Neurocardiology: translational advancements and potential.

In our original white paper published in the The Journal of Physiology in 2016, we set out our knowledge of the structural and functional organization of cardiac autonomic control, how it remodels during disease, and approaches to exploit such knowledge for autonomic regulation therapy. The aim of this update is to build on this original blueprint, highlighting the significant progress which has been made in the field since and major challenges and opportunities that exist with regard to translation. Imbalances in autonomic responses, while beneficial in the short term, ultimately contribute to the evolution of cardiac pathology. As our understanding emerges of where and how to target in terms of actuators (including the heart and intracardiac nervous system (ICNS), stellate ganglia, dorsal root ganglia (DRG), vagus nerve, brainstem, and even higher centres), there is also a need to develop sensor technology to respond to appropriate biomarkers (electrophysiological, mechanical, and molecular) such that closed-loop autonomic regulation therapies can evolve. The goal is to work with endogenous control systems, rather than in opposition to them, to improve outcomes.

Translational neurocardiology: preclinical models and cardioneural integrative aspects.

Neuronal elements distributed throughout the cardiac nervous system, from the level of the insular cortex to the intrinsic cardiac nervous system, are in constant communication with one another to ensure that cardiac output matches the dynamic process of regional blood flow demand. Neural elements in their various 'levels' become differentially recruited in the transduction of sensory inputs arising from the heart, major vessels, other visceral organs and somatic structures to optimize neuronal coordination of regional cardiac function. This White Paper will review the relevant aspects of the structural and functional organization for autonomic control of the heart in normal conditions, how these systems remodel/adapt during cardiac disease, and finally how such knowledge can be leveraged in the evolving realm of autonomic regulation therapy for cardiac therapeutics.

NO-cGMP pathway increases the hyperpolarisation-activated current, I(f), and heart rate during adrenergic stimulation.

OBJECTIVES: The role of the nitric oxide (NO)-cGMP pathway in the autonomic modulation of cardiac pacemaking is controversial and may involve an interplay between the L-type calcium current, I(CaL), and the hyperpolarisation activated current, I(f). We tested the hypothesis that following adrenergic stimulation, the NO-cGMP pathway stimulates phosphodiesterase 2 (PDE2) to reduce cAMP dependent stimulation of I(f) and heart rate (HR). METHODS: In the presence of norepinephrine (NE, 1 microM), the effects of the NO donor sodium nitroprusside (SNP) were evaluated in sinoatrial node (SAN)/atria preparations and isolated SAN cells from adult guinea pigs. RESULTS: Contrary to our hypothesis, SNP (10 and 100 microM, n=5) or the membrane permeable cGMP analogue, 8Br-cGMP (0.5 mM, n=6) transiently increased HR by 5+/-1, 12+/-1 and 12+/-2 beats/min, respectively. The guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM, n=5) abolished the increase in HR to SNP (100 microM) as did the I(f) blockers caesium chloride (2 mM, n=7) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (ZD7288, 1 microM, n=7). Addition of SNP (10 microM) also transiently increased I(f) in SAN cells (n=5). After inhibition of PDE2 with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 microM, n=5), the increase in HR to SNP in the presence of NE was significantly augmented and maintained. RT-PCR analysis confirmed the presence of PDE2 in addition to cGMP inhibited PDE3 mRNA in central SAN tissue. CONCLUSIONS: These results suggest that during adrenergic stimulation, activation of the NO-cGMP pathway does not decrease HR, but has a transient stimulatory effect that is I(f) dependent, and is limited in magnitude and duration by stimulation of PDE2.

Peripheral pre-synaptic pathway reduces the heart rate response to sympathetic activation following exercise training: role of NO.

OBJECTIVES: We tested the hypothesis that the attenuated heart rate (HR) response to sympathetic activation following swim training in the guinea pig (Cavia porcellus) results from a peripheral modulation of pacemaking by nitric oxide (NO). METHODS: Nitric oxide synthase (NOS) inhibition on the increase in heart rate with sympathetic nerve stimulation (SNS) was investigated in the isolated guinea pig double atrial/right stellate ganglion preparation from exercise trained (6-weeks swimming, n=20) and sedentary animals (n=20). Western blot analysis for neuronal nitric oxide synthase (nNOS) was performed on the stellate ganglion from both groups. RESULTS: Relative to the control group, the exercise group demonstrated typical exercise adaptations of increased ventricular weight/body weight ratio, enhanced skeletal muscle citrate synthase activity and higher concentrations of [3H]ouabain binding sites in both skeletal and cardiac tissue (P<0.05). The increase in heart rate (bpm) with SNS significantly decreased in the exercise group (n=16) compared to the sedentary group (n=16) from 30+/-5 to 17+/-3 bpm at 1 Hz; 67+/-7 to 47+/-4 bpm at 3 Hz; 85+/-9 to 63+/-4 bpm at 5 Hz and 101+/-9 to 78+/-5 bpm at 7 Hz stimulation (P<0.05). The increase in heart rate with cumulative doses (0.1-10 microM) or a single dose (0.1 microM) of bath-applied norepinephrine expressed as the effective doses at which the HR response was 50% of the maximum response (EC50) were similar in both exercise (EC50 -6.08+/-0.16 M, n=8) and sedentary groups (EC50 -6.18+/-0.07 M, n=7). Trained animals had significantly more nNOS protein in left stellate ganglion compared to the sedentary group. In the exercise group, the non-isoform selective NOS inhibitor, N-omega nitro-L-arginine (L-NA, 100 microM) caused a small but significant increase in the heart rate response to SNS. However, the positive chronotropic response to sympathetic nerve stimulation remained significantly attenuated in the exercise group compared to the sedentary group during NOS inhibition (P<0.05). CONCLUSIONS: Our results indicate that there is a significant peripheral pre-synaptic component reducing the HR response to sympathetic activation following training, although NO does not play a dominant role in this response.

Methods of surveillance for HIV infection in primary care outpatients in the United States.

Primary care outpatients provide a good sentinel population for monitoring levels and trends of HIV infection in the United States. Because a broad cross section of the population seeks primary medical care, excess blood from specimens routinely collected for other purposes is available for anonymous, unlinked HIV testing, and all age groups and both sexes can be sampled. The CDC family of surveys includes two surveys of primary care outpatients: (a) a survey of 100,000 blood specimens per year submitted by more than 6,000 primary care physicians to a national diagnostic laboratory for complete blood count or hematocrit and (b) a survey of approximately 10,000 blood specimens per year from a network of 242 primary care physicians. Each survey has different advantages: the laboratory-based survey has a large sample from a large population base, and the physician network survey has a well-defined patient population in which each patient's clinical condition can be determined. In the primary care physician network, a concurrent study of clinical patterns of disease in patients with recognized HIV infection provides additional information on the clinical syndromes associated with HIV infection and estimates of the occurrence of unrecognized HIV infection.

Neurotoxic (+)-methamphetamine treatment in rats increases brain-derived neurotrophic factor and tropomyosin receptor kinase B expression in multiple brain regions.

Methamphetamine (MA) is an abused stimulant which can result in cognitive deficits and monoamine depletions. Animal models of neurotoxic MA exposure show reductions in dopamine, serotonin, and their associated transporters. MA abuse can result in long-term attention, working memory, and executive function deficits in humans and deficits in route-based egocentric learning, novel object recognition, and novel odor preference in rodents. MA has also been shown to affect brain-derived neurotrophic factor (BDNF) in humans and rodents. This experiment examined the effects of a MA binge dosing regimen (10 mg/kg x 4 at 2 h intervals, s.c.) in Sprague-Dawley rats on BDNF, tropomyosin receptor kinase B (TrkB), and tyrosine hydroxylase (TH) mRNA expression, and plasma corticosterone. Tissues were collected 1, 7, and 24 h following the last MA dose. Expression of BDNF and TrkB mRNA was analyzed using in situ hybridization with cRNA probes. Frontal, parietal, and entorhinal cortical BDNF mRNA expression were increased by MA exposure at all time-points. Increases in BDNF mRNA were also seen in the hippocampal CA1, prefrontal cortex (PFC), piriform cortex, and locus coeruleus but only at specific times. TrkB mRNA expression was modified in several subregions of the hippocampus as well as in PFC and striatum. TH mRNA was increased at the 1 h time-point in the substantia nigra pars compacta with no differences noted at the other times. Corticosterone levels were increased at all three time-points. The findings suggest that BDNF and its receptor may be upregulated as a compensatory mechanism after MA exposure.

Nitric oxide-cGMP pathway facilitates acetylcholine release and bradycardia during vagal nerve stimulation in the guinea-pig in vitro.

1. We tested the hypothesis that nitric oxide (NO) augments vagal neurotransmission and bradycardia via phosphorylation of presynaptic calcium channels to increase vesicular release of acetylcholine. 2. The effects of enzyme inhibitors and calcium channel blockers on the actions of the NO donor sodium nitroprusside (SNP) were evaluated in isolated guinea-pig atrial-right vagal nerve preparations. 3. SNP (10 microM) augmented the heart rate response to vagal nerve stimulation but not to the acetylcholine analogue carbamylcholine (100 nM). SNP also increased the release of [3H]acetylcholine in response to field stimulation. No effect of SNP was observed on either the release of [3H] acetylcholine or the HR response to vagal nerve stimulation in the presence of the guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM). 4. The phosphodiesterase 3 (PDE 3) inhibitor milrinone (1 microM) increased the release of [3H] acetylcholine and the vagal bradycardia and prevented any further increase by SNP. SNP was still able to augment the vagal bradycardia in the presence of the protein kinase G inhibitor KT5823 (1 microM) but not after protein kinase A (PKA) inhibition with H-89 (0.5 microM) or KT5720 (1 microM) had reduced the HR response to vagal nerve stimulation. Neither milrinone nor H-89 changed the HR response to carbamylcholine. 5. SNP had no effect on the magnitude of the vagal bradycardia after inhibition of N-type calcium channels with omega-conotoxin GVIA (100 nM). 6. These results suggests that NO acts presynaptically to facilitate vagal neurotransmission via a cGMP-PDE 3-dependent pathway leading to an increase in cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels. This pathway may augment the HR response to vagal nerve stimulation by increasing presynaptic calcium influx and vesicular release of acetylcholine.

Pre-synaptic NO-cGMP pathway modulates vagal control of heart rate in isolated adult guinea pig atria.

The role of nitric oxide (NO) in the vagal modulation of heart rate (HR) is controversial. We tested the hypothesis that NO acts via a pre-synaptic, guanylyl cyclase (GC) dependent pathway. The effects of inhibiting NO synthase (NOS) and GC were evaluated in isolated atrial/right vagal nerve preparations from adult (550-750 g) and young (150-250 g) female guinea pigs. Levels of NOS protein were quantified in right atria using Western blotting and densitometry. The non-specific NOS inhibitor N- omega -nitro- L -arginine (L -NA, 100 microM, n=5) significantly reduced the negative chronotropic response to vagal nerve stimulation (VNS) at 3 and 5 Hz in the adult guinea pig. This effect was reversed with 1 m ML -arginine. Similar results were observed with the specific neuronal NOS inhibitor vinyl-N5-(1-imino-3-butenyl)- L -ornithine (L -VNIO, 100 microM, n=7). Inhibition of GC with 1H-(1,2,4)-oxadiazolo-(4, 3-a)-quinoxalin-1-one (ODQ, 10 microM, n=7) also significantly reduced the negative chronotropic response to VNS at 3 and 5 Hz in adult guinea pigs. Neither L -NA (n=6), L -VNIO (n=5) nor ODQ (n=6) changed the HR response to cumulative doses of carbamylcholine in adult guinea pig atria suggesting that the action of NO is pre-synaptic. The HR response to VNS was unaffected by L -NA (n=7) or ODQ (n=7) in young guinea pigs and Western blot analysis showed significantly lower levels of nNOS protein in right atria from young animals. These results suggest a pre-synaptic NO-cGMP pathway modulates cardiac cholinergic transmission, although this may depend on the developmental stage of the guinea pig.

Natriuretic peptides like NO facilitate cardiac vagal neurotransmission and bradycardia via a cGMP pathway.

We tested the hypothesis that natriuretic peptide receptors (NPRs) that are coupled to cGMP production act in a similar way to nitric oxide (NO) by enhancing acetylcholine release and vagal-induced bradycardia. The effects of enzyme inhibitors and channel blockers on the action of atrial natriuretic peptide (ANP), brain-derived natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) were evaluated in isolated guinea pig atrial-right vagal nerve preparations. RT-PCR confirmed the presence NPR B and A receptor mRNA in guinea pig sinoatrial node tissue. BNP and CNP significantly (P < 0.05) enhanced the heart rate (HR) response to vagal nerve stimulation. CNP had no effect on the HR response to carbamylcholine and facilitated the release of [(3)H]acetylcholine during atrial field stimulation. The particulate guanylyl cyclase-coupled receptor antagonist HS-142-1, the phosphodiesterase 3 inhibitor milrinone, the protein kinase A inhibitor H89, and the N-type calcium channel blocker omega-conotoxin all blocked the effect of CNP on vagal-induced bradycardia. Like NO, BNP and CNP facilitate vagal neurotransmission and bradycardia. This may occur via a cGMP-PDE3-dependent pathway increasing cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels.