Interventional Cardiology Journal Open Access

Myocardial ischemia and reperfusion injury (MIRI) is a serious complication after percutaneous coronary intervention, which leads to heart failure, increased infarction size, and severe arrhythmias [1,2]. Various signaling pathways were found to be participated in the process of MIRI, such as phosphatidylinositol 3-kinase (PI3K)/AKT, extracellular signal-regulated kinase (ERK), endothelial NO synthase (eNOS), etc[3-5]. Recently, more and more studies demonstrate that Na⁺/Ca²⁺/Calmodulin-dependent protein kinase II (CaMII) feedback plays vital role in heart failure [6]. Over-activity of CaMKII enhanced the late Na⁺ current(I_NaL), caused intracellular Na⁺ ([Na⁺]_i) increasing, changing the equilibrium of the Na⁺-Ca²⁺ exchanger (NCX) to impair forwardmode (Ca²⁺ extrusion), and aggravating reverse-mode (Ca²⁺ influx) exchange. Subsequently, this Ca²⁺ overload further activate CaMKII and cause a feedback loop of CaMKII/[Na⁺]_i/ [Ca²⁺]_i in heart failure. However, the role of CaMKII/[Na⁺]_i/[Ca²⁺] i feedback in MIRI is still unclear. In the present study, we took the published MIRI action potential model [7,8], 50% blocking the voltage-dependent sodium current(I_Na) and CaMKII, to observe the effect of I_Na(-) and CaMKII(-) on MIRI.

To ensure the influence of I_Na and CaMKII on MIRI, we used Roberts-Christini MIRI AP model [7,8], which combined the dynamic changes of intracellular pH (pH_i), extracellular pH(pH_e), 13 ion currents and 6 pump exchangers. All simulations were run using a pacing rate of 1 Hz. Four groups were set as: Control (MIRI only), I_Na 50% block, CaMKII 50% block and both (I_Na 50% and CaMKII 50% block), respectively.

As we showed in Figures 1A-1C, during ischemia stage, intracellular and extracellular pH were reduced to 5.8 and 6.2 from pre-ischemia stage(pH_e 7.4, pH_i 7.2), respectively. The AP amplitude was decreased alternatively, which were consistent with the previous report [9]. In Figures 1D-1G, we can see that sodium overload existed in 10-minutes ischemia stage, compared with pre-ischemia, exacerbating in 10-minutes reperfusion stage. I_Na 50% block (blue line) slightly reduced intracellular sodium concertration ([Na]_i), while activated CaMKII 50% block (green line) and both block (black line) reduced [Na]_i more significantly. Calcium overload were found in control (red line) and I_Na 50% block(blue line), while it was reduced significantly in CaMKII 50% block and both block (Figure 1H), which reveals that CaMKII is essential to keep calcium overload, not I_Na. Consistently, we found NCX current were also reduced in CaMKII 50% block and both block (Figure 1I), which suggested that NCX played downstream role of CaMKII and calcium overload.

Figure 1: Summary of simulations. Changes in (A) intracellular pH (pH_i), (B) extracellular pH (pH_e), (C) action potential, (D) intracellular sodium (Na⁺_i ), (E) maximum intracellular calcium (Ca²⁺_i), (F) I_NaCa current during simulated ischemia (gray region) and (G) Na⁺_i (H) Ca²⁺_i (I) I_NaCaat 10 minutes reperfusion during four simulations: Control(with no I_Na and CaMKII inhibition) (red), I_Na inhibition at 50 percent (blue), CaMKII inhibition at 50 percent (green) and both I_Na and CaMKII inhibition at 50 percent (dark).

To further explore the mechanisms of CaMKII and I_Na on MIRI, we analysed various currents which might be related with sodium and calcium overload. As we showed in Figures 2A-2I, during preischemia stage, we can see that Peak I_Na keeps in 350 uA/uF in normal, while it reduced half (175 uA/uF) in I_Na 50% block and both groups. All other currents, such as calcium-sodium exchanger (I_CaNa), I_NaL, background sodium current (I_Nab), sodium-potassium exchanger (I_NaK), ATP-inactivated potassium current (I_Katp), sodiumcalcium exchanger (I_NCX), sodium-bicarbonate symporter (I_NBC), sodium-exchanger (I_NHE), are nearly the same values in these four groups. During 10-minutes ischemia stage, Peak I_Na among the four groups were reduced significantly (40-60 uA/uF). Peak I_Nab increased in CaMKII 50% block and both I_Na-CaMKII block groups, while all other currents kept the simular values among four groups. During 10-minutes of reperfusion stage, Peak I_CaNa were significantly increased in CaMKII 50% block and both I_Na-CaMKII block groups, which reveals that more calcium ions were transferred from inside to outside of cells in these two groups, while other currents changed little. Therefore, I_CaNa takes responsibility for reducing calcium overload in MIRI.

Figure 2: Changes of maximum currents and exchangers during four simulations: Control (with no I_Na and CaMKII inhibition) (red), I_Na inhibition at 50 percent (blue), CaMKII inhibition at 50 percent (green) and both I_Na and CaMKII inhibition at 50 percent (dark). (A) peak I_Na, (B) peak I_CaNa, (C) peak I_NaL, (D) peak I_Nab, (E)peak I_NaCa, (F)Peak I_NaK,(G)Peak I_Katp, (H)I_NHE , (I)I_NBC during pre-ischemia(0-isc), 10 minutes ischemia(10-isc) and 10 minutes reperfusion(10-rep) stages.

As for the limitation, we used Roberts-Chritini MIRI AP model, which was the latest AP model describing the acidosis state and various currents' changes in MIRI, more information might be focused on the Markov state of ion channels.

Conclusion

The mechanisms of MIRI is complicated, considering the system is combined with various nonlinear components and frequently exhibit non-intuitive behavior, using the dynamic MIRI model showed in the present study will provide a powerful method to reveal the vague secrets. Here we used the MIRI model tof provide a solve to the interesting question, what is the focus of CaMKII/[Na⁺]_i/[Ca²⁺]_i feedback, which is more important between CaMKII and I_Na. Our study indicate that CaMKII plays a key role in calcium overload in MIRI, not I_Na, blocking of CaMKII activated I_NCX and I_CaNa currents, transferred more Ca²⁺ to the outside of cell, subsequently reducing calcium overload, and finally alleviated MIRI. Inhibited activation of CaMKII might be a potential treatment in protection against myocardial ischemia and reperfusion injury.

Acknowledgments

This study was supported by grant No. 2014A030313402 from Guangdong Scientific Foundation, No. A2014271 from Guangdong Medical Research Foundation and Project of Wenxin herbs on AF from Guangdong science and Technology Foundation (to Jianyong Qi), Special Funds of Guangdong hospital of Chinese Medicine to Minzhou Zhang 2013,2014 and Jianyong Qi 2014. Special fund to Juan Yu from 2015 Guangdong Administration of Chinese Medicine

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