In this study, VSPP and +dp/dtmax indexes were used to evaluate the systolic function of the heart. A high LVSPP value indicates strong myocardial contractility. In addition, an increase in the +dp/dtmax value indicates an increase in myocardial contractility, while a decrease in the +dp/dtmax reflects an impairment of myocardial contractility [5]. The diastolic function was evaluated by LVEDP and -dp/dtmax. LVEDP refers to the preload of the left ventricle, reflecting the diastolic function and compliance of the left ventricle. -dp/dtmax is the most sensitive index by which to reflect diastolic function [6, 7].
The experimental results showed that the left ventricular systolic and diastolic function decreased after I/R and that endurance exercise had a significant protective effect on myocardial systolic and diastolic function. This showed that the LVSSP and ±dp/dtmax of the rat heart after exercise training were significantly higher than those of the control sham operation group after 40 min of ischemia and 50 min of reperfusion, and the LVEDP was significantly lower than that of the control sham operation group after 40 min of ischemia and 50 min of reperfusion. The results also showed that exercise training could increase the anti-ischemic ability of the heart and protect the heart function during ischemia-reperfusion injury. The LVSSP and ±dp/dtmax in the train, inhibitor, and I/R model group were significantly lower than those in the train and I/R model group after 40 min of ischemia and 50 min of reperfusion, and the LVEDP after 40 min of ischemia and 50 min of reperfusion were significantly higher than those in the train and I/R model group. The results revealed that the inhibitor inhibited the action of exercise-induced protection of myocardial systolic and diastolic function during reperfusion.
Some possible reasons for the decrease in function during myocardial ischemia are that myocardial ischemia leads to the destruction of contractile components, insufficient uptake of Ca2+ by the sarcoplasmic reticulum, insufficiency of myocardial energy supply, acidosis, etc. Some possible reasons for the decline of myocardial function during reperfusion are Ca2+ overload, energy metabolism disorder, white blood cell mediated injury, etc. Endurance exercise training can alter the shape of the heart and cause the thickening of the exercise-induced myocardium, which is characterized by the increase of MHC activity of cardiac myosin, the coincidence of capillary proliferation and ratio of muscle fiber, the matching of myocardial cell growth, and non-myocardial cell growth, and the adaptable increase of the heart volume. One possible mechanism of the improvement of the anti-injury ability of the heart in I/R after the intervention of moderate-intensity endurance exercise is that the capillary hyperplasia and the increase of oxygen supply ability after exercise, thereby improving the energy supply of the heart, along with the increase of the synthesis of some other endogenous protective factors, such as NO, which in turn activates the related mediators and effectors, etc. The LVSSP and ±dp/dtmax of the heart in the train, inhibitor, and I/R model group were shown to be significantly lower than those in the train and I/R model group, and the LVEDP was significantly higher than that in the train and I/R model group. It is suggested that the inhibitor inhibits the synthesis of NO in vivo, blocks its reaction with mediators and effectors, and cancels the protective effect of endurance exercise on I/R-injured hearts, thus indicating that the protective effect of exercise adaptation to myocardial injury is at least partially mediated by the NO pathway.
The change of serum myocardial enzymes is one of the main indicators of the degree of myocardial injury. Under normal conditions, the activities of CK, LDH, and AST in serum are lower than those in the tissues. When the cell membrane is damaged and the permeability is increased, it can seep from the cell to the extracellular level [8]. The experimental results in this study showed that the activities of CK, LDH, and AST in the serum of the control I/R model group were significantly lower than those of the control sham operation group (P < 0.01). Compared with the control group, the activities of CK, LDH, and AST in the train and I/R model group were significantly higher than those of the control I/R group (P < 0.01). Compared with the exercise training model group, the activities of CK, LDH, and AST in the train, inhibitor, and I/R model group were significantly lower than those in the exercise training model group (P < 0.01 Or P < 0.05), thus indicating that I/R causes myocardial damage. It was observed that endurance exercise has a protective effect on myocardial damage caused by I/R. Finally, L-NAME inhibits the protective effect of endurance exercise on the myocardium, further indicating that NO plays an important role in the protection of exercise pre-adaptation myocardium.
The experimental results showed that, compared with the control sham operation group, the expression of iNOS protein in control I/R model group was higher than that in the control sham operation group (P < 0.05), and the expression of eNOS protein in control I/R model group was significantly higher than that in the control sham operation group (P < 0.01). Compared with the control I/R model group, the protein expressions of iNOS and eNOS in the rat myocardium of the train and I/R model group were significantly higher than those of the control I/R model group (P < 0.01), thus indicating that endurance exercise can improve the protein expression of iNOS and eNOS in the myocardium of I/R. One possible mechanism of resistance to I/R injury of moderate-intensity endurance training is to activate the expression of iNOS and eNOS, and increase the synthesis of NO through exercise. The increase of NO biosynthesis is necessary to trigger the protection of the EP delayed phase. At the same time, NO can also participate in the signal transduction pathway as a signal molecule to regulate some physiological processes and achieve the purpose of protecting the heart [9]. For example, NO can activate soluble guanylate cyclase (SGC), increase the concentration of cGMP in cells, reduce Ca2+ influx through cGMP acting on the L-calcium channel, or stimulate cGMP to reduce cAMP content and reduce myocardial contractility, thus ultimately reducing oxygen consumption and energy demand. NO can also directly act on PKC, MARK, mitochondria, etc., or on KATP, heat shock protein, sodium hydrogen exchanger, etc. ,through PKC to play a role. Compared with the train and I/R model group, the expression of iNOS in the train, inhibitor, and I/R model group significantly decreased (P < 0.01), and the expression of eNOS in the train, inhibitor, and I/R model group significantly decreased (P < 0.05). Previous studies have shown that L-NAME can slightly reduce the synthesis of endogenous NO in resting rats, including the content of NO in the liver, spleen, bone marrow, and other tissues and cells [10]. The experimental results showed that L-NAME inhibited the protein expressions of iNOS and eNOS blocked the biosynthesis of NO and caused the protective effect of endurance exercise to disappear. Therefore, it is observed that NO plays an important role in the myocardial protection induced by exercise preconditioning.
PKC is a serine threonine kinase with a single peptide chain structure, which widely exists in many tissues, organs, and cells. PKC is a key part of the NO intracellular signal transduction pathway [11, 12]. The experimental results showed that the expression of PKC in the myocardial tissue of the control I/R model group was higher than that of the control sham operation group (P < 0.01). Compared with the control I/R model group, the expression of PKC in the train and I/R model group was significantly higher than that in the control I/R model group (P < 0.01). The results showed that the expression of PKC increased at the same time of myocardial injury. Exercise had a protective effect on myocardial I/R injury and increased the PKC expression at the same time. Compared with the train and I/R model group, the expression of PKC in the train, inhibitor, and I/R model group was significantly lower than that in the train and I/R model group (P < 0.01). The results also showed that the NO inhibitor decreased the expressions of iNOS and eNOS protein, while at the same time, the protein expression of PKC also showed the same trend of decrease. One possible mechanism causing the increased trend of PKC expression at the same as that of NO expression is that exercise causes relative hypoxia of myocardial tissue and stimulates the myocardium to release endogenous trigger substances such as NO, adrenaline, bradykinin, opioid titanium, etc. These substances are coupled with Guanosine regulatory protein (G-protein) after binding with the receptor and activate the inhibitory G protein (Gi), which then decreases the activity of acylate cyclase, reduces the number of Ca2+ channels on the cell membrane, decreases the Ca2+ influx, and reduces the myocardial contractility. At the same time, the binding of the endogenous active substance with the receptor can activate another G protein (Gq). GQ is the membrane effector enzyme that activates PLC. PLC initiates the release of the intracellular calcium pool and activates PKC, and then initiates a series of downstream cascade reactions [13]. The protein expression trend of PKC was the same as that of the NO inhibitor group. This revealed that moderate-intensity endurance training stimulated cardiomyocytes to release endogenous active substances NO and NO combined with the corresponding receptors to activate Gq protein and then activate PLC. PLC decomposed PIP2 into IP3 and DAG, then activated PKC, which had a protective effect on the heart. This further illustrates that PKC is downstream of the trigger substance of NO, and plays an intermediary role in the signaling pathway of exercise preconditioning to improve myocardial ischemic tolerance.
The expression of KATP is different in various tissues. KATP in the myocardium is composed of Kir6.2 and SUR2A, KATP in smooth muscle is composed of Kir6.2 and SUR2B, KATP in pancreas β cell, brain nerve cells are composed of Kir6.2 and SUR1, and KATP in the kidney is composed of Kir1.1 and CFTR [14, 15]. In this study, the Kir6.1 and Kri6.2 gene expressions in rat myocardial tissue were consistent with those observed in previous studies.
The role of KATP in the pancreas and nervous system has been widely studied [16, 17]. KATP in the heart is normally closed. When ischemia, hypoxia, and energy shortage occur, KATP channels open to play a role in protecting the heart muscle. Previous research shows that the activity of the KATP channel depends on the number and the opening degree of the KATP channel in cells. Exercise increases the expression of Kir6.1, Kir6.2, and SUR2 in cardiomyocytes, thus indicating that increasing the expression of various subunits of the KATP channel is of great significance in improving myocardial anti-I/R injury [18]. As an effective substance, KATP is an important part of the signal transduction pathway of EP-induced myocardial protection.
The study results showed that the expressions of Kir6.1 and Kri6.2 in control I/R model group were significantly higher than those in the control sham operation group (P < 0.01), thus indicating that KATP was open to protect myocardium when the myocardial injury occurred. Compared with the control I/R model group, the expressions of Kir6.1 and Kri6.2 in the train and I/R model group were significantly higher than those in the control I/R model group (P < 0.01), thus indicating that endurance exercise increased the activity of the KATP channel and enhanced the protective effect on injured myocardium. One possible mechanism of the increase of KATP gene expression is that I/R injury causes the decrease of blood glucose level and muscle glycogen content, causes the KATP to open, leads to the outflow of K+, increases the potassium conductance of cardiomyocytes and vascular smooth muscle cells, accelerates repolarization, shortens the action potential time course, closes the Ca2+ channel, reduces Ca2+ overload in cardiomyocytes, and reduces the exchange of Na2+-Ca2+, in turn reducing myocardial injury. Previous studies have shown that I/R causes the sympathetic adrenomedullin system to secrete a large number of catecholamines, while the oxidation of catecholamines may produce oxygen free radicals [19, 20]. Oxygen free radicals may cause the loss of selective permeability of finger membrane and hinder the transport of Ca2+ ions in cells. Endurance exercise can accelerate the opening of KATP, increase the intake of catecholamine, inhibit the production of interleukin-oxygen free radicals in cells when the I/R is cleared, reduce the formation of free fatty acids and other harmful metabolites, increase the energy supply of intracellular ATP decomposition and coupling with calcium pump, and stabilize ATP production. Compared with the train and I/R model group, the expressions of Kir6.1 and Kri6.2 in the train, inhibitor, and I/R model group decreased significantly (P < 0.01). The results showed that L-NAME inhibited the expression of iNOS and eNOS protein, as well as the gene expression of mediated PKC and effector KATP.