HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats

Abstract

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 Ca(2+) 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 Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) 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 Ca(2+) 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 HIV-infected 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.

Modulation of cardiac vagal tone by bradykinin acting on nucleus ambiguus

“…Cardiac vagal neurons of nucleus ambiguus were retrogradely labeled by intrapericardial injection of rhodamine [X-rhodamine-5-(and-6)-isothiocyanate; 5(6)-XRITC], 40 µl, 0.01%, (Invitrogen, ThermoFisher Scientific, Grand Island, NY), as previously reported (Brailoiu et al, 2014a, Brailoiu et al, 2014b). 24 h after rhodamine injection, rats were euthanized by decapitation, and the brains quickly removed and immersed in ice-cold Hanks’ balanced salt solution (HBSS; Mediatech, Manassas, VA).…”

“…The intracellular Ca 2+ concentration, [Ca 2+ ] i , was assessed, as previously described, in neurons loaded with Fura-2 AM (Brailoiu et al, 2014a, Brailoiu et al, 2014b). Neurons were incubated with Fura-2 AM (5 µM in HBSS, 45 min, at room temperature).…”

“…The changes of membrane potential were assessed in neurons loaded with bis-(1,3-dibutylbarbituric acid)-trimethine-oxonol, DiBAC 4 (3), a voltage-sensitive dye, as reported (Brailoiu et al, 2014a, Brailoiu et al, 2014b). Neurons were incubated with DiBAC 4 (3) (0.5 mM in HBSS, 30 min) and the fluorescence (excitation/emission 480nm/540nm) was monitored.…”

“…Adult male rats (250–300 g) were anesthetized with ketamine hydrochloride (100–150 mg/kg) and acepromazine maleate (0.2 mg/kg), as previously reported (Brailoiu et al, 2014a, Brailoiu et al, 2014b). Nucleus ambiguus was identified based on stereotaxic coordinates (12.24 mm posterior to bregma, 2.1 mm from midline and 8.2 mm ventral to the dura mater); a guide C315G cannula was bilaterally inserted into the nucleus ambiguus.…”

“…Nucleus ambiguus was identified based on stereotaxic coordinates (12.24 mm posterior to bregma, 2.1 mm from midline and 8.2 mm ventral to the dura mater); a guide C315G cannula was bilaterally inserted into the nucleus ambiguus. A calibrated transmitter (E-mitters, series 4000; Mini Mitter, Sunriver, OR) was inserted in the intraperitoneal space, as reported (Brailoiu et al, 2014a, Brailoiu et al, 2014b). …”