase in cytosolic Ca2⫹ in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca2⫹ elevation: 1) lysosomal Ca2⫹ mobilization, 2) Ca2⫹ release via inositol 1,4,5trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca2⫹ influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca2⫹ entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIVinfected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

Peripheral neuropathy has been denoted as a putative cause of parasympathetic deregulation because of its frequent association with dysautonomia in HIV (48). The HIV invades the brain soon after systemic infection, targeting microglia and perivascular macrophages, while replicating in astrocytes (41). Whereas neurons are resistant to HIV infection, the infected brain cells may release viral components and neurotoxins that alter neuronal function (41). Antiretroviral therapy is efficient in reducing the viral load, but cannot prevent production of early viral proteins (50). Since the alteration of the sympathovagal balance occurs early in HIV infection and evolves with disease progression (4, 40), the early HIV proteins may contribute to autonomic impairment. Among the early viral components, the transactivator of transcription (Tat) protein is an important factor in the HIVinduced pathogenesis of AIDS, contributing to HIV-associated neurological and cardiovascular impairment (27, 49). Tat is expressed at high levels in the brains of HIV-1-infected individuals and has been reported to alter neuronal function (35). Moreover, Tat has recently been identified in the cerebrospinal fluid of patients with suppressed viremia due to efficient antiretroviral treatment (32). Transgenic mice expressing Tat in the myocardium present with bradycardia (22). The first 72 residues of Tat, encoded by the first exon, are critically involved in Tat neuronal effects (35). In this study, we investigate the effect of a widely used Tat fragment (1– 86) (35), referred to as Tat, on cardiac preganglionic neurons of nucleus ambiguus, which are key regulators of the parasympathetic cardiac tone (38).

autonomic cardiovascular regulation; cytosolic Ca2⫹, endoplasmic reticulum; nucleus ambiguus; transient receptor potential vanilloid type 2

MATERIALS AND METHODS

INFECTION WITH THE HUMAN IMMUNODEFICIENCY virus type 1 (HIV-1) leads to the development of HIV-dependent neurological and cardiovascular disorders (25). Autonomic imbalance and arrhythmias are often observed in HIV patients (18, 48). The mechanisms underlying autonomic dysregulation in HIVinfected patients are poorly understood. HIV has been proposed to alter the function of hypothalamic nuclei involved in sympathetic regulation and of sympathetic ganglia (18). Cardiac vagal efferent pathways are also impaired in HIV (18).

Address for reprint requests and other correspondence: G. C. Brailoiu, M.D., Dept. of Pharmaceutical Sciences, Thomas Jefferson Univ., Jefferson School of Pharmacy, Philadelphia, PA 19107 (e-mail: [email protected]). R814

Ethical approval. Animal protocols were approved by the Institutional Animal Care and Use Committee from Thomas Jefferson University and Temple University. All efforts were made to minimize the number of animals used and their suffering. Chemicals. All chemicals were from Sigma-Aldrich (St. Louis, MO), unless otherwise mentioned. Tat protein (1– 86) was from Prospec (East Brunswick, NJ). HIV-1 Tat was heat-inactivated by repeated (10 times) heating (75°C for 30 s) and cooling (4°C for 1 min), as previously reported (10). Animals. Neonatal and adult Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used in this study. Neonatal (1–2 days old) rats of either sex were used for retrograde tracing and neuronal culture, and adult male rats (250 –300 g) were used for cardiovascular measurements. Neuronal labeling and culture. Preganglionic cardiac vagal neurons of nucleus ambiguus were retrogradely labeled by intrapericardial injection of rhodamine [X-rhodamine-5-(and-6)-isothiocyanate;

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MECHANISMS OF HIV-1-TAT-INDUCED BRADYCARDIA

5(6)-XRITC], 40 ␮l, 0.01% (Invitrogen, Carlsbad, CA), as reported by Brailoiu et al. (8, 9, 12). Medullary neurons were dissociated and cultured 24 h after rhodamine injection, as previously described (8, 9, 12). In brief, the brains were quickly removed and immersed in ice-cold Hanks’ balanced salt solution (HBSS; Mediatech, Manassas, VA). Neonate rats were euthanized by decapitation. The ventral side of the medulla (containing nucleus ambiguus) was dissected and minced, and the cells were subjected to enzymatic and mechanical dissociation. Cells were filtered using a sterile 40-␮m filter/cell strainer (Falcon, Fisher Scientific, Waltham, MA) and were plated on glass coverslips in Neurobasal-A medium (Invitrogen) containing 1% GlutaMax (Invitrogen), 2% antibiotic-antimycotic (Mediatech, Herndon, VA), and 10% FBS. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. Cytosine ␤-arabino furanoside (1 ␮M) was added to the culture to inhibit glial cell proliferation (52). Calcium imaging. Measurements of intracellular Ca2⫹ concentration, [Ca2⫹]i were performed as previously described (8, 9, 12). Briefly, cells were incubated with 5 ␮M Fura-2 AM (Invitrogen) in HBSS at room temperature for 45 min and washed with dye-free HBSS. Coverslips were mounted in an open bath chamber (RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope Nikon Eclipse TiE (Nikon, Melville, NY), equipped with a Perfect Focus System and a Photometrics CoolSnap HQ2 chargecoupled device camera (Photometrics, Tucson, AZ). During the experiments, the Perfect Focus System was activated. Fura-2 AM fluorescence (emission 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR 3.1 software (Nikon). After appropriate calibration with ionomycin and CaCl2, and Ca2⫹ free and EGTA, respectively, the ratio of the fluorescence signals (340/380 nm) was converted to Ca2⫹ concentrations (28). Measurement of membrane potential. The relative changes of neuronal membrane potential were evaluated using bis-(1,3-dibutylbarbituric acid)-trimethine-oxonol, DiBAC4(3), a voltage-sensitive dye, as reported (9, 12). Neurons were incubated for 30 min in HBSS containing 0.5 mM DiBAC4(3), and the fluorescence was monitored at 0.17 Hz (excitation/emission 480 nm/540 nm). Calibration of DiBAC4(3) fluorescence was performed using the Na⫹-K⫹ ionophore gramicidin in Na⫹-free physiological solution and various concentrations of K⫹ and N-methylglucamine, as described previously (13). Surgical procedures. Adult male Sprague-Dawley rats were anesthetized with a mixture of ketamine hydrochloride (100 –150 mg/kg) and acepromazine maleate (0.2 mg/kg), as reported previously (5, 9, 12). Animals were placed into a stereotaxic instrument; a guide C315G cannula (PlasticsOne, Roanoke, VA) was bilaterally inserted into the nucleus ambiguus. The stereotaxic coordinates for identification of nucleus ambiguus were 12.24 mm posterior to bregma, 2.1 mm from midline, and 8.2 mm ventral to the dura mater. A C315DC cannula dummy (PlasticsOne) was used to prevent contamination. For transmitter implantation, a 2-cm-long incision was made along the linea alba. A calibrated transmitter (E-mitters, Series 4000; Mini Mitter, Sunriver, OR) was inserted in the intraperitoneal space, as previously described (9, 12). Subsequently, the abdominal musculature and dermis were sutured independently, and animals returned to individual cages. Telemetric heart rate monitoring. The signal generated by transmitters was collected via series 4000 receivers (Mini Mitter, Sunriver, OR), as previously described (9, 12). VitalView software (Mini Mitter) was used for data acquisition. Each data point represents the average of heart rate per 30 s. Noninvasive blood pressure measurement. In rats with cannula inserted into the nucleus ambiguus, blood pressure was noninvasively measured using a volume pressure recording sensor and an occlusion tail-cuff (CODA System, Kent Scientific, Torrington, CT), as described previously (12). One week after the insertion of the cannula, rats were exposed to handling and training every day for 1 wk. The maximum occlusion pressure was 200 mmHg, minimum pressure was

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30 mmHg, and deflation time was 10 s. Two measurements were done per 30 s (one cycle), and the average was used to calculate heart rate and systolic, diastolic, and mean arterial blood pressure. Ten acclimatization cycles were done before starting the experiments. Microinjection into nucleus ambiguus. One week after surgery (telemetric studies) or after another week of training (tail-cuff measurements), bilateral microinjections into the nucleus ambiguus were carried out using the C315I internal cannula (33 gauge, PlasticsOne) and a Neuros Hamilton syringe, model no. 7000.5 KH SYR, without animal handling. In the tail-cuff method, trained rats were in the animal holder for the duration of the experiment. For recovery, at least 2 h were allowed between two injections. Injection of L-glutamate (5 mM, 50 nl) was used for the functional identification of nucleus ambiguus (8, 9). Statistical analysis. Data were expressed as means ⫾ SE. One-way ANOVA followed by post hoc analysis using Bonferroni and Tukey tests was used to evaluate significant differences between groups. P ⬍ 0.05 was considered statistically significant. RESULTS

HIV-1 Tat increases cytosolic Ca2⫹ concentration in cardiac preganglionic neurons of nucleus ambiguus. In rhodamine-labeled neurons, Tat (500 nM) induced a fast increase in intracellular Ca2⫹ concentration, [Ca2⫹]i, followed by a rapid decrease and a plateau maintained at half maximal level (Fig. 1A). Heat-inactivated Tat (500 nM) had negligible effects on [Ca2⫹]i of cardiac vagal neurons (⌬[Ca2⫹]i ⫽ 14 ⫾ 2.1; Fig. 1, A and B). Tat (5 nM, 50 nM, 500 nM, and 5,000 nM) elevated [Ca2⫹]i with a mean amplitude of 39 ⫾ 1.8 nM, 319 ⫾ 3.4 nM, 647 ⫾ 6.3 nM, and 762 ⫾ 8.3 nM, respectively (Fig. 1B), and a calculated EC50 of 66 nM. Six cells were examined in each treatment group. Statistical significance was achieved for the latter three concentrations of Tat. Characteristic examples of changes in 340 nm/380 nm Fura-2 AM fluorescence ratio of rhodamine-labeled neurons in response to Tat (500 nM) and heat-inactivated Tat (500 nM) are shown in Fig. 1, C and D. In vitro systems test Tat effects at concentrations varying between 100 and 500 nM (1, 21, 46, 56). Since robust responses were elicited with 500 nM Tat, similar to previous reports (1, 21, 56), we used this concentration in our following experiments. Tat elicits Ca2⫹ influx in cardiac vagal neurons. We used a pharmacological approach to test the involvement of several Ca2⫹-permeable ion channels to Tat-induced Ca2⫹ increase in vagal neurons. Inhibition of N-type Ca2⫹ channels with ␻-conotoxin GVIA (100 nM, 20 min) did not significantly modify the shape of the Ca2⫹ signal produced by Tat (500 nM) in rhodamine-labeled preganglionic neurons (Fig. 2A). The amplitude and the area under curve were similarly unaffected: the amplitude of the Tat-induced response was 647 ⫾ 6.3 nM (n ⫽ 6) in the absence vs. 638 ⫾ 7.3 nM (n ⫽ 6) in the presence of N-type Ca2⫹ channel blocker (Fig. 2B); the areas under curve were 2,463 ⫾ 41 nM ⫻ min and 2,397 ⫾ 54 nM ⫻ min, respectively (Fig. 2B). Pretreatment of neurons with ␻-conotoxin MVIIC (100 nM, 20 min), a blocker of P/Q-type of Ca2⫹ channels, slightly reduced the amplitude of the Tatdependent Ca2⫹ elevation (⌬[Ca2⫹]i was 541 ⫾ 5.7 nM; n ⫽ 6), and significantly diminished the area under the curve, which measured 1,603 ⫾ 27 nM ⫻ min in this case (Fig. 2B). Since transient receptor potential vanilloid 2 (TRPV2) is a Ca2⫹permeable nonselective cation channel expressed in the nucleus ambiguus (34), we tested the effect of TRPV2 inhibitor tranilast (44, 45). In the presence of tranilast (100 ␮M, 20 min),

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Fig. 1. HIV-1 transactivator of transcription (Tat) increases [Ca2⫹]i of cardiac preganglionic nucleus ambiguus neurons in a concentration-dependent manner. A: typical examples of Ca2⫹ responses upon administration of Tat (500 nM) or heat-inactivated Tat (boiled Tat; 500 nM). B: concentration-response curve indicating the effect of Tat (5–5,000 nM; EC50 ⫽ 66 nM) and heatinactivated Tat (500 nM) on [Ca2⫹]i of cardiac vagal neurons of nucleus ambiguus; P ⬍ 0.00001 compared with basal (*) or to 500 nM Tat (#). C and D: changes in Fura-2 AM fluorescence ratio (340 nm/380 nm) of rhodamine-labeled neurons upon administration of 500 nM Tat (C) or 500 nM heatinactivated Tat (D).

Tat elicited a significantly blunted Ca2⫹ response, with a lower peak and shorter-duration plateau (Fig. 2A); the amplitude measured 411 ⫾ 5.1 nM (n ⫽ 6), and the area under the curve was 680 ⫾ 19 nM ⫻ min (Fig. 2B). When both TRPV2 and P/Q channels were inhibited, Tat no longer elicited a plateau phase, and the initial transient increase in [Ca2⫹]i was additionally reduced (Fig. 2A). ⌬[Ca2⫹]i was 359 ⫾ 4.7 nM (n ⫽ 6), and the area under the curve was 447 ⫾ 17 nM ⫻ min (Fig. 2B). The functional presence of Ca2⫹-permeable TRPV2 in rhodamine-labeled cardiac vagal neurons was detected by the Ca2⫹ response produced by application of TRPV2 agonist probenecid (100 ␮M) (2). The response to probenecid was

abolished in Ca2⫹-free saline or by the TRPV2 blocker tranilast (100 ␮M, 20 min) (Fig. 3, A and B). Tat releases Ca2⫹ from endoplasmic reticulum and lysosomal Ca2⫹ stores. In absence of extracellular Ca2⫹, Tat (500 nM) triggered a fast and transient [Ca2⫹]i elevation (Fig. 4A) by 376 ⫾ 4.2 nM (n ⫽ 6) at the peak of the response (Fig. 4B). This response was significantly reduced in amplitude compared with the effect of Tat in Ca2⫹-containing saline. Thapsigargin (1 ␮M), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2⫹ ATPase, drastically decreased the effect of Tat in Ca2⫹-free saline; ⌬[Ca2⫹]i was 79 ⫾ 2.4 nM, n ⫽ 6 neurons (Fig. 4, A and B). Blocking ryanodine receptors with ryanodine

Fig. 2. Tat promotes Ca2⫹ entry via TRPV2 and P/Q-type of Ca2⫹ channels. A: representative examples of Ca2⫹ responses triggered by Tat in the absence and presence of blockers of voltage-activated Ca2⫹ channels (Ntype, ␻-conotoxin GVIA; P/Q-type, ␻-conotoxin MVIIC) or of the nonselective cation channel TRPV2 (tranilast, Tran); inhibition of both TRPV2 and P/Q channels produced a marked decrease in Tat-induced Ca2⫹ response. B: comparison of the changes in amplitude (top) and in area under the curve (AUC; bottom) of the Ca2⫹ effects produced by Tat in the conditions mentioned in A (*P ⬍ 0.00001 compared with Tat alone).

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Fig. 3. Functional characterization of TRPV2 channels in cardiac vagal neurons. A: typical tracings of the Ca2⫹ responses induced by TRPV2 agonist probenecid alone in Ca2⫹ containing (top) and Ca2⫹-free saline (middle), or by probenecid in the presence of TRPV2 blocker tranilast (bottom). B: probenecid significantly elevates [Ca2⫹]i of cardiac parasympathetic neurons by 246 ⫾ 6 nM (n ⫽ 6); this effect is lost in Ca2⫹-free saline (⌬[Ca2⫹]i ⫽ 5 ⫾ 3.4 nM; n ⫽ 6) or in presence of tranilast (⌬[Ca2⫹]i ⫽ 19 ⫾ 4.1 nM, n ⫽ 6); P ⬍ 0.00001 compared with basal (*) or with probenecid (#).

(10 ␮M, 1 h) did not significantly interfere with the response to Tat (⌬[Ca2⫹]i was 371 ⫾ 3.9 nM; n ⫽ 6). In the presence of xestospongin C (10 ␮M, 15 min), a blocker of inositol 1,4,5trisphosphate (IP3) receptors (IP3R), rhodamine-labeled cardiac vagal neurons responded to Tat with a greatly diminished increase in [Ca2⫹]i, by 81 ⫾ 1.8 nM (n ⫽ 6) at the peak of the response (Fig. 4, A and B). A significant reduction in the Tat-mediated response was also observed in the presence of bafilomycin A1 (1 ␮M, 1 h), a V-type ATPase that inhibits lysosomal acidification (6); ⌬[Ca2⫹]i was 176 ⫾ 3.7 nM, n ⫽ 6 (Fig. 4, A and B). Pretreatment with both bafilomycin and xestospongin C largely abolished the Ca2⫹ increase produced by Tat (⌬[Ca2⫹]i) was 16 ⫾ 2.3 nM; n ⫽ 6 (Fig. 4, A and B). Tat produces a concentration-dependent depolarization of cardiac vagal neurons. Tat (500 nM) robustly depolarized cardiac-projecting parasympathetic neurons retrogradely labeled with rhodamine, an effect that lasted for the duration of the experiment (Fig. 5A). Conversely, heat-inactivated Tat (500 nM) produced negligible effects on membrane potential (⌬voltage was 0.83 ⫾ 0.1 mV, Fig. 5, A and B). Increasing concentrations of Tat (5 nM, 50 nM, 500 nM, and 5,000 nM) induced neuronal depolarizations with amplitudes of 0.6 ⫾ 0.4 mV, 4.7 ⫾ 0.6 mV, 11.9 ⫾ 0.8 mV, and 14.3 ⫾ 1.1 mV, respectively (Fig. 5B), and a calculated EC50 of 101 nM. The

responses to the latter three concentrations of Tat were significant (n ⫽ 6 neurons per each condition; P ⬍ 0.00001). Tat-induced depolarization involves TRPV2 activation. Treatment of the neurons with BAPTA-AM (200 ␮M, 20 min), a fast Ca2⫹ chelator, markedly reduced the amplitude and the duration of Tat-induced neuronal depolarization (Fig. 6A). In neurons pretreated with the TRPV2 inhibitor tranilast (100 ␮M, 20 min; Fig. 6A), Tat produced a similar, strongly diminished response. The amplitude of the depolarization elicited by Tat (500 nM) was reduced from 11.9 ⫾ 0.8 mV to 4.2 ⫾ 0.7 mV in the presence of BAPTA and to 4.1 ⫾ 0.8 mV in the presence of tranilast, while the area under the curve decreased from 73.1 ⫾ 5.4 mV ⫻ min to 7.68 ⫾ 0.41 mV ⫻ min and to 6.65 ⫾ 0.39 mV ⫻ min, respectively (Fig. 6B). Microinjection of Tat into the nucleus ambiguus produces bradycardia in conscious rats. In conscious, freely moving rats bearing cannulas implanted into the nucleus ambiguus, microinjection of control saline (50 nl) produced negligible effects on heart rate, monitored telemetrically. Microinjection of L-glutamate (5 mM, 50 nl) elicited bradycardia, without a change in blood pressure (Fig. 7A), indicating the correct placement of the cannula into the nucleus ambiguus (8, 9, 17). Two hours after L-glutamate administration, microinjection of Tat (500 fmol/50 nl) triggered a sharp and sustained decrease

Fig. 4. Tat elicits Ca2⫹ release from IP3sensitive pools and acidic Ca2⫹ stores. A: characteristic increases in [Ca2⫹]i produced by Tat (500 nM) in Ca2⫹-free saline, in the absence and presence of thapsigargin (Tg), ryanodine receptor blocker ryanodine (Ry), IP3R inhibitor xestospongin C (XeC), lysosomal disruptor bafilomycin A1 (BAF), and of a combination of the latter two. B: comparison of the effects on [Ca2⫹]i of cultured cardiac vagal neurons induced by the treatments indicated in A; P ⬍ 0.00001 compared with Tat alone (*), with Tat and with XeC ⫹ Tat treatment groups (#), or with all other conditions (⫹).

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Fig. 5. Tat depolarizes cultured cardiac preganglionic neurons of nucleus ambiguus. A: representative changes in membrane potential elicited by Tat (500 nM) or heatinactivated Tat (500 nM) in rhodamine-labeled neurons. B: concentration-response curve indicating the effect of Tat (5–5,000 nM; EC50 ⫽ 101 nM) and heat-inactivated Tat (500 nM) on the resting membrane potential of cardiac vagal neurons; P ⬍ 0.00001 compared with resting membrane potential (*) or to the effect of 500 nM Tat (#).

in heart rate (Fig. 7A). Similar responses were recorded using the tail-cuff method (Fig. 7A). The tail-cuff method allowed measurement of blood pressure; as shown in Fig. 7A, microinjection of Tat into the nucleus ambiguus had no effect on blood pressure. Heat inactivation of Tat prevented its bradycardic effects (Fig. 7A). Tat (5 fmol, 50 fmol, and 500 fmol/50 nl) decreased the heart rate of conscious rats by 6 ⫾ 1.7 beats per min (bpm), 26 ⫾ 8 bpm, and 68 ⫾ 2.1 bpm, respectively (Fig. 7B). Only the latter two effects were statistically significant (P ⬍ 0.00001). Administration of heat-inactivated Tat (500 fmol/50 nl) did not result in a significant change in heart rate (4 ⫾ 0.7 bpm). Five animals were used per each treatment group. In vivo studies evaluating Tat effects in the brain use Tat at a concentration of 3 or 4 ␮g/␮l, which is ⬎1,000 times higher than that commonly used in vitro (36, 60). Similarly, microinjection in the rat nucleus ambiguus of higher concentrations of Tat (5–500 fmol/50 nl, i.e., 0.1–10 ␮M) elicited the bradycardic responses. The explanation may reside in the fact that Tat may be locally diluted upon microinjection (60) or that Tat is not only sensitive to oxidation, but also is particularly sticky, able to bind even to silanized glass surfaces (as in the syringe barrel and needle used in stereotaxic injections) (36). Measuring actual levels of Tat in the extracellular space in the central nervous system has proven difficult because of the lack of successful ELISA strategies and because native Tat is incredibly sensitive to oxidation. Nanomolar levels of Tat have been detected in the sera of HIV-infected patients, but these values may be underestimated, since Tat can be trapped by heparan sulfate expressed on the cell surfaces (46). However,

patients with suppressed viremia due to effective antiretroviral treatment still present nanomolar levels of Tat in the cerebrospinal fluid (32). This concentration is expected to be higher near productively infected cells. As such, although Tat levels in situ (i.e., in the brain extracellular space) remain to be determined, it is conceivable that levels sufficient to promote neuronal activation may well be achieved near HIV-1-infected glia cells. Tat-induced bradycardia is mediated by TRPV2. Microinjection of TRPV2 antagonist tranilast (100 ␮M, 50 nl) alone produced a negligible increase in heart rate (Fig. 8A), with a mean amplitude of 9 ⫾ 0.7 bpm (n ⫽ 5) and with an area under the curve of 36 ⫾ 4.1 bpm ⫻ min (Fig. 8B). Concomitant microinjection of tranilast and Tat (500 fmol/50 nl) drastically reduced the amplitude and duration of Tat-induced bradycardic effect (Fig. 8A). In the presence of tranilast, Tat decreased the heart rate by 53 ⫾ 2.9 bpm (n ⫽ 5), compared with a decrease by 68 ⫾ 2.1 bpm produced by Tat alone (n ⫽ 5 rats). The areas under the curve were more dramatically affected: 52 ⫾ 4.3 bpm ⫻ min after combined tranilast and Tat administration, compared with 432 ⫾ 8.3 bpm ⫻ min produced by Tat alone (Fig. 8B). DISCUSSION

HIV-1 Tat is a 72(86)-101(104)-amino acid regulatory protein, essential for viral transcription and replication; the first 56 residues of Tat are well conserved, indicating important functional roles (20, 49). The cysteine-rich region (residues 22–37)

Fig. 6. Tat-induced depolarization is Ca2⫹dependent and TRPV2-mediated. A: representative traces depicting Tat (500 nM)-induced depolarizations in the absence (black) and presence of Ca2⫹ chelator BAPTA (light gray) or of TRPV2 blocker tranilast (Tran; dark gray). B: comparison of the amplitudes (top) and areas under curve (AUC; bottom) of Tat-triggered depolarizations in the conditions mentioned in A; *P ⬍ 0.00001 compared with Tat.

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Fig. 7. Tat microinjection into the nucleus ambiguus of conscious rats elicits a prolonged, dose-dependent decrease in heart rate. A: characteristic heart rate and blood pressure recordings after microinjection of saline, L-glutamate (L-Glu; 5 mM, 50 nl) and either Tat (500 fmol/50 nl) or boiled Tat (500 fmol/50 nl), obtained using the telemetric method (top) or the tail-cuff method (bottom). B: microinjection of Tat (5, 50, and 500 fmol) induces dose-dependent bradycardic responses, while heat-inactivated Tat (500 fmol) has negligible effects on heart rate; P ⬍ 0.00001 compared with basal heart rate (*) or to 500 fmol Tat (#). bpm, beats per min.

and the basic domain (residues 31– 61) play major roles in Tat-induced neurotoxic and excitatory effects (35). Upon release by infected microglia, macrophages, and astrocytes, Tat targets uninfected neurons to trigger Ca2⫹ responses and depolarization (7, 10, 35). These mechanisms have been implicated in both Tat-mediated neuronal responses and HIV-induced neurotoxicity (43, 46, 49). The first observation of the present study is that Tat (fragment 1– 86) produced a robust increase in [Ca2⫹]i of cardiac vagal neurons, characterized by an initial transitory peak with high amplitude, followed by a long-lasting, smaller plateau. Next, we identified that the sustained phase of the Tat-induced effect on [Ca2⫹]i was mostly dependent on Ca2⫹ entry via TRPV2 nonselective cation channels and only partially mediated by P/Q-type of voltage-activated Ca2⫹ channels. We and others reported a major role of P/Q-type of Ca2⫹ current in the physiology of cardiac parasympathetic ambiguus neurons (9, 11, 12, 31). A Tat-mediated P/Q Ca2⫹ current has also been described in suprachiasmatic nucleus neurons (19). Nonetheless, not much is known about the TRPV2 function in nucleus ambiguus neurons, despite the strong expression of this type of channel at the level of the nucleus ambiguus (34). Similarly, little evidence points to a contribution of transient receptor potential channels to Tat-induced neuronal effects. Conversely, the short-lived and high-amplitude component of the Tat-triggered Ca2⫹ response consisted of Ca2⫹ influx via TRPV2 and Ca2⫹ release from two separate intracellular Ca2⫹ pools: the lysosomes and the IP3-sensitive endoplasmic retic-

ulum stores. Tat has been reported to promote IP3 formation and/or IP3R-mediated Ca2⫹ mobilization from the endoplasmic reticulum in human fetal neurons and astrocytes (29), human macrophages (37), and human cerebrocortical terminals (23). Our finding that Tat mobilizes lysosomal Ca2⫹ stores correlates well with the fact that this protein enters neurons rapidly via receptor-mediated endocytosis, altering the endolysosomal structure and function (15, 30). Since TRPV2 are Ca2⫹-permeable nonselective cation channels modulated by Ca2⫹ and by phosphatidylinositol-4,5-bisphosphate metabolism (39, 42), we propose that HIV Tat acts first to mobilize intracellular Ca2⫹ stores and that TRPV2 stimulation occurs consequently (Fig. 9). Similar to other types of neurons (10, 35), cardiac-projecting vagal neurons of nucleus ambiguus depolarized in response to Tat application, and the effect was concentration-dependent. The depolarization occurred rapidly and presented a sustained plateau that lasted for the entire duration of the experiments. Ca2⫹ chelation or TRPV2 inhibition produced likewise reductions of the initial amplitude and abolished the sustained phase of the depolarization. The residual Tat-mediated depolarizations had largely identical kinetics and amplitudes in the presence of BAPTA-AM or tranilast pretreatment, supporting a correlation of the Ca2⫹ increase and TRPV2 activation in the mechanism. A putative explanation of the residual depolarization observed with Tat in the presence of BAPTA may lie in the ability of Tat to depolarize the neuronal cell membrane when applied extracellularly to outside-out membrane patches,

Fig. 8. Tat-induced bradycardia is TRPV2dependent. A: representative heart rate recordings after intranucleus ambiguus administration of saline, L-glutamate (L-Glu; 5 mM/50 nl) and either Tat (500 fmol/50 nl), tranilast (Tran, 100 ␮M/50 nl) or both Tat and tranilast. B: comparison of the effects on heart rate of the treatments mentioned in A, evaluated in terms of amplitude (top) and area under curve (AUC; bottom). *P ⬍ 0.00001 compared with microinjection of Tat (500 fmol/50 nl).

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Fig. 9. Proposed mechanism for Tat-induced bradycardia. In nucleus ambiguus neurons, transactivator of transcription (Tat) induces Ca2⫹ release from two pools: the lysosomes (Lys) and the endoplasmic reticulum [ER; via the inositol 1,4,5-trisphosphate receptors (IP3R)]. The consequent increase in [Ca2⫹]i activates transient receptor potential vanilloid type 2 (TRPV2) channels at the plasma membrane promoting cation entry (mostly Na⫹ and Ca2⫹) and subsequent depolarization. Depolarization triggers activation of P/Q-type of voltage-activated Ca2⫹ channels, resulting in an additional Ca2⫹ influx. This cascade converges on cardiac vagal neuron activation, increase of parasympathetic outflow to the heart, and decrease in heart rate.

and, thus, directly excite neurons on the cell surface (16). Absence of extracellular Ca2⫹ attenuated Tat-induced depolarization only by 43%, both in intact neurons and in outside-out patches (16), which is consistent with the findings of our present study. Our in vivo microinjection studies indicate that Tat administration into the nucleus ambiguus produced a strong and sustained decrease in heart rate, which was not accompanied by an effect on blood pressure. When TRPV2 channels in the nucleus ambiguus were blocked by tranilast pretreatment, Tat elicited only a transient heart rate decrease, reduced in amplitude, indicating a major contribution of TRPV2 in Tat-mediated prolonged bradycardia. Thus, we propose that Tat targets cardiac preganglionic neurons of the nucleus ambiguus to produce Ca2⫹ release from lysosomal and endoplasmic reticulum IP3-sensitive Ca2⫹ pools, resulting in intracellular Ca2⫹ increase. Further, we infer that TRPV2 activation occurs subsequently to Tat-induced stimulation of the intracellular cascade leading to IP3R-mediated Ca2⫹ release (Fig. 9). TRPV2 regulation by extracellular Ca2⫹ and phosphoinositide 4,5bisphosphate metabolism has been reported (39, 42). In addition, TRPV2 channels were recently shown to be activated downstream of a G protein- and PLC-mediated pathway (3, 42). Furthermore, AT1R stimulation by ANG II promoted IP3R-mediated Ca2⫹ elevation, which appeared to be implicated in the subsequent activation of TRPV2, since IP3R blockade prevented the TRPV2-dependent component of the Ca2⫹ response (3). As such, mobilization of Ca2⫹ from intracellular stores may precede and trigger Ca2⫹ influx via TRPV2 (3). Since TRPV2 appeared to be implicated both in the Ca2⫹ influx and in the depolarization elicited by Tat in cardiac vagal neurons, we propose that this depolarization was responsible for the activation of the P/Q-type of voltage-activated Ca2⫹ channels (Fig. 9). These intracellular events converge to neuronal activation, which is translated in vivo into a prolonged bradycardia (Fig. 9). Bradycardia is one of the factors that trigger QT interval prolongation and the consequent life-threatening polymorphic ventricular arrhythmia known as torsade de pointes (57). HIVinfected patients have a higher prevalence of QT prolongation (47, 51), along with HIV-dependent autonomic neuropathy (24, 59). Emerging evidence indicates their primary association with HIV infection rather than with the anti-HIV therapy (14,

24). Cardiovascular complications of antiretroviral treatment have been described (33, 61, 63). Particularly, patients receiving antiretroviral drugs may be at increased risk for myocardial infarction (33, 61). However, recent studies indicate that cardiovascular risk status, as estimated by Framingham risk score categories (⬍6% and ⱖ6%), remained relatively stable over 144-wk period in patients enrolled in ARIES (Atazanavir, Ritonavir, Induction with Epzicom Study; EPZ108859) (62). ARIES was a large-phase IIIb/IV treatment-simplification clinical trial in which antiretroviral therapy-naive subjects who achieved virologic suppression by week 30 on a regimen of abacavir/lamivudine plus atazanavir/ritonavir were randomized to remain on their original regimen or discontinue the ritonavir component of the regimen for up to a total of 144 wk (53–55). The effects of antiretroviral therapy on QT interval prolongation (14, 24) or on dysautonomia in HIV (14) were found insignificant. Conversely, greater QT interval duration has been reported in HIV-infected patients with asymptomatic autonomic neuropathy compared with HIV-infected patients without neurological disorder (59), indicating that dysautonomia in HIV infection has a negative impact on cardiac function. Moreover, HIV-infected individuals are at a significantly higher risk for sudden cardiac death compared with the general population with similar risk factors (58). Lethal arrhythmias account for as much as 20% of total sudden cardiac deaths in HIV-1-infected patients and are six times more prevalent in these patients than in the control population (58). Of the sudden cardiac death victims with recent laboratory investigations, over half had undetectable viral loads (58); nonetheless, patients under controlled antiretroviral therapy showing viral load suppression both in the blood and cerebrospinal fluid still present significant levels of Tat in the cerebrospinal fluid (32). Perspectives and Significance HIV-1 transactivator of transcription, or Tat, has several pleiotropic functions in addition to its transcriptional activity (27). We report here that Tat produces a sustained bradycardia by targeting neurons of nucleus ambiguus that modulate the cardiac vagal tone. We characterize the underlying cellular mechanisms of Tat-induced activation of nucleus ambiguus neurons. Our findings provide a novel mechanism for the

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cardiac autonomic dysfunction reported in HIV-infected patients (4, 18, 26, 40). GRANTS This work was supported by the National Institutes of Health Grant HL-090804 (to E. Brailoiu) from the Department of Health and Human Services. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: E.B., K.B., and G.C.B. conception and design of research; E.B., K.B., and G.C.B. performed experiments; E.B., E.D., R.A.S., and G.C.B. analyzed data; E.B., E.D., R.A.S., and G.C.B. interpreted results of experiments; E.B., E.D., and G.C.B. prepared figures; E.B., E.D., R.A.S., K.B., and G.C.B. drafted manuscript; E.B., E.D., R.A.S., K.B., and G.C.B. edited and revised manuscript; E.B., E.D., R.A.S., K.B., and G.C.B. approved final version of manuscript. REFERENCES 1. Badou A, Bennasser Y, Moreau M, Leclerc C, Benkirane M, Bahraoui E. Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway. J Virol 74: 10551–10562, 2000. 2. Bang S, Kim KY, Yoo S, Lee SH, Hwang SW. Transient receptor potential V2 expressed in sensory neurons is activated by probenecid. Neurosci Lett 425: 120 –125, 2007. 3. Barro-Soria R, Stindl J, Muller C, Foeckler R, Todorov V, Castrop H, Strauss O. Angiotensin-2-mediated Ca2⫹ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel. PLoS One 7: e49624, 2012. 4. Becker K, Gorlach I, Frieling T, Haussinger D. Characterization and natural course of cardiac autonomic nervous dysfunction in HIV-infected patients. AIDS 11: 751–757, 1997. 5. Benamar K, Addou S, Yondorf M, Geller EB, Eisenstein TK, Adler MW. Intrahypothalamic injection of the HIV-1 envelope glycoprotein induces fever via interaction with the chemokine system. J Pharmacol Exp Ther 332: 549 –553, 2010. 6. Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972–7976, 1988. 7. Brailoiu E, Brailoiu GC, Mameli G, Dolei A, Sawaya BE, Dun NJ. Acute exposure to ethanol potentiates human immunodeficiency virus type 1 Tat-induced Ca2⫹ overload and neuronal death in cultured rat cortical neurons. J Neurovirol 12: 17–24, 2006. 8. Brailoiu GC, Arterburn JB, Oprea TI, Chitravanshi VC, Brailoiu E. Bradycardic effects mediated by activation of G protein-coupled estrogen receptor in rat nucleus ambiguus. Exp Physiol 98: 679 –691, 2013. 9. Brailoiu GC, Benamar K, Arterburn JB, Gao E, Rabinowitz JE, Koch WJ, Brailoiu E. Aldosterone increases cardiac vagal tone via GPER activation. J Physiol 591: 4223–4235, 2013. 10. Brailoiu GC, Brailoiu E, Chang JK, Dun NJ. Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons. Neuroscience 151: 701–710, 2008. 11. Brailoiu GC, Deliu E, Tica AA, Chitravanshi VC, Brailoiu E. Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons. J Neurochem, 2012. 12. Brailoiu GC, Deliu E, Tica AA, Rabinowitz JE, Tilley DG, Benamar K, Koch WJ, Brailoiu E. Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats. J Neurochem 126: 739 –748, 2013. 13. Brauner T, Hulser DF, Strasser RJ. Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochim Biophys Acta 771: 208 –216, 1984. 14. Charbit B, Rosier A, Bollens D, Boccara F, Boelle PY, Koubaa A, Girard PM, Funck-Brentano C. Relationship between HIV protease inhibitors and QTc interval duration in HIV-infected patients: a crosssectional study. Br J Clin Pharmacol 67: 76 –82, 2009. 15. Chen X, Hui L, Geiger NH, Haughey NJ, Geiger JD. Endolysosome involvement in HIV-1 transactivator protein-induced neuronal amyloid beta production. Neurobiol Aging 34: 2370 –2378, 2013.

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