Mediating -Opioid-Initiated Heart Protection via the ... fileDepartment of Pathology (PJB), ......

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Mediating δ-Opioid-Initiated Heart Protection via the β2-Adrenergic Receptor:

Role of the Intrinsic Cardiac Adrenergic Cell

Short Title: Mediating Cardioprotection by the Intrinsic Cardiac Adrenergic Cell

Ming-He Huang, Hui-Qun Wang, William R Roeske, Yochai Birnbaum, Yewen Wu, Ning-Ping

Yang, Yu Lin, Yumei Ye, David J McAdoo, Michael G Hughes, Scott D Lick, Paul J Boor,

Charles Y Lui, Barry F Uretsky

Department of Internal Medicine, Cardiology Division (M-HH, YB, YW, YL, YY, CL, BFU),

Research Histopathology Core (H-QW, N-PY), Department of Neuroscience and Cell Biology

(DJM, MGH), Department of Surgery, Division of Cardiothoracic Surgery (SDL), and

Department of Pathology (PJB), University of Texas Medical Branch, Galveston, Texas

Department of Medicine, University of Arizona, College of Medicine, Sarver Heart Center

(WRR), Tucson, Arizona

Address for correspondence: Dr. Ming-He Huang, University of Texas Medical Branch,

Department of Internal Medicine, 5.106 John Sealy Annex, 301 University Boulevard,

Galveston, TX 77555-0553, Tel: (409) 772-2410, Fax: (409) 772-4982, Email:

[email protected]

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (March 16, 2007). doi:10.1152/ajpheart.01195.2006

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

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Abstract

Stimulation of cardiac β2-adrenergic receptor (β2-AR) or δ-opioid receptor (DOR) exerts a

similar degree of cardioprotection against myocardial ischemia in experimental models. We

hypothesized that δ-opioid-initiated cardioprotection is mediated by the intrinsic cardiac

adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ

hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH

immunoreactivity which was co-localized with DOR immunoreactivity in ICA cells in human

and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat

ventricular myocytes. The physiology of DOR expression was examined by determining changes

of cytosolic [Ca2+]i transients in isolated rat ICA cells using fluorescence spectrophotometry.

Exposing the selective δ-opioid agonist DPDPE to ICA cells increased [Ca2+]i transients in a

concentration-dependent manner. Such an effect was abolished by the Ca2+ channel blocker

nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine

release from ICA cells following DPDPE application. The significance of ICA cell and its

epinephrine release in δ-opioid-initiated cardioprotection was demonstrated in the rat myocardial

infarction model and ICA cell-ventricular myocyte co-culture. DPDPE administered before

coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size

by 54±15% or myocyte death by 26±4%, respectively. β2-AR blockade markedly attenuated δ-

opioid-initiated infarct-size-limiting effect and abolished δ-opioid-initiated myocyte survival

protection in rat ICA cell-myocyte co-culture. Furthermore, δ-opioid agonist exerted no myocyte

survival protection in the absence of co-cultured ICA cells during ischemia-reperfusion. We

conclude that δ-opioid-initiated myocardial infarct-size-reduction is primarily mediated via

endogenous epinephrine-β2-AR signaling pathway as a result of ICA cell activation.

Key Words: β2-adrenergic receptor, delta opioid receptor, epinephrine, ICA cell, myocardial

ischemia

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INTRODUCTION

Stimulation of β2-adrenergic receptor (β2-AR) before myocardial ischemia confers

cardioprotection via infarct-size-reduction (13, 14) and facilitated ventricular contractile

recovery (25) during ischemia-reperfusion in experimental models. Numerous studies indicate

that δ-opioid agonists precondition the heart against ischemia (2, 7, 19, 22, 23). The extent of

heart protection conferred by δ-opioids is comparable to those elicited by myocardial β2-AR

stimulation. It has been suggested that δ-opioid-initiated cardioprotection is partially mediated by

β2-AR in the murine heart (20). Although δ-opioid agonists exert potent cardioprotection, it is

unclear which type of cardiac cells actually express δ-opioid receptor (DOR). Despite receptor-

binding study of DOR (11, 27, 33) and its gene expression in human and animal hearts (2, 28),

no clear immunohistochemical evidence has demonstrated in situ DOR expression in cardiac

myocytes. The intrinsic cardiac adrenergic (ICA) cell is a neuroendocrine cell that expresses

genes and enzyme proteins for epinephrine biosynthesis (5, 9, 10). ICA cells possess an oxygen-

sensing function suggesting an intimate involvement in ischemia-reperfusion (10). In the present

study, we have hypothesized that ICA cells express DOR mediating ICA cell function. We have

also hypothesized that ICA cells participate in δ-opioid-initiated cardioprotection against

ischemia. We examined the following objectives: 1) ICA cells express DOR immunoreactivity in

human and rat hearts; 2) DOR regulates ICA cell function via [Ca2+]i transients modulation; 3) δ-

opioid stimulation enhances endogenous epinephrine release from isolated ICA cells; and 4) δ-

opioid-initiated cardioprotection depends on ICA cell activation via enhanced epinephrine

liberation and consequent myocardial β2-AR stimulation during ischemia-reperfusion in vivo and

in vitro.

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MATERIALS AND METHODS

Immunohistochemical studies: Immunohistochemical labeling of ICA cells was performed on 4-

µm paraffin sections of 4% formaldehyde-fixed cardiac tissue. Human heart tissues (n=6) were

obtained from recipient’s hearts during heart transplant surgery (n=4) or autopsy (n=2). For rat

(Sprague-Dawley) immunohistochemical studies, 4 adult rat hearts were used. The protocols for

using human and animal tissue were approved by the Institutional Review Board and the

Institutional Animal Care and the Use Committee of the University of Texas Medical Branch.

Human tissues were taken from left ventricular (LV) free wall, sinoatrial (SA) and

atrioventricular (AV) nodal regions. Immunoperoxidase (9) and immunofluorescent labeling was

performed with antibodies against tyrosine hydroxylase (TH) and phenylethanolamine-N-

methyltransferase (PNMT), markers of ICA cells (5, 9). The dilutions for mouse anti-human TH

(Neuromics, Northfield, MN) and mouse anti-rat TH (ImmunoStar Inc, Hudson, WI) were 1:40.

The dilution for rabbit anti-human PNMT (ImmunoStar Inc, Hudson, WI ) was 1:500. To co-

localize DOR and TH immunoreactivity, immunofluorescent double labeling methods were used.

The concentrations for rabbit anti-human DOR (US Biological, Swampscott, MA) and rabbit

anti-rat DOR (Calbiochem, San Diego, CA) were 1:200 and 1:250, respectively. The specificity

of mouse anti-TH and rabbit anti-DOR antibodies was tested by substituting these antibodies

with Universal Negative Controls for Mouse and Rabbit IgG (DAKO Corporation, Carinteria,

CA), respectively. Immunofluorescent double labeling was also used to determine whether ICA

cells express neuronal marker PGP 9.5 or muscle marker myosin-heavy-chain. The dilutions for

PGP 9.5 (Chemicon International Inc., Temecula, CA) and myosin-heavy-chain (Abcam Inc,

Cambridge, MA) were 1:3000 and 1:500, respectively. The double staining included four steps:

(1) rabbit anti-DOR served as the first primary antibody and was stained with goat anti-rabbit

Alexa Fluor 594 followed by amplification with donkey anti-goat Alexa Fluor 594; (2) slides

were then incubated with biotin-labeled goat anti-rabbit for 30 min to saturate unbound rabbit

IgG; (3) mouse anti-TH served as the second primary antibody and was stained sequentially with

rabbit anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 488 (Signal-Amplification

Kit for mouse antibodies, Molecular Probes, Inc., Eugene, OR). A control slide with omitted

mouse anti-TH treatment was stained with Streptavidin-Alexa Fluor 488 after step (2) to test

possible cross-reaction between goat anti-rabbit biotin used in step (2) and rabbit anti-DOR

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antibody used in step (1); (4) Slides were counterstained with DAPI. Double labeling of TH with

PNMT or TH with PGP 9.5 was performed in the same fashion. Image-IT TM FX Signal

Enhancer (Molecular Probes, Inc., Eugene, OR) and autofluorescence eliminator reagent were

used before and after staining to block nonspecific background or autofluorescence.

Immunohistochemical co-localization of TH and DOR immunoreactivity was performed in rat

hearts and in isolated rat ICA cells. Finally, to determine whether both ICA cells and ventricular

myocytes express DOR, we performed simultaneous immunofluorescent double detection of

immunoreactivity of DOR (rabbit anti-DOR diluted in 1:600) and myosin-heavy-chain (mouse

anti-myosin-heavy-chain diluted in 1:1000) in dissociated and mixed rat ventricular cardiocytes.

Detection of TH mRNA in human ICA cells by in situ hybridization: In situ hybridization was

performed on two human LV tissue samples. TH oligonucleotide probes were synthesized

(Sigma-Genosys, St. Louis, MO) per the published sequence (accession number: NM 012740).

The antisense sequence was GCATAGTTCCTGAGCT TGTCCT, and sense was

CGTATCAAGGACTCGAACAGGA. Both were labeled with fluorescein at 5'. Paraffin sections

of LV were rehydrated and treated in a microwave oven at 1000C for 6 min then postfixed with

2% paraformaldehyde followed by graded dehydration. FITC-labeled TH mRNA oligonucleotide

probes of sense and antisense were applied at the same concentration and sealed with Hybri-Well

Press-Seal Hybridization Chambers (Sigma). Hybridization was performed at 850C for 5 min

then 2.5 hr at 500C in the HybriteTM (Vysis, Inc; Downers Groove, IL). After post-hybridization

wash, goat anti-FITC was applied followed by donkey anti-goat IgG Alexa Fluor 594. The slides

were treated with Image-ITTM Enhancer before applying mouse anti-human TH antibody used

for labeling TH immunoreactivity. Dual detection of chicken anti-mouse Alexa Fluor 488 and

rabbit anti-mouse Alexa Fluor 488 was performed.

[Ca2+]i recording in isolated ICA cells: Rat ICA cell isolation was made possible by the original

observation that live ICA cells but not other cardiocytes preferentially bind to iron oxide-based

paramagnetic beads after they have been dissociated from myocardial tissue (9). Ventricular

cardiocytes were enzymatically dissociated and resuspended in 4 ml Tyrode’s solution

containing 50-µl paramagnetic beads that were not coated with any primary antibody

(Polysciences Inc., Warrington, PA). ICA cells attached to the beads were recovered by magnetic

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separation and cultured for 48-72 hr. The cultured ICA cells were loaded with 4 mmol/L fura 2-

AM for 30 min. A ratio-based microscopic fluorescent spectrometer was used to measure the

[Ca2+]i transients generated by ICA cells (10).

Western blot analysis and epinephrine assay: To determine whether both ICA cells and

ventricular myocytes express DOR protein, freshly isolated rat ICA cells and ventricular

myocytes with depletion of ICA cells were lysed separately to extract their protein. Protein of rat

brain tissue served as a positive control. Western blot analysis was performed as described (10).

The concentration for rabbit anti-DOR was 1:500. In vitro epinephrine release assay was

performed in isolated rat ICA cells. ICA cells isolated from adult rat hearts were pooled and

plated on 12-well culture plates (2 hearts /well) and cultured for 72 hr. Epinephrine release from

ICA cells in culture was determined using an HPLC-electrochemical detection system as

described (4).

Experimental Design

DOR mechanism: We studied the effects of a potent selective δ1-opioid agonist [D-Pen25]-

enkephalin (DPDPE) on [Ca2+]i transients generated by isolated ICA cells in culture. After

obtaining baseline [Ca2+]i transients of ICA cells for 15 min, DPDPE at different doses (0.1

nmol/L, 1 nmol/L, 10 nmol/L, and 100 nmol/L) was administered in random order to the ICA

cell for 15 min. Cells were then washed for 20 min with continuously recording [Ca2+]i transients

until the activity reached a plateau. The specificity of DPDPE was examined by exposing ICA

cells to the DOR antagonist naltrindole (10 µmol/L) for 10 min followed by application of

DPDPE (100 nmol/L) in the continuous presence of naltrindole for 15 min. To determine

whether the L-type Ca2+ channels were responsible for altered [Ca2+]i transients following δ-

opioid stimulation, the effects of DPDPE on ICA cells in the presence of the L-type Ca2+ channel

blocker was tested by applying nifedipine (1 µmol/L for 15 min) to the cells when the

enhancement in [Ca2+]i was elicited with DPDPE.

Effect of δ-opioid agonist on epinephrine release: After 72 hr in culture, ICA cells were switched

to 500 µL of Tyrode’s solution and incubated for 1 hr. The sample solution was collected for

basal epinephrine release. Then the culture wells were treated for 30 min with 500 µl Tyrode’s

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solution with or without DPDPE (100 nmol/L). Conditioned solutions were collected at the end

of 30 min and the culture wells were washed 3 times. After washing, 500 µl of Tyrode’s solution

was reintroduced to the culture wells for 1 hr (recovery phase). The samples were recollected and

the cells were lysed for protein quantification.

In vivo myocardial ischemia study: The rat myocardial infarct model has been described

previously (30). Briefly, following anesthesia and ventilation support, the chest is opened and the

left coronary artery encircled with a suture and ligated for 30 min. The snare is then released and

myocardium reperfused for 4 hr. The left coronary artery is then reoccluded and Evan's blue dye

injected into the right ventricle. The LV is sliced into 6-7 sections. Tissue slices are incubated for

10 min in 1% 2,3,5-triphenyl-tetrazolium-chloride (TTC), fixed in 10% formaldehyde, and

photographed to identify the ischemic myocardium at risk (uncolored by the blue dye), the

necrotic zone (unstained by TTC) and the nonischemic zones (colored by blue dye). The areas of

ischemia and necrosis in each slice are determined by planimetry, converted into percentages of

the whole for each slice, and multiplied by the weight of the slice. The examiner who analyzed

infarct size was blinded to treatment assignment. To demonstrate δ-opioid-initiated

cardioprotection, DPDPE (200 µg/kg) was intravenously infused 30 min before the coronary

artery occlusion (n=9). To determine whether the infarct-size-limiting effect of DPDPE

depended on endogenous epinephrine, the nonselective AR antagonist labetalol (2 mg/kg) was

intravenously infused over 30 min followed by DPDPE at 30 min before the coronary artery

occlusion (n=9). To determine the relative contribution of β2-AR in δ-opioid-initiated infarct-

size-reduction, the effect of DPDPE in the presence of the β2-AR antagonist ICI-118,551 (ICI,1

mg/kg) was tested (n=9). ICI was intravenously infused over 30 min followed by DPDPE

infusion at 30 min before coronary artery occlusion. The effects of saline, labetalol or ICI alone

on infarct size were determined (n= 8/group). The specificity of DPDPE was tested in the

presence of the DOR antagonist naltrindole (NTI, 200 µg/kg, n=5).

In vitro simulated ischemia in ventricular cardiocytes: To confirm the role of ICA cells in δ-

opioid-initiated cardioprotection in vitro, we utilized a simulated ischemia-reperfusion model

(18) of adult rat ICA cell-ventricular myocyte co-culture. This model allowed us to determine

ICA cell-ventricular myocyte interactions in the absence of sympathetic nerve innervation. ICA

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cell-myocyte co-culture was prepared by directly plating the dissociated ventricular cardiocytes

to 24-well plates for 24 hr without undergoing magnetic ICA cell purification. Ischemia was

induced by layering mineral oil (0.5 ml for 2 hr) over a thin film of hypoxic media (pre-bubbled

with N2 gas) covering the cells followed by 60 min of "reperfusion" in normal Tyrode’s solution.

The treatments included 1) saline, 2) labetalol (1 µmol/L), 3) ICI (1 µmol/L), 4) NTI (10

µmol/L), 5) DPDPE (100 nmol/L), 6) labetalol+DPDPE, 7) ICI+ DPDPE, and 8) NTI+DPDPE.

Saline, labetalol, ICI, NTI, and DPDPE were applied individually to the cell culture for 30 min

before ischemia. For drug combination treatment, labetalol, ICI and NTI were added

individually10 min prior to the application of DPDPE. Finally, we tested whether DPDPE

exerted any myocyte protection in the absence of co-cultured ICA cells. The ICA cell-free

myocytes were prepared by depleting the ICA cells from dissociated mixed ventricular

cardiocytes with the magnetic purification method. This ICA cell depletion procedure was

repeated 4 times to ensure maximum effect. Myocyte death was quantified by counting trypan

blue-stained cells and expressed as a percentage of the total cells counted. All experiments (n= 5

rats/group) were carried out at 37°C. The examiner who counted cells was blinded to the

treatment assignment.

Data analysis: For semi-quantification of ICA cells in human LV tissue, ICA cells were defined

as cells displaying TH immunofluorescence. Those cardiocytes with nuclei stained with DAPI

(blue fluorescence) without TH signal were classified as non-ICA cells. The percentage of

ICA/non-ICA cells was counted from 20 consecutive views (100X lens) for each slide. At least 3

slides were examined for each LV sample. Data are presented as mean ± SD. For quantification

of [Ca2+]i transients, the firing frequency of [Ca2+]i spikes of ICA cells was determined based on

an average of the last 5-min duration for the baseline and each drug treatment. The outcome

measure was the number of spikes/min. ANOVA was used for analyzing changes in [Ca2+]i

transients, epinephrine release, and myocardial ischemia studies with the Bonferroni adjustment

for intra-group comparisons. Paired t-test was used to determine the effects of naltrindole and

nifedipine on DPDPE-induced [Ca2+]i changes. The significance level α is 0.05.

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RESULTS

Identification of human ICA cells ICA cells were identified in human hearts (Figure 1). In situ

expression of TH mRNA was identified in ICA cells but not in the sympathetic nerve endings

(Figure 2). ICA cells expressed TH, PNMT, and PGP 9.5 immunoreactivity (Figure 2) and were

distributed diffusely throughout the LV. Perivascular distribution was a common feature.

Abundant ICA cells were also observed in the smooth muscle layer of epicardial coronary artery

(Figure 1E). The density of ICA cells varied considerably constituting as many as 20% to as little

as 0% of total cardiocytes/microscopic view (100X lens) with an average of 7±2%. ICA cells

were identified in the SA (Figure 1G) and AV nodal regions (data not shown).

Identification of DOR in ICA cells DOR immunoreactivity was exclusively co-localized with

TH in human and rat ICA cells (Figures 3 and 4). Ventricular myocytes expressed myosin-

heavy-chain immunoreactivity (Figure 4G). Over 90% of isolated ICA cells co-expressed TH

and DOR immunoreactivity yielding extremely high ICA cell purity. DOR-positive ICA cells

constituted approximately 13±4% of total rat ventricular cardiocytes based on the cell counting.

No DOR immunoreactivity was identified in human and rat ventricular myocytes in situ and in

vitro. No immunoreactivity was detected in IgG control slides. The control slides for double

labeling that was stained with only Streptavidin-Alexa Fluor 488 after step 2 showed no cross-

reactivity to DOR confirming immunohistochemical specificity of DOR expression in ICA cells.

Western blot analysis detected DOR protein in rat ICA cell isolates, which was identical to that

found in rat brain. No DOR protein was detected in ventricular myocytes in the absence of ICA

cells following ICA cell depletion (Figure 4H).

Modulation of [Ca2+]i transients and epinephrine release of rat ICA cells Application of

DPDPE to ICA cells increased [Ca2+]i transient spikes (n=11 cells) in a concentration-dependent

manner (Figure 5A). Increases in [Ca2+]i transients were sometimes preceded by a brief

quiescent phase. DPDPE (100 nmol/L) did not change [Ca2+]i transient activity significantly in

the presence of the DOR antagonist naltrindole (10 µmol/L, n=4, p<0.01). DPDPE-enhanced

[Ca2+]i transients were abolished in the presence of nifedipine (1 µmol/L, n=4, p<0.01). Basal

endogenous epinephrine release was detected from isolated ICA cells in culture. Application of

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DPDPE (100 nmol/L) increased epinephrine release 2.4-fold (p<0.01, n=4 duplicates from 8 rats,

Figure 5B). Norepinephrine was not detected during the baseline, DPDPE’s treatment or during

the recovery phase.

δ-opioid-initiated myocardial and myocyte protection: Intravenous infusion of DPDPE prior to

coronary artery occlusion reduced LV infarct size by 54±15% compared to control (Figure 6A).

Infusion of labetalol prior to ischemia tended to increase (statistically insignificant) the infarct

size compared to control. DPDPE-initiated infarct-size-reduction was markedly attenuated in the

rats pretreated with labetalol. The β2-AR blocker ICI alone significantly increased infarct size

compared to control. The infarct size following DPDPE infusion in the presence of labetalol or

ICI was not significantly different than those treated with saline infusion. There was no

significant difference in body and LV weight among animal groups. There was a small reduction

in arterial blood pressure in the rats following labetalol infusion

In vitro simulated ischemia-reperfusion markedly increased myocyte death compared to the non-

ischemic state (p<0.001). Pretreatment of cardiocytes with DPDPE reduced ischemia-induced

cell death by 26±4% (p<0.001, n=5 rats). In the presence of labetalol, ICI, or NTI, DPDPE no

longer exerted protection on ischemia-induced myocyte death (Figure 6B). In myocytes in the

absence of co-cultured ICA cells, ischemia resulted in 95±2% myocyte death compared to that of

56±3% in ICA cell-ventricular myocyte co-culture (p<0.001). Application of DPDPE prior to the

ischemia exerted no myocyte protection during subsequent ischemia-reperfusion in ventricular

myocytes in the absence of co-cultured ICA cells (Figure 6C). A 48% and 70% increase

(p<0.001) in myocyte death was seen at rest and during ischemia-reperfusion, respectively, in

myocytes without co-cultured ICA cells, compared to those with co-cultured ICA cells.

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DISCUSSION

We have demonstrated DOR expression in ICA cells in human and rat hearts. Consistent with

immunohistochemical findings, the physiological evidence provided herein demonstrates that

[Ca2+]i transients generated by ICA cells can be modified by δ-opioid stimulation. Activation of

ICA cells enhances endogenous epinephrine release and β2-AR stimulation that is essential for

the δ-opioid-initiated myocardial protection against ischemia.

ICA cells in the human heart: Human ventricular ICA cells express TH mRNA that encodes the

rate-limiting enzyme for epinephrine biosynthesis. The abundant ICA cells present in the SA and

AV nodes is consistent with an animal study reporting an intimate anatomic relationship between

ICA cells and cardiac pacemaker and conduction systems (5) suggesting a regulatory role of ICA

cells in impulse generation and conduction in human heart. The presence of ICA cells in

transplanted human LV myocardium (Figure 1H) is consistent with the concept that ICA cells

may provide an alternative adrenergic supply in the compromised sympathetic innervation (8).

This notion has been substantiated by the finding that the epinephrine producing gene PNMT

mRNA is upregulated in ventricular tissue of transplanted human hearts (6).

Cellular origin of DOR: The present study has located DOR immunoreactivity exclusively to the

ICA cells in human and rat hearts. Recently, Patel et al (18) reported DOR immunoreactivity in

the T-tubules of isolated rat ventricular myocytes in culture. However, the lack of corroborative

immunohistochemical evidence of in situ DOR immunoreactivity in rat myocardial tissue section

in that study (18) has made their claim inconclusive. The unequivocal in situ and in vitro

immunohistochemical evidence of DOR expression by ICA cells (Figures 3 and 4) is consistent

with our Western blot analysis demonstrating abundant DOR protein expression by ICA cells but

not myocytes (Figure 4H).

DOR signaling in ICA cells: Ca2+ influx is a fundamental mechanism for neurotransmitter

release from neuroendocrine cells (15). Increase in [Ca2+]i transients in ICA cells following δ-

opioid stimulation was mediated through Ca2+ influx via L-type Ca2+ channels, since the increase

in [Ca2+]i was abolished in the presence nifedipine (Figure 5). The cAMP-PKA pathway is the

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best-characterized mechanism underlying opioid signaling in catecholaminergic neurons in the

locus ceruleus (12). Opioid exposure to these neurons up-regulates cAMP-pathway resulting in

increased Ca2+ influx (12, 16). DOR stimulation also activates cAMP pathway in olfactory bulb

neurons (17) and CHO cell line expressing human DOR (21). PKA phosphorylates L-type Ca2+

channels and enhances Ca2+ influx (26). The dependence of δ-opioid-enhanced [Ca2+]i transients

on L-type Ca2+ channels in ICA cells suggests an involvement of the cAMP-PKA pathway.

Mechanism involved in δ-opioid-initiated cardioprotection: We have demonstrated that δ-opioid-

initiated cardioprotection depends on ICA cells activation. Unlike sympathetic nerve endings that

release norepinephrine (a potent β1-AR agonist with negligible β2-AR effect), ICA cells release

epinephrine that is a potent endogenous β2-AR agonist. This is of particular importance since β2-

AR is essential in mediating cardioprotection against ischemia (14, 25) presumably via its anti-

apoptotic effect (3, 31). The increased infarct size in the presence of β2-AR blockade compared

to saline or labetalol (Figure 6A) is probably due to a fact that selective β2-AR blockade creates

unopposed β1-AR stimulation as a result of sympathetic nervous activation. Myocardial β1-AR

stimulation exerts detrimental proapoptotic effect (3, 31) hence increased infarct size. It appears

that β2-AR blockade did not completely abolish DPDPE-initiated infarct-size-limiting effect in

vivo, although this incompleteness is not statistically significant. Nevertheless, this incomplete

infarct-size-limiting effect is unlikely due to a direct δ-opioid effect on myocytes, as DPDPE-

initiated myocyte protection is lacking in the presence of β2-AR blockade in ICA cell-myocyte

co-culture or in myocytes in the absence of co-cultured ICA cells (Figure 6 B and 6C). It is well

known that systemic δ-opioid infusion elicits complex neuronal responses resulting in secondary

cardiovascular effects via non-adrenergic pathways. For instance, δ-opioid stimulation of sensory

neurons in dorsal root ganglia increases release of calcitonin gene-related peptide (1), a

neuropeptide exerting infarct-size-limiting effect during ischemia-reperfusion (29). It might also

be possible that enhanced epinephrine release from ICA cells might activate β-AR located on the

cardiac afferent nerve endings eliciting cardiocardiac reflex resulting in secondary cardiac effects

(24). Such secondary effects were not present in ICA cell-ventricular myocyte co-culture model

allowing direct assessment of δ-opioid signaling mechanisms in ICA cells. It is interesting to

note that in the absence of co-cultured ICA cells, ~95% myocytes died during ischemia-

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reperfusion, suggesting a crucial role of ICA cells in maintaining myocyte vitality during

hypoxia. This finding is consistent with a murine study that targeted disruption of TH gene

causes lethal cardiomyopathy and fetal demise in the uterus where the hypoxic distress is

frequently encountered (32).

Limitations of the study: We acknowledge several limitations in the present study. The 7% of

ICA cells identified in human LV samples is relatively low compared to 13% in rat LV samples.

Since the human samples were largely derived from diseased hearts with end-stage heart failure,

it might not truly represent the percentage of ICA cells in normal human heart. The cellular

mechanisms underlying δ-opioid regulation of ICA cell [Ca2+]i transients were not explored.

Although DPDPE induces robust epinephrine release from isolated ICA cells in vitro, we cannot

totally exclude the possibility that endogenous epinephrine derived from the sources other than

ICA cells might also contribute, in part, to β2-AR-mediated cardioprotection in vivo. Finally, the

signaling pathways of β2-AR-mediated cardioprotection by epinephrine were not further

characterized. We presumed that pertussis toxin-sensitive Gi pathway was likely involved in β2-

AR-mediated cardioprotection as proposed previously (3, 31).

In conclusion, we have localized DOR expression in ICA cells in human and rat hearts. δ-opioid

stimulation of ICA cells enhances endogenous epinephrine liberation resulting in myocardial β2-

AR-mediated cardioprotection against ischemia. This study has demonstrated a novel δ-opioid

signaling and its cardioprotective mechanism that is dependent on neuroendocrine function of

ICA cells.

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ACKNOWLEDGEMENTS

We acknowledge the support of Gordon Ewy, MD in the early stage of this project. This study

was supported by the John Sealy Memorial Foundation from the University of Texas Medical

Branch.

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FIGURE AND TABLE LEGENDS

Figure 1. Immunoperoxidase (panels A and C) and immunofluorescent (other panels) labeling of

ICA cells in human hearts. ICA cells expressing TH immunoreactivity (red) are distributed

diffusely throughout the LV myocardium. Perivascular location is a frequent feature of ICA

cells. Arrow (C) denotes a terminal arteriole. Panel E demonstrates abundant ICA cells in the

smooth muscle layers of epicardial circumflex coronary artery. TH-expressing sympathetic nerve

fiberss (arrows in D and G) can occasionally be seen in the field. Inserts are magnified ICA cell

images in panels B and D (arrows). TH immunoreactivity (green) was identified in ICA cells and

sympathetic nerve fibers in the SA nodal tissue (G). ICA cells are seen in transplanted human

LV tissue (H). All the scale bars =10 µm except for panel B (20µm).

Figure 2. Expression of TH mRNA and other neuroendocrine markers in human ICA cells. Two

ICA cells (A) that exhibit TH immunoreactivity (green) express TH mRNA (red, B) detected by

in situ hybridization in LV tissue. Inserts are magnified images of ICA cells. Panel C shows TH

immunoreactivity exhibited by a bundle of sympathetic nerve fibers that express no TH mRNA

(D). Panels E and F co-localize the immunoreactivity of TH (green) and PNMT (red) in an ICA

cell. Insert (F) is the overlay of E and F showing the co-localization of TH and PNMT. Panels G

and H co-localize the immunoreactivity of TH (green) and general neuronal marker PGP 9.5

(red) in a cluster of ICA cells. Scale bars =10 µm.

Figure 3. Expression of DOR immunoreactivity in human ICA cells. Immunofluorescent co-

localization of TH (green, A) and DOR immunoreactivity (red, B) in an ICA cell in human LV

tissue. Panel C: superimposed images of A and B exhibiting co-localized TH and DOR

immunoreactivity with TH concentrated on the opposite end. Inserts are magnified images.

Panels D and E show another ICA cell co-expressing immunoreactivity of TH and DOR. Panel F

demonstrates expression of DOR immunoreactivity (brown) by a perivascularly distributed ICA

cell in LV tissue by immunoperoxidase labeling (insert is the magnified cell image). Panel G

shows a TH-expressing sympathetic nerve fiber (green) in LV tissue. Double staining of the

same slide with anti-DOR antibody detects no DOR immunoreactivity in this nerve fiber and

ventricular myocytes (H). Scale bars =10 µm.

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Figure 4. DOR expression in rat and human ICA cells. Immunofluorescent double labeling co-

localizes DOR (red, A) and TH (green, B) immunoreactivity in a cluster of ICA cells in rat

ventricular tissue. Panel C shows dissociated rat ventricular cardiocytes with only ICA cell

(arrow) exhibiting DOR immunoreactivity (green). Myocytes which display myosin-heavy-chain

immunoreactivity (red) express no detectable DOR immunoreactivity. Panel D shows >90%

isolated rat ICA cells expressing DOR immunoreactivity (green). Panels E and F show

immunofluorescent double labeling co-localizes the DOR (red) and TH (green)

immunoreactivity in isolated rat ICA cells. Insert (E) is the overlay of E-F showing co-localized

TH and DOR immunoreactivity in ICA cells. Panel G shows DOR activity (green) in two ICA

cells (arrow) abutted on myocytes expressing myosin-heavy-chain immunoreactivity (red) in

human LV tissue section. Calibration bar =10 µm. Panel H: Western blot analysis (repeated

twice) detects DOR protein in rat ICA cell isolates (lane 1) and brain tissue (lane 2) but not in

ventricular myocytes in the absence ICA cells (lane 3). Protein loading was 25 µg/lane. The

equivalency of protein loading was verified by the levels of β-actin.

Figure 5. Panel A: Modulation of [Ca2+]i transients generated by ICA cells by DPDPE. Bar

graph: DPDPE elicits a concentration-dependent increase (y-axis in fold) in [Ca2+]i transients

(n=11 cells). Top tracing shows that DPDPE (100 nmol/L) increases [Ca2+]i transients in an ICA

cell in culture. Middle tracing shows that increase in [Ca2+]i transients elicited by DPDPE is

abolished in the presence of nifedipine. Bottom tracing shows that DPDPE fails to increase

[Ca2+]i transients in the presence of the selective DOR antagonist naltrindole (NTI, 10 µmol/L).

Vertical scale bars represent 340/380 [Ca2+]i ratio. Panel B: DPDPE enhances epinephrine

release from ICA cells in culture. Basal epinephrine release at 1 hr is not different between the

two groups before the treatment of DPDPE or vehicle. Application of DPDPE (+DPDPE, 100

nmol/L for 30 min) to ICA cells increases epinephrine release by 2.4 fold (n=4 duplicates from 8

rats). There is no increase in epinephrine release when ICA cells are exposed to vehicle solution

(-DPDPE, n=4 duplicates from 8 rats). Enhanced epinephrine release persists during recovery

phase after the removal of DPDPE. **: p<0.01, *: p<0.05.

Figure 6. Panel A: Effects of DPDPE (200 µg/kg) on infarct-size-reduction in the absence and

presence of labetalol (Lab, 2 mg/kg), ICI-118,551 (ICI, 1 mg/kg), or NTI (200 µg/kg) in vivo.

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Effects of labetalol, ICI, or saline (S) on infarct size are presented. Infarct size in DPDPE group

is markedly smaller that those of all other groups (p<0.001). Panel B: Effects of DPDPE (100

nmol/L) on ischemia-reperfusion-induced ventricular myocyte death in the absence and presence

of labetalol (1 µmol/L), ICI (1 µmol/L), or NTI (10 µmol/L) in ICA cell-ventricular myocyte co-

culture (n= 5/group). The myocyte death in ischemic group is significantly higher than that of

nonischemic one (p<0.001). Among ischemic groups, the myocyte death in DPDPE group is

significantly lower than those of all other groups (p<0.001). Panel C: Effects of DPDPE on

ischemia-reperfusion-induced cell death in ventricular myocytes in the absence of co-cultured

ICA cells (n=5/group). *: p<0.05, **: p<0.01, ***: p<0.001, ns: nonsignificant.

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C

A

D

F

G H

B

E

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A B

C D

E F

G H

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A

B

C F

D

E

G H

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A B

C D

E F1 2 3

DOR

H

β-actin

G

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Nifedipine

DPDPE

DPDPE NTI

DPDPE

10 min

Baseline ± DPDPE Recovery

A

B

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**ICI vs. (Lab+DPDPE)

***ICI vs. (ICI+DPDPE)

**ICI vs. Saline

PIntra-group

**Lab vs. ICI+DPDPE

*ICI vs. NTI

*ICI vs. Lab

*Saline vs.(NTI+DPDPE)

**Saline vs. (ICI+DPDPE)

*Saline vs. Lab

***Saline vs. ICI

PIntra-group

Panel B statistics

Panel A statistics

*** ns

***

A

B

C

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