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Original Basic Science—General

Multiple Progressive Thermopreconditioning Improves Cardiac Ischemia/Reperfusion-induced Left Ventricular Contractile Dysfunction and Structural Abnormality in Rat

Chen, Yueh-Hsi Bsc1; Chiang, Chih-Yao MD, PhD1,2,3; Chang, Tzu-Ching PhD1; Chien, Chiang-Ting PhD1

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doi: 10.1097/TP.0000000000003176
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Abstract

Introduction

Ischemic heart disease secondary to acute myocardial infarction or S-T-segment elevation myocardial infarction usually results from acute thrombotic occlusion of a coronary artery and is a leading cause of death worldwide.1 Timely reperfusion is necessary to decrease the cell death resulting from cardiac ischemia. Coronary artery reperfusion is currently the most effective therapy for acute myocardial infarction; however, the process of restoring blood flow to the ischemic myocardium itself can induce myocardial damages, also known as myocardial ischemia/reperfusion (I/R) injury.2 In addition, I/R injury is an unavoidable event that occurs during the process of organ transplantation or cardiac surgery. Sudden restoration of blood flow to ischemic myocardium may induce the myocardial I/R injury through oxidative stress, apoptosis, autophagy, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction.3-5 In addition, the severity of myocardial infarction depends on the functional and structural integrity of coronary microcirculation.1 We aim to establish an efficient and noninvasive strategy to attenuate cardiac I/R-induced dysfunction via the preservation of contractile function and structural integrity of the damaged heart.

Several preconditioning techniques enhanced cellular defense proteins like Hsp-70, Bcl-2, and Bcl-xL to protect the cardiovascular system against oxidative stress induced injury.3,6 In the classical in vivo ischemic preconditioning and postconditioning models, a reduction in the myocardial infarct size was usually selected as the relevant end point in most cases, but this was often not consistently correlated with a corresponding ventricular function.7 Pharmacologic preconditioning by granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine, was recently used to treat patients of acute myocardial infarction and could endow cardioprotection against myocardial I/R through the enhanced phosphorylation and association of Stat3 with HSF18 and mobilization of progenitor cells.9 However, controversy exists, as some patients developed proinflammatory and prothrombotic effects because G-CSF induces cardiac thrombosis through an inflammation–thrombosis interaction.4,9 Therefore, in this study, we aimed to determine the cardiac G-CSF expression and location in response to I/R injury.

On the other hand, thermopreconditioning was the most accessible method for inducing cardiovascular resistance and tolerance.3,6,10,11 Furthermore, in human trials or animal studies, high frequency of sauna, thermal therapy, or triple progressive thermopreconditioning (3PTP) with gradual increase in body surface area by 3 steps of 42°C bathing could provide more efficient protection on improvement of myocardial perfusion and vascular endothelial dysfunction.3,12,13 3PTP therapies prevented whole-body hyperthermia and evoked adverse effects including acute myocardial and cerebral infarction induced by abrupt transient changes in blood pressure, heart rate, blood viscosity, fibrinolytic activity, and platelet function.3,14 Increasing the frequency of thermal preconditioning like 3PTP further enforced the cardiovascular protection by decreasing oxidative stress, endoplasmic reticulum stress, autophagy, apoptosis, and leukocyte infiltration through MnSOD, Hsp-70, Bcl-2, and Bcl-xL upregulation more than single treatment of thermal preconditioning.3,6 The effect of 3PTP has been reported to reduce infarct size3 and to depress FeCl3-induced thrombosis in rats.6 However, the direct evidence for the improvement of cardiac contractile function by 3PTP has not been determined. In the present study, we explore whether 3PTP preserves cardiac contractile function and structural integrity after cardiac I/R injury. The possible response of cardiac G-CSF is also determined in the 3PTP-treated I/R heart.

METHODS AND MATERIALS

Animals

Thirty-two male Wistar rats (200–220 g) aged 8 mo were purchased from BioLASCO Taiwan Co. Ltd. (Taipei) and housed in the Experimental Animal Center of National Taiwan Normal University. Food and water were provided ad libitum. All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan Normal University (No. 102008) and were in accordance with the guidelines of the Ministry of Science and Technology of Republic of China. All efforts were made to minimize animal suffering.

Setup for PTP

The model for established progressive thermopreconditioning (PTP) in the rat was demonstrated in Figure 1A and was clearly described previously.6 In brief, these animals were progressively immersed into the 42°C water bath at line 1 (~15% of total body surface area) for 5 min, line 2 (~40% of total body surface area) for 5 min, and line 3 (~70% of total body surface area) for 5 min. Three consecutive cycles of PTP (3PTP) were performed within 8 h, and the interval time between 2 PTP sessions was 4 h. Our serial studies3,6 demonstrated that 72 h after 3PTP exerts higher protection than single PTP against the cardiovascular injury. We divided these rats into 4 groups (n = 8 in each group): (1) sham control exposed to air (non-3PTP); (2) I/R of non-3PTP control (I/R); (3) 72 h after 3 consecutive cycles of PTP (3PTP); and (4) 3PTP with I/R injury (3PTP-I/R). The designed protocol was indicated in Figure 1B.

FIGURE 1.
FIGURE 1.:
The setup for 3PTP treatment in the rat. Graphic description of model operation (A). The protocols in the Sham, 3PTP, I/R, and 3PTP with I/R injury (3PTP-I/R) rats (B). 3PTP induction is performed 3 d before I/R injury, and the I/R injury includes 60 min of ischemia and 240 min of reperfusion. I/R-induced typical S-T segment elevation indicated by black arrows occurred both in I/R and 3PTP-I/R rats (C). Statistical data of R-R and P-R interval were indicated between I/R and 3PTP-I/R groups (D). *P < 0.05 as compared with the respective baseline level. # P < 0.05 as compared with the I/R group. 3PTP, triple progressive thermopreconditioning; I/R, ischemia/reperfusion.

Induction of Myocardial Infarction

Myocardial infarction was induced by ligation of the left anterior descending (LAD) coronary artery for 60 min followed by 240 min of reperfusion. In brief, the left anterior descending coronary artery close to its origin, approximately 3 mm away from the left coronary ostium, was ligated with 6-0 Prolene, and a slipknot was tied to establish reversible coronary artery occlusion. The successful performance of coronary occlusion and reperfusion was considered successful if: (1) the color of the infarcted area changed from red to white; (2) the left atrium was enlarged just after the coronary artery was ligated; and (3) the S-T segment was elevated by > 0.2 mm after left coronary artery ligation (Figure 1C). Myocardial ischemia was confirmed by the presence of regional cyanosis and S-T segment elevation on the electrocardiography (ECG).

Measurement of Hemodynamic and ECG Parameters

Under urethane (1.2 g/kg, ip) anesthesia, the rat’s trachea was intubated for artificial ventilation (Small Animal Ventilator Model 683) with 50 breaths/min, a tidal volume of 8 mL/kg, and a positive end-expiratory pressure of 5 cm H2O. Hemodynamic examination was performed as previously described.15 Hemodynamic parameters in the left ventricle, including the heart rate, left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), left ventricular pressure (LVP) (LVDP = LVSP − LVEDP), maximal rate of rise in LVP (+dp/dt), and maximal rate of fall in LVP (−dp/dt), were measured and recorded by a PE50 tubing containing heparinized saline with a pressure transducer introduced into the left ventricle via the right carotid artery. Indices of contractility and relaxation in the left ventricular function and the maximal rates of +dP/dt and −dP/dt were determined in I/R and 3PTP-I/R rats. The right, left foreleg- and left hindleg-limb leads with electrodes placed in a lead 2-like position were attached for ECG measurement. We also measured R-R interval (index for heart rate) and P-R interval (index for atrioventricular conduction velocity) from ECG tracings. All the parameters were simultaneously recorded with an iWorx 214 data recorder (IX-214; iWorx Systems, Inc.). After the assessment of the basal parameters, myocardial infarction was performed to assess the hemodynamic parameters at the stages of ischemia and reperfusion. At least 6 consecutive cardiac cycles were measured for each animal. A Powerlab data-acquisition system (ADI Instruments) was used for acquiring data throughout the experiment.

Heart Microcirculation Determination

A full-field laser perfusion imager (MoorFLPI, Moor Instruments Ltd., Devon, United Kingdom) was adapted to continuously quantitate the cardiac microcirculatory blood flow intensity in the rat as described previously.3 The imager uses laser speckle contrast imaging, which exploits the random speckle pattern generated when the heart is illuminated by a laser light. The contrast image is processed to produce a 16-color coded image that correlates with blood flow in the heart such as blue is defined as low flow and red as high flow. The negative control value is set at 0 perfusion unit (blue color) and the positive value is at 1000 perfusion unit (red color). The perfusion units were real-time analyzed by the MoorFLPI software.

Infarct Size Calculation

Infarct size was measured as described previously.3 In brief, after I/R injury, 5 mL of methyl blue was injected through the jugular vein and the heart was removed 2 min later. The heart was serially sliced from base to apex at a 3-mm interval. These slices were incubated at 37°C for 20 min in a 2% 2,3,5-triphenyltetrazolium chloride (Sigma–Aldrich) to distinguish the infarct (white) from the viable (red) myocardial area. The heart slice images were taken with a digital camera (Nikon) and projected at 5-fold magnification. The area of each region was calculated with Image-Pro Plus 6.0 (IPP 6.0; Roper Industries, Sarasota, FL) on the scanned image, and the data are presented as the proportion of infarction area to the whole area of the heart.

Western Blotting

The total proteins obtained from the infarct areas of left heart ventricle after myocardial I/R were homogenized with a prechilled mortar and pestle in extraction buffer, which consisted of 10 mmol/L Tris-HCl (pH 7.6), 140 mmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 g/mL pepstatin A, and 10 g/mL aprotinin. The mixtures were homogenized completely by vortexing and kept at 4°C for 30 min. The homogenate was centrifuged at 12 000g for 12 min at 4°C, the supernatant was collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Expression of rabbit monoclonal Hsp-70 (ab45133, Abcam, 1:5000), rabbit monoclonal Bax (ab32503, Abcam, 1:5000), rabbit polyclonal Bcl-2 (ab196495, Abcam, 1:1000), rabbit polyconal Bag3 (NBP2-27398, NOVUS, 1:2000), rabbit monoclonal Beclin-1 (3495, Cell Signaling, 1:1000), rabbit monoclonal LC3-II (3868, Cell Signaling, 1:1000), and mouse monoclonal β-actin (A5441, Sigma–Aldrich, 1:10 000) was determined by a Western blot technique. A protein sample (80 μg) was mixed with 1× sample buffer, resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membrane (Millipore, Billerica). The blot was blocked by Hyblock (Hycell, Taipei, Taiwan) for 1 min and incubated with primary antibodies overnight at 4oC. Detection of signals was measured by Western Lightning plus-ECL (PerkinElmer, Waltham).

In Situ Demonstration of Oxidative Stress and Apoptosis Formation

We compared the degree of oxidative stress by ED-1 and G-CSF stains and apoptosis by poly(ADP-ribose) polymerase 1 (PARP1) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Histochemical staining was performed on formalin fixed, paraffin-embedded heart sections with or without I/R injury by hematoxylin and eosin, mouse monoclonal ED-1 (MCA34/R, BioRAD, 1:100), goat polyclonal G-CSF (sc-49679, Santa Cruz, 1:500), rabbit monoclonal PARP1 (ab191217, Abcam, 1:500), and TUNEL stains (BioVision, CA). Briefly, paraffin sections were deparaffinized with xylene and rehydrated in an alcohol series and water. Cardiac sections were subjected to antigen retrieval and were blocked with a peroxidase-blocking reagent. Each section was incubated with the respective primary antibody overnight at 4°C. After washing, the section was incubated with Envision system-horseradish peroxidase-labeled polymer (Dako, Glostrup, Denmark) for 1 h at room temperature. The sections were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Dako) and counterstained with hematoxylin. Apoptotic cells in the heart were identified by TUNEL staining. The TUNEL method for the in situ apoptotic assay was performed according to the method of Gavrieli et al16 with minor modifications. The number of positive ED-1 and TUNEL stained cells was evaluated by counting stained cells per high power field (×400) in at least 20 randomly selected fields. The percentage of positive stained area in the brown color of PARP1 and G-CSF stained was analyzed in the section area. Twenty high-power (×400) fields were randomly selected for each section, and the value of each oxidative stress was analyzed using a Sonix Image Setup (Sonix Technology Co., Ltd., Hinschu, Taiwan, Republic of China) containing image analyzing software Carl Zeiss AxioVision Rel.4.8.2 (Future Optics Sci. & Tech. Co., Ltd., Hangzhou, China).

Statistical Analysis

All data were expressed as the mean ± SEM. The measures of interest are the comparisons of the parameters in the rats that underwent coronary ligation with or without 3PTP. Two-way ANOVA was used for establishing differences among groups followed by Tukey’s post hoc test. The columns were defined by the treatment (with or without 3PTP) and the rows were defined by the presence or absence of artery ligation. Confidence interval was placed at 95%, so that differences were adapted as significant for P < 0.05. We used Sigma Plot 12.0 for graphs preparation. All statistical analyses were performed using the SPSS software system.

RESULTS

3PTP-induced Model

As shown in Figure 1A, the rat was immersed into 42°C water bath progressively as described in the Methods and Materials section. Figure 1B demonstrated the protocol that the 3PTP treatment was performed 3 d before I/R injury for achieving the maximal protective effect.3,6 The baseline of ECG wave was similar in sham and 3PTP groups without I/R injury (upper panel of Figure 1C). During ischemic or reperfusion period, marked S-T segment elevation (Figure 1C) and prolonged R-R (a decrease in heart rate) and P-R interval (a delay in atrioventricular conduction velocity) (Figure 1D) in the ECG wave were found in I/R group. As compared with the I/R group, cardiac ischemia or reperfusion injury also evoked a similar degree of S-T segment elevation, and P-R interval elongation in 3PTP-I/R rats. However, the increased R-R interval during the ischemic period was partly inhibited in the 3PTP-I/R rats as compared with the I/R group. ECG showed that the impairment in left ventricular contractile performance and conduction velocity during I/R injury was comparable in I/R and 3PTP-I/R groups. However, 3PTP seems to play a protective role in prevention of cardiac ischemia increased R-R interval (bradycardia).

3PTP Improves Ventricular Hemodynamics

We successfully developed a technique for direct measurement of cardiac hemodynamics including LVP, left ventricular systolic blood pressure (LVSBP), and LVEDP in the anesthetized rats (Figure 2A). The determination of LVSBP, LVEDP, and ±dp/dt was indicated in Figure 2B. The baseline level of LVSBP, LVEDP, and the calculated +dp/dt and –dp/dt were all similar between I/R and 3PTP-I/R groups (Figure 2C, D, and E). In response to I/R injury, the markedly elevated LVEDP and depressed +dp/dt and –dp/dt were found in both I/R and 3PTP-I/R rats. However, the increased degree of LVEDP (Figure 2C) during I/R periods was significantly attenuated in 3PTP-I/R versus I/R groups. I/R injury also significantly depressed the level of +dp/dt (Figure 2D) and −dp/dt (Figure 2E) in 3PTP-I/R versus I/R groups. However, the degree of reperfusion injury-decreased +dp/dt value was significantly improved in 3PTP-I/R rats as compared with I/R rats implicating 3PTP partly preserving the ventricular contractile function. The data of I/R depressed −dp/dt value were not different between I/R and 3PTP-I/R (Figure 2E) implying that 3PTP does not confer protection in the ventricular relaxing function.

FIGURE 2.
FIGURE 2.:
The 3PTP effect on I/R-induced cardiac contractile dysfunction. The original graphs of I/R effect on left ventricular pressure are shown in I/R and 3PTP-I/R rats (A). In response to I/R, the left ventricular systolic blood pressure (LVSBP), end-diastolic pressure of left ventricular pressure (LVEDP), and dp/dt determination are demonstrated (B). LVEDP is significantly depressed in the 3PTP-I/R rats as compared with I/R rats (C). The statistic data of +dp/dt level are significantly decreased in I/R and 3PTP-I/R groups (D). However, after reperfusion 4 h, the +dp/dt value in 3PTP-I/R group is significantly higher than that in the I/R group (D). Compared with baseline, the statistic data of −dp/dt value is significantly decreased in both I/R and 3PTP-I/R groups (E). #P < 0.05 as compared with respective baseline level. *P < 0.05 and **P < 0.01 as compared with the I/R group. 3PTP, triple progressive thermopreconditioning; +dp/dt, maximal rate of rise in left ventricular pressure; −dp/dt, maximal rate of fall in left ventricular pressure; I/R, ischemia/reperfusion.

3PTP Improved I/R Depressed Microvascular Blood Flow and Decreased I/R-induced Infarct Size

A typical perfusion image in response to I/R from respective 1 I/R or 3PTP-I/R rat was indicated in Figure 3A. The microvascular cardiac blood flow (upper panel of I/R and 3PTP-I/R) and whole heart image (lower panel of I/R and 3PTP-I/R) displayed full cardiac blood flow and red color at baseline in I/R and 3PTP-I/R rats (Figure 3A). I/R by LAD ligation caused a markedly decreased blood perfusion (pink or green color in the base of the left ventricle) in the indicated area of I/R group. However, the microvascular blood flow perfusion was still well maintained and the regional cyanosis of infarct area of the heart was greatly depressed in the 3PTP-I/R rat (Figure 3A). The statistical data showed that in response to I/R, cardiac microvascular blood flow was significantly decreased at ischemic 5 min and 1 h and reperfusion 5 min and 4 h in the I/R and 3PTP-I/R groups versus the baseline level of respective groups (Figure 3C). However, the degree of I/R depressed microvascular blood flow was significantly improved at ischemic 1 h and reperfusion 5 min of the 3PTP-I/R group.

FIGURE 3.
FIGURE 3.:
The 3PTP effect on I/R affected cardiac blood flow. The cardiac microcirculation (A and C) and infarct size (B and D) were detected in I/R heart. The cardiac microcirculation of the selected areas indicated with yellow color square (A) is shown in response to ischemia and reperfusion from 1 I/R and 3PTP-I/R rat. Statistic data of cardiac microcirculation indicated by perfusion unit (PU) from 6 rats in I/R and 3PTP-I/R groups are depicted (C). Graph B showed the infarct areas indicated by yellow arrows in the hearts from Sham, 3PTP, I/R, and 3PTP-I/R group. Statistic data of infarct size from 6 rats in I/R and 3PTP-I/R groups are depicted (D). *P < 0.05 compared with baseline value. #P < 0.05 compared with respective I/R group. 3PTP, triple progressive thermopreconditioning; I/R, ischemia/reperfusion.

The typical 2,3,5-triphenyltetrazolium chloride stains of 5 sections from 4 groups of rats were demonstrated in Figure 3B. It is obvious that the rats that underwent LAD ligation had an infarct and that those with LAD ligation did not have infarct. We compared the infarct size in the rats that underwent the ligation with versus without 3PTP preconditioning. In the I/R and 3PTP-I/R rats, the normal area indicated by blue color and infarct size indicated by white color from apex to base of the damaged heart was clearly observed in I/R and 3PTP-I/R hearts (Figure 3B). Our data showed that the increased white color infarct size in sections I, II, III, and V was significantly reduced in 3PTP-I/R hearts as compared with that of I/R hearts (Figure 3D). This data implicated that 3PTP affords cardioprotection to reduce I/R-enhanced infarct size.

3PTP Reduces Cardiac I/R-enhanced Erythrocyte Extravasation, Leukocyte Infiltration, Structural Disruption, PARP1/TUNEL-related Apoptosis Formation, and Inflammatory G-CSF Stain and ED-1 (Macrophage) Infiltration

I/R would increase erythrocytes extravasation in the damaged tissue and evoke oxidative stress, vasoconstriction, and spasm of the coronary artery through the action of active hemoglobin released from the hypoxic erythrocytes.17,18 The G-CSF induction by a G-CSF inducer facilitated infarct expansion in association with the promotion of monocyte recruitment in the infarcted region during the early phase of acute myocardial infarction.19 In addition, I/R evoked oxidative stress like apoptosis-related mechanism in the damaged heart. We therefore determined PARP1 expression and TUNEL positive cells in the damaged heart.

As shown in Figure 4A, I/R enhanced cardiac erythrocyte extravasation, leukocyte and ED-1 infiltration, structural disruption in the area of coronary artery and cardiomyocytes, PARP1/TUNEL apoptosis formation, and G-CSF stain in I/R and 3PTP-I/R groups as compared with their baseline control. However, 3PTP treatment efficiently and significantly ameliorated cardiac I/R-enhanced erythrocyte extravasation, leukocyte infiltration, structural disruption in the area of coronary artery and cardiomyocytes, PARP1/TUNEL-related apoptosis formation, inflammatory G-CSF stain, and ED-1 (macrophage) infiltration (Figure 4B). These data implicated that 3PTP exerts a cardiac protection against I/R-induced oxidative stress, inflammation, apoptosis, and structural damage.

FIGURE 4.
FIGURE 4.:
The 3PTP effect on I/R-induced heart inflammation erythrocyte (enlarged images of black squares) and leukocyte extravasation, histological structure of coronary artery and myocyte, apoptosis level with TUNEL assay, G-CSF expression, and ED1 levels in the rat hearts with or without 3PTP pretreatment (A). The statistical data of (A) are presented in (B). *P < 0.05 vs Sham group. #P < 0.05 vs I/R group. 3PTP, triple progressive thermopreconditioning; G-CSF, granulocyte colony-stimulating factor; I/R, ischemia/reperfusion; PARP1, poly(ADP-ribose) polymerase 1; TUNEL, transferase-mediated dUTP-biotin nick end labeling.

3PTP Enhances Hsp-70 and Preserves Bcl-2 Proteins in the Heart

In the baseline level, 3PTP treatment significantly enhanced cardiac Hsp-70 (Figure 5A) and partly but not significantly increased Bag3 (Figure 5B) expression as compared with sham control. Cardiac I/R significantly depressed Hsp-70 expression (Figure 5A), decreased Bag3 (Figure 5B), and increased the Bax/Bcl-2 ratio (Figure 5C) as compared with sham control group in the I/R group. However, the degree of downregulation of cardiac protective Hsp-70 and Bag3 expression and the increase of the proapoptotic Bax/Bcl-2 ratio was significantly attenuated in 3PTP-I/R group as compared with that in the I/R group.

FIGURE 5.
FIGURE 5.:
The 3PTP effects on apoptosis-related proteins Hsp-70 (A), Bag3 (B), Bax/Bcl-2 (C), Beclin-1 (D), and LC3-II (E) expression in the I/R heart are determined with Western blot (upper panel). The statistical analysis of densitometry are shown in each lower panel. *P < 0.05 vs Sham group. #P < 0.05 vs I/R group. 3PTP, triple progressive thermopreconditioning; I/R ischemia/reperfusion; ns, not significant.

To determine the role of autophagy in response to 3PTP and I/R injury, autophagy related proteins, Beclin-1 expression and LC3-II/LC3-I expression, were determined by Western blot from the infarct area of the I/R heart. Our data showed that Beclin-1 expression (Figure 5D) and the LC3-II/LC3-I (Figure 5E) ratio were not affected by 3PTP pretreatment or I/R injury in our model with 1-h ischemia and 4-h reperfusion between I/R and 3PTP-I/R groups.

DISCUSSION

Our serial studies demonstrated that whole-body hyperthermia induces abrupt hypertension/hypotension, tachycardia, and oxidative stress.6,16 3PTP significantly attenuates the exacerbated alteration of hemodynamics and oxidative stress3,6,20 and thereby confers cardiovascular protection against arterial thrombosis6 and cardiac I/R injury.3 The present study further explored that 3PTP preserved post-I/R cardiac contractile function possibly through the upregulation of Bag3-mediated mechanisms to preserve cardiac structural integrity and contractile function and to depress I/R-enhanced infarct size and apoptosis amount.

A brief period of postexplant ischemia with enhancement by topical heating (“backtable preconditioning”) could be a simple and effective means of improving the functional recovery of heart transplants through protein kinase C or mitochondrial potassium channel.21 How can 3PTP maintain and preserve cardiac LVEDP and +dp/dt in response to I/R injury? This may be due to the structural integrity of the coronary artery and the surrounded myocardium with less fragmentation in the 3PTP-I/R group as compared with the I/R group. Molecular chaperones and cochaperones like stress-inducible Hsp-70 and constitutively expressed Hsc-70 have critical roles in regulation of protein folding, aggregation, and misfolded proteins degradation.22 Bag3 and Hsc-70/Hsp-70 can interact to stabilize myofibril structure and inhibit myofibrillar degeneration in response to mechanical stress.22 The HIV has been reported to contribute to cardiomyopathy. Upregulation of Bag3 expression decreased O2•− levels and restored contraction amplitudes to normal in the HIV-1 transgenic mice myocytes posthypoxia/reoxygenation.23 Hypoxia/reoxygenation significantly reduced neonatal mouse ventricular cardiomyocytes Bag3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux.24 Our data suggest that I/R significantly reduced cardiac Bag3 expression leading to structural disruption, whereas 3PTP enhanced Hsp-70 and partially preserved Bag3 resulting in the preservation of structural integrity as compared with I/R heart.

Reduction in I/R injury and enhancement in organ protection can be obtained by ischemic preconditioning, postconditioning, pharmacologic preconditioning,17,25 and antioxidant-based preservation solution.26 Pharmacologic preconditioning or postconditioning with G-CSF has been applied to treat acute myocardial infarction.8,9 G-CSF treatment preserves high +dP/dt and low LVEDP and improves early postinfarct ventricular expansion through promotion of reparative collagen synthesis in the infarcted area.27 G-CSF pretreatment before occlusion decreased infarct size via the increased Bcl-2 and Bcl-xL protein expression.28 However, the adverse effects by G-CSF might induce cardiac thrombosis through an inflammation–thrombosis interaction.4,9 Our data indicated that highly expressed G-CSF appeared in I/R cardiomyocyte in Figure 4. Preconditioning of G-CSF at a sublethal dose might reduce cardiac injury and infarct size. G-CSF modifies wound healing by promoting monocytopoiesis and infiltration of monocytes and macrophages into injured tissue.19 In this study, we suggest that excess G-CSF production by cardiac I/R might lead to myocardial injury and promote monocyte/macrophage infiltration, whereas the excess G-CSF expression and monocyte/macrophage infiltration can be attenuated by 3PTP. Our previous research implicated that 3PTP conferred vascular protection against FeCl3-induced arterial inflammation and thrombosis.6

In Figure 4, our data showed the increased erythrocyte extravasation in the I/R heart tissue. The increased erythrocyte extravasation may through the Fe-heme toxicity lead to secondary hypoxia and further damage. The increase in NO consumption converted the bioactivity of serotonin from a vasodilator to a vasoconstrictor in isolated coronary arterioles and accelerated NO consumption by hemoglobin-Fe(II)NO-bearing erythrocytes that may exacerbate ischemia-mediated vasospasm.29 Active free-hemoglobin release from erythrocytes has shown to induce oxidative-stress damage in acute coronary syndromes.18 Our results also demonstrated that 3PTP treatment can efficiently attenuate the erythrocyte extravasation in the damaged cardiac tissue implicating the possible anti-inflammation and antispasm effect by 3PTP.

Mitochondria play an important role in cardiac contractile capability and in regulation of apoptotic pathway.30 Cardiac mitochondrial protection increased the LVSP and +dp/dt and decreased LVEDP, the leakage of cytochrome c protein from the mitochondria to the cytoplasm, and cardiac apoptosis.30 Increased Bax or decreased Bcl-2 and Bcl-xL opened the mitochondria permeability transition pore through the outer mitochondrial voltage-dependent anion conductance channel to release cytochrome C into cytosol.31 Increased mitochondrial Bcl-2/Bax ratio in the cardiac tissue in our present study may block the voltage-dependent anion conductance channel opening to decrease cytosloic Cytochrome C release subsequently reducing apoptosis formation in the I/R tissue. Our previous study evidenced that 3PTP enhances several defense mechanisms (upregulating MnSOD, Bcl-2, and Bcl-xL) to counteract I/R-induced mitochondrial dysfunction by preserving mitochondrial Bcl-2 expression and decreasing cytosloic cytochrome C release.3 In the present study, we further found that the upregulation of Bag3 in the 3PTP rat heart (Figure 5B). Mitochondrial abnormalities impact the development of myofibrillar myopathies. One recent report suggested that Bag3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in Bag3 expression impact cardiomyocyte function.32 Taken together, we can find that 3PTP could protect mitochondrial function and structure in the organ/tissue through the wide-range defense mechanisms including the upregulating Hsp-70, MnSOD, Bcl-2, Bcl-xL, and Bag3.

In comparison to our 3PTP model in rats, the frequency of sauna or infrared sauna bathing has been reported to improve the prediction of the long-term risk for fatal cardiovascular diseases mortality and to improved cardiac function.33,34 Sauna bathing refers to the process of physiotherapy with steam in a closed room. The temperature of sauna steam might be over 60°C in the air but lower to 30°C on the floor. Compared to sauna bathing, our 3PTP model in water bath has the advantages of more average thermo distribution and conduction. Because of average thermodistribution and conduction, the temperature of 3PTP is lower than sauna bath and reduces the heat shock risk of cardiovascular system. 3PTP enhanced the cardiovascular protection including antioxidant, anti-inflammatory, and antiapoptotic mechanisms.3,6 3PTP via PI3K/Akt signaling pathway upregulates Hsp-70 and eNOS expression and increases NO production.6 In clinical aspects, we may adapt 3PTP for inducing cardiovascular protection in the patients 3 d before a performance of cardiac surgery. For prevention of abrupt hemodynamic fluctuation during whole-body hyperthemia, a safe strategy for progressive water bath immersion at 42°C or lower like our 3PTP model is recommended. Because the cardiovascular protection by single PTP may be limited for a shorter duration and lower temperature (<42°C), 3PTP with the increased frequency for thermopreconditioning confers further cardiovascular protection against thrombus formation and myocardial infarction.3,6 We suggest that introducing the 3PTP procedure in the human subjects with cardiovascular diseases or heart surgery patients requires the standard procedures of clinical trials. We suggest immersion to water bath progressive to the knee, belly, and breast each 5-min period. However, humans are much larger then rats and, thus, the thermal diffusion is completely different. We will test/optimize a suitable condition on larger animals in future.

In conclusion, a modified 3PTP provides a wide-range defense mechanism and strategy to provide cardiac protection against I/R injury possibly by the Bag3-mediated preservation of structural and functional integrity in the damaged heart.

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