| Ischemia-reperfusion injury often occurs in clinic, including thrombolysis after acute myocardial infarction, blood vessel formation and extracorporeal circulation. It causes arrhythmias, heart dysfunction and cell death. A number of studies reveal that intracellular Ca2+ overload is an important cause for ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. It is accepted that intracellular Ca2+ overload causes contracture. This not only disrupts the cell to cell contact, and the integrity of sarcolemmal membrane, but also causes a secondary massive Ca2+ influx, which exacerbates contracture, and leads to heart injury. In contrast, in isolated cardiac cells, which are free from mechanical interactions with adjacent cell, injuries still occur upon exposing to calcium overload. Therefore, it is highly likely that other mechanisms are involved in calcium overload-induced heart injury.Calpain is a group of Ca2+-dependent intracellular cysteine proteases. During myocardial ischemia and reperfusion, calpain is translocated to sarcolemmal membrane, cleaves its substrates, such as a-fodrin, and Na+/K+-ATPase, and lead to damage on cell structure and function. Taking into consideration the documentation that contracture is a cause of intracellular Ca2+ overload, it is necessary to illustrate the relationship between calpain and contracture in calcium overload-induced heart injury.Clinical studies have revealed that patients with postoperative atrial fibrillation are accompanied with high serum chloride concentrations following coronary artery bypass grafting surgery. Laboratory studies have demonstrated that interfering with transmembrane Cl- flux with a Cl- channel blocker, such as SITS or NPPB, protects cardiac cells against anoxia/re-oxygenation injury. However, the injury mechanisms are not yet clear. In view of the importance of Ca2+ overload in the ischemia-reperfusion injury, whether the cardiac injury induced by abnormal of transmembrane Cl- flux is relevant to calcium overload or calpains warrants an investigation.Aims 1. To determine the relationship between calpain and contracture in Ca2+ overload-induced heart injury. 2. To determine whether calpain mediated the heart injury caused by Cl-.Methods Part 1: To determine the relationship between calpain and contracture in Ca2+ paradox-induced heart injury SD male rats of 250-300 g body weight were heparinized and decapitated, their hearts were quickly removed and perfused with normal Krebs-Henseleit(KH) solution in Langendorff apparatus. To study the effects of the drugs on the normal heart, the experiments were randomly divided into: 1. Control group: the hearts were perfused with normal KH solution. 2. The MDL28170 control group: the hearts were treated with 10 mmol/L MDL28170 for 8 min followed by perfusion with normal KH solution. 3. KB-R7943 control group: the hearts were treated with 10 mmol/L KB-R7943 for 8 min followed by perfusion with normal KH solution. To study the effects of MDL28170 and KB-R7943 on 3-min Ca2+ paradox-induced heart injury, the experiments were randomly divided into: 1. 3-min Ca2+ paradox group: the hearts were successively perfused with Ca2+-free KH solution for 3 min and KH solution containing 1.25 mmol/L Ca2+ for 30 min. 2. 3-min Ca2+ paradox with 10 mmol/L MDL28170 group: MDL28170 was administered during a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal Ca2+-containing KH solution. 3. 3-min Ca2+ paradox with 10 mmol/L KB-R7943 group: KB-R7943 was administeredduring a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal Ca2+-containing KH solution. To study the effects of MDL28170 and KBR-7943 on 5-min Ca2+ paradox-induced heart injury, the experiments were randomly divided into: 1. 5-min Ca2+ paradox group: the hearts were successively perfused with Ca2+-free KH solution for 5 min and KH solution containing 1.25 mmol/L Ca2+ for 30 min. 2. 5-min Ca2+ paradox with 10 mmol/L MDL28170 group: MDL28170 was administeredduring a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal Ca2+-containing KH solution. 3. 5-min Ca2+ paradox with 10 mmol/L KB-R7943 group: KB-R7943 was administeredduring a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal Ca2+-containing KH solution. The perfusion durations are same in all groups. Left ventricular end-diastolic pressure(LVEDP) and left ventricular pressure(LVP) were recorded. The LVEDP was used to reflect contracture, and left ventriculardeveloped pressure(LVDP) was calculated to reflect the systolic function(n=6). At the end of experiments, TTC staining were preformed to measure the myocardial injury area(n=6). During the first 2 minutes of Ca2+ repletion, the coronary effluent was also collected and lactate dehydrogenase(LDH) and troponin I(Tn I) were measured(n=6). In addition, after 2 min of Ca2+ repletion heart tissues were collected to measure calpain activity, calpain translocation and the degradation of a-fodrin(n=4).Part 2: To determine whether calpain mediated the heart injury caused by ClSD male rats were heparinized and decapitated, their hearts were quickly removed and perfused with normal KH solution in Langendorff apparatus. To observe the effects of the drugs on normal hearts, the experiments were divided into: 1. Control group: the hearts were perfused with normal KH solution. 2. Drugs control group: the hearts were treated with 10 or 5 mmol/L NPPB, DIDS, 90 mmol/L Cl-(replacement Cl- with gluconate) for 8 min followed by perfusion with normal KH solution. To observe the effects of the drugs on myocardial ischemia-reperfusion injury, the experiments were divided into: 1. I/R group: the hearts were subiected to global no-flow ischemia for 45 min and reperfusion for 2 h. 2. I/R with NPPB, DIDS or 90 mmol/L Cl- group: the hearts were administered with 5 mmol/L NPPB, 5 mmol/L DIDS or 90 mmol/L Cl- for 3 min before ischemia and for the first 5 min of reperfusion. 3. I/R with MDL28170 group: MDL28170 were administered with 3min before ischemia and for the first 5 min of reperfusion. To observe the effects of the drugs on Ca2+ paradox-induced injury, the experiments were divided into: 1. Ca2+ paradox group: the hearts were successively perfused with Ca2+-free KH solution for 3 min and KH solution containing 1.25 mmol/L Ca2+ for 30 min. 2. Ca2+ paradox with drugs group: The drugs were administered during a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal, Ca2+-containing KH solution.3. Ca2+ paradox with MDL28170 group: MDL28170 was administeredduring a 1-min perfusionperiod prior to treatment with Ca2+-free KH solution, during the entire period of Ca2+-free KH solutionand for the first 2 minutes of treatment with the normal, Ca2+-containing KH solution. The perfusion durations are same in all groups. LVEDP and LVP were recorded, and LVEDP and the calculated LVDP were used to reflect cardiac function(n=10). Cell death were evaluated by measuring the level of LDH, myocardial injury area, the apoptotic index, and caspase-3 activity(n=6). The calpain translocation and degradation of a-fodrin were detected to indicate calpain activity(n=4).Results Part 1: The relationship between calpain and contracture in Ca2+ paradox-induced heart injury 1. At the end of experiments compared with control group, calpain inhibitor MDL28170 and the Na+/Ca2+ exchanger inhibitor KB-R7943 had no effects on the injury area and function in normal hearts. 2. The heart in 3-min Ca2+ paradox group exhibited increased LVEDP, suggesting occurrence of contracture. It also displayed no contractile performance, characterized by diminishing LVDP, no viable tissue as shown by TTC staining and an increase release of LDH in the coronary effluent. In addition, the calpain activity, the percentage of the membrane anchorage of calpain, and the degradation of a-fodrin, a calpain substrate, were significantly increased. 3. MDL28170 had no effects on LVEDP in 3-min Ca2+ paradoxical hearts, but it reduced myocardial injury area and the release of LDH, and improved contractile function recovery. Compared to MDL28170, KB-R7943 significantly decreased the level of LVEDP, which was accompanied with less cell death and LDH release, and better contractile function recovery. Both MDL28170 and KB-R7943 reduced the activity of calpain, the percentage of the membrane anchorage of calpain, and the degradation of a-fodrin.4. Compared with 3-min Ca2+ paradox, 5-min Ca2+ paradox did not impose any obvious alterations in TTC staining and LDH release. It also caused an increase in the level of LVEDP, although the extent was lower than that in 3-min Ca2+ paradox group. 5. Either MDL28170 or KB-R7943 did not reduce the level of LVEDP in 5-min Ca2+ paradoxical hearts; they did not rescue contractile function. In contrast, they still preserved tissue surviving, which was accompanied with decreases in the activity of calpain, the percentage of the membrane anchorage of calpain and the degradation of a-fodrin. 6. The amount of troponin I in the coronary effluent in both 3-min and 5-min Ca2+ paradox group was increased, and troponin I release in 5-min Ca2+ paradox group was higher than that in 3-min Ca2+ paradox group. KB-R7943 significantly reduced the loss of troponin I in both 3-min and 5-min Ca2+ paradoxical hearts. However, MDL28170 had no effect on troponin I release.Part 2: The role of calpain in Cl--induced heart injury 1. In the normal control hearts NPPB at 10 mmol/L slightly but signigicantly increased the size of the myocardial injury area; DIDS at 10 mmol/L irreversibly increased LVEDP. But NPPB and DIDS at 5 mmol/L had no effects on myocardial injury area and function in normal control hearts. Treatment with 90 mmol/L Clhad no effects on the normal hearts. 2. Similar to MDL28170, blockade of transmembrane Cl- flux with either 5 mmol/L NPPB, 5 mmol/L DIDS, or 90 mmol/L Cl- reduced myocardial injury area and the release of LDH, the apoptotic indices and caspase-3 activity.Furthermore, all three treatments improved function recovery in ischemia-reperfused hearts. They also decreased the level of LVEDP, but the difference between the NPPB treatment group and I/R group did not reach statistical significance. 3. Similar to MDL28170, blockade of transmembrane Cl- flux with either 5 mmol/L NPPB, 5 mmol/L DIDS, or 90 mmol/L Cl- reduced myocardial injury area and the release of LDH, the apoptotic indices and caspase-3 activity.Moreover, all three treatments decreased the level of LVEDP, which was accompanied with improved function recovery in Ca2+ paradoxical hearts. 4. Compared with control group, the hearts subjected to ischemia-reperfusion or Ca2+ paradox exhibited an increase in the percentage of the membrane anchorage of calpain, and the degradation of a-fodrin, a substrate of calpain. More importantly, NPPB, DIDS, 90 mmol/L Cl-, as well as MDL28170 reduced these changes.Conclusions 1. The results suggest that in addition to contracture, activation of calpain is an important pathway in mediating intracellular Ca2+ overload-induced heart injury. Contracture is the main cause for contractile dysfunction, whereas calpain is involved in cell death. 2. The data provide evidence suggesting that the blockade of transmembrane Cl- flux mitigates I/R-induced cardiac injury via the inhibition of calpain activity. |
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