This is the first study to assess the role of Nav channels in the AB neuron excitability and baroreflex sensitivity in CHF state. The present study showed that: (1) expression of Nav channel mRNA and protein was lower in the NGs from CHF rats than that from sham rats. (2) Nav current density and cell excitability were reduced in A- and C-type AB neurons from CHF rats, compared with that from sham rats; (3) CHF attenuated the aortic baroreflex sensitivity; (4) Nav channel activator (rATX II) significantly enhanced the Nav current density and cell excitability of AB neurons, and improved the baroreflex sensitivity in CHF rats. These results suggest that CHF-induced low expression and hypoactivation of Nav channels mediate the depressed AB neuron excitability and subsequently contribute to the blunted aortic baroreflex sensitivity.
Morphological study has demonstrated that there is no difference in total fiber density, A-type fiber density, C-type fiber density, and ratio of A-/C-type fiber density in carotid sinus nerve between sham and CHF dogs (Wang et al., 1996
). Our present study also suggests that there is no change in either total neuron number or the ratio of A-type/C-type neurons in the NGs from CHF rats, compared with those in sham rats. These results provide the important information that the depressed baroreflex in CHF might be not due to the structural changes in the AB neurons but most likely reflect functional changes at the cellular and molecular levels.
In general, the sodium channels in primary afferent neurons can be separated into TTX-s and TTX-r Nav
channels. In the dorsal root ganglia, only the TTX-s Nav
channels carry the Nav
currents in larger diameter low-threshold mechano-sensitive neurons, whereas the TTX-s and TTX-r Nav
channels are expressed in smaller diameter nociceptive neurons (Arbuckle and Docherty, 1995
; Caffrey et al., 1992
; Tate et al., 1998
). In the present study, we sought to measure whether the three Nav
channel subunits (Nav
1.8, and Nav
1.9) mainly expressed in the NG (Kwong et al., 2008
) were also differentially distributed in the A-type and C-type NG neurons. The immunofluorescent double-staining is a unique tool to qualify the protein colocalization although it is not reliable enough to perform a quantitative measurement. The results from immunofluorescent and electrophysiological observations indicate that TTX-s Nav
1.7) are expressed in A-type and C-type NG neurons but TTX-r Nav
1.8 and Nav
1.9) are located only in C-type NG neurons. More importantly, CHF significantly decreased the mRNA and protein expression levels of three Nav
channel subunits ( and ) and subsequently blunted the Nav
currents in the AB afferent neurons ( and ).
The mechanisms responsible for mediating afferent sensitivity of barosensitive neurons to pressure are complex and not thoroughly understood. The process of translating changes in arterial wall tension into impulse traffic to the nucleus tractus solitarii (the first site of baroreceptor neuron contacting with central nervous system) involves two broad functional steps: 1) mechanotransduction which is governed by the properties of mechanosensitive ion channels in the nerve terminal and the mechanical properties of the coupling of the arterial wall to the sensory terminal; and 2) spike initiation which is governed by the excitability of membrane voltage sensitive ion channels that influence the electrical (cable) properties of the axonal projection and cell body. All of these factors could be (and likely are) altered in CHF. It is generally assumed that the blunted sensitivity results from an impairment of mechanotransduction at the sensory terminals. In the present study, we observed that the mRNA and protein expression levels and the current density of Nav channels were reduced in the AB neurons from CHF rats. The cell excitability of AB neurons was also suppressed in CHF rats, and a Nav channel activator (rATX II) significantly increased the Nav current density and cell excitability of AB neurons from CHF rats (–). Based on these results, we can assume that CHF-lowered expression and activation of Nav channels contributes to the suppressed cell excitability of AB neurons in CHF state (the second process above).
Many studies have used the responses of blood pressure and heart rate to electrical stimulation of baroreceptor-containing nerve for the evaluation of the baroreflex sensitivity in rats (Fan and Andresen, 1998
; Salgado et al., 2007
; Tang and Dworkin, 2007
). Electrical Stimulation of the rat aortic depressor nerve has several advantages to examine the baroreflex function in the present study. First, the rat aortic depressor nerve contains only baroreceptor afferent fibers and no chemoreceptor afferent fibers to transmit the chemoreceptor information (Fan et al., 1996
; Kobayashi et al., 1999
; Sapru et al., 1981
; Sapru and Krieger, 1977
); secondly, the baroreflex induced by stimulating rat aortic depressor nerve is measured without the baroreceptor ending in the reflex arc, which allows us to specifically examine the role of electrical excitability of AB in the baroreflex function (second process above); thirdly, by varying the frequency of stimulus, one can differentially activate A- and C- afferent fibers, and thus evaluate the relative contribution of each to the altered Nav
channel and baroreflex function in CHF. In the present study, the baroreflex responses of blood pressure and heart rate to the electrical stimulation of the baroreceptor nerve are significantly depressed in CHF rats (). In addition, our present study also found that microinjection of rATX II (Nav
channel activator) into the NG significantly improved the baroreflex sensitivity induced by aortic depressor nerve stimulation in CHF rats (). The fact is that nodose neurons are found to influence the conduction and frequency of the electrical impulses in the baroreceptor central axons projecting to the central nervous system when electrical signals in the baroreceptor peripheral axons reach the nodose neurons (Ducreux et al., 1993
). One recent review paper (Browning, 2003
) has concluded that the excitability of vagal afferent neurons has dramatic consequences for the regulation and modulation of vago-vagal reflex. Furthermore, Devor has reported that electrical excitability of the soma in the dorsal root ganglia may be required to insure the reliable afferent electrical impulses transmitted to the spinal cord (Devor, 1999
). These results, taken together, demonstrate that the Nav
channel dysfunction of AB neurons contributes to the blunted baroreflex sensitivity through attenuating the AB neuron excitability in CHF rats, which is indirectly confirmed by the finding that rATX also increases the baroreflex sensitivity induced by aortic depressor nerve stimulation in sham rats ().
We do realize, however, that a disadvantage of the electrical stimulation technique is that it does not represent a physiological substrate for baroreceptor activation. Thus results from reflex experiments evoked by the electrical stimulation needed to be tempered by this limitation. In future, we will further address this issue using baroreflex evoked by changes in arterial blood pressure. However, we also understand that a major limitation to this approach (blood pressure-mediated baroreflex sensitivity) is that possible alterations in the mechanotransduction process at the baro-sensory nerve terminal may also play a role in the suppressed baroreceptor function in response to pressure changes.
Although rATX II significantly increased Nav current density and cell excitability of AB neurons, and improved aortic baroreflex sensitivity in CHF rats, it did not completely normalize these functions towards the level seen in sham rats. Modulating ion channel function usually includes acutely influencing the activation of ion channels and chronically altering the expression of ion channels. Our present study showed the low mRNA and protein expression levels of Nav channels in nodose neurons from CHF rats ( and ), which could explain the above results because of the inability of rATX to improve the expression of Nav channels. In addition, it is not possible to identify the contribution of the various Nav channel subunits to the cell excitability and baroreflex sensitivity from the present study because no specific Nav channel activators are available for Nav1.7, Nav1.8, and Nav1.9. Further study is needed to explore these outstanding issues.
From the findings that the expression of the three Nav channel subunits is decreased in A-type and C-type nodose neurons from CHF rats (), we cannot clearly confirm the low expression of the Nav channels in the AB neurons from CHF rats due to the limitation of the method (DiI labelling was lost from cells during the immunofluorescent staining procedure and could not be used as a marker of AB neurons with immunofluorescent staining). However, it is reasonable to assume that the protein expression of the Nav channels are lowered in the AB neurons from CHF rats because the AB neurons are a part of the nodose neurons and the whole cell patch-clamp data indicate that the Nav current density is reduced in A-type and C-type AB neurons from CHF rats ( and ).
In the present study, the Nav
currents were recorded in the isolated primary AB neurons cultured in the medium for 4–24 h. It is possible that the culture conditions (such as nerve growth factor in the medium) altered the electrophysiological characteristics of the Nav
channels in the AB neuron cells. However, this is not likely because our preliminary data have found that the Nav
currents recorded in the acutely dissociated neurons are same as that obtained in the AB neurons cultured in the medium at 24 h regardless of sham or CHF rats, which is consistent with data from Kwong’s study (Kwong et al., 2008
The mechanism(s) responsible the down-regulation of the Nav
channel expression and function in the AB neurons from CHF rats are not understood. One possible candidate is angiotensin II (Ang II)-superoxide-nuclear factor kappa B (NFκB) signaling pathway. CHF elevated endogenous Ang II levels in animal models and in humans (Li et al., 2006
; Roig et al., 2000
; Schunkert et al., 1993
). Ang II can induce superoxide production in many cell types through NADPH oxidase activation and mitochondrial dysfunction (Touyz and Berry, 2002
; Zhang et al., 2007
). Recent study has shown that reactive oxygen species down-regulates cardiac Nav
1.5 channel expression via NFκB activation (Shang et al., 2008
). Further study is needed to explore how the Nav channel expression and activation are reduced in the AB neurons from CHF rats.
In conclusion, our results suggest that reduced expression and activation of Nav channels mediates the suppressed cell excitability of AB neurons and subsequently contributes to the blunted baroreflex sensitivity in CHF state. The results of this study provide important information that improving the Nav channel function in the baroreceptor neurons is a new therapeutic strategy for the baroreflex impairment in CHF.