Effects Of CaMKⅡ_δ Ablation On Triggered Arrhythmia And Its Related Mechanism

Epidemiological data shows that fatal ventricular arrhythmias initiated by abnormal impulses is the major cause of sudden cardiac death (SCD) in heart failure (HF) patients. Recent studies indicate that, under the condition of heart failure or sustainedβ-adrenergic receptor stimulation, overexpressed Ca2+/calmodulin-dependent protein kinase (CaMKII) becomes the primary path for ICa-L and intracellular Ca2+ homeostasis regulation in heart.CaMKII overexpression is closely related to triggered arrhythmia in structral heart disease. In vivo and in vitro evidence have proved that acute CaMKII inhibition could improve calcium handling and reduce abnormal impulses in some pathological conditions. However, results of chronic inhibition may different from acute inhibition. The effects of long-term CaMKII inhibition on triggered arrhythmia may be more valuable and need to be elucidated.Based on findings above, we used a CaMKIIδablated mice model in our study, and investigated long-term effects of decreased CaMKIIδactivity on triggered impulses and its related mechanism in normal and heart failure LV myocytes. Part I Effects of CaMKIIδablation on triggered impulses in normal myocytesPurpose:To study the long-term effects of decreased CaMKIIδactivity on triggered impulses in normal LV myocytes.Methods:LV myocytes were isolated enzymatically from CaMKIIδ-knockout (KO) and C57BL/6 (WT) mice. CaMKII activities were then identified by CaMKII activity kit (Promega). For triggering EAD, a simulation model of stretch-activated channel (SAC) currents was coupled with LV myocytes by patch-clamp system. ICa-L and INCX were also recorded under the whole-cell mode in patched cells. Using Fura-2 as intracellular Ca2+ indicator, levels of intracellular diastolic Ca2+, calcium transients and sarcoplasmic reticular Ca2+ leak were tested by Ionoptix calcium imaging system. Western blot analysis were used for testing expression levels of Calcium channel and NCX.Results:In normal condition, KO myocytes showed significant higher EAD inducibility than WT myocytes (94% vs.13%, p<0.05) and need much lower Gsac values for inducing EAD (3.4±0.3 vs.7.6±0.9 ns,* p<0.05). Peak current density remarkably increased both in ICa-L (-7.2±0.3 pA/pF vs.-6.2±0.2 pA/pF, p<0.05) and INCx (-0.52±0.11 pA/pF vs.-0.30±0.06 pA/pF, p<0.05), and increased channel protein levels were also showed respectively. EAD inducibility in KO myocytes did not changed when replace Ca2+ with Ba2+ in perfusion solution and could be completely inhibited by 2uM specific calcium channel blocker nifedipine. Intracellular calcium imaging results showed that SR leak level in KO myocytes was signigicantly lower than WT myocytes, and no difference in intracellular diastolic Ca2+ concentration and calcium transients were found between KO and WT myocytes. Conclusions:Reduced physiological CaMKII8 activity could increase protein expression of NCX and calcium channel, and up-regulate current density of ICa-L and INCX. These changes contributed to the increased electrical instability in CaMKII-KO myocytes. Part II Effects of CaMKIIδablation on triggered impulses in failing myocytesPurpose:To study the long-term effects of decreased CaMKIIδactivity on triggered impulses in myocytes with pathological condition.Methods:Surgical constriction of the thoracic aorta was used for inducing pressure overload heart failure in KO and WT mice.4 weeks after surgery, echocardiographic measurements were conducted for evaluation of heart function. Mice with EF value<50% were enrolled into experiment. CaMKII activities were then identified by CaMKII activity kit (Promega). For triggering EAD, a simulation model of stretch-activated channel (SAC) currents was coupled with LV myocytes by patch-clamp system. Ica-L and INCX were also recorded under the whole-cell mode in patched cells. Using Fura-2 as intracellular Ca2+ indicator, levels of intracellular diastolic Ca2+, calcium transients and sarcoplasmic reticular Ca2+ leak were tested by Ionoptix calcium imaging system. Western blot analysis was used for testing protein level of Calcium channel and NCX channel.Results:In failing LV myocytes, KO-HF myocytes showed much lower EAD inducibility level than WT-HF myocytes (50% vs.84.2%, p<0.05) and need significant higher Gsac level for inducing EAD (15.7±2.1 ns vs.7.2±0.5ns, p<0.05). Compared to WT-HF myocytes, KO-HF myocytes showed a reduced Ica-L peak current density without changing in channel protein level. For NCX, both of INcx density and protein level were significantly reduced in KO-HF myocytes. Compared to WT-HF myocytes, intracellular calcium imaging results showed a reduced SR leak level, and no change in intracellular diastolic Ca2+ concentration, calcium transients in KO-HF myocytes.Conclusions:Under the pathological condition of heart failure, long-term reduce of CaMKIIδactivity could sigificantly lead to decreased ICa-L and INCX desity, and attenuate calcium leak from sarcoplasmic reticular, which lead to improved intracellular Ca2+ homeostasis and electrical stability in failing myocytes.