Preemptive, but not reactive, spinal cord stimulation

Am J Physiol Heart Circ Physiol 292: H311–H317, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00087.2006.

Preemptive, but not reactive, spinal cord stimulation mitigates transient ischemia-induced myocardial infarction via cardiac adrenergic neurons E. M. Southerland,1 D. M. Milhorn,1 R. D. Foreman,2 B. Linderoth,2,3 M. J. L. DeJongste,4 J. A. Armour,5 V. Subramanian,6 M. Singh,6 K. Singh,6 and J. L. Ardell1 1

Department of Pharmacology, East Tennessee State University, Johnson City, Tennessee; 2Department of Physiology, Oklahoma University Health Science Center, Oklahoma City, Oklahoma; 3Department of Neurosurgery, Karolinska Institutet, Stockholm, Sweden; 4Department of Cardiology, Thorax Center, University Hospital of Groningen, Groningen, The Netherlands; 5Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada; and 6Department of Physiology, East Tennessee State University, Johnson City, Tennessee Submitted 21 January 2006; accepted in final form 7 August 2006

INTEGRATED CONTROL OF regional cardiac function represents the dynamic interplay between local factors, such as the FrankStarling mechanism, the cardiac nervous system, and circulating hormones (e.g., angiotensin II, epinephrine), to modulate cardiac tissues (2, 5). The evolution of cardiac pathology, such as myocardial ischemia, is associated with remodeling of these elements such that imbalance of neurohumoral control, especially excessive sympathetic efferent neuronal activation, can induce adverse cardiac events (5, 10, 27). The reason that cardiac pathology can be effectively managed by ␤-adrenoceptor blockade and/or angiotensin-converting enzyme inhibitors (10, 18) may be related to the fact that these agents act not only directly on cardiomyocytes but also indirectly via the cardiac

nervous system (5). Specifically, subpopulations of neurons within the intrathoracic cardiac neuronal hierarchy possess adrenoceptors (2, 5), and modulation of their activity can have an impact on the evolution of cardiac pathology (10, 29). Acute coronary artery syndromes (ACS) represent multifaceted processes involving both direct effects on cardiac myocytes and their modulation by neurohumoral factors (18, 26). A major adverse consequence of ACS is angina pectoris (12). Although early revascularization by percutaneous transmural interventions has become the primary therapy to alleviate adverse consequences of ACS (17), adjunct pharmacological therapies such as those aimed to resolve or prevent clot formation (18), ␤-adrenoceptor blockers (18, 23), or adenosine (35) can contribute to further infarct reduction. Electrical neuromodulation using spinal cord stimulation (SCS) has been shown to represent a safe, long-term adjunct therapy for patients with chronic angina pectoris refractory to standard treatments (20). Electrical stimuli delivered to the upper thoracic spinal cord segments suppress pain associated with myocardial ischemia (20). Clinically, SCS also exhibits anti-ischemic properties that include increased exercise tolerance (15), diminished ST segment deviation during stress (7, 15), and improved myocardial lactate metabolism (20). Clinical studies indicate that anginal pain can still be evoked in the presence of SCS when stress (e.g., exercise) is of sufficient magnitude to induce myocardial ischemia (20). The mechanisms whereby this mode of therapy produces its beneficial effects are, as yet, poorly understood. SCS influences the processing of information within the central nervous system (8). Animal studies indicate that SCS may inhibit spinothalamic tract neurons (8). It also influences information processing within the intrathoracic cardiac nervous system (2, 13), reducing basal activity and blunting reflex activation of intrinsic cardiac neurons that can occur when transducing regional ventricular ischemia (13). These stabilizing effects are eliminated by transection of neuronal connections between the spinal cord and the intrathoracic cardiac nervous system (13). Since sympathetic efferent and afferent projections are disrupted by such transections (2) and since catecholamines can induce cardioprotection against transient myocardial ischemiainduced apoptosis via both ␣1- and ␤-adrenergic receptor mechanisms (26), we hypothesize that early-phase cardiopro-

Address for reprint requests and other correspondence: J. L. Ardell, Dept. of Pharmacology, East Tennessee State Univ., James H. Quillen College of Medicine, Johnson City, TN 37614-0577 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

␣1-adrenoceptor; ␤-adrenoceptor; ventricular infarction; neuromodulation

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Southerland EM, Milhorn DM, Foreman RD, Linderoth B, DeJongste MJL, Armour JA, Subramanian V, Singh M, Singh K, Ardell JL. Preemptive, but not reactive, spinal cord stimulation mitigates transient ischemia-induced myocardial infarction via cardiac adrenergic neurons. Am J Physiol Heart Circ Physiol 292: H311–H317, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00087.2006.—Our objective was to determine whether electrical neuromodulation using spinal cord stimulation (SCS) mitigates transient ischemia-induced ventricular infarction and, if so, whether adrenergic neurons are involved in such cardioprotection. The hearts of anesthetized rabbits, subjected to 30 min of left anterior descending coronary arterial occlusion (CAO) followed by 3 h of reperfusion (control), were compared with those with preemptive SCS (starting 15 min before and continuing throughout the 30-min CAO) or reactive SCS (started at 1 or 28 min of CAO). For SCS, the dorsal C8-T2 segments of the spinal cord were stimulated electrically (50 Hz, 0.2 ms, 90% of motor threshold). For preemptive SCS, separate groups of animals were pretreated 15 min before SCS onset with 1) vehicle, 2) prazosin (␣1-adrenoceptor blockade), or 3) timolol (␤-adrenoceptor blockade). Infarct size (IS), measured with tetrazolium, was expressed as a percentage of risk zone. In controls exposed to 30 min of CAO, IS was 36.4 ⫾ 9.5% (SD). Preemptive SCS reduced IS to 21.8 ⫾ 6.8% (P ⬍ 0.001). Preemptive SCS-mediated infarct reduction was eliminated by prazosin (36.6 ⫾ 8.8%) and blunted by timolol (29.4 ⫾ 7.5%). Reactive SCS did not reduce IS. SCS increased phosphorylation of cardiac PKC. SCS did not alter blood pressure or heart rate. We conclude that preemptive SCS reduces the size of infarcts induced by transient CAO; such cardioprotection involves cardiac adrenergic neurons.

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tection induced by preemptive SCS is dependent to a considerable extent on adrenergic efferent neurons. MATERIALS AND METHODS

Fig. 1. Protocols for the in situ rabbit heart experiments in which hearts in each respective group were exposed to regional ischemia (open areas) with or without spinal cord stimulation (SCS). The control group (protocol 1) consisted of 30 min of left coronary artery occlusion (CAO) followed by a 3-h reperfusion period. Similarly, 30 min of CAO and 3 h of reperfusion stress were utilized to evaluate all neuromodulation treatments (protocols 2–7). For the 3 preemptive SCS groups, SCS was delivered at frequencies of 50 Hz (protocols 2– 4) or 5 Hz (subgroup, protocol 3.1). For protocol 3, the arrow indicates the time when pretreatment with the adrenoceptor blocking agents prazosin or timolol occurred. For the reactive SCS groups, SCS (50 Hz) commenced 1 min after CAO onset (protocols 5 and 7) or at 28 min of CAO (protocol 6). For protocol 5, SCS terminated at 1 min of reperfusion. For protocols 6 and 7, SCS was maintained until the end of the 3-h reperfusion period. AJP-Heart Circ Physiol • VOL

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Subjects. One hundred thirty-four New Zealand White rabbits of either sex, weighing between 1.95 and 3.55 kg, were used in these acute studies. This animal model was chosen because their hearts possess minimal collateral blood flow and thus have a distinct and homogeneous risk zone to evaluate therapeutic interventions for transient myocardial ischemia (22). All experiments were performed in accordance with the guidelines for animal experimentation described in the “Guiding Principals for Research Involving Animals and Human Beings” (1). The Institutional Animal Care and Use Committee of the East Tennessee State University approved these experiments. Surgical preparation. Rabbits were anesthetized with intravenous pentobarbital sodium (30 mg/kg iv, supplemented as needed; i.e., 2 mg/kg iv if the animal responded to noxious stimuli or control arterial blood pressure increased). The trachea was intubated via a cervical incision, and mechanical ventilation was initiated and maintained with a positive pressure ventilator (MD Industries, Mobile, AL) using 100% O2. Core body temperature was maintained at 38°C via a heating pad. The right carotid artery and jugular vein were each cannulated for blood pressure monitoring and administration of additional anesthesia and drugs, respectively. Heart rate was assessed from a lead II electrocardiogram. All hemodynamic data were recorded concurrently on a Gould model TA6000 recorder. A laminectomy was performed at the T1 level, followed by the subdural placement of two plate electrodes (2 ⫻ 3 mm) slightly to the

left of the midline at the C8 and T2 level. SCS was delivered via these indwelling electrodes connected to a Grass S88 stimulator (Grass Instruments, Quincy, MA) using a constant current stimulus isolation unit (Grass PSIU 6G). The parameters used to stimulate the spinal cord were 5 or 50 Hz at 0.2-ms duration. To determine the adequate stimulus intensity, we progressively increased current intensity until minor muscle contractions were induced in the left upper forelimb (motor threshold, MT). Current intensity used for the experimental protocols was set at 90% of MT; this intensity averaged 0.84 ⫾ 0.34 (SD) mA. Stability of the stimuli intensity was confirmed by repeat MT determinations at the end of the experimental protocols. A thoracotomy was performed in the left, fourth intercostal space. Subsequently, the pericardium was opened to expose the heart. A 2-0 silk suture on a curved, tapered needle was passed around the left anterior descending coronary artery at a level one-third of the distance from the left ventricular base to apex. Regional cyanosis and bulging occurred when the ends of the suture were pulled through a small polyethylene tube to form a snare, secured by clamping the tube with a hemostat. Cyanosis and regional diskinesia were evinced in the territory downstream to that vessel occlusion site; these changes disappeared immediately upon reperfusion. Myocardial tissue perfused by the snared coronary artery was considered the zone at risk; the remainder of the left ventricle was considered to be the non-risk zone. A 20-min stabilization period preceded the onset of each experimental protocol. Experimental protocols. Figure 1 summarizes the experimental protocols employed for each of the 10 groups of rabbits studied. Animals in the control occlusion group [protocol 1: control coronary

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weighed and then frozen at ⫺20°C. The heart was then cut into 2-mm-thick slices, parallel to the atrioventricular groove. Tissue slices so obtained were incubated for 20 min at 37°C in 1% triphenyltetrazolium chloride (TTC) and sodium phosphate buffer (pH 7.4). These tissue slices were then placed in 10% formalin to improve the contrast between stained and unstained tissue. Areas of infarction (TTC negative), risk zone (negative fluorescence under UV light), and non-risk zone (positive fluorescence under UV light) were traced to plastic overlays. These areas of interest were then measured using computer-assisted planimetry (Image Research). The infarct and risk zones were calculated by multiplying each area by the tissue thickness, and their products were summed. Infarct size is expressed as a percentage of the risk zone. Phosphorylation of PKCs. Four additional animals were prepared to evaluate the capacity of SCS to induce phosphorylation of left ventricular PKC. After laminectomy and electrode placement, the treatment group (n ⫽ 2) underwent 46 min of SCS (50 Hz, 200 ␮s, 90% MT) followed by 30-min recovery, whereas the sham group (n ⫽ 2) served as a 76-min time control. At the end of these experiments, left ventricular tissues were flash frozen for subsequent analysis. From these left ventricle samples, tissue lysates were prepared in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, and 0.5% Nonidet P-40) using a homogenizer. Equal amounts of proteins (50 ␮g) were precleared from endogenous rabbit IgG by incubating the lysates with protein A-agarose beads. The lysates were resolved using 10% SDS-polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride membrane. The membranes were blocked in Tris-buffered saline containing 5% milk and 0.1% Tween 20 and then incubated with primary antibodies (phospho-PKC, 1:1,000; or phospho-PKC-␨, 1:1,000; Cell Signaling Technology, Beverly, MA). Protein loading was normalized using ␤-actin immunostaining. Statistical analysis. All data are presented as means (SD). Sigmastat 3.1 (Systat Software) with one-way analysis of variance with post hoc comparisons (Holm-Sidak test) was used to test for differences within and between groups. A significance of P ⬍ 0.05 was utilized. RESULTS

Hemodynamic variables. For untreated controls (Fig. 1, protocol 1), heart rate was not different among baseline, CAO, or reperfusion (261.6 ⫾ 25.2, 263.0 ⫾ 21.6, and 262.0 ⫾ 18.5 beats/min). In these same animals, although CAO did not significantly reduce blood pressure (77.5 ⫾ 15.0 mmHg baseline, 74.6 ⫾ 13.1 mmHg CAO), blood pressure in reperfusion was significantly reduced (69.0 ⫾ 11.6 mmHg) from baseline and CAO levels. For the selective autonomic blockers, prazosin pretreatment induced a significant decrease in basal mean arterial blood pressure (86.5 ⫾ 8.3 vs. 66.8 ⫾ 8.1 mmHg). Timolol pretreatment reduced heart rate (untreated controls: 234.2 ⫾ 21.7 beats/min; treated: 214.1 ⫾ 16.4 beats/min); these changes were sustained throughout the subsequent observation periods. Within-group comparisons for preemptive neuromodulation (Table 1) indicate that SCS by itself induced no significant change in heart rate or blood pressure. During the reperfusion period, blood pressure was reduced from baseline values in the vehicle control and timolol-pretreated groups. For reactive SCS, heart rate did not change significantly throughout the protocols (data not shown), whereas blood pressure was significantly reduced from baseline level during CAO for reactive SCS protocols 5 (84.5 ⫾ 14.6 to 79.2 ⫾ 13.4 mmHg) and 7 (81.9 ⫾ 12.4 to 77.3 ⫾ 8.9 mmHg). By 2 h of reperfusion, blood pressure was further reduced from both

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arterial occlusion (CAO); n ⫽ 33] were subjected to 30 min of regional ventricular ischemia followed by a 3-h reperfusion period. The duration of arterial occlusion was chosen as sufficient to elicit an infarct size (IS) of approximately one-third the risk zone in untreated controls (35). Eleven animals in the control group underwent a dorsal laminectomy before the induction of regional myocardial ischemia, and 23 did not have cord surgery. Preemptive electrical neuromodulation. Animals included in this part of the study were subdivided into six separate groups; each was subjected to 30 min of CAO followed by a 3-h reperfusion period. For these animals, a SCS stimulation frequency of 50 Hz was employed, a frequency that has been shown to provide therapeutic benefits (20). In protocol 3.1, SCS was applied at 5 Hz to test the cardioprotective effects of decade lower stimulus frequency. Preemptive protocol 2 (n ⫽ 9) involved 5 min of SCS followed by a 10-min rest period before the longer duration of SCS (32 min) was applied. In this group, the 30-min period of CAO began 1 min after the onset of the longer duration SCS (Fig. 1). Animals in protocol 3 (n ⫽ 35) were subjected to 46 min of SCS. In this group, the 30-min period of ischemia was initiated 15 min after SCS began and was terminated 1 min before SCS was completed. In 24 of these animals, SCS was delivered at a frequency of 50 Hz (protocol 3), and in 11 animals, SCS was delivered at a frequency of 5 Hz (protocol 3.1). To determine whether adrenergic receptor blockade can affect preemptive SCS-induced cardioprotection, we subjected 17 additional animals to protocol 3 (50 Hz) 15 min after either the ␣1-adrenoceptor blocking agent prazosin (0.15 mg/kg iv; n ⫽ 8) or the ␤-adrenoceptor blocking agent timolol (2 mg/kg iv; n ⫽ 9) was administered. The prazosin dose was selected because of its effectiveness in blocking the hypertensive response to intravenous injections of phenylephrine (100 ␮M, 1 ml). The dose of timolol blocked the hypotensive and tachycardic response to intravenous isoproterenol (100 ␮M, 1 ml). These receptor-blocking agents exhibit no membrane stabilizing effects (30). To determine whether additional epochs of preemptive SCS augmented cardioprotection to transient myocardial ischemia, we subjected animals in preemptive protocol 4 (n ⫽ 11) to 30 min of SCS followed by a 15-min period of rest. Thereafter, SCS was again initiated and maintained for 46 min. In this group, the 30-min ischemic period began 15 min after the second SCS commenced. Nine animals were excluded from final analysis because of myocardial ischemia-induced terminal ventricular fibrillation (VF) events and thus failure to complete the entire protocol. Terminal VF was induced in 9.1% of the control group (n ⫽ 3), 6.8% of the animals receiving preemptive SCS (5 animals: 3 from protocol 3 sham treatment plus 1 each from protocol 3 with prazosin or timolol pretreatment), and 4.3% of the animals receiving reactive SCS (1 animal from protocol 6). Reactive electrical neuromodulation. Animals included in this part of the study were subdivided into three separate groups; each was subjected to 30 min of CAO followed by a 3-h reperfusion period. For these animals, SCS was delivered at a stimulation frequency of 50 Hz at 90% MT. For protocol 5 (n ⫽ 8), 30 min of SCS commenced 1 min after onset of the 30-min CAO and terminated at 1 min of reperfusion. For animals in protocols 6 (n ⫽ 6) and 7 (n ⫽ 9), SCS was initiated at 28 or 1 min of CAO, respectively, and maintained throughout reperfusion. These time points for onset of reactive SCS during CAO were chosen to characterize the effects of early- and late-onset neuromodulation therapy on the ability to modulate IS. IS measurement. At the end of each experiment, the hearts were rapidly excised, mounted on a modified Langendorff apparatus, and perfused with room temperature 0.9% saline to remove blood from the coronary circulation. The same coronary artery site previously occluded was subsequently reoccluded. Thereafter, 2- to 9-␮m fluorescent polymer microspheres (Duke Scientific, Palo Alto, CA) were injected into the coronary artery perfusate to demarcate the ventricular region at risk. After both atria were removed, the rest of the heart was

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Table 1. Hemodynamic data for preemptive SCS neuromodulation, protocol 3 Preblock Baseline

Postblock Baseline

SCS

SCS ⫹ CAO

240.1⫾30.3 242.0⫾20.5 212.3⫾12.9*‡

240.5⫾27.5 241.8⫾23.7 211.0⫾10.9*‡

2-h Reperfusion

Heart rate, beats/min SCS ⫹ vehicle SCS ⫹ prazosin SCS ⫹ timolol

239.7⫾29.7 241.9⫾21.0 234.2⫾21.7

240.3⫾29.0 245.1⫾24.8 214.1⫾16.4*‡

246.6⫾31.2 245.0⫾23.3 210.2⫾9.8*‡

Blood pressure, mm Hg SCS ⫹ vehicle SCS ⫹ prazosin SCS ⫹ timolol

75.2⫾15.6 86.5⫾8.3 82.4⫾9.7

75.3⫾15.5 66.8⫾8.1* 78.1⫾9.8

75.2⫾16.2 67.8⫾7.6* 75.4⫾10.2*

72.1⫾13.0 66.6⫾7.0* 72.9⫾8.0*†

67.6⫾13.0*† 63.5⫾6.9* 63.9⫾6.6*†

baseline and CAO levels in all reactive SCS groups to a similar level that averaged 71.3 ⫾ 9.6 mmHg across all three groups. Effects of SCS on IS. Body weight and left ventricular risk zones were similar in all experimental groups (data not shown). Figure 2 summarizes IS, expressed as a percentage of the zone at risk, in rabbits subjected to 30-min periods of regional ischemia without (control) vs. those with preemptive neuromodulation (protocols 1– 4). In control hearts, IS averaged 36.4 ⫾ 9.5%; no significant difference is identified when

animals with (36.7 ⫾ 9.5%) or without a laminectomy (36.2 ⫾ 9.8%) are compared. Preemptive SCS impacted on IS induced by 30 min of ischemia with 3 h of reperfusion. As summarized in Fig. 2, although a short duration of preemptive SCS at 50 Hz (protocol 2: 5 min of SCS, 10 min rest, followed by SCS concurrent with coronary occlusion) reduced IS marginally (29.9 ⫾ 9.0%; P ⬍ 0.08) compared with control, SCS (protocol 3: 50 Hz starting 15 min before occlusion and maintained throughout the occlu-

Fig. 2. Infarct size (IS) plotted as a percentage of the risk zone for control animals and for rabbits with preemptive SCS (c.f., Fig. 1). Data points represent individual animals, and the points with error bars indicate mean (SD) data for each group. Control animals (protocol 1) are subdivided into those with no cord surgery or laminectomy controls (surg. control; included placement of SCS electrodes). *P ⬍ 0.05 compared with control. #P ⬍ 0.05 compared with protocol 3.1. AJP-Heart Circ Physiol • VOL

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Values are means (SD). SCS, 46 min of spinal cord stimulation (C8-T2 at 50 Hz, 0.2 ms, 90% motor threshold) with 30 min of coronary artery occlusion (CAO) starting at 15 min after SCS onset. Prazosin or timolol was administered intravenously 15 min before baseline. Within group comparisons: *P ⬍ 0.05 compared with preblock baseline; †P ⬍ 0.05 compared with postblock baseline. Between group comparisons: ‡P ⬍ 0.05 compared with vehicle and prazosin pretreatment groups.

SPINAL CORD ACTIVATION INDUCES CARDIOPROTECTION

Fig. 3. IS plotted as a percentage of risk zone for control animals subjected to ischemia (control CAO) and animals with 50-Hz preemptive SCS (c.f. Fig 1, protocol 3). The preemptive SCS groups received vehicle or selective adrenergic blockade (prazosin or timolol) 15 min before onset of SCS. Data points represent individual animals, and the points with error bars indicate mean (SD) data for each group.*P ⬍ 0.05 compared with CAO alone (protocol 1). #P ⬍ 0.05 compared with protocol 3 vehicle control. AJP-Heart Circ Physiol • VOL

Fig. 4. IS plotted as a percentage of the risk zone for control animals and for rabbits with reactive SCS (c.f., Fig. 1). Data points represent individual animals, and points with error bars indicate mean (SD) data for each group. There was no significant infarct reduction from control animals (protocol 1) in response to any of the reactive SCS neuromodulation treatments evaluated (protocols 5–7).

16.0-s duration), and three animals with reactive SCS (23.3 ⫾ 18.7-s duration). Animals with terminal VF events were excluded from subsequent data analysis. DISCUSSION

This study demonstrates for the first time that preemptive electrical neuromodulation therapy with SCS reduces the size of infarcts induced by transient ventricular ischemia. These data also indicate that such SCS-induced cardioprotection involves cardiac adrenergic neurons, acting via ␣1- and ␤-adrenoceptors, and that preemptive SCS increases phosphorylation of cardiac PKC pathways. The capacity of preemptive SCS to reduce IS depends on the duration and frequency of the stimulus applied to the dorsal aspect of the upper thoracic

Fig. 5. Effects of preemptive SCS on phosphorylation of left ventricular (LV) PKC. From flash-frozen LV samples, tissue lysates were prepared, loaded on 10% SDS gels, and analyzed by Western blotting using phospho-PKC (p-PKC) primary antibodies. Equal loading of proteins in each lane was normalized using actin immunostaining.

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sion) reduced IS significantly (21.8 ⫾ 6.7% of the risk zone; P ⬍ 0.001) compared with control. In contrast, no change in IS was evident in protocol 3.1, when SCS was delivered at 5 Hz (IS ⫽ 36.1 ⫾ 9.1%). The addition of a second 30-min cycle of preemptive SCS before CAO (protocol 4) did not confer any additional cardioprotection compared with protocol 3 (IS ⫽ 27.9 ⫾ 9.0%). As summarized in Fig. 3, the capacity of preemptive SCS to reduce IS was abolished by pretreatment with prazosin (IS ⫽ 36.6 ⫾ 8.8%) and significantly attenuated following pretreatment with timolol (IS ⫽ 29.4 ⫾ 7.5%). In contrast to the infarct reduction induced by preemptive SCS, reactive SCS did not reduce IS (Fig. 4). In control hearts, IS averaged 36.4 ⫾ 9.5%. In animals with SCS initiated 1 min into CAO, IS averaged 41.3 ⫾ 12.0% when SCS terminated at 1 min of reperfusion (protocol 5). SCS initiated at 28 min of CAO and maintained throughout the reperfusion period (protocol 6) was ineffectual in reducing IS (38.1 ⫾ 9.1%). SCS initiated at 1 min of CAO and maintained throughout 3 h of reperfusion (protocol 7) likewise did not reduce IS (36.5 ⫾ 9.8%). Effects of SCS on PKC phosphorylation. Phosphorylation of total myocardial PKC and PKC-␨ increased in the SCS treatment group compared with tissues derived from sham animals (Fig. 5, n ⫽ 2 for each group). Compared with sham controls at 30 min post-SCS, phospho-PKC increased 2.75 ⫾ 0.02-fold and phospho-PKC-␨ increased 9.19 ⫾ 0.07-fold (data not shown). Effects of SCS on VF. Myocardial ischemia induced VF in 3 of 33 animals in the control group, in 5 of 73 animals with preemptive SCS, and in 1 of 22 animals with reactive SCS. For preemptive SCS, all five terminal VF events occurred in protocol 3, with three in the vehicle control group and 1 each in the prazosin and timolol pretreatment groups. All terminal VF events occurred during CAO. Transient periods of VF (33.0 ⫾ 19.0-s duration) were noted in two animals in the control group, four animals with preemptive SCS (46.8 ⫾

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long-lasting stabilizing effects on the intrinsic cardiac nervous system by suppressing its excessive reflex activation during focal myocardial ischemia (13). As such, SCS may blunt reflex activation of sympathetic efferent neurons elicited by acute myocardial ischemia as first described by Malliani et al. (19) in 1969. Thus we propose that there are at least two separate processes involved in SCS-mediated effects in reducing IS, as characterized in this study. The first involves a low-level neurally dependent catecholamine release into the cardiac interstitium that, when evoked before coronary occlusion, will activate cardiomyocyte PKC pathways to induce protection. The second acts via the cardiac nervous system to limit reflex activation of cardiac sympathetic efferent neurons during the ischemic insult itself. In support of the second proposed mechanism, Cardinal et al. (7) recently demonstrated that sympathetic efferent neuron-dependent ST segment deviations in the stressed myocardium are mitigated by SCS. The specific descending neural pathways that are modified by SCS to affect peripheral neuronal and myocyte interactions remain poorly defined. SCS modulation of the intrinsic cardiac nervous system is eliminated by bilateral ansae transection, a surgical procedure that eliminates afferent and efferent sympathetic axons coursing between spinal cord neurons to intrathoracic neurons (13). For instance, SCS may induce neuropeptide release from cardiac sympathetic afferent nerve terminals (e.g., CGRP, substance P, opiates) to affect adjacent neuronal and/or myocyte function (9, 11). Activation of sympathetic efferent preganglionic neurons may also alter the processing of information within the intrathoracic sympathetic nervous system (24). With respect to SCS-mediated effects inducing IS reduction, data presented in this study indicate that this involves cardiac neuronal ␣1-adrenoceptors, with secondary contributions by ␤-adrenoceptors. Different populations of cardiac adrenergic neurons are known to possess both these receptor subtypes (4). Activation (or blockade) of either can modify regional cardiac function (4) and alter neurotransmitter release from sympathetic efferent nerve terminals (29). Neuromodulation therapy and arrhythmias. In addition to effects on cardiomyocyte viability, myocardial ischemia impacts cardiac electrical function. It is a classic concept that excessive sympathetic neuronal activity can increase dispersion of ventricular electrical events that ultimately result in VF secondary to excessive local release of catecholamines (14). The clinical importance of this is emphasized by the effectiveness of ␤-adrenergic blockade in reducing sudden cardiac death in patients post-myocardial infarction (18, 23). In the current study, preemptive SCS did not reduce the incidence of sudden cardiac death associated with transient myocardial ischemia in the rabbit model. However, it should be considered that the lack of collateral blood flow in the risk zone in the rabbit model (22) will impact local neural and myocyte function and as such are not necessarily reflective of what may occur in humans. Indeed, in a canine model with regional supply/demand imbalance induced by a chronic ameroid constrictor, sympathetic efferent neuron-dependent ST segment deviations in the stressed myocardium are mitigated by SCS (7). Thoracic SCS can also reduce the potential for ischemic induced ventricular tachycardia/VF in a canine model of healed myocardial infarction and pacing-induced heart failure (16). Together, these data indicate that neuromodulation therapy

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spinal cord. In accord with data derived from experimental animals (13), this form of therapy exerts minimal effects on basal cardiac function. Preemptive SCS demonstrates frequency dependence, as evidenced by differences in cardioprotection induced by 5 vs. 50 Hz electrical stimuli applied to the spinal cord at the same relative intensity and for the same duration. In humans, the higher frequency is associated with anti-ischemic effects and long-lived angina relief (20). Preemptive SCS also demonstrated a threshold effect in as much as 5 min of SCS with 10 min of rest was insufficient to impart a significant benefit compared with the 40% reduction in IS evoked when SCS was initiated at same time point (15 min before coronary occlusion) and continuously maintained until 1 min of reperfusion. Interestingly, adding a 30-min cycle of preemptive SCS 15 min before longer duration (46 min) SCS did not provide additional cardioprotection for infarct reduction. In contrast, reactive SCS was not effective in reducing IS, even when initiated within 1 min of coronary occlusion onset and maintained during reperfusion. Late-onset SCS neuromodulation, initiated 2 min before coronary reperfusion, was likewise ineffective in reducing IS. It remains to be determined whether neuromodulation therapy initiated postischemia is cardioprotective. Catecholamines and cardioprotection. Adrenergic receptors, coupled to distinct signal transduction pathways, affect cardiac myocytes and the neurons that regulate them (26, 34). For cardiomyocytes, PKC activation represents a major mediator in preconditioning (26, 35); it can be activated via multiple pathways that include ␣1-adrenergic receptors (32). Exogenous activation of ␣1-adrenoceptors induces early- and late-phase preconditioning for reduction of IS and stunning (6, 21, 28, 31). During early-phase preconditioning, such activation also reduces ischemia-reperfusion-induced cardiac arrhythmias (33). ␤-Adrenoceptor activation can also limit IS to transient myocardial ischemia via a non-PKC pathway that involves PKA and p38 MAPK (26). Although endogenous release of norepinephrine following tyramine administration can also reduce IS (6, 31), and depletion of cardiac catecholamines using reserpine raises PC threshold (3), most data indicate that the major myocyte triggers for ischemic preconditioning do not include catecholamines (35). Yet, as demonstrated in this study, preemptive SCS reduces IS induced by transient myocardial ischemia. Such protection involves neurally dependent activation of cardiac PKC pathways coupled to ␣1-adrenergic receptors. SCS-mediated reductions in IS also involve ␤-adrenoceptors, although the specific cellular pathways and relevant subtype-dependent effects remain to be determined. In addition to direct effects on cardiomyocytes, we propose that ␣1- and ␤-adrenergic receptors can also modulate the cardiac nervous system to effect cardiomyocyte viability. Neuromodulation and cardioprotection. A key issue with regard to understanding the mechanisms whereby SCS exerts its beneficial cardiac effects relates to the fact that the processing of information arising from the ischemic myocardium by the intrinsic cardiac nervous system can be modified by electrical neuromodulation therapy. Electrical activation of neurons in the upper thoracic spinal cord modifies the processing of information within not only the central nervous system (8) but also the intrinsic cardiac nervous system (13). SCS does not appear to interfere with primary efferent neuronal control of nodal tissues in the heart (25) but does exert immediate and

SPINAL CORD ACTIVATION INDUCES CARDIOPROTECTION

may have impact on multiple aspects of the adverse cardiac pathology associated with myocardial ischemia. Perspectives. Data derived from these experiments indicate that preemptive neuromodulation therapy in addition to its antianginal properties is effective in mediating ventricular infarct reduction consequent to transient myocardial ischemia. The ineffectiveness of reactive SCS to reduce IS in the acute setting represents a limitation. However, in clinical practice, SCS has been shown to be a long-term adjunct therapy for patients with chronic angina pectoris (20). It should be considered that as an unrecognized benefit to chronic SCS therapy, these patients may experience a relative state of cardioprotection to transient periods of myocardial ischemia.

The financial support of the National Institutes of Health (J. L. Ardell, R. D. Foreman, and K. Singh), American Heart Association (J. L. Ardell, D. M. Milhorn), Department of Veterans Affairs Merit Review Grant (K. Singh), and Canadian Institutes of Health Research (J. A. Armour) is gratefully acknowledged. REFERENCES 1. American Physiological Society. Guiding principals for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281–R283, 2002. 2. Ardell JL. Intrathoracic neuronal regulation of cardiac function. In: Basic and Clinical Neurocardiology, edited by Armour JA and Ardell JL. New York: Oxford University Press, 2004, p. 118 –152. 3. Ardell JL, Yang XM, Barron BA, Downey JM, Cohen MV. Endogenous myocardial norepinephrine is not essential for ischemic preconditioning in rabbit heart. Am J Physiol Heart Circ Physiol 270: H1078 – H1084, 1996. 4. Armour JA. Intrinsic cardiac neurons involved in cardiac regulation possess ␣1-, ␣2-, ␤1-, and ␤2-adrenoceptors. Can J Cardiol 13: 277–284, 1997. 5. Armour JA. Cardiac neuronal hierarchy in health and disease. Am J Physiol Regul Integr Comp Physiol 287: R262–R271, 2004. 6. Bankwala Z, Hale SL, Kloner RA. ␣-Adrenoceptor stimulation with exogenous norepinephrine or release of endogenous catecholamines mimics ischemic preconditioning. Circulation 90: 1023–1029, 1994. 7. Cardinal R, Ardell JL, Linderoth B, Vermeulen M, Foreman RD, Armour JA. Spinal cord activation differentially modulates ischemic electrical responses to different stressors in canine ventricles. Auton Neurosci 111: 37– 47, 2004. 8. Chandler MJ, Brennan TJ, Garrison DW, Kim KS, Schwartz PJ, Foreman RD. A mechanism of cardiac pain suppression by spinal cord stimulation: implications for patients with angina pectoris. Eur Heart J 14: 96 –105, 1993. 9. Croom JE, Foreman RD, Chandler MJ, Barron KW. Cutaneous vasodilation during dorsal column stimulation is mediated by dorsal roots and CGRP. Am J Physiol Heart Circ Physiol 272: H950 –H957, 1997. 10. Dell’Italia LJ, Ardell JL. Sympathetic nervous system in the evolution of heart failure. In: Basic and Clinical Neurocardiology, edited by Armour JA and Ardell JL. New York: Oxford University Press, 2004, p. 340 –367. 11. Eliasson T, Mannheimer C, Waagstein F, Andersson B, Berg CH, Augustinsson LE, Hedner T, Larson G. Myocardial turnover of endogenous opioids and CGRP in the human heart and the effects of spinal cord stimulation on pacing-induced angina pectoris. Cardiology 89: 170 –177, 1998. 12. Foreman RD. Mechanisms of cardiac pain. Annu Rev Physiol 61: 143– 167, 1999. 13. Foreman RD, Linderoth B, Ardell JL, Barron KW, Chandler MJ, Hull SS, TerHorst GJ, DeJongste MJL, Armour JA. Modulation of intrinsic cardiac neurons by spinal cord stimulation: implications for therapeutic use in angina pectoris. Cardiovasc Res 47: 367–375, 2000. 14. Han J, Garcia de Jalon P, Moe GK. Adrenergic effects on ventricular vulnerability. Circ Res 14: 516 –524, 1964.

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