Isoprenaline Enhancement Of Sarcoplasmic Reticulum Calcium Leak In Cardiac Myocytes Is Mediated By Calcium/Calmodulin-Dependent Protein Kinase Ⅱ

Objective: To explore whether sarcoplasmic reticulum (SR) calcium leak in intact rat ventricular myocytes depends on calcium/ calmodulin-dependent protein kinaseⅡ(CaMKⅡ) or protein kinase A (PKA).Methods: 1. We take the"four-step"enzyme digestion method to isolate Spraque-Dawley(SD) rats'ventricular myocytes. The"four-step"included perfusion with Tyde's solution (NT) without Ca²⁺, 60μmol/ L Ca²⁺ NT and 180μmol/L Ca²⁺ NT respectively, incubation with KB solution in room temperature. 2. There were five groups in our study: the control group, isoprenaline (ISO) group, CGP group, H-89 group, KN-93 group. After loaded with Fura-2/AM, the isolated ventricular myocytes were perfused with ISO. In this way, we analogy the state of over activatedβ-adrenergic receptors in chronic heart failure. 3. The calcium fluorescence intensity ratio was measured by calcium imaging techniques. After myocytes were perfused with drugs of different groups, Ratio 1(R1) was first measured. Then we added tetracaine, which is the inhibitor of SR calcium release channel, and got R2 value. After rapidly washed out tetracaine, caffeine was added to trigger the SR calcium release, and we got the value of R3. (R1-R2) value is on behalf of myocardial diastolic calcium leak, (R3-R1) value represents the capacity of SR calcium load, the ratio between the two represents the level of calcium leak.Results: 1.There was no statistical significance among five groups in SR calcium capacity induced by caffeine. 2. The drop level of calcium fluorescence intensity ratio induced by tetracaine (ΔFratio:0.014±0.0025 vs. 0.004±0.0011,P<0.01) and the level of calcium leak (%, 44.3±5.32 vs. 20.3±7.33,P<0.05) are obviously increased in ISO group compared with control group. The level of calcium fluorescence intensity ratio induced by tetracaine (ΔFratio:0.007±0.0019 vs.0.014±0.0025, P<0.01) and calcium leak (%, 23.6±7.49 vs. 44.3±5.32, P<0.05) in CGP group decreased much more than ISO group. There is no statistical significance between CGP group and control group in above indexes. 3. Both the drop level of calcium fluorescence intensity ratio induced by tetracaine (ΔFratio: 0.014±0.0031 vs. 0.004±0.0011,P<0.01) and the level of calcium leak (%:47.9±6.78 vs. 20.3±7.33,P<0.05) are obviously increased in H-89 group compared with control group. There is no statistical significance between H-89 and ISO group in above indexes. 4. Both the level of calcium fluorescence intensity ratio decreased by tetracaine (ΔFratio: 0.006±0.0027 vs.0.014±0.0025, P<0.01) and the level of calcium leak (%, 25.9±9.92 vs. 44.3±5.32, P<0.05) are obviously decreased in KN-93 group compared with ISO group. There is no statistical significance between KN-93 and control group in above indexes.Conclusions: The increased myocytes'diastolic SR calcium leak induced by ISO is mediated by CaMKII, not PKA dependent.