Articles in PresS. Am J Physiol Heart Circ Physiol (March 27, 2015). doi:10.1152/ajpheart.00850.2014
1 2
Full Title: Interleukin-6 mediates exercise preconditioning against myocardial ischemia reperfusion injury
3
Short title: Interleukin-6 and exercise preconditioning
4 5 6
Authors: 1Graham Ripley McGinnis Ph.D., 1Christopher Ballmann M.Ed., 1Bridget Peters M.Ed., 2Gayani Nanayakkara Ph.D., 3Michael Roberts Ph.D., 2Rajesh Amin Ph.D., 1John C. Quindry Ph.D.
7 8 9 10
1
Auburn University School of Kinesiology, Cardioprotection Laboratory
2
Auburn University Harrison School of Pharmacy, Department of Drug Discovery and Development 3
Auburn University School of Kinesiology, Molecular and Applied Sciences Laboratory
11 12 13 14 15
Corresponding Author:
16
John C. Quindry, PhD, FACSM
17 18 19 20 21
Cardioprotection Laboratory School of Kinesiology Auburn University Auburn, AL 36830 Phone (334) 844-1421; fax (334) 844-1467
22
Email: [email protected]
23 24
1
Copyright © 2015 by the American Physiological Society.
25
Abstract:
26
Interleukin-6 (IL-6) is a pleiotropic cytokine that protects against cardiac ischemia-reperfusion
27
(IR) injury following pharmacologic and ischemic preconditioning (IPC), but the affiliated role
28
in exercise preconditioning is unknown. Our study purpose was to characterize exercise induced
29
IL-6 cardiac signaling (Aim 1) and evaluate myocardial preconditioning (Aim 2). In aim 1, C57
30
and IL-6-/- mice underwent 3 days of treadmill exercise for 60 min*day-1 at 18 m*min-1. Serum,
31
gastrocnemius and heart were collected PRE, POST, 30, and 60 minutes following the final
32
exercise session and analyzed for indices of IL-6 signaling. For Aim 2, a separate cohort of
33
exercise preconditioned (C57 EX and IL-6-/- EX) and sedentary (C57 SED and IL-6-/- SED) mice
34
received surgical IR injury (30 minutes I, 120 minutes R) or a time matched sham operation
35
(SH). Ischemic and perfused tissues were examined for necrosis, apoptosis and autophagy. In
36
Aim 1, serum IL-6 and IL-6R, gastrocnemius and myocardial IL-6R were increased following
37
exercise in C57 mice only. p-STAT3 was increased in gastrocnemius and heart in C57 and IL-6-/-
38
mice post exercise, while myocardial iNOS and COX-2 were unchanged in the exercised
39
myocardium. Exercise protected C57 EX mice against IR induced arrhythmias and necrosis,
40
while arrhythmia score and infarct outcomes were higher in C57 SED, IL-6-/- SED and IL-6-/- EX
41
mice as compared to SH. C57 EX mice expressed increased p-p44/42 MAPK (Thr202/Tyr204)
42
and p-p38 MAPK (Thr180/Tyr182) compared to IL-6-/- EX mice, suggesting pathway
43
involvement in exercise preconditioning. Findings indicate exercise exerts cardioprotection via
44
IL-6, and strongly implicates protective signaling originating from the exercised skeletal muscle.
45
Key Words: Cardioprotection, Exercise, Myokine, Myocardial Infarction
46 47
Introduction:
2
48
Cardiovascular disease (CVD) remains a leading cause of morbidity in the US and most
49
predominately manifests as the myocardial infarction, or ischemia reperfusion (IR) injury (57).
50
Clinical outcomes subsequent to IR accrue in a time-dependent fashion(6, 27, 44) beginning with
51
ventricular arrhythmias and rapidly proceeding to irreversible cell death by apoptosis and
52
necrosis (7). Autophagic processes also determine cellular fates during myocardial IR injury (17,
53
20, 21). Clinical countermeasures to the cellular pathology that underpin IR require novel
54
understanding of robust and sustainable approaches to cardiac preconditioning via
55
pharmacologic and lifestyle countermeasures. Over the last 30 years ischemic preconditioning
56
(IPC) (46) has been the primary investigative avenue to uncover viable countermeasures to IR
57
injury. Exercise has emerged in recent years as an alternative scientific model that exhibits
58
mechanistic differences from IPC (15, 53), yet is just as effective as a cardioprotective stimulus.
59
From a phenomenological perspective exercise preconditioning provides a more sustained
60
window of protection than IPC (36), and protects the aged myocardium (52) whereas IPC does
61
not (58). Understanding the endogenous mechanisms of exercise-induced cardioprotection has
62
been the subject of intense inquiry in recent years (10, 15, 24, 25, 29, 37, 50, 55), yet remains
63
incompletely understood. Several essential mediators to the exercised heart include endogenous
64
antioxidants (16, 24, 25, 51), and ATP-sensitive potassium channels (KATP channels) (5, 55, 56).
65
Recent findings suggest that exercise induced preconditioning is triggered by receptor mediated
66
events including cardiac production of endogenous opioid compounds (11, 42). Given recent
67
interest in acute exercise and skeletal muscle derived “myokines,” there is a rationale to believe
68
exercise preconditioning may occur through paracrine and endocrine like processes originating
69
in the exercised muscle, though this process remains untested (39, 47-49, 61). In the context of
70
exercise, interleukin 6 (IL-6) is perhaps the most notable myokine with cardioprotective potential
3
71
(30, 49, 61), and has been implicated as an essential trigger of IPC (9, 40, 59). Just as intriguing
72
as IPC, and perhaps with more immediate clinical ramifications, remote preconditioning of the
73
heart is an established physiologic response to intermittent ischemia of limb skeletal muscle (8).
74
Given that muscle derived compounds are cardioprotective against myocardial infarction (4, 31),
75
there is reason to suspect that a similar phenomenon may occur due to exercise.
76
Mechanistic studies of IL-6 induced cardioprotection have been carried out both in vivo (9) and
77
in vitro (59), pharmacologically and via IPC. IPC induced signaling through the Janus kinase-
78
signal transducer and activator of transcription (JAK/STAT) pathway reduced necrosis following
79
30 minute regional ischemia (9). The infarct sparing effects were abrogated in IL-6-/- mice,
80
demonstrating an essential role for IL-6 in IPC (9). Bolli, et al. developed a sophisticated mouse
81
model with a cardiac specific, inducible STAT3 deletion and demonstrated the importance of
82
STAT3 signaling in IPC (3). Smart et al. further demonstrated IL-6 induced cardioprotection
83
against IR injury in vitro. They observed a reduction in cellular damage, preservation of cell
84
viability, and Ca2+ homeostasis following IR (59). The protection afforded to the cardiomyocytes
85
was nitric oxide (NO) and phosphotidyl-inositol 3-kinase (PI3-K) dependent, and was abolished
86
by iNOS and PI3-K inhibitors (59).
87
This collective understanding serves as the scientific rationale for the postulate that exercise
88
induced increases in circulating IL-6, largely attributed to increased synthesis and release from
89
exercising skeletal muscle, is sufficient to activate protective pathways in the myocardium.
90
Therefore, the purpose of this study was to investigate the acute post-exercise period of IL-6
91
signaling in the heart, and to study the role of IL-6 in exercise induced cardioprotection against
92
IR injury using an IL-6 knockout mouse model. In that regard, it was hypothesized that exercise
4
93
will increase IL-6 signaling in the heart and decrease IR induced apoptotic and necrotic tissue
94
death in an IL-6 dependent fashion.
5
95
Methods:
96
Animals
97
Male mice (56 C57, C57BL/6J and 48 IL-6-/- from the same background strain, B6;129S2-
98
Il6tm1Kopf/J) were used to complete the two aims of this study. Auburn University IACUC
99
approval was granted prior to the start of the investigation and in accordance with National
100
Institutes of Health guidelines for the care and use of laboratory animals. Animals were housed
101
at the Auburn University Biological Research Facility on a 12:12 reversed light:dark cycle with
102
access to water and rodent chow ad libitum. In Aim 1, 64 mice (C57, n = 32 and IL-6-/-, n = 32)
103
were assigned to exercise or sedentary treatments to evaluate the acute myokine response to
104
exercise in the blood, skeletal muscle and heart (Figure 1). Exercised mice were habituated to
105
treadmill exercise for 10, 20, 30, and 40 minutes on consecutive days, followed by a rest day and
106
then performed the 3 day treatment of exercise for 60 min●day-1, at 18 m●min-1 and 0% grade.
107
Exercise was performed on a calibrated motorized rodent treadmill (Columbus Instruments,
108
Columbus, OH) and sedentary mice, used as pre-exercise controls, spent a time matched duration
109
on the treadmill at 0 m●min-1. Under isofluorane anesthesia, tissue was collected from sedentary
110
mice (PRE) and exercised mice immediately post (POST), 30 minutes (30), or 60 minutes post
111
exercise (60). Blood serum, gastrocnemius, soleus, extensor digitorum longus (EDL), and hearts
112
were snap frozen for subsequent analysis. Mice in Aim 2 (C57 n = 24 and IL-6-/- n = 16)
113
followed an identical habituation and preconditioning exercise protocol and received an in vivo
114
ischemia reperfusion injury 24 hours following the final exercise session.
115
in vivo Ischemia Reperfusion Injury
116
Twenty-four hours following the final exercise session, mice received surgically induced IR
117
injury or a time matched sham operation. Mice were anesthetized using sodium pentobarbital (50
6
118
mg/kg) and a tracheotomy and left thoracotomy were performed. Mice were supported with a
119
pressure driven mechanical ventilator (Kent Scientific, Torrington, CT), and connected to limb
120
lead electrodes integrated into a physiological data acquisition system (Biopac, Santa Barbara,
121
CA) in order to record ECG activity. The left anterior descending (LAD) coronary artery was
122
occluded with a sterile surgical suture passed through polyethylene tubing, creating a reversible
123
ligature. Regional ischemia was administered for 30 minutes followed by 120 minutes
124
reperfusion. At the conclusion of IR, the ligature was re-established and 4% Evan’s Blue dye
125
was injected via left ventricular cardiac puncture allowing for visualization of the area at risk
126
(AAR). An additional group of C57 mice (n=8) received a time equivalent sham operation.
127
Electrocardiogram data were collected and analyzed for ventricular arrhythmias using Biopac
128
software and evaluated using the A Score method(43), a scoring system designed to categorize
129
severity of IR based on the incidence of premature ventricular contractions (PVC’s), episodes
130
and duration of ventricular fibrillation (VF) and tachycardia (VT), and accounting for mortality.
131
To assess myocardial necrosis, hearts were excised, sectioned into 2 mm transverse cross
132
sections and incubated with 1% triphenol-tetrazolium chloride (TTC) for 15 minutes at 37ºC. To
133
assess apoptosis and autophagy, the ischemic and perfused areas of the myocardium were
134
separated and snap frozen in liquid nitrogen for Western Blotting and PathScan analysis.
135
Serum IL-6 ELISA
136
Serum IL-6 was quantified using a commercially available ELISA (Invitrogen KMC0062)
137
following manufacturer protocol. Briefly, blood from Aim 1 was collected via direct cardiac
138
puncture and allowed to clot. Samples were centrifuged for 10 minutes at 10,000 x g at 4ºC and
139
stored at -80ºC until analysis. Serum samples were run in duplicate with the absorbance read at
140
450 nm and plotted against a standard curve of mouse IL-6 (r2 = 0.998).
7
141
PCR
142
Approximately 20 mg of myocardial, gastrocnemius, soleus, and EDL tissue was used for RNA
143
isolation using 500 μL Ribozol® RNA Extraction Reagent (Amresco N580), per manufacturer
144
instructions. RNA was precipitated with 500 μL Isopropanol with 0.5 μL Glycogen, ethanol
145
washed, and reconstituted with DEPC H2O. A 3% agarose gel was run to verify extracted RNA
146
purity. Isolated RNA (1 μg) was converted to cDNA using a Verso cDNA kit (ThermoScientific
147
AB-1453/B) with a reverse transcription cycle of 30 minutes at 42ºC. The resulting cDNA (50
148
ng/μL) was diluted to a final concentration of 5 ng/μL and stored at -20ºC. Primer efficiency
149
curves were run to select optimum cDNA concentrations and ensure single amplification
150
products. PerfeCTa® SYBR Green Super Mix (Quanta BioSciences 95054) was combined with
151
forward and reverse primers (Table 1) and 25 ng cDNA. qPCR conditions were 95ºC for 2
152
minutes, followed by 35 cycles of denaturing at 95ºC for 30 seconds, re-annealing at 62ºC for 30
153
seconds, and extension at 72ºC for 5 minutes. Relative mRNA expression was calculated using
154
the ΔΔCt method for n=7-9 observations/group.
155
Western Blotting
156
Serum, gastrocnemius and heart (Aim 1) samples were homogenized in lysis buffer with
157
phosphatase (GBiosciences Phosphatase Arrest II, 1:100) and protease (Sigma #P2714, 1:10)
158
inhibitors to acquire a whole muscle homogenate. Cytosolic and nuclear fractions were obtained
159
using a nuclear isolation kit (ThermoScientific, Waltham, MA) following manufacturer
160
instructions. Ischemic and perfused regions of hearts (Aim 2) were homogenized in PathScan®
161
Sandwich ELISA Lysis buffer containing protease and phosphatase inhibitors. Samples were
162
normalized for protein concentration, diluted with Laemmli sample buffer with 2-
163
mercaptoethanol, and heated at 95ºC for 5 minutes.
8
164
Proteins were separated on 6% (iNOS), 10% (IL-6R, COX-2, [P]-STAT3, [P]-Akt, Atg3, Atg5,
165
Beclin 1) or 18% (LC3II/I) polyacrylamide gels and transferred onto methanol activated PVDF
166
membranes. Membranes were exposed to primary antibodies (IL-6R, Santa Cruz #374259
167
1:1000 in 5% NFDM; P-STAT3, Cell Signaling #9145; STAT3, Cell Signaling #4904; P-Akt,
168
Cell Signaling #9271; Akt, Cell Signaling #9272; iNOS, Cell Signaling #13120; COX-2, Cell
169
Signaling #12282; Atg3, Cell Signaling #3415; Atg5, Cell Signaling #12994; Beclin 1, Cell
170
Signaling #3495; LC3II/I, Cell Signaling #12741, 1:1000 in 5% BSA, and αTubulin,
171
Developmental Studies Hybridoma Bank #12G10, 1:1000 in 5% NFDM) overnight at 4ºC.
172
Membranes were incubated in mouse or rabbit targeted secondary antibodies (Cell Signaling
173
#7076 and #7074, respectively, 1:2000 in BSA) for 1 hour at RT, and blots imaged with
174
Luminata Forte HRP substrate (Millipore). Images were captured with a ChemiDoc-It® Imager
175
(UVP, Upland, CA) and analyzed using NIH ImageJ software.
176
PathScan
177
PathScan slides were assembled in gaskets and blocked for 15 minutes at RT. Ischemic and
178
perfused homogenates, diluted to 1.0 mg*mL-1, were added to wells and incubated overnight at
179
4ºC with rocking. Wells were incubated with a detection antibody followed by HRP-linked
180
Streptavidin with four 5 minute washes separating each step. Slides were exposed with
181
LumiGLO®/Peroxide substrate and quantified using UVP software, and normalized to the total
182
intensity of each sample.
183
Statistical Analysis
184
All values are presented as means ± standard error (SEM). A 2 (genotype) x 4 (time) ANOVA
185
was used to analyze effects of genotype and exercise on mRNA and protein expression following
186
exercise. Arrhythmia scores were categorized using a non-parametric scoring method, and were
9
187
compared using a Kruskal-Wallis test for non-parametric data. Repeated measures ANOVA was
188
used to analyze group differences in AAR, infarct size (necrosis), and apoptosis with Tukey’s
189
HSD post hoc analysis to evaluate significant differences when appropriate. Significance was set
190
a priori at p ≤ 0.05.
191
10
192
Results:
193
Aim 1
194
Serum exercise response
195
Consistent with previous findings, serum IL-6 was increased approximately 4.5 fold 30 minutes
196
post-exercise (from 1.7±0.4 pg/L to 7.5±2.4 pg/L, Figure 2). Exercise increased the soluble form
197
of the IL-6 receptor (sIL-6R) in serum 4.5±0.4, 4.5±0.8, and 5.0±0.6 fold at POST, 30, and 60,
198
respectively, in C57 mice only (interaction effect, p=0.003). Pre exercise sIL-6R levels were
199
similar between mouse strains, but significantly higher in C57 mice compared to IL-6-/- mice at
200
all sample times post exercise (Figure 3).
201
Gastrocnemius exercise response
202
Western blotting and RT-PCR was used to examine the expression of proteins associated with
203
IL-6 signaling in response to the acute exercise stimulus in gastrocnemius and the myocardium.
204
Mixed gastrocnemius was assayed as an index of skeletal muscle IL-6 signaling (Figure 4).
205
Exercise elicited an increase in gastrocnemius IL-6R expression (strain main effect, p
1 2
Full Title: Interleukin-6 mediates exercise preconditioning against myocardial ischemia reperfusion injury
3
Short title: Interleukin-6 and exercise preconditioning
4 5 6
Authors: 1Graham Ripley McGinnis Ph.D., 1Christopher Ballmann M.Ed., 1Bridget Peters M.Ed., 2Gayani Nanayakkara Ph.D., 3Michael Roberts Ph.D., 2Rajesh Amin Ph.D., 1John C. Quindry Ph.D.
7 8 9 10
1
Auburn University School of Kinesiology, Cardioprotection Laboratory
2
Auburn University Harrison School of Pharmacy, Department of Drug Discovery and Development 3
Auburn University School of Kinesiology, Molecular and Applied Sciences Laboratory
11 12 13 14 15
Corresponding Author:
16
John C. Quindry, PhD, FACSM
17 18 19 20 21
Cardioprotection Laboratory School of Kinesiology Auburn University Auburn, AL 36830 Phone (334) 844-1421; fax (334) 844-1467
22
Email: [email protected]
23 24
1
Copyright © 2015 by the American Physiological Society.
25
Abstract:
26
Interleukin-6 (IL-6) is a pleiotropic cytokine that protects against cardiac ischemia-reperfusion
27
(IR) injury following pharmacologic and ischemic preconditioning (IPC), but the affiliated role
28
in exercise preconditioning is unknown. Our study purpose was to characterize exercise induced
29
IL-6 cardiac signaling (Aim 1) and evaluate myocardial preconditioning (Aim 2). In aim 1, C57
30
and IL-6-/- mice underwent 3 days of treadmill exercise for 60 min*day-1 at 18 m*min-1. Serum,
31
gastrocnemius and heart were collected PRE, POST, 30, and 60 minutes following the final
32
exercise session and analyzed for indices of IL-6 signaling. For Aim 2, a separate cohort of
33
exercise preconditioned (C57 EX and IL-6-/- EX) and sedentary (C57 SED and IL-6-/- SED) mice
34
received surgical IR injury (30 minutes I, 120 minutes R) or a time matched sham operation
35
(SH). Ischemic and perfused tissues were examined for necrosis, apoptosis and autophagy. In
36
Aim 1, serum IL-6 and IL-6R, gastrocnemius and myocardial IL-6R were increased following
37
exercise in C57 mice only. p-STAT3 was increased in gastrocnemius and heart in C57 and IL-6-/-
38
mice post exercise, while myocardial iNOS and COX-2 were unchanged in the exercised
39
myocardium. Exercise protected C57 EX mice against IR induced arrhythmias and necrosis,
40
while arrhythmia score and infarct outcomes were higher in C57 SED, IL-6-/- SED and IL-6-/- EX
41
mice as compared to SH. C57 EX mice expressed increased p-p44/42 MAPK (Thr202/Tyr204)
42
and p-p38 MAPK (Thr180/Tyr182) compared to IL-6-/- EX mice, suggesting pathway
43
involvement in exercise preconditioning. Findings indicate exercise exerts cardioprotection via
44
IL-6, and strongly implicates protective signaling originating from the exercised skeletal muscle.
45
Key Words: Cardioprotection, Exercise, Myokine, Myocardial Infarction
46 47
Introduction:
2
48
Cardiovascular disease (CVD) remains a leading cause of morbidity in the US and most
49
predominately manifests as the myocardial infarction, or ischemia reperfusion (IR) injury (57).
50
Clinical outcomes subsequent to IR accrue in a time-dependent fashion(6, 27, 44) beginning with
51
ventricular arrhythmias and rapidly proceeding to irreversible cell death by apoptosis and
52
necrosis (7). Autophagic processes also determine cellular fates during myocardial IR injury (17,
53
20, 21). Clinical countermeasures to the cellular pathology that underpin IR require novel
54
understanding of robust and sustainable approaches to cardiac preconditioning via
55
pharmacologic and lifestyle countermeasures. Over the last 30 years ischemic preconditioning
56
(IPC) (46) has been the primary investigative avenue to uncover viable countermeasures to IR
57
injury. Exercise has emerged in recent years as an alternative scientific model that exhibits
58
mechanistic differences from IPC (15, 53), yet is just as effective as a cardioprotective stimulus.
59
From a phenomenological perspective exercise preconditioning provides a more sustained
60
window of protection than IPC (36), and protects the aged myocardium (52) whereas IPC does
61
not (58). Understanding the endogenous mechanisms of exercise-induced cardioprotection has
62
been the subject of intense inquiry in recent years (10, 15, 24, 25, 29, 37, 50, 55), yet remains
63
incompletely understood. Several essential mediators to the exercised heart include endogenous
64
antioxidants (16, 24, 25, 51), and ATP-sensitive potassium channels (KATP channels) (5, 55, 56).
65
Recent findings suggest that exercise induced preconditioning is triggered by receptor mediated
66
events including cardiac production of endogenous opioid compounds (11, 42). Given recent
67
interest in acute exercise and skeletal muscle derived “myokines,” there is a rationale to believe
68
exercise preconditioning may occur through paracrine and endocrine like processes originating
69
in the exercised muscle, though this process remains untested (39, 47-49, 61). In the context of
70
exercise, interleukin 6 (IL-6) is perhaps the most notable myokine with cardioprotective potential
3
71
(30, 49, 61), and has been implicated as an essential trigger of IPC (9, 40, 59). Just as intriguing
72
as IPC, and perhaps with more immediate clinical ramifications, remote preconditioning of the
73
heart is an established physiologic response to intermittent ischemia of limb skeletal muscle (8).
74
Given that muscle derived compounds are cardioprotective against myocardial infarction (4, 31),
75
there is reason to suspect that a similar phenomenon may occur due to exercise.
76
Mechanistic studies of IL-6 induced cardioprotection have been carried out both in vivo (9) and
77
in vitro (59), pharmacologically and via IPC. IPC induced signaling through the Janus kinase-
78
signal transducer and activator of transcription (JAK/STAT) pathway reduced necrosis following
79
30 minute regional ischemia (9). The infarct sparing effects were abrogated in IL-6-/- mice,
80
demonstrating an essential role for IL-6 in IPC (9). Bolli, et al. developed a sophisticated mouse
81
model with a cardiac specific, inducible STAT3 deletion and demonstrated the importance of
82
STAT3 signaling in IPC (3). Smart et al. further demonstrated IL-6 induced cardioprotection
83
against IR injury in vitro. They observed a reduction in cellular damage, preservation of cell
84
viability, and Ca2+ homeostasis following IR (59). The protection afforded to the cardiomyocytes
85
was nitric oxide (NO) and phosphotidyl-inositol 3-kinase (PI3-K) dependent, and was abolished
86
by iNOS and PI3-K inhibitors (59).
87
This collective understanding serves as the scientific rationale for the postulate that exercise
88
induced increases in circulating IL-6, largely attributed to increased synthesis and release from
89
exercising skeletal muscle, is sufficient to activate protective pathways in the myocardium.
90
Therefore, the purpose of this study was to investigate the acute post-exercise period of IL-6
91
signaling in the heart, and to study the role of IL-6 in exercise induced cardioprotection against
92
IR injury using an IL-6 knockout mouse model. In that regard, it was hypothesized that exercise
4
93
will increase IL-6 signaling in the heart and decrease IR induced apoptotic and necrotic tissue
94
death in an IL-6 dependent fashion.
5
95
Methods:
96
Animals
97
Male mice (56 C57, C57BL/6J and 48 IL-6-/- from the same background strain, B6;129S2-
98
Il6tm1Kopf/J) were used to complete the two aims of this study. Auburn University IACUC
99
approval was granted prior to the start of the investigation and in accordance with National
100
Institutes of Health guidelines for the care and use of laboratory animals. Animals were housed
101
at the Auburn University Biological Research Facility on a 12:12 reversed light:dark cycle with
102
access to water and rodent chow ad libitum. In Aim 1, 64 mice (C57, n = 32 and IL-6-/-, n = 32)
103
were assigned to exercise or sedentary treatments to evaluate the acute myokine response to
104
exercise in the blood, skeletal muscle and heart (Figure 1). Exercised mice were habituated to
105
treadmill exercise for 10, 20, 30, and 40 minutes on consecutive days, followed by a rest day and
106
then performed the 3 day treatment of exercise for 60 min●day-1, at 18 m●min-1 and 0% grade.
107
Exercise was performed on a calibrated motorized rodent treadmill (Columbus Instruments,
108
Columbus, OH) and sedentary mice, used as pre-exercise controls, spent a time matched duration
109
on the treadmill at 0 m●min-1. Under isofluorane anesthesia, tissue was collected from sedentary
110
mice (PRE) and exercised mice immediately post (POST), 30 minutes (30), or 60 minutes post
111
exercise (60). Blood serum, gastrocnemius, soleus, extensor digitorum longus (EDL), and hearts
112
were snap frozen for subsequent analysis. Mice in Aim 2 (C57 n = 24 and IL-6-/- n = 16)
113
followed an identical habituation and preconditioning exercise protocol and received an in vivo
114
ischemia reperfusion injury 24 hours following the final exercise session.
115
in vivo Ischemia Reperfusion Injury
116
Twenty-four hours following the final exercise session, mice received surgically induced IR
117
injury or a time matched sham operation. Mice were anesthetized using sodium pentobarbital (50
6
118
mg/kg) and a tracheotomy and left thoracotomy were performed. Mice were supported with a
119
pressure driven mechanical ventilator (Kent Scientific, Torrington, CT), and connected to limb
120
lead electrodes integrated into a physiological data acquisition system (Biopac, Santa Barbara,
121
CA) in order to record ECG activity. The left anterior descending (LAD) coronary artery was
122
occluded with a sterile surgical suture passed through polyethylene tubing, creating a reversible
123
ligature. Regional ischemia was administered for 30 minutes followed by 120 minutes
124
reperfusion. At the conclusion of IR, the ligature was re-established and 4% Evan’s Blue dye
125
was injected via left ventricular cardiac puncture allowing for visualization of the area at risk
126
(AAR). An additional group of C57 mice (n=8) received a time equivalent sham operation.
127
Electrocardiogram data were collected and analyzed for ventricular arrhythmias using Biopac
128
software and evaluated using the A Score method(43), a scoring system designed to categorize
129
severity of IR based on the incidence of premature ventricular contractions (PVC’s), episodes
130
and duration of ventricular fibrillation (VF) and tachycardia (VT), and accounting for mortality.
131
To assess myocardial necrosis, hearts were excised, sectioned into 2 mm transverse cross
132
sections and incubated with 1% triphenol-tetrazolium chloride (TTC) for 15 minutes at 37ºC. To
133
assess apoptosis and autophagy, the ischemic and perfused areas of the myocardium were
134
separated and snap frozen in liquid nitrogen for Western Blotting and PathScan analysis.
135
Serum IL-6 ELISA
136
Serum IL-6 was quantified using a commercially available ELISA (Invitrogen KMC0062)
137
following manufacturer protocol. Briefly, blood from Aim 1 was collected via direct cardiac
138
puncture and allowed to clot. Samples were centrifuged for 10 minutes at 10,000 x g at 4ºC and
139
stored at -80ºC until analysis. Serum samples were run in duplicate with the absorbance read at
140
450 nm and plotted against a standard curve of mouse IL-6 (r2 = 0.998).
7
141
PCR
142
Approximately 20 mg of myocardial, gastrocnemius, soleus, and EDL tissue was used for RNA
143
isolation using 500 μL Ribozol® RNA Extraction Reagent (Amresco N580), per manufacturer
144
instructions. RNA was precipitated with 500 μL Isopropanol with 0.5 μL Glycogen, ethanol
145
washed, and reconstituted with DEPC H2O. A 3% agarose gel was run to verify extracted RNA
146
purity. Isolated RNA (1 μg) was converted to cDNA using a Verso cDNA kit (ThermoScientific
147
AB-1453/B) with a reverse transcription cycle of 30 minutes at 42ºC. The resulting cDNA (50
148
ng/μL) was diluted to a final concentration of 5 ng/μL and stored at -20ºC. Primer efficiency
149
curves were run to select optimum cDNA concentrations and ensure single amplification
150
products. PerfeCTa® SYBR Green Super Mix (Quanta BioSciences 95054) was combined with
151
forward and reverse primers (Table 1) and 25 ng cDNA. qPCR conditions were 95ºC for 2
152
minutes, followed by 35 cycles of denaturing at 95ºC for 30 seconds, re-annealing at 62ºC for 30
153
seconds, and extension at 72ºC for 5 minutes. Relative mRNA expression was calculated using
154
the ΔΔCt method for n=7-9 observations/group.
155
Western Blotting
156
Serum, gastrocnemius and heart (Aim 1) samples were homogenized in lysis buffer with
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phosphatase (GBiosciences Phosphatase Arrest II, 1:100) and protease (Sigma #P2714, 1:10)
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inhibitors to acquire a whole muscle homogenate. Cytosolic and nuclear fractions were obtained
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using a nuclear isolation kit (ThermoScientific, Waltham, MA) following manufacturer
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instructions. Ischemic and perfused regions of hearts (Aim 2) were homogenized in PathScan®
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Sandwich ELISA Lysis buffer containing protease and phosphatase inhibitors. Samples were
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normalized for protein concentration, diluted with Laemmli sample buffer with 2-
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mercaptoethanol, and heated at 95ºC for 5 minutes.
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Proteins were separated on 6% (iNOS), 10% (IL-6R, COX-2, [P]-STAT3, [P]-Akt, Atg3, Atg5,
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Beclin 1) or 18% (LC3II/I) polyacrylamide gels and transferred onto methanol activated PVDF
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membranes. Membranes were exposed to primary antibodies (IL-6R, Santa Cruz #374259
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1:1000 in 5% NFDM; P-STAT3, Cell Signaling #9145; STAT3, Cell Signaling #4904; P-Akt,
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Cell Signaling #9271; Akt, Cell Signaling #9272; iNOS, Cell Signaling #13120; COX-2, Cell
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Signaling #12282; Atg3, Cell Signaling #3415; Atg5, Cell Signaling #12994; Beclin 1, Cell
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Signaling #3495; LC3II/I, Cell Signaling #12741, 1:1000 in 5% BSA, and αTubulin,
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Developmental Studies Hybridoma Bank #12G10, 1:1000 in 5% NFDM) overnight at 4ºC.
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Membranes were incubated in mouse or rabbit targeted secondary antibodies (Cell Signaling
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#7076 and #7074, respectively, 1:2000 in BSA) for 1 hour at RT, and blots imaged with
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Luminata Forte HRP substrate (Millipore). Images were captured with a ChemiDoc-It® Imager
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(UVP, Upland, CA) and analyzed using NIH ImageJ software.
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PathScan
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PathScan slides were assembled in gaskets and blocked for 15 minutes at RT. Ischemic and
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perfused homogenates, diluted to 1.0 mg*mL-1, were added to wells and incubated overnight at
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4ºC with rocking. Wells were incubated with a detection antibody followed by HRP-linked
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Streptavidin with four 5 minute washes separating each step. Slides were exposed with
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LumiGLO®/Peroxide substrate and quantified using UVP software, and normalized to the total
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intensity of each sample.
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Statistical Analysis
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All values are presented as means ± standard error (SEM). A 2 (genotype) x 4 (time) ANOVA
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was used to analyze effects of genotype and exercise on mRNA and protein expression following
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exercise. Arrhythmia scores were categorized using a non-parametric scoring method, and were
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compared using a Kruskal-Wallis test for non-parametric data. Repeated measures ANOVA was
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used to analyze group differences in AAR, infarct size (necrosis), and apoptosis with Tukey’s
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HSD post hoc analysis to evaluate significant differences when appropriate. Significance was set
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a priori at p ≤ 0.05.
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Results:
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Aim 1
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Serum exercise response
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Consistent with previous findings, serum IL-6 was increased approximately 4.5 fold 30 minutes
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post-exercise (from 1.7±0.4 pg/L to 7.5±2.4 pg/L, Figure 2). Exercise increased the soluble form
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of the IL-6 receptor (sIL-6R) in serum 4.5±0.4, 4.5±0.8, and 5.0±0.6 fold at POST, 30, and 60,
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respectively, in C57 mice only (interaction effect, p=0.003). Pre exercise sIL-6R levels were
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similar between mouse strains, but significantly higher in C57 mice compared to IL-6-/- mice at
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all sample times post exercise (Figure 3).
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Gastrocnemius exercise response
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Western blotting and RT-PCR was used to examine the expression of proteins associated with
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IL-6 signaling in response to the acute exercise stimulus in gastrocnemius and the myocardium.
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Mixed gastrocnemius was assayed as an index of skeletal muscle IL-6 signaling (Figure 4).
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Exercise elicited an increase in gastrocnemius IL-6R expression (strain main effect, p
