Basis for the preferential activation of cardiac sympathetic nerve activity in heart failure. Results

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1 Basis for the preferential activation of cardiac sympathetic nerve activity in heart failure Rohit Ramchandra a, Sally G. Hood a, Derek A. Denton b,1, Robin L. Woods a,2, Michael J. McKinley a, Robin M. McAllen a, and Clive N. May a,1 a Howard Florey Institute, University of Melbourne, Parkville, Victoria 31, Australia; and b Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 34, Australia Contributed by Derek A. Denton, November 23, 28 (sent for review June 3, 28) In heart failure (HF), sympathetic nerve activity is increased. Measurements in HF patients of cardiac norepinephrine spillover, reflecting cardiac sympathetic nerve activity (), indicate that it is increased earlier and to a greater extent than sympathetic activity to other organs. This has important consequences because it worsens prognosis, provoking arrhythmias and sudden death. To elucidate the mechanisms responsible for the activation of in HF, we made simultaneous direct neural recordings of and renal SNA () in two groups of conscious sheep: normal animals and animals in HF induced by chronic, rapid ventricular pacing. In normal animals, the level of activity, measured as burst incidence (bursts of pulse related activity/1 heart beats), was significantly lower for (3 5%) than for (94 2%). Furthermore, the resting level of, relative to its maximum achieved while baroreceptors were unloaded by reducing arterial pressure, was set at a much lower percentage than. In HF, burst incidence of increased from 3 to 91%, whereas burst incidence of remained unaltered at 95%. The sensitivity of the control of both and by the arterial baroreflex remained unchanged in HF. These data show that, in the normal state, the resting level of is set at a lower level than, but in HF, the resting levels of SNA to both organs are close to their maxima. This finding provides an explanation for the preferential increase in cardiac norepinephrine spillover observed in HF. arterial baroreflex renal sympathetic nerve activity sheep Heart failure (HF) is associated with an increase in sympathetic nerve activity (SNA) that shows a large regional heterogeneity in the extent to which activity increases. The level of SNA to different organs may be estimated by measurements of regional norepinephrine (NE) spillover, and it has been observed that in HF there is a greater increase in NE spillover from the heart than the kidney, and no change from the gut (1, 2). This increase in cardiac NE spillover occurs at an earlier stage of HF than it does to other organs (3, 4), again suggesting a preferential stimulation of cardiac SNA () in HF. The increase in in HF is detrimental as it can promote the progression of the disease and leads to development of arrhythmias and sudden death (5). The mechanisms accounting for the increase in in HF are poorly understood. One explanation for the preferential increase in cardiac NE spillover in HF is that, in the normal state, the resting levels of and renal SNA () are similar, but in HF, sympathetic activity increases more to the heart than to the kidney. An alternative explanation is that the resting level of is set at a lower level compared with that to other organs, such the kidney, and in HF, both and increase to near maximum levels. Both explanations seem plausible, but the resting levels of and have not been simultaneously recorded in the normal state and compared with activities of these nerves in HF. To establish which of these possibilities is correct, we simultaneously recorded the resting levels of sympathetic activity to the heart and kidney in conscious normal healthy sheep and in sheep with HF induced by rapid ventricular pacing. Results Resting Hemodynamics and and in Normal and Heart Failure Sheep. Left ventricular ejection fraction and fractional shortening, measured in conscious sheep by echocardiography, gradually decreased over 6 8 weeks of rapid ventricular pacing at 2 22 beats/min. At 1 2 days prior to implantation of cardiac sympathetic recording electrodes, HF animals had an ejection fraction of 36 1%, a reduction of 47 3% from prepacing levels (P.1), and a fractional shortening of 17 1%, a reduction of 35 3% from prepacing levels (P.1). Ventricular dilatation in the HF group was shown by the increase in left ventricular diastolic diameter, from 3..1 cm to cm, in HF animals. Additional evidence that the sheep were in HF was the finding of a significantly higher central venous pressure (CVP) (P.5), significantly higher heart rate (HR) (P.5), and a significantly lower mean arterial pressure (P.5) than in normal animals (Table 1). In the normal state the resting level of was considerably less than that for, whereas in HF the levels of activity in both nerves were similar and close to their maxima (Fig. 1). The grouped data indicate that the resting burst incidence (bursts of pulse-related activity/1 heart beats) of was 3 5%, which was significantly lower than the burst incidence of 94 2% in (P.1) (Fig. 2 and Table 1). HF was associated with a 3-fold increase in burst incidence of to almost a burst every heart beat (Table 1). Direct comparisons between the levels of nerve activity, measured as total discriminated spikes/second above threshold, in different animals have to be interpreted cautiously because the absolute size of the signal is affected by physical factors related to the electrode placement. There were, however, significantly greater mean levels of both and in HF sheep compared with those in normal sheep (P.5) (Table 1). The burst frequencies (bursts of activity/min) of both nerves were significantly elevated during HF (Table 1). In HF, the increase in was because of increases in both burst frequency and burst incidence, whereas the increase in was because of an increase in burst frequency. Baroreflex Control of Heart Rate in Normal Sheep and Heart Failure Sheep. Consistent with our previous study of the baroreflex control of HR (6), the top plateau of HR, the maximum HR Author contributions: D.A.D., R.L.W., M.J.M., R.M.M., and C.N.M. designed research; R.R. and S.G.H. performed research; R.R., S.G.H., and C.N.M. analyzed data; and R.R., D.A.D., R.L.W., M.J.M., R.M.M., and C.N.M. wrote the paper. The authors declare no conflict of interest. 1 To whom correspondence may be addressed. ddenton@unimelb.edu.au or clive.may@florey.edu.au. 2 Present address: Department of Epidemiology and Preventative Medicine, Monash University, Clayton, Victoria 38, Australia. 29 by The National Academy of Sciences of the USA PNAS January 2, 29 vol. 16 no. 3 cgi doi pnas

2 Table 1. Resting levels of cardiovascular variables in normal and HF groups from 5 min of control data Cardiovascular variables Normals HF Mean Arterial Pressure, mmhg * Heart rate, beats per min * Central venous pressure, mmhg *, spikes(s) * Cardiac burst incidence, bursts per 1 heart beats * Cardiac burst frequency, bursts per minute *, spikes(s) * Renal burst incidence, bursts per 1 heart beats Renal burst frequency, bursts per minute * Values are mean SEM, n 7 in both groups. *, P.5 between groups., P.5 between cardiac and renal variables in normal animals. reached when arterial pressure was reduced with sodium nitroprusside, was significantly lower in the HF group (124 5 beats/min) compared with the normal group ( beats/min) (P.5). In contrast, the bottom plateaus of HR, reached during infusion of phenylephrine, were similar in both groups (Table 2). In addition, the sensitivity of the control of HR by the arterial baroreflex, as indicated by the maximum slope of the relation between HR and arterial pressure, was reduced in HF animals ( beats/min/mmhg) compared with normal animals ( beats/min/mmhg) (Table 2). AP mmhg CVP mmhg RN spikes Hz CN spikes Hz Time (sec) Time (sec) Fig. 1. Raw traces from one normal animal (Left) and one HF animal (Right) indicating the lower burst incidence of compared with in normal animals and the similar burst incidences in both nerves in HF. AP, arterial pressure. Baroreflex Control of and in Normal Sheep and Heart Failure Sheep. In normal animals, the resting level of SNA, as a percentage of its maximum level when the inhibitory influence of baroreceptors was removed by reducing arterial pressure with sodium nitroprusside, was significantly lower for ( %) than for ( %) (P.1) (Fig. 3 and Table 2). The sensitivity of the arterial baroreflex control of, as shown by the steeper maximum slope, was significantly greater than that for. The threshold at which was abolished by rising diastolic blood pressure was also significantly lower than that for. The curves relating diastolic blood pressure to burst incidence are shown in Fig. 3. In the HF group, the maximum slopes of the baroreflex relations between diastolic blood pressure and normalized were not significantly different compared with the normal group (Fig. 3 and Table 2). The maximum slope of the arterial baroreflex relation between normalized and diastolic blood pressure also showed no significant difference between the two groups of animals (Fig. 3 and Table 2). The resting level of, as a percentage of the maximum level during baroreceptor unloading, was significantly higher in the HF group ( %) compared with the normal group ( %) (P.1) (Table 2). The resting level of as a percentage of the maximum was not significantly raised in the HF group compared with normal animals (Table 2). In agreement with our previous finding (6), spectral analysis showed no difference in the low-frequency power of HR variability between the normal and HF groups. In addition, there was no correlation between low-frequency power of HR Burst Incidence (bursts/1 heart beats) Normal * * Heart Failure Fig. 2. Burst incidence in individual normal and HF animals (n 7 in each group). Note the lower burst incidence of compared with in the normal animals and the increase in burst incidence of during HF. *, significantly different to burst incidence of normal animals. Error bars, SEM. PHYSIOLOGY Ramchandra et al. PNAS January 2, 29 vol. 16 no

3 Table 2. Baroreflex parameters for normal and HF animals Baroreflex parameters Normals HF sbp vs. HR Maximum slope, beats per min, mmhg * Top plateau, beats per min * Bottom plateau, beats per min dbp vs. normalized SNA Maximum slope, %max per mmhg Threshold, mmhg * Saturation, mmhg Resting SNA level, % of maximum * Values are mean SEM, n 7 in both groups. sbp, systolic blood pressure; dbp, diastolic blood pressure. *, P.5 between groups., P.5 between nerves. variability and burst frequency in normal animals (data not shown). Discussion The major finding of this study was the significantly lower resting level of compared with in the normal state and the similar levels of activity in both nerves in HF. In addition to the different resting levels in normal animals, the sensitivity of the arterial baroreflex control for was significantly higher than that for. In HF, both nerve activities were increased to similar levels as a percentage of their maxima, with no change in the sensitivity of their control by the arterial baroreflex. Our finding offers an explanation for the preferential increase in cardiac NE spillover observed in human HF patients (1, 2). Studies in human HF patients have generally observed a 3- to 5-fold increase in cardiac NE spillover (4, 7, 8). Most of these patients had lower ejection fractions than our HF sheep and were SNA (% of maximum) SNA (% of maximum) Normal animal - baroreflex curve HF animal - baroreflex curve Burst incidence Burst incidence Normal animal - burst incidence curve HF animal - burst incidence curve Fig. 3. Comparison of arterial baroreflex relations of SNA to diastolic blood pressure (dbp) in normal and HF sheep. (Left) Baroreflex curves for (dotted) and (solid line) in normal animals (Top) and HF animals (Bottom). The SNA is presented as the percentage of maximum activity measured when arterial pressure was reduced with sodium nitroprusside. (Right) Relation of burst incidence (bursts/1 heart beats) of (dotted) and (solid line) to diastolic blood pressure (dbp) in normal animals (Top) and HF animals (Bottom). Circles, the resting points measured during control conditions; error bars, SEM. on medication without which cardiac NE spillover might have been even higher. Our sheep received no medication, and directly recorded was 3-fold higher in HF than in the normal state. Although it is difficult to make direct comparisons between the human and animal studies, it is likely that the impaired neuronal reuptake of NE in HF makes an additional contribution to the preferential increase in cardiac NE spillover measured in human HF (4, 7). Future studies that incorporate simultaneous recordings of and cardiac NE spillover are needed to clarify this issue. Studies examining differential control of and have been conducted primarily in anesthetized animals (9 11). These studies have failed to observe a difference between the resting levels of and. Anesthesia can influence the resting level of nerve activity as well as dampen baroreflex responses (12), and it is likely that the main differences between our study and other studies are because of anesthesia and perhaps the after effects of surgery. and have been recorded in conscious cats (2 6 days after surgery), but no consistent differences between their resting activity levels were observed (13, 14). In our study, the animals were allowed at least three days to recover from surgery, after which resting arterial pressure and HR had reestablished their normal levels. The reasons for the differences between our study and the one in conscious cats are unclear and it is possible that the difference may be attributed to the different species being studied. Our finding in conscious sheep that the sensitivity of the baroreflex control of is greater than that for confirms similar findings in the conscious cat (14) and anesthetized rabbit (15). The incidence of sympathetic bursts reflects the inherent generation of synchronized discharges of activity (16), whereas the burst amplitude has been proposed to reflect the relative number of activated nerve fibers (17). Our finding that the resting burst incidences of the cardiac and renal nerves are significantly different from each other in normal animals suggests that the generation of cardiac and renal bursts may be regulated by separate central circuits in the brainstem. Previous studies have suggested that there is a topographical organization of the presympathetic neurons in the rostral ventral lateral medulla (RVLM) (18, 19) with respect to vasomotor control in different organs, which could lead to differentiated responses to specific stimuli (2, 21). In addition to tonic drive to from the RVLM (19, 2, 22), recent studies have indicated that disinhibition of neurons in the rostral medullary raphé leads to increased with little accompanying vasomotor response, by a mechanism independent of the RVLM (22). It is possible, therefore, that the presympathetic neurons in the medullary raphé may be involved in setting the higher level of in HF. Our results cgi doi pnas Ramchandra et al.

4 showing different resting levels of and provide further evidence of the heterogeneity of the control of sympathetic outflow to different organs. Altered neural control by the arterial baroreflex has been offered as a possible explanation for the increased SNA in HF (23 25). Our study indicates that the increase in SNA to the heart or the kidney in this sheep model of HF is not because of desensitization of the arterial baroreflex. This lack of change in the baroreflex control of in HF confirms previous findings in another group of animals (6). The normal control of by the arterial baroreflex indicates that the impaired response of HR must be because of depressed parasympathetic control, as described (26, 27). Studies in humans (28, 29) and animal models of HF (3, 31) support the concept that the arterial baroreflex control of SNA is not altered in HF. However, there is evidence from studies in rabbits that the baroreflex control of is desensitized in HF (24, 25, 32). Our results indicate that the sympathoexcitation at this stage of HF in this sheep model cannot be attributed to desensitization of the arterial baroreflex. Our study was conducted in conscious animals with stable pacing-induced HF, without the complication of medication. Although all animal models have limitations, the animal model of pacing-induced HF has been used extensively and shows the symptoms of HF seen clinically, including decreased ventricular function and intense neurohumoral activation (3, 33). Importantly, the level of neurohumoral activation in pacing models of HF closely parallels that seen in human HF patient populations (34). The degree of HF in our sheep corresponds to the New York Heart Association (NYHA) class II. We cannot rule out that with worsening HF and further decreases in ejection fraction, there may be a desensitization of the arterial baroreflex. It should be noted that previous studies have indicated differences in the control of muscle sympathetic nerve activity between sexes (35, 36). Because our study was conducted in ewes, our interpretations are therefore limited to the female sex. In conclusion, we have shown that in the normal state the resting level of was significantly lower than that of in conscious sheep. In addition, the arterial baroreflex control of was more sensitive than that of. During HF, the activity in both nerves was increased to similar levels as a percentage of their maxima, with no change in control by the arterial baroreflex. Our finding offers an explanation for the preferential increase in cardiac NE spillover observed in human HF patients. Methods Adult merino ewes (35 49 kg body wt) were housed in individual metabolism cages in association with other sheep. Experiments were conducted on conscious sheep accustomed to laboratory conditions and human contact (n 7 in each group). All experiments were approved by the Animal Experimentation Ethics Committee of the Howard Florey Institute. Surgical Procedures. Before the studies, sheep underwent two or three aseptic surgical procedures, each separated by two weeks recovery. Anesthesia was induced with i.v. sodium thiopental (15 mg/kg) and after intubation was maintained with % isoflurane/o 2. During the first surgery all sheep were prepared with a carotid arterial loop. In a second group of sheep to be induced into HF a pacemaking lead (Medtronic Inc.) was inserted into the right ventricle as described (6). In a separate further operation, intrafascicular electrodes were implanted in the left cardiothoracic nerves as described (37), and intrafascicular electrodes were also implanted in the left renal nerves (38). Experiments were conducted on standing, conscious sheep, and to minimize any effect of surgical stress experiments were not started until the third day after implantation of the electrodes. In cases where implantation of nerve electrodes did not yield good sympathetic recordings (n 2 in each group), the animals were allowed to recover from the surgery for at least three weeks and the implantation was undertaken in the right cardiothoracic and the right renal sympathetic nerves. In all operations, animals were treated with antibiotics (Ilium Propen; procaine penicillin, Troy Laboratories Ptd Ltd or Mavlab) at the start of surgery and then for 2 days after the operation. Postsurgical analgesia was maintained with intramuscular injection of flunixin meglumine (1 mg/kg) (Troy Laboratories or Mavlab) at the start of surgery, then 4 and 16 h after surgery. On the day before implantation of recording electrodes, sterile venous and arterial cannulae were inserted as described (6). Experimental Protocols. Experiments were conducted on two groups of sheep, a group in the normal state and a group in HF. Ejection fraction was measured by using short axis M-wave echocardiography on conscious sheep lying on their right side. After placement of ventricular pacing leads, a basal measurement was made before the start of ventricular pacing, at 2 22 beats/min for 7 8 weeks. The development of HF was monitored weekly by using echocardiography, which was done with the pacing switched off. Sheep were considered to be in HF when ejection fraction had fallen to 4%. All experiments were conducted with the pacing switched off. and were recorded differentially between the pair of electrodes with the best signal-to-noise ratio. The signals were amplified (1, ) and filtered (bandpass 4 1, Hz), displayed on an oscilloscope, and passed through an audio amplifier and loud speaker. Sympathetic nerve activity (5, Hz), arterial blood pressure (1 Hz), and CVP (1 Hz) were recorded on computer by using a CED micro 141 interface and Spike 2 software (Cambridge Electronic Design). At 3 4 days after surgical implantation of cardiac and renal sympathetic nerve electrodes, a 5-min recording of resting,, CVP, and AP was made in conscious sheep in the normal state and in sheep in HF. After this, baroreflex curves were generated by measuring,, and HR responses to increases and decreases in arterial pressure induced by i.v. administration of incremental doses of phenylephrine and sodium nitroprusside as described (6). To confirm that this five-min recording was representative of baseline, repeated recordings were made in 5 of the 7 animals in each group. In normal animals, 5-min recordings repeated on 3 separate days showed comparable burst incidences (32 4 vs vs. 24 3% for ; 94 2 vs vs. 92 3% for ). Recordings in HF animals also showed good reproducibility over 3 days (92 2 vs vs. 89 4% for ; 96 1 vs vs. 97 2% for ). Data Analysis. Nerve recordings were obtained from the left sides of five sheep and the right sides of two sheep in each group. There were no differences in the responses between two sides in either group. Previous studies have recorded left and right simultaneously and found no differences between the resting levels or baroreflex control of on the two sides (39, 4). Hence data collected from both the left and right sides were grouped together to give n 7 in each group. Data were analyzed on a beat-to-beat basis by using custom written routines in the Spike 2 program as described (6). For each heartbeat, the program determined diastolic, systolic and mean arterial pressure, heart period, and the number of discriminated spikes above threshold between the following diastolic pressures, a measure of burst size. The threshold was set just above background so that spikes from small bursts were counted. The background noise was taken as the spikes per second during the highest dose of phenylephrine, when SNA was abolished, and this was subtracted from the data collected on the day. Baroreceptor relations were constructed from data collected during infusion of phenylephrine and sodium nitroprusside as described (6), and the threshold and saturation points of the baroreflex curves were calculated (41). Power spectral density was calculated for both the normal and the HF sheep for HR by using custom written routines in Spike 2 (6). Data are expressed as means SEM and were analyzed by using unpaired Student s t test (SigmaStat, ver 2.3, Access Softek). P.5 was considered statistically significant. ACKNOWLEDGMENTS. The authors thank Tony Dornom for technical assistance and David Trevaks for Spike 2 programming. This work was supported by National Health and Medical Research Council (NHMRC) of Australia Grants and 5924, National Heart Lung and Blood Institute Grant 5-R1- HL , the Harold and Leila Mathers Charitable Trust, The Search Foundation, the Robert J. and Helen C. Klegberg Foundation, NHMRC Research Fellowships (to C.N.M.), (to M.J.M.), and (R.M.M.), and National Heart Foundation of Australia Postdoctoral Research Fellowship PF7M3293 (to R.R.). PHYSIOLOGY Ramchandra et al. PNAS January 2, 29 vol. 16 no

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