Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Short communication
Firing patterns of muscle sympathetic neurons during apnea in chronic heart failure patients and healthy controls Petra Zubin Maslov a, J. Kevin Shoemaker b,c, Zeljko Dujic a,⁎ a b c
Department of Physiology, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia Neurovascular Research Laboratory, School of Kinesiology, Western University, London, Ontario, Canada Department of Physiology and Pharmacology, Western University, London, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 2 September 2013 Accepted 30 September 2013 Keywords: Action potential detection Sleep apnea Cardiovascular disease
a b s t r a c t In the present study we investigated the influence of end-expiratory breathing cessation on firing activity of muscle sympathetic fibers in 6 stable chronic heart failure (CHF) patients and in 6 healthy age and gender matched controls. Integrated multi-unit bursts, as well as action potentials (APs), were identified from multi-unit muscle sympathetic nerve activity (MSNA) recordings during baseline and during functional residual capacity (FRC) apnea. Compared with controls, CHF patients had higher burst frequency and AP firing frequency (P b 0.05) at baseline. FRC apnea caused an increase in the number of APs per multi-unit sympathetic burst, in the AP frequency (P b 0.05) and in the number of active clusters per multi-unit sympathetic burst in both groups (controls P b 0.06, CHF group P = 0.1). The data suggest a comparable pattern of sympathetic activation associated with breath hold in healthy middle-aged individuals and in stable CHF patients. Thus, recruitment patterns for this stress are not affected by CHF despite their elevated sympathetic state. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
Voluntary apnea is a powerful sympathetic stimulus through multiple mechanisms including hypoxia, hypercapnia and an increased central drive-to-breathe (Steinback et al., 2010a). Similar pathophysiological mechanisms are coupled with sleep-disordered breathing that occurs in 3–14% of middle aged population and in 30–50% chronic heart failure (CHF) patients (Augostini, 2012). Sleep apnea is recognized as an important risk factor for the development of hypertension as well as for progression of cardiac dysfunction in patients with CHF (Sin et al., 1999). During sleep apnea, tonic inhibition of sympathetic outflow by pulmonary stretch receptors ceases, while stimulation of peripheral and central chemoreceptors by hypoxia and hypercapnia further augments sympathetic nerve activity (Somers et al., 1995; Floras, 2009). Baseline sympathetic outflow increases in CHF patients and more if these patients experience sleep apnea. Cycles of apnea and arousal during the night expose the failing heart and peripheral circulation to repetitive norepinephrine release far greater than needed for circulatory homeostasis (Floras, 2009). The ability of sleep apnea to increase sympathetic outflow in CHF patients who already have marked elevations in neuronal activity, suggests that these patients retain the ability to increase sympathetic drive during the apneas. However, the underlying firing activity of the sympathetic neurons that occurs during sleep apnea in CHF patients is not completely understood. Previous studies with single-unit recordings of sympathetic fibers
⁎ Corresponding author. Tel.: +385 21 557 906; fax: +385 21 465 304. E-mail address: [email protected] (Z. Dujic).
have shown increased multiple firing of the same sympathetic neuron during voluntary apnea (Elam et al., 2002). We have recently shown that stable CHF patients express a present, but diminished ability to recruit additional postganglionic sympathetic neurons in a response to hemodynamic stress such as premature ventricular contraction (PVC) (Maslov et al., 2012). In the present study voluntary cessation of breathing at functional residual capacity (FRC) level served as a model of physiological stimulus that can affect firing pattern of sympathetic neurons in stable CHF patients and in healthy,
Table 1 Hemodynamic parameters during baseline and during end-expiratory apnea. CHF patients (N = 6)
MAP (mm Hg) SBP (mm Hg) DBP (mm Hg) HR (bpm) SV (mL) CO (L/min) TPR (a.u.)
Controls (N = 6) Baseline
End-exp apnea
Baseline
End-exp apnea
82 ± 9⁎⁎ 126 ± 15 64 ± 8 68 ± 5 88 ± 28 5.9 ± 2 15 ± 18
91 ± 7⁎ 139 ± 15⁎ 71 ± 9⁎ 68 ± 8 81 ± 24 5.6 ± 2 18 ± 7⁎
96 ± 7 146 ± 17 70 ± 5 62 ± 5 115 ± 16 7±1 13 ± 1
108 ± 9⁎ 164 ± 14⁎ 81 ± 10⁎ 65 ± 13 105 ± 20 7±1 16 ± 4⁎
Values are mean ± SD. MAP, mean arterial pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; SV, stroke volume; CO, cardiac output; TPR, total peripheral resistance. ⁎ P b 0.05 compared to baseline. ⁎⁎ P b 0.05 compared to controls.
1566-0702/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autneu.2013.09.016
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
age and gender matched controls. We hypothesized that despite chronic sympathoexcitation, stable CHF patients will manifest further activation of sympathetic reserve during end-expiratory apnea through increased firing of already active sympathetic fibers and through the recruitment of latent subpopulation of sympathetic fibers. For this purpose we used previously described technique that enables the identification and morphological classification of action potentials from raw, filtered signal of multi-unit muscle sympathetic nerve activity (MSNA) recordings (Salmanpour et al., 2010; Steinback et al., 2010b; Breskovic et al., 2011).
67
Data were obtained from six stable CHF patients and from six healthy control subjects matched for gender and age. Inclusion and exclusion criteria for CHF group were the same as for our previous studies (Maslov et al., 2012; Zubin et al., 2013). CHF patients were recruited from the Department of Cardiology, University Hospital of Split. Healthy volunteers free of cardiovascular disease were matched for age and gender and recruited as control group. All subjects gave written informed consent to participate in the study that was conducted in accordance with the Declaration of Helsinki and was approved by research ethics board at The University of Split, School of Medicine.
Fig. 1. Change in action potential (AP) parameters and mean burst area/min from baseline (0 s) to apnea at functional residual capacity (FRC) level. White circles represent chronic heart failure (CHF) patients and black circles represent controls. Values are means, bars represent SD. *, P b 0.05 compared to baseline for CHF group; †, P b 0.05 compared to baseline for control group.
68
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
Hemodynamic parameters were measured continuously using finger photoplethysmography (Finometer, Finapress Medical Systems, Arnhem, The Netherlands) and multiunit MSNA was obtained using microneurography (Hagbarth and Vallbo, 1968). Total peripheral resistance (TPR) was calculated as mean arterial pressure (MAP) divided by cardiac output (CO). After instrumentation and 15 min of normal breathing, subjects were instructed to exhale until FRC lung level and to cease breathing as long as possible. MSNA was assessed in the right peroneal nerve by microneurography (Hagbarth and Vallbo, 1968). An active tungsten microelectrode with 2MΩ impedance was inserted percutaneously into the peroneal nerve while the reference electrode was inserted 1–3 cm from the recording site. The nerve signal was amplified (100 000 times), band pass filtered (band pass 700–2000 HZ), rectified, integrated using 0.1 s time constant, sampled at 10 000 Hz (Powerlab/ 16SP; ADInstruments) and stored for subsequent analysis using Chart software (version 5.5.6.7). Data were analyzed during 1 min of baseline and throughout the FRC apnea. Action potentials (APs) were detected and extracted from the raw, filtered MSNA signal using the technique reported previously (Salmanpour et al., 2010; Steinback et al., 2010b; Breskovic et al., 2011). Integrated muscle sympathetic nerve activity (MSNA) was expressed as number of sympathetic bursts per minute (burst frequency), as number of sympathetic bursts per 100 heart beats (burst incidence) and as mean burst area per minute. AP data were quantified as number of APs within a sympathetic burst (AP/burst) and as number of APs per minute (AP frequency). Extracted APs were then ordered based on peak-to-peak amplitude and grouped into clusters as previously described (Salmanpour et al., 2010). Results are expressed as mean ± SD and the level of significance is set at P = 0.05. Differences in hemodynamic and sympathetic parameters between the groups at baseline were tested using Student's two tailed, unpaired t-test. A Wilcoxon test was used to assess the difference in hemodynamic and sympathetic parameters between the baseline and FRC apnea in both groups. All analyses were performed with Statistica 7.0 software (Statsoft, Inc., Tulsa, USA).
ECG
Breath hold duration at FRC level lasted 35 ± 20 s in CHF patients, and 36 ± 16 s in controls. Compared with controls, CHF patients had higher baseline burst frequency (32± 8 vs. 50±17, controls vs. CHF, respectively, P b 0.05), burst incidence (50 ± 15 vs. 75 ± 28, controls vs. CHF, respectively, P b 0.05), mean burst area/min (2.05 ± 0.6 vs. 4.1 ± 2, controls vs. CHF, respectively, P b 0.05), AP firing frequency (195 ± 64 vs. 631 ± 382 spikes/min, controls vs. CHF, respectively, P b 0.05), and APs/burst (6 ± 2 vs. 12 ± 7, controls vs. CHF, respectively, P b 0.05). Compared to baseline, end-expiratory apnea caused a significant increase in MAP, systolic blood pressure (SBP), diastolic blood pressure (DBP) and TPR in both groups (Table 1). Fig. 1 shows that the FRC breath-hold caused an increase in AP/burst and AP frequency in both groups (P b 0.05). In controls, activation of additional sympathetic neurons occurred during FRC apnea (3 ± 1 active cluster at baseline vs. 4 ± 1 during FRC apnea, P b 0.06). The same trend of change toward increase in the number of additional active sympathetic neurons was observed in CHF group but did not reach traditional statistical significance (4 ± 1 active cluster at baseline vs. 5 ± 2 during FRC apnea P = 0.1). All subjects held their breath until the level of maximal tolerance and the change in hemodynamic and sympathetic parameters from the baseline to FRC apnea was comparable between the groups (Figs. 2 and 3). The main finding of the present study was that both, the control group and patients with stable CHF augmented sympathetic nerve activity during end-expiratory breath hold through at least two mechanisms: first, through an increase in the number of APs within a sympathetic burst, and second, through an increase in AP firing frequency. Importantly, the increase in AP/burst included the activation of additional clusters of sympathetic neurons that were not active during baseline. Thus, it appears that the firing strategies of sympathetic fibers during the chemoreflex stress elicited by breath hold were similar between the CHF patients and healthy controls. An increase in hemodynamic and sympathetic parameters between the baseline and the FRC apnea was comparable between the groups suggesting similar level of chemoreflex stress during the FRC apnea.
0.6V
0.2V
BP
250mmHg
60mmHg
Resp.
7.5V
Belt 7.0V
MSNA
2V
Integ. 0V
MSNA
2V
Raw -2V
Fig. 2. Original recording of hemodynamic and sympathetic responses during end-expiratory apnea in one CHF patient. BP, blood pressure, MSNA, muscle sympathetic nerve activity.
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
ECG
69
0.6V 0.4V
BP
250mmHg
80mmHg
Resp.
6.5V
Belt 3.5V
MSNA
2V
Integ. 0V
MSNA
2V
Raw -2V
Fig. 3. Original recording of hemodynamic and sympathetic responses during end-expiratory apnea in one control subject. BP, blood pressure, MSNA, muscle sympathetic nerve activity.
Macefield et al. used their single-unit recording methodology (Macefield et al., 1994) to observe the repeated firing of the same sympathetic fibers within a cardiac interval in patients with obstructive sleep apnea (OSAS), but not in the CHF patients with sinus rhythm (Macefield et al., 1999). Single-unit approach determines whether or not a single neuron becomes more or less active, but cannot determinate the activity of latent population of sympathetic neurons that become active only during physiological stress such as sudden drop in MAP (Maslov et al., 2012) or hypoxia and/or hypercapnia (Breskovic et al., 2011). The approach used in the current study and in our previous studies, quantifies the APs of all sympathetic fibers contributing to a burst with determination of the AP size and appearance of new and larger APs during recording (Steinback et al., 2010b). With the use of this technique we have previously shown that, in young healthy subjects, FRC apnea caused an increase in AP/burst, AP frequency and recruitment of previously silent, larger neurons (Breskovic et al., 2011). If firing properties of the stable CHF patients were compared to older healthy individuals from the current study, and to young healthy individuals from the previous study (Breskovic et al., 2011), it seems that CHF patients retain the ability to increase sympathetic activity, as a response to breathing cessation, through the same mechanisms as healthy individuals: increase in AP/ burst, AP firing frequency and recruitment of previously silent, larger neurons. The current observations provide additional information on pathophysiology of sympathetic nervous system in stable CHF patients during breathing cessation. Specifically, despite markedly higher baseline levels of MSNA, these patients exhibit the potential to increase further their sympathetic outflow during volitional apneas. However, it should be emphasized that a volitional end-expiratory apnea does not have the same physiological background regarding the respiratory responses seen in OSA. Our experimental model of apnea is more similar to central sleep apnea rather than OSA, in which inspiratory efforts are made against a collapsed upper airway. Better understanding of firing pattern of sympathetic nervous system during central sleep apnea is important as it is recognized to have pivotal role in cardiovascular deterioration and mortality in CHF patients with sleep-related disturbances.
Acknowledgments The authors thank Ante Obad, Nediljko Pivac and Darija Bakovic for their help in recruitment of CHF patients. We also thank Toni Breskovic and Branka Runtic for their assistance in data collection. References Augostini, R., 2012. A novel approach to the treatment of central sleep apnea in patients with heart failure. Herzschrittmacherther. Elektrophysiol. 23, 9–13. Breskovic, T., Steinback, C.D., Salmanpour, A., Shoemaker, J.K., Dujic, Z., 2011. Recruitment pattern of sympathetic neurons during breath-holding at different lung volumes in apnea divers and controls. Auton. Neurosci. 164, 74–81. Elam, M., McKenzie, D., Macefield, V., 2002. Mechanisms of sympathoexcitation: single-unit analysis of muscle vasoconstrictor neurons in awake OSAS subjects. J. Appl. Physiol. 93, 297–303. Floras, J.S., 2009. Sympathetic nervous system activation in human heart failure: clinical implications of an updated model. J. Am. Coll. Cardiol. 54, 375–385. Hagbarth, K.E., Vallbo, A.B., 1968. Pulse and respiratory grouping of sympathetic impulses in human muscle-nerves. Acta Physiol. Scand. 74, 96–108. Macefield, V.G., Wallin, B.G., Vallbo, A.B., 1994. The discharge behaviour of single vasoconstrictor motoneurones in human muscle nerves. J. Physiol. 481 (Pt 3), 799–809. Macefield, V.G., Rundqvist, B., Sverrisdottir, Y.B., Wallin, B.G., Elam, M., 1999. Firing properties of single muscle vasoconstrictor neurons in the sympathoexcitation associated with congestive heart failure. Circulation 100, 1708–1713. Maslov, P.Z., Breskovic, T., Brewer, D.N., Shoemaker, J.K., Dujic, Z., 2012. Recruitment pattern of sympathetic muscle neurons during premature ventricular contractions in heart failure patients and controls. Am. J. Physiol. Regul. Integr. Comp. Physiol. 303, R1157–R1164. Salmanpour, A., Brown, L.J., Shoemaker, J.K., 2010. Spike detection in human muscle sympathetic nerve activity using a matched wavelet approach. J. Neurosci. Methods 193, 343–355. Sin, D.D., Fitzgerald, F., Parker, J.D., Newton, G., Floras, J.S., Bradley, T.D., 1999. Risk factors for central and obstructive sleep apnea in 450 men and women with congestive heart failure. Am. J. Respir. Crit. Care Med. 160, 1101–1106. Somers, V.K., Dyken, M.E., Clary, M.P., Abboud, F.M., 1995. Sympathetic neural mechanisms in obstructive sleep apnea. J. Clin. Invest. 96, 1897–1904. Steinback, C.D., Breskovic, T., Frances, M., Dujic, Z., Shoemaker, J.K., 2010a. Ventilatory restraint of sympathetic activity during chemoreflex stress. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1407–R1414. Steinback, C.D., Salmanpour, A., Breskovic, T., Dujic, Z., Shoemaker, J.K., 2010b. Sympathetic neural activation: an ordered affair. J. Physiol. 588, 4825–4836. Zubin, M.P., Breskovic, T., Shoemaker, J.K., Olson, T.P., Johnson, B.D., Eterovic, D., Dujic, Z., 2013. Firing patterns of muscle sympathetic neurons during short-term use of continuous positive airway pressure in healthy subjects and in chronic heart failure patients. Respir. Physiol. Neurobiol. 187, 149–156.
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Short communication
Firing patterns of muscle sympathetic neurons during apnea in chronic heart failure patients and healthy controls Petra Zubin Maslov a, J. Kevin Shoemaker b,c, Zeljko Dujic a,⁎ a b c
Department of Physiology, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia Neurovascular Research Laboratory, School of Kinesiology, Western University, London, Ontario, Canada Department of Physiology and Pharmacology, Western University, London, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 2 September 2013 Accepted 30 September 2013 Keywords: Action potential detection Sleep apnea Cardiovascular disease
a b s t r a c t In the present study we investigated the influence of end-expiratory breathing cessation on firing activity of muscle sympathetic fibers in 6 stable chronic heart failure (CHF) patients and in 6 healthy age and gender matched controls. Integrated multi-unit bursts, as well as action potentials (APs), were identified from multi-unit muscle sympathetic nerve activity (MSNA) recordings during baseline and during functional residual capacity (FRC) apnea. Compared with controls, CHF patients had higher burst frequency and AP firing frequency (P b 0.05) at baseline. FRC apnea caused an increase in the number of APs per multi-unit sympathetic burst, in the AP frequency (P b 0.05) and in the number of active clusters per multi-unit sympathetic burst in both groups (controls P b 0.06, CHF group P = 0.1). The data suggest a comparable pattern of sympathetic activation associated with breath hold in healthy middle-aged individuals and in stable CHF patients. Thus, recruitment patterns for this stress are not affected by CHF despite their elevated sympathetic state. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
Voluntary apnea is a powerful sympathetic stimulus through multiple mechanisms including hypoxia, hypercapnia and an increased central drive-to-breathe (Steinback et al., 2010a). Similar pathophysiological mechanisms are coupled with sleep-disordered breathing that occurs in 3–14% of middle aged population and in 30–50% chronic heart failure (CHF) patients (Augostini, 2012). Sleep apnea is recognized as an important risk factor for the development of hypertension as well as for progression of cardiac dysfunction in patients with CHF (Sin et al., 1999). During sleep apnea, tonic inhibition of sympathetic outflow by pulmonary stretch receptors ceases, while stimulation of peripheral and central chemoreceptors by hypoxia and hypercapnia further augments sympathetic nerve activity (Somers et al., 1995; Floras, 2009). Baseline sympathetic outflow increases in CHF patients and more if these patients experience sleep apnea. Cycles of apnea and arousal during the night expose the failing heart and peripheral circulation to repetitive norepinephrine release far greater than needed for circulatory homeostasis (Floras, 2009). The ability of sleep apnea to increase sympathetic outflow in CHF patients who already have marked elevations in neuronal activity, suggests that these patients retain the ability to increase sympathetic drive during the apneas. However, the underlying firing activity of the sympathetic neurons that occurs during sleep apnea in CHF patients is not completely understood. Previous studies with single-unit recordings of sympathetic fibers
⁎ Corresponding author. Tel.: +385 21 557 906; fax: +385 21 465 304. E-mail address: [email protected] (Z. Dujic).
have shown increased multiple firing of the same sympathetic neuron during voluntary apnea (Elam et al., 2002). We have recently shown that stable CHF patients express a present, but diminished ability to recruit additional postganglionic sympathetic neurons in a response to hemodynamic stress such as premature ventricular contraction (PVC) (Maslov et al., 2012). In the present study voluntary cessation of breathing at functional residual capacity (FRC) level served as a model of physiological stimulus that can affect firing pattern of sympathetic neurons in stable CHF patients and in healthy,
Table 1 Hemodynamic parameters during baseline and during end-expiratory apnea. CHF patients (N = 6)
MAP (mm Hg) SBP (mm Hg) DBP (mm Hg) HR (bpm) SV (mL) CO (L/min) TPR (a.u.)
Controls (N = 6) Baseline
End-exp apnea
Baseline
End-exp apnea
82 ± 9⁎⁎ 126 ± 15 64 ± 8 68 ± 5 88 ± 28 5.9 ± 2 15 ± 18
91 ± 7⁎ 139 ± 15⁎ 71 ± 9⁎ 68 ± 8 81 ± 24 5.6 ± 2 18 ± 7⁎
96 ± 7 146 ± 17 70 ± 5 62 ± 5 115 ± 16 7±1 13 ± 1
108 ± 9⁎ 164 ± 14⁎ 81 ± 10⁎ 65 ± 13 105 ± 20 7±1 16 ± 4⁎
Values are mean ± SD. MAP, mean arterial pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; SV, stroke volume; CO, cardiac output; TPR, total peripheral resistance. ⁎ P b 0.05 compared to baseline. ⁎⁎ P b 0.05 compared to controls.
1566-0702/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autneu.2013.09.016
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
age and gender matched controls. We hypothesized that despite chronic sympathoexcitation, stable CHF patients will manifest further activation of sympathetic reserve during end-expiratory apnea through increased firing of already active sympathetic fibers and through the recruitment of latent subpopulation of sympathetic fibers. For this purpose we used previously described technique that enables the identification and morphological classification of action potentials from raw, filtered signal of multi-unit muscle sympathetic nerve activity (MSNA) recordings (Salmanpour et al., 2010; Steinback et al., 2010b; Breskovic et al., 2011).
67
Data were obtained from six stable CHF patients and from six healthy control subjects matched for gender and age. Inclusion and exclusion criteria for CHF group were the same as for our previous studies (Maslov et al., 2012; Zubin et al., 2013). CHF patients were recruited from the Department of Cardiology, University Hospital of Split. Healthy volunteers free of cardiovascular disease were matched for age and gender and recruited as control group. All subjects gave written informed consent to participate in the study that was conducted in accordance with the Declaration of Helsinki and was approved by research ethics board at The University of Split, School of Medicine.
Fig. 1. Change in action potential (AP) parameters and mean burst area/min from baseline (0 s) to apnea at functional residual capacity (FRC) level. White circles represent chronic heart failure (CHF) patients and black circles represent controls. Values are means, bars represent SD. *, P b 0.05 compared to baseline for CHF group; †, P b 0.05 compared to baseline for control group.
68
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
Hemodynamic parameters were measured continuously using finger photoplethysmography (Finometer, Finapress Medical Systems, Arnhem, The Netherlands) and multiunit MSNA was obtained using microneurography (Hagbarth and Vallbo, 1968). Total peripheral resistance (TPR) was calculated as mean arterial pressure (MAP) divided by cardiac output (CO). After instrumentation and 15 min of normal breathing, subjects were instructed to exhale until FRC lung level and to cease breathing as long as possible. MSNA was assessed in the right peroneal nerve by microneurography (Hagbarth and Vallbo, 1968). An active tungsten microelectrode with 2MΩ impedance was inserted percutaneously into the peroneal nerve while the reference electrode was inserted 1–3 cm from the recording site. The nerve signal was amplified (100 000 times), band pass filtered (band pass 700–2000 HZ), rectified, integrated using 0.1 s time constant, sampled at 10 000 Hz (Powerlab/ 16SP; ADInstruments) and stored for subsequent analysis using Chart software (version 5.5.6.7). Data were analyzed during 1 min of baseline and throughout the FRC apnea. Action potentials (APs) were detected and extracted from the raw, filtered MSNA signal using the technique reported previously (Salmanpour et al., 2010; Steinback et al., 2010b; Breskovic et al., 2011). Integrated muscle sympathetic nerve activity (MSNA) was expressed as number of sympathetic bursts per minute (burst frequency), as number of sympathetic bursts per 100 heart beats (burst incidence) and as mean burst area per minute. AP data were quantified as number of APs within a sympathetic burst (AP/burst) and as number of APs per minute (AP frequency). Extracted APs were then ordered based on peak-to-peak amplitude and grouped into clusters as previously described (Salmanpour et al., 2010). Results are expressed as mean ± SD and the level of significance is set at P = 0.05. Differences in hemodynamic and sympathetic parameters between the groups at baseline were tested using Student's two tailed, unpaired t-test. A Wilcoxon test was used to assess the difference in hemodynamic and sympathetic parameters between the baseline and FRC apnea in both groups. All analyses were performed with Statistica 7.0 software (Statsoft, Inc., Tulsa, USA).
ECG
Breath hold duration at FRC level lasted 35 ± 20 s in CHF patients, and 36 ± 16 s in controls. Compared with controls, CHF patients had higher baseline burst frequency (32± 8 vs. 50±17, controls vs. CHF, respectively, P b 0.05), burst incidence (50 ± 15 vs. 75 ± 28, controls vs. CHF, respectively, P b 0.05), mean burst area/min (2.05 ± 0.6 vs. 4.1 ± 2, controls vs. CHF, respectively, P b 0.05), AP firing frequency (195 ± 64 vs. 631 ± 382 spikes/min, controls vs. CHF, respectively, P b 0.05), and APs/burst (6 ± 2 vs. 12 ± 7, controls vs. CHF, respectively, P b 0.05). Compared to baseline, end-expiratory apnea caused a significant increase in MAP, systolic blood pressure (SBP), diastolic blood pressure (DBP) and TPR in both groups (Table 1). Fig. 1 shows that the FRC breath-hold caused an increase in AP/burst and AP frequency in both groups (P b 0.05). In controls, activation of additional sympathetic neurons occurred during FRC apnea (3 ± 1 active cluster at baseline vs. 4 ± 1 during FRC apnea, P b 0.06). The same trend of change toward increase in the number of additional active sympathetic neurons was observed in CHF group but did not reach traditional statistical significance (4 ± 1 active cluster at baseline vs. 5 ± 2 during FRC apnea P = 0.1). All subjects held their breath until the level of maximal tolerance and the change in hemodynamic and sympathetic parameters from the baseline to FRC apnea was comparable between the groups (Figs. 2 and 3). The main finding of the present study was that both, the control group and patients with stable CHF augmented sympathetic nerve activity during end-expiratory breath hold through at least two mechanisms: first, through an increase in the number of APs within a sympathetic burst, and second, through an increase in AP firing frequency. Importantly, the increase in AP/burst included the activation of additional clusters of sympathetic neurons that were not active during baseline. Thus, it appears that the firing strategies of sympathetic fibers during the chemoreflex stress elicited by breath hold were similar between the CHF patients and healthy controls. An increase in hemodynamic and sympathetic parameters between the baseline and the FRC apnea was comparable between the groups suggesting similar level of chemoreflex stress during the FRC apnea.
0.6V
0.2V
BP
250mmHg
60mmHg
Resp.
7.5V
Belt 7.0V
MSNA
2V
Integ. 0V
MSNA
2V
Raw -2V
Fig. 2. Original recording of hemodynamic and sympathetic responses during end-expiratory apnea in one CHF patient. BP, blood pressure, MSNA, muscle sympathetic nerve activity.
P. Zubin Maslov et al. / Autonomic Neuroscience: Basic and Clinical 180 (2014) 66–69
ECG
69
0.6V 0.4V
BP
250mmHg
80mmHg
Resp.
6.5V
Belt 3.5V
MSNA
2V
Integ. 0V
MSNA
2V
Raw -2V
Fig. 3. Original recording of hemodynamic and sympathetic responses during end-expiratory apnea in one control subject. BP, blood pressure, MSNA, muscle sympathetic nerve activity.
Macefield et al. used their single-unit recording methodology (Macefield et al., 1994) to observe the repeated firing of the same sympathetic fibers within a cardiac interval in patients with obstructive sleep apnea (OSAS), but not in the CHF patients with sinus rhythm (Macefield et al., 1999). Single-unit approach determines whether or not a single neuron becomes more or less active, but cannot determinate the activity of latent population of sympathetic neurons that become active only during physiological stress such as sudden drop in MAP (Maslov et al., 2012) or hypoxia and/or hypercapnia (Breskovic et al., 2011). The approach used in the current study and in our previous studies, quantifies the APs of all sympathetic fibers contributing to a burst with determination of the AP size and appearance of new and larger APs during recording (Steinback et al., 2010b). With the use of this technique we have previously shown that, in young healthy subjects, FRC apnea caused an increase in AP/burst, AP frequency and recruitment of previously silent, larger neurons (Breskovic et al., 2011). If firing properties of the stable CHF patients were compared to older healthy individuals from the current study, and to young healthy individuals from the previous study (Breskovic et al., 2011), it seems that CHF patients retain the ability to increase sympathetic activity, as a response to breathing cessation, through the same mechanisms as healthy individuals: increase in AP/ burst, AP firing frequency and recruitment of previously silent, larger neurons. The current observations provide additional information on pathophysiology of sympathetic nervous system in stable CHF patients during breathing cessation. Specifically, despite markedly higher baseline levels of MSNA, these patients exhibit the potential to increase further their sympathetic outflow during volitional apneas. However, it should be emphasized that a volitional end-expiratory apnea does not have the same physiological background regarding the respiratory responses seen in OSA. Our experimental model of apnea is more similar to central sleep apnea rather than OSA, in which inspiratory efforts are made against a collapsed upper airway. Better understanding of firing pattern of sympathetic nervous system during central sleep apnea is important as it is recognized to have pivotal role in cardiovascular deterioration and mortality in CHF patients with sleep-related disturbances.
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