Effects of CaMKII phosphorylation on myocardial ischemia-reperfusion injury in rats / 中华急诊医学杂志

Objective:To explore the effects of calcium/calmodulin dependent protein kinase II (CaMKII) on myocardial ischemia-reperfusion injury in vitro, and apoptosis and autophagy of myocardial cells in isolated rats.Methods:Seventy female SD rats (250-280 g) with normal electrocardiogram were selected to establish the myocardial IR injury model by Langendorff perfusion system. These SD rats were randomly divided into five groups (n=14): cardiac ischemia reperfusion group (IR group), CaMKII phosphorylation activator group (IR+ isoproterenol group), CaMKII phosphorylation inhibitor analogue group (IR+KN92 group), CaMKII phosphorylation inhibitor group (IR+KN93 group), and control group. After reperfusion, the left ventricular function and myocardial morphology were measured to assess the myocardial injury, and TUNEL was performed to assess the apoptosis index. Western blot was used to determine the phosphorylation levels of CaMKII and PLN (p-CaMKII/CaMKII and p-PLN/PLN), and the expression levels of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3, and autophagy marker proteins LC3II/LC3I, Beclin-1 and P62.Results:Compared with the control group, the left ventricular function of the IR group was decreased, morphological arrangement of myocardial fibers was disordered, and the apoptosis index was increased. The levels of p-CaMKII/CaMKII, p-PLN/PLN, cleaved caspase-3, Bax/Bcl-2, LC3II/LC3I, and Beclin-1 were increased significantly, while the level of P62 was decreased significantly, and apoptosis and autophagy were increased significantly (all P<0.05). Compared with the IR group, the myocardial damage of rats in the IR+KN93 group was significantly improved, the apoptosis index was decreased, and the expression of p-CaMKII/CaMKII, p-PLN/PLN, Cleaved Caspase-3, Bax/Bcl-2, LC3II/LC3I and Beclin-1 were significantly decreased and the level of p62 was remarkable increased, and apoptosis and autophagy decreased significantly (all P< 0.05). Compared with the IR group, the left ventricular function was further decreased in the IR+ isoproterenol group, while the levels of apoptosis and autophagy were further increased ( P < 0.05), while there was no significant difference in myocardial indexes between the IR+ KN92 group and the IR group ( P > 0.05). Conclusions:Inhibition of CaMKII phosphorylation attenuates isolated myocardial ischemia-reperfusion injury by reducing apoptosis and autophagy.

Role of CaMK II in pancreatic injury in mice with severe acute pancreatitis / 南方医科大学学报

OBJECTIVE@#To investigate the expression of Ca2+/calmodulin-dependent protein kinase II (CaMK Ⅱ) in pancreatic tissues of mice with severe acute pancreatitis (SAP) and explore the protective effect of KN93, a CaMK Ⅱ inhibitor, against pancreatic injury in SAP and the possible mechanism.@*METHODS@#Thirty-six healthy male C57 mice were randomly divided into sham operation group, SAP group, KN93 group and SAP + KN93 group (n=9). Serum and pancreatic tissue samples were collected 24 h after modeling. The pathological changes in the pancreatic tissues were observed using HE staining. Serum lipase and amylase activities and the levels of inflammatory factors were detected using ELISA. Western blotting was used to detect the expressions of CaMK Ⅱ, p-CaMK Ⅱ, p-NF-κB, MAPK and p-MAPK in mouse pancreas.@*RESULTS@#Compared with those in sham operation group, the expressions of p-CaMK Ⅱ, p-NF-κB and p-MAPK were significantly increased in SAP group (P < 0.05). KN93 treatment obviously alleviated pathological injuries of the pancreas in SAP mice, and significantly lowered serum levels of lipase, amylase and inflammatory factors (TNF-α and IL-6) and phosphorylation levels of NF-κB, ERK and MAPK proteins (P < 0.05).@*CONCLUSION@#The activity of CaMK Ⅱ is significantly increased in the pancreatic tissue of SAP mice. KN93 can alleviate pancreatic injury and inflammation in SAP mice possibly through the ERK/MAPK signaling pathway.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure / 中华急诊医学杂志

Objective To observe the effect of KN93, a CaMK Ⅱ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMK Ⅱ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis.Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observedin rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA.(2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrementof intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decayprocess of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMK Ⅱ may be a new therapeutic target for arrhythmias in the heart failure.

Effect of Ca2+/calmodulin-dependent protein kinaseⅡinhibitor KN-93 on late sodium current in rabbits model of heart failure / 中国病理生理杂志

AIM:To investigate the change of late sodium current (INaL) and the effect of Ca2+/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on INaLin the cardiomyocytes after isoproterenol-induced heart fai-lure (HF) in rabbits. METHODS:The rabbit model of HF was induced by injecting isoproterenol (300 μg·kg-1· d-1) for 15 d. One month later, all rabbits received by echocardiography and HE staining to observe the morphological changes of myocardium for evaluating the HF model. The protein expression of NaV1.5, CaMKⅡδ and phosphorylated CaMKⅡδ was determined by Western blot. The ventricular myocytes were isolated from the rabbits of normal saline(NS) group and HF group by Langendorff perfusion, and the whole-cell patch-clamp technique was used to record INaL. RE-SULTS:Compared with NS group,the heart rate in HF group was increased (P<0.01), the ventricular cavity was en-larged (P<0.05),and the cardiac function was decreased(P<0.01). Compared with NS group,the cardiomyocytes in HF group arranged in disorder, vacuolar degeneration and myocardial interstitial edema were observed, and fibrous tissue increased. The protein levels of NaV1.5,CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group(P<0.01). INaLin HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of INaLin HF group and NS group was significantly increased, but that in HF group increased more obviously than that in NS group (P<0.01). After ATXⅡ had induced stable current, we added KN-93 into NS group and HF group,and we found that the ATXⅡ-increased INaLin NS group and HF group was signifi-cantly decreased(P<0.05).CONCLUSION:CaMKⅡinhibitor KN-93 inhibits the increase in INaLin HF rabbits,which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on INaL.

Ca2+/calmodulin-dependent protein kinase Ⅱ inhibitor K N-93 aggravates the calcium paradox-induced heart injury / 中国药理学通报

Aim ToinvestigatetheeffectsofCa2+/calmodulin-dependent protein kinase Ⅱ inhibitor KN-93 on calcium overload-induced heart injury.Methods Thirty-twoisolatedratheartswererandomlydivided into the control group,KN-93 control group,calcium paradox group,and calcium paradox with KN-93 treat-ment group.Left ventricular pressure were recorded, and the heart function was evaluated by the left ventric-ular end-diastolic pressure (LVEDP ) and developed pressure (LVDP).Coronary flow (CF)were collect-ed,and lactate dehydrogenase (LDH)content was de-termined.Triphenyltetrazolium chloride staining was usedtomeasuretheinfarctsize.Results Compared with the control group,KN-93 at 2. 5 μmol·L-1 had no effects on coronary flow,cardiac performance and cell death at the end of perfusion in normal rats (P>0. 05 );The hearts of calcium paradox exhibited a de-crease in LVDP and CF,meanwhile an increase in LV-EDP,LDH,and infarct size of 18 ±7. 2% (P <0. 01).2. 5 μmol·L-1 KN-93 further increased the levels of LVEDP,LDH and infarct size (P<0. 01)in Ca2+paradoxical hearts,while it provoked the decline intheCFandLVDP(P<0.01).Conclusion The data demonstrates that KN-93 aggravates heart injury in calcium paradox,it also suggests that CaMKⅡ is in-volved in the Ca2+overload-induced heart injury.

The effects of calmodulin kinase Ⅱ inhibitor on hypertrophic cardiac myocytes / 中华急诊医学杂志

Objective To investigate the effect of the calmodulin kinase Ⅱ Inhibitor KN-93 on L-typecalcium current(ICa,L)and intracellular calcium concentration([Ca2+]i)in hypertrophic cardiac myocytes.Methods Forty-eight female New Zealand white rabbits were randomized(random number)into four groups(12 animals in each group):the sham operation group(sham group),the left ventricular hypertrophy group(LVH group),the myocardial hypertrophy + KN-93 group(KN-93 group),and the myocardial hypertrophy + KN-92 group(KN-92 group).Myocardial hypertrophy in the rabbits was established by coarctation of the abdominal aorta.In the sham group,the abdominal aorta was dissociated without coarctation.Eight weeks after coarctation,single ventricular myocytes were isolated by enzymaticdissociation,and ICa,L was recorded using perforated-patch recording(PPR)techniques.[Ca2+]i was measured using single-cell calcium imaging with the fluorescence calcium indicator dye fura-2/AM.Results Cardiac hypertrophy was successfully established after 8 weeks of coarctation of the abdominal aorta.The peak ICa,L in the LVH group and the sham group was(1.38 ± 0.3)nA and(0.87 ± 0.1)nA at 0 mV,respectively(P ＜0.01,n =12).There was no significant difference in Ica,L density between the LVH group and the sham group[(6.7 ±1.0)pA/pF vs.(6.3±0.7)pA/pF; P≥0.05,n=12].The addition of either KN-92(0.5 μmol/L)or KN-93(0.5 μmol/L)to the perfusing solution caused a modest steady-state inhibition of peak ICaL(9.4％ ±2.8％,KN-92; 10.5％ ±3％,KN-93)(P≥0.05,n =12)at 0 mV.However,at a higher concentration(1 μmol/L),KN-93 more potently inhibited peak ICa,L(40％±4.9％)compared to KN-92(13.4％ ± 3.7％ ; P ＜ 0.01,n =12).Resting[Ca2+]i levels in fura-2-loaded myocytes isolated from the sham,LVH,KN-92,and KN-93 groups were(98 ± 12.3)nmol/L,(154 ± 26.2)nmol/L,(147 ± 29.6)nmol/L,and(108 ± 21.2)nmol/L,respectively.Conclusions The CaMK Ⅱ specific inhibitor,KN-93,can effectively block ICa,L and reduce intracellular calcium overload in hypertrophic cardiac myocytes.This action may account for the antiarrhythmic effect of KN-93 in hypertrophic ventricular myocardium.

Effects of CaMK Ⅱ inhibitor KN93 on remifentanil-induced hyperalgesia / 中华行为医学与脑科学杂志

ObjectiveTo study the effects of intrathecal injection of CaMK Ⅱ inhibitor KN93 on the hyperalgesia induced by remifentanil in a incision pain model.MethodsSixty SD rats were divided randomly into 5 groups ( n =12):C group (control) ; (I) group ( incision pain ) ; R group ( incision pain + remifentanil ) ; DMSO group ( incision pain + remifentanil + DMSO) and KN93 group ( incision pain + remifentanil).In group R,DMSO and KN93,remifentanil (0.04 mg/kg,1 mg remifentanil was dissolved in 40 ml NS ) needed to be infused subcutaneously 30 min at the moment of surgery.Group DMSO and group KN93 were respectively intrathecal injected 20 μl DMSO( 10％ ) and 20 μl KN93 (50 μg/20 μl,dissolved in 10％ DMSO).Each rat received tests of the paw mechanical withdrawal threshold (PMWT) and the paw withdrawal thermal latency (PWTL) at the times of 24 h before and 2h,6h,24h,48h after surgery.ResultsCompared with the group C,the PWTL( ( 11.24 ± 0.69) s,(10.36 ±0.29)s,(11.29 ±1.12)s,(12.21 ±0.75)s) and PMWT( (25.5 ± 1.20)s,(24.92 ± 1.98)s,(25.47± 1.54 ) s,( 27.14 ± 1.04 ) s) of the group I were significantly decreased after surgery (P ＜ 0.05 ).Compared with the group Ⅰ,the PWTL( (8.48 ±0.72)s,(8.58 ±0.45)s,(8.46 ±0.92)s,(9.07±0.79) s and PMWT( (21.2± 2.42 ) s,( 19.58 ± 1.12 ) s,( 21.87 ± 1.56 ) s,( 22.26 ± 1.64 ) s ) of the group R were significantly decreased after surgery (P ＜ 0.05 ).Compared with the group R,the PWTL( ( 13.32 ± 0.73 ) s,( 11.79 ± 0.32) s,( 11.86 ±0.98)s,(12.76 ±0.82)s) and PMWT((29.75 ±1.38)s,(28.27± 1.16)s,(26.5 ± 1.02)s,(27.79 ± 1.22)s) of the group KN93 were significantly increased (P ＜ 0.05 ).ConclusionIntrathecal injection KN93 could relieve the hyperalgesia induced by remifentanil

Ca~(2+)/calmodulin-dependent kinaseⅡare involved in tumor necrosis factor α-induced cardiomyocyte hypertrophy in rats / 中国药理学通报

Aim To investigate whether Ca~(2+)/calmodulin-dependent kinase Ⅱ(CaMKⅡ)contribute to tumor necrosis factor α(TNF-α)-induced cardiomyocyte hypertrophy.Methods The protein content was assayed with Lowry's method.The cardiomyocytes volumes were measured by computer photograph analysis system.The protein synthesis was assayed with[~3H]-lencine incorporation method.[Ca~(2+)]_i transient was measured by Till image system by cell-loading Fura-2/AM.The expression of CaMKⅡδ_B was determined by Western blot.Results ① TNF-α significantly induced the increase of protein content, [~3H]-leucine incorporation and cell size;These responses were significantly suppressed by KN93, a selective CaMKⅡ inhibitor.② TNF-α increased the amplitude of the spontaneous Ca~(2+) transients in cultured ventricular myocytes from the neonatal rat;CaMKⅡ inhibitor KN93 can suppress the elevation induced by TNF-α.③ TNF-α significantly increased the expression of CaMKⅡδ_B.Concluslon CaMKⅡ signal pathway are involved in TNF-α-induced cardiomyocyte hypertrophy in rats.

Effect of intrathecal injection of KN93, a potent inhibitor of CaMKⅡ, on pain behavior in a mouse model of bone cancer pain / 中华行为医学与脑科学杂志

Objective To investigate the role of Ca2+/calmodulin-dependent protein kinase Ⅱ on pain behavior in a mouse model of bone cancer pain. Methods 40 male C3H/HeN mice were divided randomly into 5 groups:sham group (S group, n=8) ,control group (C group, n=8) and KN93 treat group (T1, n=8;T2, n=8;T3, n = 8 ). Group C and T were induced mouse models of bone cancer pain by intra-left-femur inoculations of osteolytic NCTC2472 cells while group S were injected only α-MEM. On the 14 d after inoculations,group S and C received intrathecal injection of 20% DMSO 5 μl . While group T1, T2, T3 received intrathecal injection of KN93 15nmol,30nmol,60nmol which dissolved in 5 μl 20% DMSO respectively. Mice received pain behavior tests including quantification of spontaneous flinches, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) before and at 0.5 h,2 h,4 h,8 h after administration. Results Treatment with KN93(15 nmol) have no effect on bone cancer pain,while treatment with KN93(30 nmol,60 nmol) can dose-dependently reverse quantification of spontaneous flinches, mechanical allodynia and thermal hyperalgesia which were induced by bone cancer pain, At 0. 5h after administration, the quantification of spontaneous flinches of the two groups ( (7.25 + 1.49 ), (4. 12 + 1.36 ) ) were decreased when compared with control group ( 11.62 + 1.92 ),PWMT((1.28 +0.14)g;(1.75 +0.46)g),PWTL((14.64 +2.12) s; (16.85 + 1.61)s)were increased when compared with control group ( (0.47 + 0. 16) g, ( 11.32 + 1.68 ) s) (P ＜ 0.05 ＝. The effect lasts for at least 4 h and disappears at 8 h. Conclusion CaMK Ⅱ may play an important role in the mechanism of bone cancer pain. Intrathecal KN93 injection can effectively attenuated bone cancer pain.

Ca~(2+)/calmodulin-dependent kinaseⅡare involved in tumor necrosis factor ?-induced cardiomyocyte hypertrophy in rats / 中国药理学通报

Aim To investigate whether Ca2+/calmodulin-dependent kinase Ⅱ(CaMKⅡ)contribute to tumor necrosis factor ?(TNF-?)-induced cardiomyocyte hypertrophy.Methods The protein content was assayed with Lowry's method.The cardiomyocytes volumes were measured by computer photograph analysis system.The protein synthesis was assayed with[3H]-lencine incorporation method.[Ca2+]i transient was measured by Till image system by cell-loading Fura-2/AM.The expression of CaMKⅡ?B was determined by Western blot.Results ① TNF-? significantly induced the increase of protein content,[3H]-leucine incorporation and cell size;These responses were significantly suppressed by KN93,a selective CaMKⅡ inhibitor.② TNF-? increased the amplitude of the spontaneous Ca2+ transients in cultured ventricular myocytes from the neonatal rat;CaMKⅡ inhibitor KN93 can suppress the elevation induced by TNF-?.③ TNF-? significantly increased the expression of CaMKⅡ?B.Concluslon CaMKⅡ signal pathway are involved in TNF-?-induced cardiomyocyte hypertrophy in rats.