medication effects on human cardiovascular regulation are difficult to predict based on preclinical and clinical investigations .
drugs could interfere with cardiovascular regulation in brain or periphery directly or through secondary baroreflexmediated adjustments in heart rate ( hr ) and vascular tone .
highfidelity phenotyping during infusion of vasoactive medications can be used to detect drug effects on human cardiovascular control that could otherwise go undetected .
hcn4 generates  funny  pacemaker f currents ( if ) promoting slow diastolic depolarization and cardiac rhythm generation.1 intracellular cyclic adenosine monophosphate ( camp ) modulates if 
 2 , and adrenergic stimulation augments camp , thereby increasing hcn4 conductivity and hr .
given hcn4 's important role in autonomic hr adjustments , pharmacological hcn4 inhibition could profoundly affect human cardiovascular regulation .
in fact , chronotropic competence is reduced3 or absent4 in mutated hcn4 channels unresponsive to camp .
conversely , blockade of hcn4 channels on baroreceptor sensory neurons increased their excitability,5 , 6 which would tend to facilitate afferent baroreflex signaling and baroreflex function .
the issue is relevant because ivabradine7 , 8 is used in the treatment of heart failure9 and angina pectoris10 ; however , the influences of hcn4 on human cardiovascular autonomic regulation have not been studied , aside from noninvasive baroreflex assessment of profound sympathetic activation.11 ivabradine is an openchannel blocker12 producing 
inhibition,13 thus ivabradine may be more effective at increased hr.14 we tested the hypothesis that pharmacological hcn4 blockade with ivabradine affects baroreflex hr regulation in healthy participants such that hr is reduced at a given blood pressure ( bp ) .
moreover , we assessed whether ivabradine would interfere with baroreflex regulation of hr and sympathetic efferent traffic to resistance vessels and whether or not baroreflex bp buffering is perturbed .
finally , we evaluated whether or not in  vivo responses mirror ivabradine 's usedependent pharmacology .
healthy men ( aged 1840  years ) with a body mass index between 18 and 30  kg / m and a resting hr 60  beats per minute ( bpm ) were eligible for this study .
preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements .
all procedures were in accordance with the helsinki declaration of 1975 ( as revised in 1983 ) . a national competent authority and the local internal review board approved the studies .
this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) .
randomization for study drug sequence ( randomization block size : 6 ) and manufacturing and labeling of blinded medications was done centrally by the pharmacy of the charit medical university .
medications were prepared as identical capsules in neutral containers labeled with the randomization code and a visit identifier .
adherence to randomized medication sequence was secured by following the predefined numeric sequence of the visits .
consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school .
on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5  mg ) or matching placebo 13 hours and 1  hour before testing .
testing was conducted between 8 and 11  am in a quiet laboratory at an ambient temperature of 22 to 23c .
recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) .
we obtained blood samples after at least 30  minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) .
muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) .
we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30  minutes
after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) .
dose incrementation was stopped after the maximum dose of 2.1  g / kg per minute had been reached , sbp had changed by > 25  mm  hg , or hr had dropped < 40  bpm .
main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest .
exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity .
the latter was defined as sbp change per dose increment of sodium nitroprusside and phenylephrine , respectively .
ivabradine is expected to change hr directly , but it may do so also indirectly via baroreflexes through changes in bp . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure  1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) .
resting sbp of both visits ( vertical dotted lines in the figure  1 ) were averaged .
the average served as shared common ( standard ) pressure ( vertical solid line in the figure ) .
this standard pressure was used as input ( abscissa value ) to the baroreflex curve function to calculate normalized rri and hr for each visit separately ( ordinate values ) . 

the example assumes lower sbp on ivabradine than on placebo ( 118  vs 122  mm  hg ) .
the solid vertical line represents mean sbp ( 120  mm  hg ) , which determines  normalized rri  values .
without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values .
hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure .
baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm  hg bins separately for placebo and ivabradine . related bins for all participants
, a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted .
note that the bins for lowest and highest bp are least occupied by values , thus their weight in calculations of curvefitting parameters and confidence bands are small ( figure  6a and 6b ) .
with sd of 6.7  bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan
, primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results ,
several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) .
exploratory variables have been tested with t tests for paired data and correlation analysis without adjustments for multiple testing .
healthy men ( aged 1840  years ) with a body mass index between 18 and 30  kg / m and a resting hr 60  beats per minute ( bpm ) were eligible for this study .
preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements .
all procedures were in accordance with the helsinki declaration of 1975 ( as revised in 1983 ) . a national competent authority and the local internal review board approved the studies .
this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) .
randomization for study drug sequence ( randomization block size : 6 ) and manufacturing and labeling of blinded medications was done centrally by the pharmacy of the charit medical university .
medications were prepared as identical capsules in neutral containers labeled with the randomization code and a visit identifier .
adherence to randomized medication sequence was secured by following the predefined numeric sequence of the visits .
consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school .
on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5  mg ) or matching placebo 13 hours and 1  hour before testing . the washout period between study days was at least 3  weeks .
testing was conducted between 8 and 11  am in a quiet laboratory at an ambient temperature of 22 to 23c .
recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) .
we obtained blood samples after at least 30  minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) .
muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) .
we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30  minutes
, we obtained resting baseline recordings in the supine position . after taking blood samples and measuring cardiac output
, we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) .
dose incrementation was stopped after the maximum dose of 2.1  g / kg per minute had been reached , sbp had changed by > 25  mm  hg , or hr had dropped < 40  bpm .
main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest .
exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity .
the latter was defined as sbp change per dose increment of sodium nitroprusside and phenylephrine , respectively .
ivabradine is expected to change hr directly , but it may do so also indirectly via baroreflexes through changes in bp . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure  1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) .
resting sbp of both visits ( vertical dotted lines in the figure  1 ) were averaged .
the average served as shared common ( standard ) pressure ( vertical solid line in the figure ) .
this standard pressure was used as input ( abscissa value ) to the baroreflex curve function to calculate normalized rri and hr for each visit separately ( ordinate values ) . 

the example assumes lower sbp on ivabradine than on placebo ( 118  vs 122  mm  hg ) .
the solid vertical line represents mean sbp ( 120  mm  hg ) , which determines  normalized rri  values . without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values .
hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure .
baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm  hg bins separately for placebo and ivabradine . related bins for all participants
using graphpad prism 5 , a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted .
note that the bins for lowest and highest bp are least occupied by values , thus their weight in calculations of curvefitting parameters and confidence bands are small ( figure  6a and 6b ) .
with sd of 6.7  bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan
, primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results ,
several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) .
exploratory variables have been tested with t tests for paired data and correlation analysis without adjustments for multiple testing .
we screened 26 men : 23 met inclusion and exclusion criteria and entered the study .
twentyone men completed both study visits ( 2 dropped out ) ( figure  2 ) .
microneurographic recordings were obtained in 18 participants on both days . in 3 participants , we failed to obtain a stable nerve recording position at the second experimental day .
the characteristics of the participants were as follows : age 26.84.2  years , height 1.820.06  m , body mass 79.810.5  kg , body mass index 24.13.0  kg / m , bp 1308/728  mm  hg , and hr 67.45.8  bpm .
ic indicates informed consent ; iva , ivabradine ; plc , placebo experimental data are shown in table . if blockade reduced resting hr with and without normalization for prevailing bp ( figure  3a ) ( for hr normalization , see methods ) .
bp and stroke volume remained unchanged with a trend toward reduced cardiac output ( 7.6% ) and increased total peripheral resistance ( 5.6% ) on ivabradine .
sbp sensitivity to sodium nitroprusside infusion was similar on both study days with a trend toward reduced sensitivity ( improved bp buffering ) to the vasoconstrictor phenylephrine with if blockade ( figure  5 ) .
ivabradine did not alter pharmacological baroreflex sensitivities , such as the cardiac parasympathetic and vasoconstrictor sympathetic baroreflex gains . if blockade dampens the increase in msna burst frequency during higher sodium nitroprusside infusion rates , namely , when high vasoconstrictor activity and elevated hr cooccur ( figure  6a ) ; however , hrindependent msna parameters ( ie , msna burst incidence and total activity ) were not altered by hcn4 blockade during pharmacological baroreflex testing ( data not shown ) .
upward shift of the cardiac baroreflex curve to longer rris ( figure  6b ) .
consequently , rri lengthening by ivabradine was virtually independent of the prevailing hr during pharmacological baroreflex testing .
hemodynamic , sympathetic , hormonal , and baroreflex parameters au indicates arbitrary units ; brg , baroreflex gain ( ie , baroreflex sensitivity ) ; co , cardiac output ; dbp , diastolic blood pressure ; hr , normalized ( baroreflexcorrected ) heart rate ; hrp , normalized heart rate during parasympathetic activation ; mbp , mean blood pressure ; msna , muscle sympathetic nerve activity ; ntp , sodium nitroprusside ; phe , phenylephrine ; sbp , systolic blood pressure ; sv , stroke volume ; tpr , total peripheral resistance .
b , ivabradine 's use dependence : hr lowering is more pronounced in participants with higher resting hr . with resting values of 56.5  bpm , ivabradine has no effect ( unresponsive hr ) .
c , effects of hcn4 inhibition on hr during parasympathetic activation ( hrp ) through baroreflex loading with phenylephrine .
hrp was assessed as hr at the largest individual phenylephrine dose that was reached on both study days .
d , participants with lower hrp on placebo exhibited a smaller reduction in hrp with ivabradine .
the observation is consistent with ivabradine 's usedependent pharmacology ; however , parasympathetic activation shifted the unresponsive hr to 44.2  bpm , which is 12  bpm lower compared with resting conditions ( see panel b ) .
b , individual changes in msna burst frequency , burst incidence , and total msna by ivabradine compared with placebo .
total msna has been rescaled ( 20 ) for visual range harmonization among the 3 msna measures ( also see table ) .
au indicates arbitrary units ; hb , heart beats ; msna , muscle sympathetic nerve activity .  

effects of hcn4 inhibition on bp sensitivity to infusion of the vasoconstrictor phenylephrine  ( upper panel ) and the vasodilator sodium nitroprusside  ( lower panel ) during placebo ( open circles ) and ivabradine ( closed circles ) . 
note the trend toward improved baroreflex buffering of bp increases on ivabradine ( upper panel ) .
a , sympathetic baroreflex : although ivabradine may dampen the increase in msna burst frequency during nitroprusside infusion ( ie , when high vasoconstrictor activity and elevated hr cooccur ) , baroreflex curves are virtually identical if hrindependent msna measures ( burst incidence and total activity ) are used ( curves not shown ) .
b , cardiac baroreflex : ivabradine shifts (  offsets  ) the baroreflex curve upward ( to longer rris ) with virtually no effect on slope or range .
note that the bands do not overlap with high blood pressure , indicating that baroreflexmediated hr slowing is intensified by ivabradine .
dbp indicates diastolic blood pressure ; hr , heart rate ; msna , muscle sympathetic nerve activity ; rri , rr interval ; sbp , systolic blood pressure .
participants with higher resting hr on placebo exhibited more pronounced hr slowing on ivabradine , showing  use dependence 
we observed a similar pattern during baroreflex loading with phenylephrine ( figure  3c and 3d ) ; however , the hr at which ivabradine did not slow hr further was only 44.2  bpm ( ie , 12  bpm lower during parasympathetic activation ) .
together , figure  3b and 3d visualize ivabradine 's usedependent characteristics ; however , they also show , as does figure  6b , that ivabradine and parasympathetic activation are additive in terms of hr reduction .
we observed that hcn4 blockade with ivabradine reduces hr , leaving baroreflex hr and msna regulation intact .
although hcn4 is involved in transducing autonomic activity at the sinus node level , baroreflex hr regulation was preserved on ivabradine .
the cardiac baroreflex curve was parallelshifted toward lower hr without reductions in baroreflex gain or range .
similarly , baroreflex hr slopes determined noninvasively by crossspectral analysis or the sequence method were unchanged with ivabradine in a human model of cardiac sympathetic activation.11 because msna discharges occur during diastole when arterial baroreceptors are unloaded , we expected reduced msna frequencies with compensatory increases in sympathetic action potentials within bursts ; however , ivabradineinduced hr reduction was not associated with resting msna changes .
moreover , sympathetic baroreflex regulation was virtually identical on placebo and on ivabradine considering hrindependent msna measurements .
finally , bp responses to vasoactive medications did not increase on ivabradine , suggesting that baroreflex bp buffering remained fully functional.19 
  together with previous studies in genetic animal models and in patients with familial sinus bradycardia due to hcn4 mutations,3 , 20 , 21 , 22 , 23 our study provides new insight into hcn4 's role in hr regulation . in patients with the 573x mutation
, hcn4 does not respond to camp.3 nevertheless , hr increased 50  bpm during exercise .
likewise , hr acceleration to adrenoreceptor stimulation was intact in adult hcn4deleted mice24 and in a heterozygous knockin model rendering hcn4 camp insensitive.4 on ivabradine , chronotropic competence in terms of hr acceleration and deceleration over a wide hr range assessed through baroreflex loading and unloading was unchanged .
consequently , if can not be the crucial mechanism transducing autonomic signals at the level of the sinus node .
cardiac pacemaker models are composed of several clocks,25 , 26 raising questions about hcn4 's dominant role in sinoatrial node automatism and rate regulation .
instead , hcn4 may defend the sinus node against excess hyperpolarization,27 preventing bradycardia . despite its usedependent pharmacology,12 indicated by larger hr reduction in persons with higher resting hr , ivabradine aggravated baroreflexmediated bradycardia in our study .
moreover , in the signify study , almost 10 000 patients with stable coronary artery disease received ivabradine doses up to 10  mg twice daily .
bradycardia and atrial fibrillation occurred more often in the ivabradine group.28 together with our mechanismoriented study , these observations translate the preclinical concept that if is a defense mechanism against excess bradycardia to humans . using a similar approach , we previously observed that 2adrenoreceptor stimulation with clonidine also disables physiological safeguards maintaining hr.29 
  our study has potential limitations .
we did not investigate responses to ivabradine under steady state conditions ; however , we showed earlier that acute dosing 13 hours and 1  hour before measurements was sufficient to attain ivabradine plasma concentrations in a therapeutic range.11 furthermore , the drug 's bradycardic activity consists of an initial rapid and subsequent relatively long duration of action caused by the ndealkylated metabolite and the parent drug , respectively.30 on the other hand , the 3week washout period was more than twice the time required to avoid carryover , according to the kinetics of the bradycardic effect with repeated oral administration.30 ivabradine 's pharmacological interaction with hcn4 channels complicates the interpretation of our study . as an openchannel blocker , for example , ivabradine features use dependence with trapping of the drug in the closed hcn4 channel.8 furthermore , hcn4 is also expressed in the nervous system , particularly in the thalamus,31 in spinal interneurons,32 and in baroreflex afferents.5 , 6 msna is a sensitive readout for changes in central autonomic regulation.33 , 34 , 35 because msna did not change , confounding effects by neural hcn4 inhibition is unlikely ; however , diseases could alter the ivabradine response .
blunted baroreflex function in type 1 diabetic rats , for example , was associated with increased hcn expression and camp sensitivity.6 our findings in healthy participants can not be simply extrapolated to patients .
finally , given the potential influence of respiration on postganglionic sympathetic discharge , it would have been desirable to also record and analyze breathing , yet others did not observe effects of ivabradine on respiratory rate either in healthy men36 or in patients with chronic obstructive pulmonary disease.37 

highfidelity physiological phenotyping provides pharmacological insight that could not be gained by routine hr and bp measurements .
we observed that hcn4 blockade with ivabradine reduced hr but did not impair sympathetic or parasympathetic baroreflex function .
this feature preserves baroreflexdependent buffering of bp changes and aggravates parasympathetically mediated hr reduction ; therefore , ivabradine 's use dependence is not protective against marked bradycardia .
moreover , our findings provide a better understanding of hcn4 's physiology in humans , which could be applied to elucidate diseases associated with perturbed hr regulation . excess hcn4 conductivity , for example , could contribute to postural tachycardia syndrome , a condition characterized by sinus tachycardia with standing and a mismatch between cardiac and vascular sympathetic activation.38 , 39 indeed , hcn4 inhibition may improve symptoms of postural tachycardia syndrome.40 although gainoffunction mutations in the hcn4 gene have not been discovered , functional hcn4 gain of function has been described in right atrial samples from patients with chronic atrial fibrillation.41 patients with heart failure or coronary artery disease , who are currently being considered for ivabradine treatment , also show redistribution of sympathetic activity toward the heart with an associated increase in hr.42 , 43 

this study was funded by a grant of the german research foundation ( deutsche forschungsgemeinschaft , dfg ) awarded to jordan ( jo 284/81 ) .
tank , heusser , and may were funded by a grant of the german aerospace center ( deutsches zentrum fr luft und raumfahrt , dlr , 50 wb1117 ) .
 backgroundsympathetic and parasympathetic influences on heart rate ( hr ) , which are governed by baroreflex mechanisms , are integrated at the cardiac sinus node through hyperpolarizationactivated cyclic nucleotide  gated channels ( hcn4 ) . 
 we hypothesized that hcn4 blockade with ivabradine selectively attenuates hr and baroreflex hr regulation , leaving baroreflex control of muscle sympathetic nerve activity intact.methods and resultswe treated 21 healthy men with 27.5  mg ivabradine or placebo in a randomized crossover fashion . 
 we recorded electrocardiogram , blood pressure , and muscle sympathetic nerve activity at rest and during pharmacological baroreflex testing . 
 ivabradine reduced normalized hr from 65.98.1 to 58.46.2  beats per minute ( p<0.001 ) with unaffected blood pressure and muscle sympathetic nerve activity . 
 on ivabradine , cardiac and sympathetic baroreflex gains and blood pressure responses to vasoactive drugs were unchanged . 
 ivabradine aggravated bradycardia during baroreflex loading.conclusionshcn4 blockade with ivabradine reduced hr , leaving physiological regulation of hr and muscle sympathetic nerve activity as well as baroreflex blood pressure buffering intact . 
 ivabradine could aggravate bradycardia during parasympathetic activation.clinical trial registrationurl : http://www.clinicaltrials.gov . 
 unique identifier : nct00865917 . 
Clinical Trial Registration
Introduction
Methods
Participants
Study Design and Protocol
End Points
HR Normalization
Merged Baroreflex Curves
Statistics
Results
Discussion
Conclusion
Sources of Funding
Disclosures