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Cardiac-generated sympathetic stress alters heart-brain communication, reduces EEG-
1
theta activity, and increases locomotor behavior
2
3
Jacopo Agrimi
a, b
, Danilo Menicucci
c
, Marco Laurino
d
, Chelsea D Mackey
b
, Laila Hasnain
b
, Sneha
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Dodaballapur
b
, Ross A McDevitt
h
, Donald B Hoover
e, g
, Angelo Gemignani*
c
, Nazareno Paolocci*
5
a, f
, Edward G Lakatta*
b
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a) Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
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b) Laboratory of Cardiovascular Sciences, National Institute on Aging, National Institutes of
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Health Biomedical Research Center (BRC), Baltimore, MD, USA
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c) Department of Surgical, Medical, Molecular Pathology, and Critical Care Medicine,
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University of Pisa, Pisa, Italy
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d) Institute of Clinical Physiology, National Research Council, Pisa, Italy
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e) Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee
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State University, Johnson City TN.
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f) Department of Biomedical Sciences, University of Padova, Padova, Italy
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g) Center of Excellence in Inflammation, Infectious Disease and Immunity, East Tennessee
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State University, Johnson City, TN
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h) The Comparative Medicine Section, National Institute on Aging, National Institutes of
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Health, Baltimore, MD, USA.
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*Contributed equally
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Keywords: Adenylyl cyclase type 8, EEG Theta Rhythm, Granger Causality, Locomotor Activity,
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Heart-Brain Communication, Sympathetic Stress
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Correspondence to:
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Nazareno Paolocci, M.D., Ph.D.
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Division of Cardiology, Traylor 911
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Johns Hopkins Hospital
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720 Rutland Avenue
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Baltimore, MD, 21212
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npaoloc1@jhmi.edu
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Edward G. Lakatta, M.D.
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Laboratory of Cardiovascular Sciences,
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National Institute on Aging,
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National Institutes of Health Biomedical Research Center (BRC),
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Baltimore, MD, 21224
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lakattae@grc.nia.nih.gov
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2
Abstract
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Brain modulation of myocardial activity via the autonomic nervous system is increasingly well
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characterized. Conversely, how primary alterations in cardiac function, such as an intrinsic increase
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in heart rate or contractility, reverberate on brain signaling/adaptive behaviors - in a bottom-up
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modality - remains largely unclear. Mice with cardiac-selective overexpression of adenylyl cyclase
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type 8 (TGAC8) display increased heart rate and reduced heart rhythm complexity associated with
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a nearly abolished response to external sympathetic inputs. Here, we tested whether chronically
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elevated intrinsic cardiac performance alters the heart-brain informational flow, affecting brain
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signaling and, thus, behavior. To this end, we employed dual lead telemetry for simultaneous
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recording of EEG and EKG time series in awake, freely behaving TGAC8 mice and wild-type (WT)
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littermates. We recorded EEG and EKG signals, while monitoring mouse behavior with established
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tests. Using heart rate variability (HRV) in vivo and isolated atria response to sympathomimetic
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agents, we first confirmed that the TGAC8 murine heart evades autonomic control. The EEG
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analysis revealed a substantial drop in theta-2 (4-7 Hz) activity in these transgenic mice. Next, we
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traced the informational flow between EKG and EEG in the theta-2 frequency band via the Granger
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causality statistical approach and we found a substantial decrement in the extent of heart/brain
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bidirectional communication. Finally, TGAC8 mice displayed heightened locomotor activity in
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terms of behavior, with higher total time mobile, distance traveled, and movement speed while
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freezing behavior was reduced. Increased locomotion correlated negatively with theta-2 waves
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count and amplitude. Our study shows that cardiac-born persistent sympathetic stress disrupts
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the information flow between the heart and brain while influencing central physiological patterns,
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such as theta activity that controls locomotion. Thus, cardiac-initiated disorders, such as
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persistently elevated cardiac performance that escapes autonomic control, are penetrant enough
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to alter brain functions and, thus, primary adaptive behavioral responses.
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60
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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3
Introduction
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According to the somatic marker theory postulated by Antonio Damasio (following the thoughts
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of one of the fathers of psychophysiology, William James), afferent somatic signals arising from
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the body's peripheral districts are integrated into higher brain regions, particularly the
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ventromedial prefrontal cortex (VMPFC), and they can influence complex behaviors, such as
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decision making
1,2
. The bidirectional relationship between the heart and the brain falls entirely
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within this loop. Indeed, besides the autonomic nervous system's descending control on cardiac
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function, information is processed by the intrinsic cardiac nervous system (the so-called "little
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brain of the heart") that communicates back to the brain through ascending fibers located in the
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spinal cord and vagus nerve
3
. These afferent impulses reach relay stations such as the medulla,
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hypothalamus, thalamus, and, ultimately, the cerebral cortex
3
, carrying sensory information that
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can initiate centrally-directed behaviors
4
.
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Cardiac rhythmicity is under continuous surveillance and control of the two branches of
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the autonomic nervous system (ANS)
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. A balanced influence of sympathetic and parasympathetic
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efferent fiber discharge over cardiac pacemaker cells located in the sinoatrial and atrioventricular
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nodes is ultimately needed to generate the final heart beating frequency (HR)
5
. However, there
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are clinical situations in which the heart escapes this autonomic control. In this sense, the
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transplanted heart that harbors severed efferent and afferent autonomic fibers is emblematic in
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that it evades both sympathetic and parasympathetic surveillance almost entirely
6
. These
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individuals experience persistently elevated HR at rest and cannot adjust their myocardial
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performance to an increased workload, such as during exercise
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. What is more, after heart
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transplantation, 63% of heart recipients face anxiety and depression, especially during the first
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post-transplant year
8
. Despite numerous observational clues suggesting that peripheral changes
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in cardiac activity can influence behavior, definitive experimental evidence proving this point is
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lacking, along with the nature of the mechanism(s) eventually underlying this bottom-up
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phenomenon and most behavioral patterns that are affected
9
.
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Transgenic mice that overexpress adenylyl cyclase (AC) type 8 (TGAC8) in a cardiac-
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selective manner display persistent elevated HR, reduced HR variability (HRV), and increased
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4
contractility owing to enduringly high intrinsic cardiac cAMP-PKA-Ca2
+
signaling
10,11
. The TGAC8
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heart flees top-down autonomic surveillance by blocking beta-adrenergic signaling and
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catecholamine production to escape harmful additional sympathetic stress. In the present study,
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we sought to determine whether persistent cardiac-initiated stress also disrupts bottom-up
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signaling from the heart to the brain, leading to altered brain activity and behavioral sequelae.
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2. Materials and Methods
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2.1 Animals
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All studies were executed in agreement with the Guide for the Care and Use of Laboratory Animals
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published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996). The
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experimental procedures were approved by the Animal Care and Use Committee of the National
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Institutes of Health (protocol #441-LCS-2016). A breeder pair of TG
AC8
mice, generated by ligating
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the murine α-myosin heavy chain promoter to a cDNA coding for human AC8
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, were a gift from
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Nicole Defer/Jacques Hanoune, Unite de Recherches, INSERM U-99, Hôpital Henri Mondor, F-
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94010 Créteil, France. Wild type (WT) littermates, bred from the C57BL/6 background, were used
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as controls.
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2.2 Evaluation of isolated atrial function and response to adrenergic agents
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To evaluate adrenergic responses of the sinoatrial node (SAN) in the absence of autonomic inputs,
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we isolated the cardiac atria leaving the SAN intact. TGAC8 and control mice were deeply
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anesthetized with isoflurane and euthanized by decapitation. Hearts were removed rapidly and
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transferred to oxygenated (95% O
2
, 5% CO
2
), cold (4°C) Krebs-Ringer bicarbonate buffer (pH 7.35
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to 7.4) of the following composition (mM): 120 NaCl, 4.7 KCl, 1.2 KH
2
PO
4
, 25 NaHCO
3
, 2.5 CaCl
2
,
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1.2 MgCl
2
, and 11.1 D-glucose. The entire atrium was dissected from the ventricles, and each side
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was impaled with a small metal hook (#28 trout hooks) with attached 5-0 sutures. The left atrium
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was anchored to the bottom of a vertical support rod in a 15 mL tissue bath, and the right atrium
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was attached to a 25-g force transducer (World Precision Instruments, Sarasota, FL). Krebs-Ringer
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buffer in the tissue bath was oxygenated continuously and maintained at 37°C. Spontaneous
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atrial contractions were recorded at a resting tension of 0.3 to 0.5 g using a ML224 Bridge
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Amplifier (ADInstruments, Colorado Springs, CO), a PowerLab/8SP, and a computer running Lab
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Chart version 7.3.8. Buffer was replaced at 10 min intervals, and three times after recovery from
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drug treatment. Baseline atrial rate and force of contractions were recorded after a 30 min
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stabilization period. Then responses to a maximally effective concentration of the tyramine (10-4
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M final concentration), which stimulates release of norepinephrine from noradrenergic nerves,
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and the directly acting sympathomimetic, L-isoproterenol, were recorded
12
. Treatments were
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separated by at least 30 min to preclude desensitization.
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2.2.1 Drugs
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L-isoproterenol hydrochloride and tyramine hydrochloride were purchased from Sigma-Aldrich
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(St. Louis, MO).
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2.3 Telemetry double implant to simultaneously monitor the EEG and EKG
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Telemetric radio transmitters (F20-EET; Data Sciences International (DSI), St. Paul, MN) were
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surgically implanted in young (3-4 months) WT and TG
AC8
mice as described
13
. Briefly two surface
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electrodes, a positive electrode (parietal cortex; AP, -2.0 mm; L, 2.0 mm) and a reference electrode
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(cerebellum; AP, -6.0 mm; L, 2.0 mm), were passed subcutaneously to the cranial base and placed
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directly on the dura mater. Two additional bipotential electrodes were routed subcutaneously via
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a vertical midline incision overlying the abdomen with leads situated in the right upper chest and
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Figure 1 (A) Schematic of F20-EET double implant for telemetry recording in mice. (B) Representative images of EEG
recording in a WT and TGAC8 mouse. (C) Representative images of EKG recording in a WT and TGAC8 mouse.
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