Kv4.3 Inhibits Oxidation-dependent CaMKII Activation And Its Mechanisms

Background Ca²⁺/Calmodulin（Ca M）dependent protein kinase II（CaMKII）,activated by Ca²⁺overload and oxidative stress,has been recognized as a novel therapeutic target for heart failure（HF）.Kv4.3 protein binds to CaMKII at the Ca²⁺/Ca M binding site and subsequently suppresses the Ca²⁺/Ca M-dependent CaMKII activation.However,further researches are required to test whether Kv4.3also inhibits the oxidative stress-dependent CaMKII activation.Moreover,Kv4.3expression is decreased in HF along with an increased oxidation of CaMKII.Therefore,it is necessary to test whether restoring Kv4.3 expression inhibits the oxidative activation of CaMKII and improves cardiac function in failing heart.Materials and Methods Firstly,adult mice cardiomyocytes were treated with 4-aminopyridine and Kv4.3 antisense adenovirus,respectively,to dissociate the Kv4.3-CaMKII complex and lower the expression of Kv4.3.After H₂O₂ exposure,Western blot was used to measure the oxidation and activity（phosphorylation）of CaMKII in cardiomyocytes.Co-immunoprecipitation was then applied to test whether Kv4.3 binding induces the oxidation or phosphorylation of CaMKII,followed by the use of fluorescence spectrum to determine the conformational change of CaMKII after interaction with Kv4.3 and/or Ca M.These assays aimed to elucidate the underlying mechanisms of Kv4.3 mediated inhibition of CaMKII oxidation and oxidation-induced CaMKII activation.Finally,HF mice were transfected with Kv4.3or Kv4.2 adeno-associated virus（AAV）,and reactive oxygen species（ROS）and the oxidation and activity of CaMKII were measured.Meanwhile,echocardiography and dual-excitation fluorescence photomultiplier system were used to examine cardiac function and cardiomyocytes contraction/relaxation,respectively.Results H₂O₂-induced CaMKII oxidation was enhanced after dissociating the Kv4.3-CaMKII complex by 4-aminopyridine,as well as the overall CaMKII activity and the CaMKII activity in sarcoplasmic reticulum region.Similar results were obtained in cardiomyocytes transfected with Kv4.3 antisense adenovirus.In co-immunoprecipitation,Kv4.3 bind to CaMKII but not to the oxidative or phosphorylated CaMKII.Fluorescence spectrum indicated that the conformational change induced by Kv4.3 binding was different from that by Ca M binding:the former prevented the unfolding of CaMKII autoinhibitory domain,while the latter led to the opening of CaMKII autoinhibitory domain.Although ROS level remained unchanged in HF mice transfected with AAV-Kv4.3,the oxdation level of CaMKII significantly decreased,while this effect was not observed in mice transfected with AAV-Kv4.2.In HF mice,Kv4.3 or Kv4.2 restoration both suppressed the overall CaMKII activity and the CaMKII activity in sarcoplasmic reticulum region,along with the enhancement in systolic function of whole heart and cardiomyocytes.These salutary effects were more pronounced in HF mice transfected with AAV-Kv4.3.In addition,restoration of Kv4.3 expression did not affect the diastolic function of HF mice.Conclusions Kv4.3 suppresses the oxidation and oxidation-induced activation of CaMKII through binding to the Ca²⁺/Ca M binding site,which subsequently keeps the autoinhibitory domain of CaMKII from unfolding.Restoration of Kv4.3 expression reduces the oxidation and oxidation-induced activation of CaMKII in failing heart,which can improve cardiac systolic function without impairment of diastolic function.