The Cardioprotective Effects of Late-Phase Remote Preconditioning of Trauma Depends on Neurogenic Pathways and the Activation of PKC and NF-κB (But Not iNOS) in Mice

Abstract

Background:

A superficial abdominal surgical incision elicits cardioprotection against cardiac ischemia–reperfusion (I/R) injury in mice. This process, called remote preconditioning of trauma (RPCT), has both an early and a late phase. Previous investigations have demonstrated that early RPCT reduces cardiac infarct size by 80% to 85%. We evaluated the cardioprotective and molecular mechanisms of late-phase RPCT in a murine I/R injury model.

Methods:

Wild-type mice, bradykinin (BK) 2 receptor knockout mice, 3M transgenic mice (nuclear factor κB [NF-κb] repressor inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha [IκBα^{(S32A, S36A, Y42F)}]), and inducible nitric oxide synthase (iNOS) knockout mice were analyzed using a previously established I/R injury model. A noninvasive abdominal surgical incision was made 24 hours prior to I/R injury and the infarct size was determined at 24 hours post-I/R injury.

Results:

The results indicated that a strong cardioprotective effect occurred during late-phase RPCT (58.42% ± 1.89% sham vs 29.41% ± 4.00% late RPCT, mean area of the infarct divided by the mean area of the risk region; P ≤ .05; n = 10). Furthermore, pharmacological intervention revealed the involvement of neurogenic signaling in the beneficial effects of late RPCT via sensory and sympathetic thoracic nerves. Pharmacological experiments in transgenic mice-implicated BK receptors, β-adrenergic receptors, protein kinase C, and NF-κB but not iNOS signaling in the cardioprotective effects of late RPCT.

Conclusion:

Late RPCT significantly decreased myocardial infarct size via neurogenic transmission and various other signaling pathways. This protective mechanism differentiates late and early RPCT. This study describes a new cardiac I/R injury prevention method and refines the concept of RPCT.

Keywords

myocardial ischemia reperfusion injury remote nonischemic preconditioning cardioprotection mechanism signal transduction

Introduction

Cardiac ischemia–reperfusion (I/R) injury is a major global health problem and common cause of postcardiac arrest morbidity and mortality. In 1986, Murry et al first reported that ischemic preconditioning (IPC)¹ was a powerful cardioprotective strategy. Since then, various signaling molecules have been implicated in IPC, including bradykinin (BK), opioids, adenosine, and norepinephrine (NE).^2
–4 Remote IPC (RIPC), another cardioprotective technique, has been demonstrated to elicit remote cardioprotection following ischemia of the kidneys, limbs, liver, skeletal muscle, and brain.⁵ Although RIPC has emerged as a powerful and simple method for the amelioration of I/R injury to the myocardium, the use of this mechanism as a cardioprotective clinical strategy to attenuate the pathophysiological consequences of I/R injury is limited because RIPC is initiated by a brief ischemic stimulus, suggesting that transient ischemia or the interruption of blood flow is a requisite trigger for the cardioprotection elicited by RIPC. Remote preconditioning of trauma (RPCT) has been widely studied since Ren first reported this phenomenon in 2004 in a mouse model of I/R.⁶ The RPCT results in an 80% to 85% reduction in infarct size, making this strategy the most powerful currently employed cardioprotective technique that has been successfully applied in multiple species.^7,8 Importantly, the cardioprotection induced by RPCT is initiated by superficial skin incision on the abdomen (peripheral nociception). Similar to IPC, RPCT has an early and a late phase. However, the early and late phases of RPCT differ from the corresponding phases of IPC⁹ because they are tumor necrosis factor α (TNF-α) independent.⁶ We previously reported that the early phase of RPCT requires neurogenic signaling involving the spinal nerves as well as the activation of cardiac sensory and sympathetic nerves. Previous studies have demonstrated that BK-dependent activation, adenosine triphosphate-sensitive potassium (K_ATP) channels, and the repression of protein kinase C (PKC)-δ or PKC-∊ are involved in RPCT-elicited cardioprotection.¹⁰

In the present study, late RPCT was administered in the form of an abdominal incision 24 hours before cardiac I/R injury, and the cardioprotective effects of RPCT were analyzed at 24 hours after I/R. Late-phase RPCT provided a longer period for the body to respond to the abdominal incision, differentiating this phase from early RPCT and the associated acute reaction. Knowledge of the relationships between the mechanisms of late and early RPCT is currently limited. Understanding the precise mechanisms of this powerful cardioprotective phenomenon may lead to the development of novel and effective therapeutic approaches to reduce cardiac damage after I/R injury.

Methods

Animals

The mice were maintained according to the guidelines for the administration of laboratory animal research and the Institutional Animal Care and Use Committee of Harbin Medical University in China, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, revised 1985), and the Position of the American Heart Association on Research Animal Use (1984). Wild-type (WT) mice (B6129SF2/J F2), BK receptor 2 (BK2R) knockout mice, inducible nitric oxide synthase (iNOS) knockout mice, and 3M inhibitor of nuclear factor of kappa light polypeptide (NF-κB) gene enhancer in B-cells inhibitor alpha (IκBα ^{(S32A, S36A, Y42F)}) transgenic mice (3M transgenic [Tg] mice)¹¹ were obtained from the Harbin Medical University Animal Center. Male and female animals were equally distributed among the groups, and all of the mice used in this study were between 10 and 16 weeks of age. Post hoc analyses confirmed previous results that indicated the absence of gender-related differences in RPCT in mice. The research protocol was approved by the institutional ethics committee for the administration of laboratory animals of Harbin Medical University, China.

Mouse Model and Surgical Procedures

The mice were anesthetized using sodium pentobarbital (90 mg/kg, intraperitoneally [ip]). A 2-cm transverse incision was made on the abdominal midline. The incision extended through the skin and muscle and into the peritoneum and was immediately closed using 7-0 polypropylene sutures. The bleeding was minimal (<50 μL).^6,10 After 24 hours, the mice were anesthetized using the same method, intubated with polyethylene 90 tubing, and ventilated using a mouse mini ventilator (Harvard Apparatus, Holliston, Massachusetts). Blood pressure and electrocardiogram (Digi-Med Sinus Rhythm Analyzer, Micro-Med, Inc, Louisville, Kentucky) data were recorded 10 minutes before and during ischemia and for 10 minutes after reperfusion. The ventilation conditions used included a rate of 100 ± 5 breaths/min and tidal volume of 2.2 mL. The blood gas analysis performed after open chest surgery and during the 45-minute I/R using a 248 pH/Blood Gas Analyzer (Essex Co., 9 2DX, UK) revealed the following values: pH 7.38 ± 0.01, PCo ₂ 26.9 ± 1.3, and Po₂ 236.3 ± 19.87 (n = 10). The body temperature of the mice was maintained at 36 ± 1°C.⁶ A 1.5-cm incision between the second and third ribs exposed the left anterior descending (LAD) coronary artery while avoiding rib and sternal resection as well as retraction and rotation of the heart. An 8-0 nylon suture was placed around the LAD 2 mm from the tip of the left auricle, and a piece of soft silicon tubing (0.64 millimeter inner diameter, 1.19 millimeter outer diameter) was placed over the artery. Coronary artery occlusion was achieved by tightening, and then the thoracic wall was closed by the 7-0 nylon suture 45 minutes after coronary artery occlusion, and the animal was subsequently allowed to recover. The experimental design is outlined in Figure 1. The mice received the abdominal incision 24 hours prior to a 45-minute occlusion of the coronary artery. The infarct size was measured 24 hours after cardiac reperfusion. One group of control mice were not subjected to abdominal incision (sham). A second group of control mice did not receive abdominal incisions or pharmacological or surgical intervention (sham + treatment). The late RPCT mice received pharmacological or vehicle treatment (late RPCT + treatment or vehicle).

Figure 1.

Schematic representation of the experimental design. The time courses of the pharmacological manipulations, surgical procedures, and ischemia–reperfusion (I/R) are shown for the 5 experimental groups (sham, remote preconditioning of trauma [RPCT], RPCT + treatment, sham + treatment, and RPCT + vehicle).

The mice were divided into 39 groups (the 37 groups in Table 1 + the apoptotic nuclei and DNA fragmentation groups). Figure 2 shows groups 1 and 2 (n = 10) and the apoptotic nuclei (n = 9) and DNA fragmentation groups (n = 9). Figure 3 shows groups 4 to 12 (n = 10). Figure 4 shows groups 13 to 21 (n = 10). Figure 5 shows groups 22 to 37, including groups 22 to 25 (n = 15), 26 to 29 (n = 10), 30 to 33 (n = 15), and 34 to 37 (n = 10).

Table 1.

Infarct/LV, Risk/LV, and Infarct/Risk of the Experimental Groups Used in This Study.^a

Group	Procedure	Infarct/LV	Risk/LV	Infarct/Risk
1	Sham	31.73 ± 0.79	54.32 ± 2.43	58.42 ± 1.89
2	Late RPCT	17.45 ± 1.21	59.35 ± 3.54	29.41 ± 4.00
3	WT sham	29.41 ± 2.05	49.55 ± 1.88	59.37 ± 1.98
4	WT late RPCT	12.84 ± 1.88	45.10 ± 2.04	28.49 ± 3.63
5	BK2R KO sham	21.60 ± 2.95	42.40 ± 1.65	51.05 ± 1.72
6	BK2R KO late RPCT	24.34 ± 0.57	48.80 ± 2.55	49.89 ± 1.21
7	Sham	29.07 ± 1.54	50.21 ± 7.56	57.91 ± 1.96
8	Propranolol late RPCT	30.54 ± 1.66	53.35 ± 1.54	57.17 ± 1.96
9	Vehicle late RPCT	11.95 ± 1.43	40.12 ± 3.20	29.79 ± 2.04
10	Sham	32.34 ± 2.66	55.27 ± 1.77	58.52 ± 2.28
11	Chelerythrine late RPCT	25.60 ± 0.92	49.96 ± 3.49	51.25 ± 1.20
12	Vehicle late RPCT	16.52 ± 1.83	58.34 ± 1.10	28.32 ± 1.43
13	Sham	36.05 ± 1.32	62.32 ± 3.56	57.78 ± 1.80
14	Late RPCT	14.25 ± 0.56	49.47 ± 1.44	28.81 ± 4.12
15	Vehicle late RPCT	13.68 ± 1.34	47.38 ± 2.35	28.88 ± 1.93
16	Sham Hex	33.04 ± 0.87	55.66 ± 1.77	59.37 ± 1.98
17	Hex late RPCT	28.87 ± 1.58	52.40 ± 2.52	55.10 ± 1.50
18	Sham at C7 + late RPCT	14.31 ± 1.85	58.10 ± 1.64	24.64 ± 2.17
19	Transection at C7 + late RPCT	11.53 ± 2.44	45.11 ± 4.22	25.56 ± 1.94
20	Sham at T7 + late RPCT	14.76 ± 1.76	57.35 ± 1.86	25.74 ± 1.56
21	Transection at T7 + late RPCT	28.59 ± 2.62	50.12 ± 2.82	57.05 ± 1.96
22	Sham	32.52 ± 1.37	57.34 ± 2.34	56.76 ± 2.13
23	Sham + PDTC	26.68 ± 2.64	49.96 ± 3.49	53.42 ± 1.97
24	PDTC + late RPCT	30.73 ± 2.49	58.34 ± 1.10	52.69 ± 2.04
25	Vehicle late RPCT	12.52 ± 1.54	45.92 ± 2.32	27.28 ± 1.73
26	WT sham	31.05 ± 1.78	56.23 ± 3.65	55.22 ± 1.92
27	WT late RPCT	13.06 ± 1.54	45.92 ± 2.32	28.45 ± 3.67
28	Sham 3M	18.54 ± 2.38	49.33 ± 1.75	37.59 ± 1.26
29	3M late RPCT	21.22 ± 1.64	52.95 ± 2.77	40.08 ± 1.20
30	Sham	34.49 ± 2.05	61.44 ± 0.88	56.14 ± 1.57
31	Sham + AG	25.05 ± 1.80	47.45 ± 1.38	52.81 ± 2.07
32	AG + late RPCT	13.58 ± 1.50	53.61 ± 2.82	25.34 ± 1.55
33	Vehicle late RPCT	14.55 ± 1.66	54.28 ± 4.22	26.87 ± 1.50
34	WT sham	27.50 ± 1.90	46.67 ± 1.66	58.93 ± 1.87
35	WT late RPCT	14.21 ± 0.83	49.89 ± 2.78	28.49 ± 3.63
36	iNOS KO sham	29.87 ± 1.08	54.46 ± 3.77	54.85 ± 1.02
37	iNOS KO late RPCT	16.15 ± 2.02	58.43 ± 1.48	27.64 ± 2.76

Abbreviations: AG, aminoguanidine; BK2R KO, BK2R knockout mice; C, cervical; T, thoracic; 3M Tg, 3M transgenic mice; infarct/LV, mean area of the infarct divided by the mean area of the left ventricle; infarct/risk, mean area of the infarct divided by the mean area of the risk region; iNOS KO, iNOS knockout mice; late RPCT, late phase of remote preconditioning of trauma; risk/LV, mean area of the risk region divided by the mean area of the left ventricle; WT, wild-type mice; SE, standard error.

^aThe data are presented as the mean ± SE. The final column shows infarct size as the percentage of the risk region between the experimental group and the control group.

Figure 2.

Late remote preconditioning of trauma (RPCT) protects against cardiac ischemia reperfusion (I/R) injury. A, The infarct size was determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining (red). Quantification revealed a reduction in infarct size in the late RPCT mice compared with the sham controls (*P ≤ .05, n = 10). B, The number of deoxynucleotidyl transferase deoxyuridine-triphosphatase nick-end labeling (TUNEL)-positive (green fluorescence) apoptotic nuclei was significantly decreased in the late RPCT mice compared with the sham controls (*P ≤ .05, n = 9). C, Analysis of DNA fragmentation revealed a significant reduction in apoptosis in the late RPCT group compared with the sham group (*P ≤ .05 vs control; n = 9).

Figure 3.

The cardioprotective effects of late remote preconditioning of trauma (RPCT) are dependent on bradykinin receptor 2 (BK2R), β-adrenergic, and protein kinase C (PKC) signaling. A, The cardioprotective effects of late RPCT were blocked in BK2R knockout mice (*P ≤ .05 vs wild-type late RPCT; n = 10). B, Propranolol treatment prevented late RPCT-mediated cardioprotection (*P ≤ .05 vs vehicle late RPCT; n = 10). C, Treatment with the PKC inhibitor chelerythrine prevented cardioprotection after late RPCT (*P ≤ .05 vs vehicle late RPCT; n = 10).

Figure 4.

Neurogenic transmission is required for late RPCT-induced cardioprotection. A, Hexamethonium administration prevented the cardioprotective effects of late RPCT (*P ≤ 0.05 vs vehicle late remote preconditioning of trauma (RPCT); n = 10). B, The cardioprotective effects of late RPCT were blocked by spinal cord transection at the T7 vertebral level but not at the C7 vertebral level (*P ≤ .05 vs sham; n = 10).

Figure 5.

Nuclear factor κB (NF-κB), not inducible nitric oxide synthase (iNOS) signaling, is involved in the cardioprotective effects of late remote preconditioning of trauma (RPCT). A, The cardioprotective effects of late RPCT were blocked by administration of the NF-κB blocker PDTC (*P ≤ .05 pyrrolidine dithiocarbamate (PDTC) vs vehicle late RPCT; n = 15). B, Infarct size was not significantly different in 3M transgenic (Tg) mice compared with sham controls (P = .17 3M sham vs 3M late RPCT; n = 10). C, The iNOS inhibitor aminoguanidine (AG) did not prevent the cardioprotective effects of late RPCT (*P ≤ .05 vs vehicle late RPCT; n = 15). D, The cardioprotective effects of late RPCT were not blocked in iNOS knockout mice (*P ≤ .05 vs iNOS knockout sham; n = 10).

Pharmacological Intervention

The ganglionic blocker hexamethonium (Hex, 20 mg/kg, intravenously [iv]),¹² the β-adrenergic blocker propranolol (2 mg/kg, iv),^13,14 the PKC inhibitor chelerythrine (5 mg/kg, iv),¹⁰ the NF-κB blocker pyrrolidine-dithiocarbamic acid (120 mg/kg, ip), and the iNOS inhibitor aminoguanidine (AG, 150 mg/kg, ip) were administered via abdominal injection 15 minutes prior to the abdominal incision. All of the drugs were dissolved in physiological saline. For all of the agents, the vehicle control was the vehicle used for each drug.

Assessment of Infarct Size and Apoptosis in the Myocardium

Forty-eight hours after surgery, the mice were treated with phenobarbital sodium (90 mg/kg, ip), and the hearts were dissected out and perfused with phosphate-buffered saline (PBS). After the PBS perfusion, a 2% 2,3,5-triphenyltetrazolium chloride staining solution was added to label ischemic tissue, and nonischemic tissue was stained with 5% phthalocyanine blue. Next, the harvested hearts were fixed in 10% formalin for histological examination and to determine the size of the infarct.^6,10,15 Midventricular cardiac tissue was sectioned to assess infarct size, and terminal deoxynucleotidyl transferase deoxyuridine-triphosphatase nick-end labeling (TUNEL) staining was performed. Apoptotic DNA fragmentation was assessed in situ using the DeadEnd Fluorometric TUNEL system (Promega, Madison, Wisconsin), followed by staining with an anti-α-sarcomeric actin antibody (Sigma, St Louis, Missouri), and the nuclear dye 4′,6-diamidino-2-phenylindole (Invitrogen, Life Technologies, Grand Island, New York).^16,17 The TUNEL-positive (green) nuclei were expressed as a percentage of total nuclei, based on the number counted in 10 randomly selected microscopic fields from midventricular cardiac sections (5 sections, with approximately 400 nuclei/animal). The rate of apoptosis was determined using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Applied Science, Indianapolis, Indiana). The apoptosis kit quantified histone-complexed DNA fragments (nucleosomes) in the cytoplasm of apoptotic cells using a sandwich ELISA. The results were normalized to internal standards and expressed as fold increases over the control.^18,19

Statistical Analysis

For the parameters that required quantification and evaluation of statistical significance, the results were expressed as the mean ± standard error of mean. Statistical significance was determined using Student t test (2-tailed distribution and 2 samples of unequal variance) with Bonferroni correction. For multiple group comparisons, 1-way analysis of variance, followed by Fisher post hoc test, was used. P ≤ .05 was considered statistically significant.

Results

Late RPCT Reduces Infarct Size and Apoptosis After I/R

A significant reduction in infarct size was observed after late RPCT (Figure 2A; 58.42% ± 1.89% sham vs 29.41% ± 4.00% late RPCT; P ≤ .05; n = 10). Apoptosis was assessed after I/R using TUNEL staining and an ELISA-based nucleosome assay in both the sham and the late RPCT hearts. The proportion of TUNEL-positive nuclei significantly decreased in the myocardia of the mice subjected to late RPCT compared with the proportion of TUNEL-positive nuclei in the sham controls (Figure 2B; 32.08% ± 1.05% vs 17.88% ± 0.65%; P ≤ .05; n = 9). The quantitative apoptosis assay detected the levels of mono- and oligonucleosomes in the apoptotic myocardial cells and showed a significant decrease in myocardial apoptosis in the late RPCT-treated mice compared with the myocardial apoptosis observed in the mice of the sham group (Figure 2C).

Bradykinin receptor 2, β-AR, and PKC Signaling Participate in the Cardioprotective Effects of Late RPCT

We investigated whether the signaling pathways involved in the cardioprotective effects of early RPCT (BK2R, β-AR, and PKC signaling¹⁰) also mediate the cardioprotective effects of late RPCT. Late RPCT did not protect against myocardial infarction in the BK2R knockout mice (Figure 3A; 51.05% ± 1.72% BK2R knockout sham vs 49.89% ± 1.21% BK2R knockout with late RPCT; P > .05; n = 10).

To assess whether late RPCT requires β-adrenergic signaling, the mice were treated with the β-AR antagonist propranolol (2 mg/kg) or vehicle 15 minutes prior to the abdominal incision (Figure 3B). Propranolol treatment completely inhibited the cardioprotective effect of late RPCT (57.17% ± 1.96% propranolol vs 29.79% ± 2.04% vehicle control; P ≤ .05; n = 10). To assess the involvement of PKC signaling, the mice were treated with chelerythrine (5 mg/kg, iv) or vehicle 15 minutes prior to the abdominal incision, and the infarct size was assessed 24 hours after reperfusion (Figure 3C). Blocking PKC abolished late RPCT-induced cardioprotection (51.25% ± 1.20% chelerythrine vs 29.79 ± 2.04 vehicle control, P ≤ .05; n = 10).

Cardioprotection After Late RPCT Depends on Neurogenic Transmission

To determine whether neurogenic transmission via the sympathetic nervous system was required for the late RPCT-induced cardioprotection against I/R injury, the sympathetic ganglionic blocker Hex (20 mg/kg) was administered prior to the abdominal incision. Hex blocked the cardioprotective effects of late RPCT (Figure 4A; 29.41% ± 4.43% late RPCT vs 55.10% ± 1.50% late RPCT Hex; P ≤ .05; n = 10), indicating that neurogenic transmission played an important role. Next, the spinal cord was transected at two different vertebral levels (C7 and T7) before late RPCT was induced (Figure 4B). Spinal cord transection at C7 had no effect on the effects of late RPCT (Figure 4B; 24.64% ± 2.17% sham C7 vs 25.56 %± 1.94% transected C7; P ≤ .05; n = 10). In contrast, transection at T7 abolished the cardioprotective effect of late RPCT against I/R injury (Figure 4B; 25.74% ± 1.56% sham T7 vs 57.05% ± 1.96% transected T7; P ≤ .05; n = 10).

Nuclear factor-κB, not iNOS, is Required for the Cardioprotective Effects of Late RPCT

To examine the role of the transcription factor NF-κB in mediating the cardioprotective effects of late RPCT, the mice were treated with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 120 mg/kg, ip) or vehicle 15 minutes before the abdominal incision. Pyrrolidine dithiocarbamate treatment completely blocked the cardioprotective effects of late RPCT (Figure 5A; 52.69% ± 2.04%, PDTC vs 27.28% ± 1.73% vehicle control, P ≤ .05; n = 15). In contrast, the infarct size in 3M Tg mice, in which NF-κB activation is blocked by cardiac-specific expression of dominant-negative IκBα, did not significantly differ from the infarct size in the sham mice (Figure 5B; 37.59% ± 1.26% 3M sham vs 40.08% ± 1.20% 3M late RPCT, P = .17; n = 10).

Finally, to determine whether iNOS was required for the cardioprotective effects of late RPCT, the mice were treated with the iNOS inhibitor AG (150 mg/kg, ip) or vehicle, and infarct size was assessed after I/R injury (Figure 5C). Aminoguanidine treatment did not abolish the cardioprotective effects of late RPCT (25.34% ± 1.55% AG vs 26.87 ± 1.50 sham, P = .48; n = 15). We also examined iNOS knockout mice, and the cardioprotective effect of late RPCT was intact in the knockout mice (Figure 5D; 28.49% ± 3.63% WT late RPCT vs 27.64% ± 2.76% iNOS knockout late RPCT; P = .85; n = 10), indicating that iNOS was not required for the beneficial effects of late RPCT during I/R injury.

Discussion

Ren first described RPCT in 2004. Because the abdominal incision is administered 24 hours prior to I/R injury in late RPCT, late RPCT avoids acute stress reactions. Therefore, late RPCT is a superior therapeutic approach for the treatment of cardiac arrest-induced tissue injury. It is well established that a combination of necrotic cell death and apoptosis are responsible for the acute loss of myocardial cells after I/R.^18,20
–22 In a previous study, we found that activation of a specific skin nerve field (abdominal skin) through C fiber nociceptors could protect against cardiac I/R injury.¹⁰ The fact that RPCT did not depend on transient ischemic trigger or the interruption of blood flow in organs or tissues fits with this finding. Moreover, we also showed that RPCT was TNF-α independent.⁶ In addition, in the present study, we found that the late phase of RPCT required the activation of the transcription factor NF-κB but was independent of iNOS (unlike IPC and RIPC). These results indicate that the mechanism of RPCT is unique and distinct from those elucidated for IPC and RIPC and thus represent a new mechanism of cardioprotection. However, Kharbanda et al reported that remote ischemia preconditioning induced by transient limb ischemia (cuff inflation and deflation of upper arm) reduced endothelial I/R injury in humans and reduced experimental myocardial infarct size.²³ This result showed that transient limb ischemia was a simple preconditioning stimulus with important potential clinical applications. Although this preconditioning stimulus depended on transient local tissue ischemia, there was no trauma and it was easier to operate than the skin incision we used in this study. Thus, limb ischemia elicited by a noninvasive blood pressure cuff inflation/deflation was much better feasible than abdominal skin incision. To remain consistent with previous studies of RPCT and with the focus on the late phase of RPCT, in the present study, an abdominal skin incision was selected as the remote protective stimulus. Indeed, we were not sure whether thoracotomy could elicit cardioprotection. However, we hypothesized that thoracotomy might constitute a serious injury in mice subjected to 45 minutes of cardiac ischemia and would likely decrease the survival rate at 24 hours after I/R injury. In addition, an abdominal skin incision is easier to achieve and provides greater potential clinical feasibility compared with thoracotomy. Thus, an abdominal skin incision was the better choice for the remote stimulus in the present study. In future studies, we will investigate the relationship between remote cardioprotection and remote cardiac injury to determine the types of remote trauma that induce cardioprotection and cardiac injury and characterize the underlying mechanisms. Although we admitted that abdominal incision would never be considered as an acceptable therapy for patients with cardiac disease, it provided a new trigger (peripheral nociception) that elicited remote cardioprotection through activation of C fibers in the skin. If we could find new stimulation methods replacing skin incision to activate C fibers, it would be acceptable for clinic. In our follow-up study of RPCT, we demonstrated that application of topical capsaicin elicited cardioprotection via stimulation of peripheral nociception. This result was of important significance because it showed a potential clinical cardioprotective strategy that was nontraumatic peripheral nociception instigated by chemical stimulation of sensory C fibers in the skin. However, we didn’t know whether late RPCT could elicit remote cardioprotection. Thus, abdominal incision was still selected as the basic stimulus to investigate remote cardioprotection in this study.

In the present study, we analyzed myocardial apoptosis using TUNEL staining and evaluated DNA fragmentation based on the formation of DNA nucleosome ladders during late RPCT. We determined that the cardioprotective effects of late RPCT were due, at least in part, to a reduction in cellular apoptosis and cell death after I/R injury.

Because the cardioprotective effects of RPCT are induced through remote injury, we hypothesized that the protective effects might be mediated via humoral or neurogenic pathways. Studies have shown that remote IPC is initiated through a neurogenic ganglionic mechanism.²⁴ In a previous study, we demonstrated that the ganglionic blocker Hex (which inhibits neurotransmission from sympathetic and parasympathetic preganglionic nerves) abolished the cardioprotective effect of RPCT after I/R injury.¹⁰ In the present study, we demonstrated that a similar neurogenic mechanism was required for late RPCT. In contrast, spinal cord transection at the C7 level had no effect on the effects of late RPCT, but transection at the T7 level completely abolished the effects of late RPCT. These data support the important conclusion that the thoracic spinal cord is required for late RPCT and that the brain is not involved in this neuromodulation-induced cardioprotection. Therefore, both early and late RPCT require combined sympathetic and parasympathetic preganglionic neurogenic transmission at the level of the thoracic spinal cord.

The results of the previous study revealed that several endogenous factors/mediators of myocyte and endothelial and neural origins such as extracellular signal regulators, including a number of G protein-coupled receptors (BK, AR, adenosine, and opioid receptors), contribute to the cardioprotection elicited by IPC and RIPC.²⁵ Bradykinin is released from sympathetic nerve endings in the heart during brief or RIPC. Bradykinin receptors 1 and 2 (BK1R and BK2R) are 2 of the major receptor subtypes that mediate the action of BK. The BK2R positively mediates most of the physiological functions of kinins, whereas BK1R seems to play an injurious role in myocardial I/R. Previous studies have shown that the activation of BK2R plays a significant role in both brief and remote IPC and is known to trigger NE release from cardiac sympathetic nerves.²⁶ Bradykinin can be generated in adrenergic nerve endings by the kallikrein-kinin system, activating BK2R in sympathetic nerve terminals and sensitive C fiber endings in an autocrine and paracrine fashion, respectively. Activated C fibers release calcitonin gene-related peptide and substance P, which then act at their respective receptors on sympathetic nerve terminals to evoke NE release.^10,27 Evidence strongly suggests that the release of NE from cardiac sympathetic nerve endings during ischemia²⁸ activates α- or β-AR,²⁶ leading to cardiac preconditioning in vivo and in vitro.^13,29 In the present study, we investigated whether BK2R and β-AR are involved in the mechanisms of late RPCT. We found that late RPCT induced cardioprotective effects that were abolished in the BK2R knockout mice and that the β-AR blocker propranolol completely abolished the cardioprotective effect of late RPCT.

The PKC signaling pathway plays an integral role in many signaling cascades that govern cellular behavior.³⁰ In the normal myocardium, PKC-∊ forms signaling complexes with at least 36 different proteins classified as structural elements, signaling molecules, and stress-responsive proteins.³¹ Protein kinase C-∊-dependent cardioprotection induces dynamic modulation of these complexes, suggesting a functional role for these complexes in the genesis of protection against I/R injury.^32,33 We previously reported that the activation of PKC-∊ and the inhibition of PKC-δ after early RPCT were BK2R dependent. We demonstrated that the cardioprotective effects of early RPCT were dependent on PKC, BK2R, and mitochondrial adenosine triphosphate-sensitive potassium (mitoK_ATP) as well as potentially on sarcolemmal adenosine triphosphate-sensitive potassium channels. In the present study, we observed that the PKC blocker chelerythrine completely blocked late RPCT-induced cardioprotection against I/R injury.

Tumor necrosis factor α is an important upstream activator of NF-κB that regulates iNOS gene expression¹¹ and plays a role in IPC and I/R injury.³⁴ In a previous study, we reported that iNOS was required for late IPC.³⁵ Therefore, it is conceivable that TNF-α activates NF-κB-dependent iNOS expression in late IPC.^36,37 However, the data obtained in our previous study did not indicate that TNF-α was involved in the mediation of early or late-phase RPCT,⁶ suggesting that the mechanisms regulating RPCT are unique and distinct from the mechanisms underlying brief and remote IPC. The results of the present study indicated that late RPCT required NF-κB activation. However, late RPCT was iNOS independent, indicating that NF-κB may activate an unknown protein that then mediates the cardioprotective effects of late RPCT.

The cardioprotective phenomenon elicited by late RPCT is an innovative advance of the concept of manipulating skin nociceptors and neurogenic signaling to affect remote organ function, including cardioprotection against ischemic injury. Late RPCT appears to be particularly promising when the acute stress reactions induced by early RPCT are also considered. Both brief and remote IPC strategies (except limb ischemia) are limited by the requirement of an ischemic stimulus to a second organ. Because it employs stimulation of peripheral nociception, late RPCT may be a clinically useful option to prevent I/R injury after elective cardiac arrest during cardiac surgery. We have collected strong evidence to support our hypothesis (Figure 6) that a remote, superficial abdominal surgical incision activates a neurogenic signal that is transmitted via C fibers and activates spinal nerves higher in the spinal cord, resulting in the activation of the cardiac sympathetic system, which involves the release of BK and NE and the activation of BK2R and β-AR. In turn, this triggers PKC activation within cardiomyocytes, protecting against cardiac I/R injury. The activation of this signaling pathway by early RPCT leads to cardioprotection via mitoK_ATP channels, whereas late RPCT activates a signaling pathway that results in NF-κB activation, which likely induces the transcription of an as yet undefined protein with cardioprotective properties. However, requisite explanation should be offered. There was not enough evidence to show the sequence of the signaling step in this study. We built the integrated hypothesis of the mechanism of late RPCT with the help of the previous study. In future study, we will investigate the late RPCT deeply and refine the concept of RPCT.

Figure 6.

Schematic representation of the mechanisms and signaling pathways involved in the cardioprotective effects of late-phase remote preconditioning of trauma (RPCT). Abdominal incision activates the cardiac sympathetic nervous system through C fibers and spinal nerves, leading to the release of bradykinin (BK) and norepinephrine (NE). Bradykinin and NE initiate intracellular signaling in target myocardial cells via bradykinin receptor 2 (BK2R) and β-adrenergic receptor (β-AR), either in parallel or via cross signaling, resulting in protein kinase C (PKC) activation. These signaling cascades ultimately result in nuclear factor κB (NF-κB) activation and the increased transcription of an unknown protein that mediates the cardioprotective effects of late RPCT.

Footnotes

Authors’ Contribution

Y. Song contributed to conception and design and analysis, drafted and critically revised the manuscript, gave final approval and agrees to be accountable for all aspects of work ensuring integrity and accuracy. X. Ren contributed to conception and design, critically revised the manuscript, gave final approval and agrees to be accountable for all aspects of work ensuring integrity and accuracy. Y. Ye contributed to acquisition, analysis and interpretation. P. Li contributed to acquisition and interpretation. Y. Zhao contributed to analysis and interpretation. Q. Miao contributed to analysis. D. Hou contributed to interpretation.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by a grant from the National Natural Science Foundation of China (81470425).

References

Murry

Jennings

Reimer

. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74(5):1124–1136.

Schultz

JEJ

Hsu

Gross

. Ischemic preconditioning and morphine-induced cardioprotection involve the delta (δ)-opioid receptor in the intact rat heart. J Mol Cell Cardiol. 1997;29(8):2187–2195.

Auchampach

Gross

. Adenosine A1 receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol. 1993;264(5 pt 3):h1327–h1327.

Oxman

Arad

Klein

Avazov

Rabinowitz

. Limb ischemia preconditions the heart against reperfusion tachyarrhythmia. Am J Physiol. 1997;273(4 pt 2):h1707–h1712.

Heusch

Bøtker

Przyklenk

Redington

Yellon

. Remote ischemic conditioning. JACC. 2015;65(2):177–195.

Ren

Wang

Jones

. TNF-α is required for late ischemic preconditioning but not for remote preconditioning of trauma. J Surg Res. 2004;121(1):120–129.

Gross

Baker

Moore

Falck

Nithipatikom

. Abdominal surgical incision induces remote preconditioning of trauma (RPCT) via activation of bradykinin receptors (BK2R) and the cytochrome P450 epoxygenase pathway in canine hearts. Cardiovasc Drugs Ther. 2011;25(6):517–522.

Redington

Disenhouse

Strantzas

. Remote cardioprotection by direct peripheral nerve stimulation and topical capsaicin is mediated by circulating humoral factors. Basic Res Cardiol. 2012;107(2):1–10.

Jones

Flaherty

Tang

. Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism. J Mol Cell Cardiol. 1999;31(8):1469–1481.

10.

Jones

Fan

Liao

. Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling. Circulation. 2009;120(suppl 11):s1–s9.

11.

Pasparakis

Alexopoulou

Episkopou

Kollias

. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med. 1996;184(4):1397–1411.

12.

Weinbrenner

Nelles

Herzog

Sárváry

Strasser

. Remote preconditioning by intrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway. Cardiovasc Res. 2002;55(3):590–601.

13.

Frances

Nazeyrollas

Prevost

. Role of β1-and β2-adrenoceptor subtypes in preconditioning against myocardial dysfunction after ischemia and reperfusion. J Cardiovasc Pharmacol. 2003;41(3):396–405.

14.

Wajima

Beguier

Yaguchi

. Effects of cariporide (HOE642) on myocardial infarct size and ventricular arrhythmias in a rat ischemia/reperfusion model: comparison with other drugs. Pharmacology. 2004;70(2):68–73.

15.

Guo

Qiu

Tang

Yang

Bolli

. Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol. 1998;275(4 pt 2):h1375–h1387.

16.

Fan

Yuan

Song

. Small heat-shock protein Hsp20 attenuates beta-agonist-mediated cardiac remodeling through apoptosis signal-regulating kinase 1. Circulation. 2006;99(11):1233–1242.

17.

Zhou

Fan

Ren

. Overexpression of histidine-rich Ca-binding protein protects against ischemia/reperfusion-induced cardiac injury. Cardiovasc Res. 2007;75(3):487–497.

18.

Fan

Ren

Qian

. Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury. Circulation. 2005;111(14):1792–1799.

19.

Ren

Wang

. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation. 2009;119(17):2357–2366.

20.

Nicolaou

Rodriguez

Ren

. Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac function and protects against ischemia/reperfusion injury. Circ Res. 2009;104(8):1012–1020.

21.

Qian

Ren

Wang

. Blockade of Hsp20 phosphorylation exacerbates cardiac ischemia/reperfusion injury by suppressed autophagy and increased cell death. Circ Res. 2009;105(12):1223–1231.

22.

Ibáñez

Heusch

Ovize

Van de Werf

. Evolving therapies for myocardial ischemia/reperfusion Injury. JACC. 2015;65(14):1454–1471.

23.

Kharbanda

Mortensen

White

. Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation. 2002;106(23):2881–2883.

24.

Gho

Schoemaker

van den Doel

Duncker

Verdouw

. Myocardial protection by brief ischemia in noncardiac tissue. Circulation. 1996;94(9):2193–2200.

25.

Heusch

. Molecular basis of cardioprotection signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res. 2015;116(4):674–699.

26.

Seyedi

Win

Lander

Levi

. Bradykinin B2-receptor activation augments norepinephrine exocytosis from cardiac sympathetic nerve endings mediation by autocrine/paracrine mechanisms. Circ Res. 1997;81(5):774–784.

27.

Seyedi

Maruyama

Levi

. Bradykinin activates a cross-signaling pathway between sensory and adrenergic nerve endings in the heart: a novel mechanism of ischemic norepinephrine release? J Pharmacol Exp Ther. 1999;290(2):656–663.

28.

Mackins

Kano

Seyedi

. Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion. J Pharmacol Exp Ther. 2006;116(4):1063–1070.

29.

Asimakis

Inners-McBride

Conti

Yang

. Transient β adrenergic stimulation can precondition the rat heart against post-ischaemic contractile dysfunction. Cardiovasc Res. 1994;28(11):1726–1734.

30.

Churchill

Mochly-Rosen

. The roles of PKC delta and lunate epsilon isoenzymes in the regulation of myocardial ischemia/reperfusion injury. Biochem Soc Trans. 2007;35(pt 5):1040–1042.

31.

Akhter

Milano

Shotwell

. Transgenic mice with cardiac overexpression of α1B-ARsin vivo α1-AR-mediated regulation of β-adrenergic signaling. J Biol Chem. 1997;272(34):21253–21259.

32.

Dorn

Mochly-Rosen

. Intracellular transport mechanisms of signal transducers. Annu Rev Physiol. 2002;64:407–429.

33.

Dorn

Force

. Protein kinase cascades in the regulation of cardiac hypertrophy. J Clin Invest. 2005;115(3):527–537.

34.

Xuan

Tang

Banerjee

. Nuclear factor-κB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res. 1999;84:1095–1109.

35.

Guo

Jones

Xuan

. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA. 1999;96(20):11507–11512.

36.

Dawn

Xuan

Marian

. Cardiac-specific abrogation of NF-kappaB activation in mice by transdominant expression of a mutant Ikappa. J Mol Cell Cardiol. 2001;33(1):161–173.

37.

Brown

McGuinness

Wright

. Cardiac-specific blockade of NF-κB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo. Am J Physiol Heart Circ Physiol. 2005;289(1):h466–h476.