Role Of Heterogeneous Sympathetic Hyperinnervation And Calcium Regulatory Protein Expression Alterations In Long-term Left Atrial Overload-Induced Atrial Fibrillation

Objective: The mechanisms of atrial electrophysiological changes and atrial fibrillation(AF) induced by long-term left atrial pressure overload are not fully understood, and the role of sympathetic nervous system and calcium regulatory proteins and their phosphorylation level changes are also need to be evaluated. Therefore, left ventricular hypertrophy and heart failure rabbit models were made by abdominal aortic coarctation. Our study is to evaluate the effects of long-term left atrial pressure overload on atrial electrophysiological parameters and AF inducibility. We also observe the changes of sympathetic nerve distribution and calcium regulatory proteins, such as cardiac ryanodine receptor(RyR2) and its phosphorylation state (RyR2-2809P), FKBP12.6, L-type calcium channel, SERCA_2a, phospholamban (PLB) and its phosphorylation state (PLBp-Ser¹⁶). Hence, the main objective of our study is to evaluate the role of left atrial sympathetic innervation and calcium regulatory protein expression alterations in long-term left atrial overload-induced atrial fibrillation.Methods: 42 male japanese rabbits were randomized into Sham group(n=14) without abdominal aortic coarctation, left ventricular hypertrophy group(LVH group)(n=16) with 50-60% abdominal aortic coarctation and heart failure group(HF group)(n=12) with 70-80% abdominal aortic coarctation. Echocardiographic parameters such as left atrial diameter(LAD), left ventricular end diastolic diameter(LVED), interventricular septum diastolic thickness(IVSDT), left ventricularposterior wall diastolic thickness (LVPWDT), right ventricular diameter(RVD) and left ventricular ejection firaction(LVEF) were assessed 8 weeks after operation. 8 animals were selected randomly from Sham and LVH group to perform Langendorff-perfiised electrophysiological study and measure sinus cycle length(SCL), wenckbach cycle length of AV conduction(AVWCL), atrial effective refractory period(AERP), dispersion of AERP(AERPD) and intra-atrial conduction time(IACT) and AF inducibility. The remaining rabbits which included 6 from Sham group, 8 from LVH group and 5 from HF group were used in hemodynamic measurements to assess heart rate (HR), systolic blood pressure(SBP), diastolic blood pressure(DBP), left ventricular end diastolic pressure(LVEDP), LW+dp/dtmaii and -dp/dtmax. Then, blood sample were obtained to measure atrial natriuretic peptide(ANP) and norepinephrine(NE), and atrial and left pulmonary superior vein(LPSV) tissue were excised. Westernblot analyses were performed to test P-ARK1, LTCCs, FKBP12.6, RyR2-5029, RyR2-2809P, SERCA2a, PLB and PLBp-Ser16 protein expressions. Pathological examination and Tyrosine hydroxylase(TH) immunohistochemistry test were performed, and atrial tissue cyclic adenosine monophosphate(cAMP) level was also measured.Results: 1) Echocardiographic results 8 weeks after operation: Compared with Sham group, LAD, IVSDT and LVPWDT in LVH group were increased significantly, and LAD, LVED and RVD in HF group were also increased significantly;compared with LVH group, HF group had increased LAD, LVED and RVD, decreased LVEF. 2) Hemodynamic measurements: Compared with Sham group, SBP^ DBP^ LV+dp/dtmax and -dp/dtmax in LVH group were increased significantly, and SBP and LVEDP in HF group were also increased significantly, LY+dp/dtmax and -dp/dtmsx were decreased significantly;compared with LVH group, HF group had decreased SBP, DBP,LV+dp/dtmax and -dp/dtmaK, increased LVEDP. 3) Electrophysiological study results:Compared with Sham group, High left AERP(77.75±14.36 vs 96.50±8.47ms, /><0.01)was shortened, AERPD (32.75±12.65 vs 12.63±7.31ms, PO.01) and IACT(39.11±2.99 vs 25.60±3.70ms, P<0.01) was increased in LVH rabbits. There was alsosignificant increase in vulnerability to AF in LVH group(4/8 vs 0/8, PO.05). 4)Plasma ANP , NE and atrial tissue cAMP levels: ANP in HF group was higher thanLVH and Sham group;NE in HF and LVH group were higher than Sham group, andNE in HF group was also higher than LVH group;cAMP in Sham group was higherthan LVH and HF. 5) Pathological examination and immunohistochemistry testresults: (T)Left atrial cross sectional area(LACSA) in LVH and HF group werehigher than Sham group. ?Ostial LPSV diameter and vessel wall thickness in LVHand HF group were larger than Sham group. ?Left atrial collagen volumefraction(CVF) and LAACVF in LVH and HF group were higher than Sham group.?TH immunohistochemistry test: Left atrial nerve density were more heterogeneousin LVH group compared with Sham group;left atrial max nerve density and nerveheterogeneity were higher in HF group than Sham group. LPSV max and mean nervedensity were lower in LVH group than Sham and HF group. 6) Westernblot analysis:?P-ARK1 protein expressions in LVH and HF group were higher than Sham group.?LTCCs protein expressions in LVH and HF were lower than Sham group.(3)FKBP12.6 protein expressions in LVH and HF group were higher than Shamgroup, and FKBP12.6 in HF group was lower than LVH group. ?Compared withSham group, RyR2-5029 protein expression in LVH was increased significantly,RyR2-5029 protein expression in HF was decreased significantly, and RyR2-5029protein expression in HF was lower than LVH group. (§)RyR2-2809P proteinexpressions in LVH and HF group were higher than Sham group. ?RyR2 relativephosphorylation levels in LVH and HF group were higher than Sham group, and RyR2 relative phosphorylation level in HF group was higher than LVH group. (7)SERCA2a protein expression in HF group was lower than Sham and LVH group. (§)PLB and PLBp-Ser16 protein expressions in LVH and HF group were lower than Sham group, and PLB and PLBp-Ser16 protein expressions in HF group were lower than LVH group. (9)PLB relative phosphorylation levels in LVH and HF group were lower than Sham group, and PLB relative phosphorylation level in HF group was lower than LVH group.Conclusion: 1) Long-term left atrial pressure over-load can shorten high left AERP, prolong AERPD and IACT, increase AF inducibility. 2) Long-term left atrial pressure over-load can induce left atrial myocardial hypertrophy, atrial interstitial fibrosis and a series of pathologic changes named as atrial structural remodeling. 3) Long-term left atrial pressure over-load can cause left atrial heterogeneous sympathetic hyperinnervation, decreased LTCCs and SERCA2a protein expressions, increased RyR2 relative phosphorylation level, decreased PLB relative phosphorylation level, which may be associated with AF. 4) Long-term left atrial pressure over-load can increase ostial LPSV diameter accompanied with left atrial dilation, which may participate in AF development.