The influence of ischemic pre-conditioning and inhibition of the ubiquitin proteasome system on cardiac ischemic injury in a murine model

Abstract

Myocardial infarction (MI) is a state when the flow of blood stops in a coronary artery or a succeeding vessel resulting in hypoxia leading to damage in the downstream cardiac tissue. Ischemia/reperfusion (I/R) injury leads to significant tissue damage and initiation of inflam-matory reactions. The roles of nuclear factor (NF)-κB and hypoxia inducible factor (HIF) in myocardium are still under discussion. It has been reported, that reduction of NF-κB activity by attenuation of the ubiquitin proteasome system (UPS) exerts bbeneficial effects on I/R injury in the myocardium. Furthermore, preceding short periods of ischemia before I/R injury like in Angina pectoris are known to protect the heart. Based on these findings, we hypothesized that repetitive short cycles of I/R will raise the cardiac HIF-levels and attenuate NF-κB levels, thus reduce inflammation. It was also hypothesized that pre- and post-conditioning with bortezomib (BZ), a blocker of UPS, will decrease ischemic injury, help to preserve cardiac function and facilitate remodeling after I/R.<br /> 9-12 weeks old female C57BL/6 mice underwent LAD constriction or sham surgery. Mice were divided into 5 different groups. Sham group; 60 min group treated with 15 min sham ischemia daily for 6 days prior to 1 x 60 min ischemia; repetitive group treated with 15 min ischemia daily for 6 days prior to 1 x 60 min ischemia, repetitive + BZ group received the same protocol complemented with 3 doses of BZ; 60 min ischemia + BZ group received the same treatment as the 60 min group complemented with 3 doses of BZ. 24 h post I/R injury changes were monitored in all groups. 21 d post ischemia success of remodeling was investigated in the sham, the 60 min, the 60 min + BZ and the repetitive groups.<br /> 24 h after I/R, 60 min group exhibited the biggest infarct size while all other groups developed significantly smaller infarct size. The 60 min group exhibited significantly reduced end-systolic pressure and ejection fraction as well as reduced dP/dt_max and dP/dt_min accompanied by an increased end-systolic as well as end-diastolic volume. All pre-conditioned groups exhibited less impaired cardiac function than the 60 min group. Interestingly, short periods of I/R, BZ treat-ment and combination of both in advance significantly reduced the malignant influence of I/R on the cardiac performance. Among the pre-conditioned groups the repetitive I/R treatment resulted in the best hemodynamic function 24 h after I/R.<br /> The investigation of inflammatory mediators 24 h after I/R revealed that all I/R groups exhibited increased levels of HIF-1α being highest in both BZ groups. IL-6 and IL-1β as well as ICAM-1 were expressed at the significantly highest levels in the 60 min + BZ group. <br /> Evaluation of the scar size after 21 d after I/R revealed that the scars of the animals in the 60 min and in the 60 min + BZ groups were significantly larger than in the sham and in the repetitive group, whereas the scar size did not differ significantly between later groups. Evaluation of the cardiac function revealed totally different results than after 24 h post I/R. Significantly reduced ejection fraction as well as decreased dP/dt_max and dP/dt_min was found in the 60 min group in comparison to the sham group. It is to be noted here that the end-systolic and end-diastolic pressures were compensated in 60 min group and in all other experimental groups. Surprisingly the 60 min + BZ group developed only slightly better cardiac function than the 60 min group. The repetitive group exhibited the most well preserved cardiac function in comparison to the I/R groups. Ejection fraction, end-systolic and end-diastolic pressure, and volume as well as dP/dt_max, dP/dt_min and cardiac output were found to be better preserved in the repetitive group than in any other experimental group. <br /> Taken together, the pre-conditioning with repetitive ischemia was successful in improving cardiac performance even in the long term. This study is the first one to report the upregulation of HIF-1α as result of BZ and repetitive I/R treatment. All experimental groups which were exposed to I/R exhibited increased HIF levels. In general, we did not gain the desired attenuation in inflammation by the BZ treatment. Also the BZ treatment was not found to be as successful as the repetitive treatment in long term. However, repetitive treatment cannot be applied in clinical setting. Thus, possibly another specific way to increase HIF level may help or a new protocol of BZ treatment has to be established in order to reduce I/R injury.