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Changes in Calcium Cycling and Its Mechanisms in Sinoatrial-node underlying the TAC-induced Heart Failure in Mouse

Abstract : Heart failure (HF) is the final outcome of several chronic diseases that affect the heart function, and manifests as the inability of the heart to maintain blood flow to meet the body's needs first at effort and then at rest. HF is usually accompanied by rhythm abnormalities including sinus node dysfunction.Heart rhythm is fixed by the automatic activity of the primary pacemaker, the sinoatrial node (SAN), which fix the heart rhythm by automatically generating action potentials (AP). The firing of the AP depends on the diastolic depolarization, which brings the membrane potential to the AP threshold. This depolarization is caused by the opening of ion channels located at the sarcolemmal membrane (membrane clock), and at the sarcoplasmic reticulum (SR) (calcium clock), working together in what it has been named coupled-clock. Although some studies have pointed to alteration of the membrane clock in HF, the calcium clock mechanisms is understudied.Thus, in this thesis we aim to analyse the calcium clock in an experimental model of HF by transverse aortic constriction (TAC) in the mouse. Control animals were subjected to sham surgery. HF was determined by reduced ejection fraction by M-mode echocardiography. Congestive HF was determined by enhanced lung weight/tibia length. Electrocardiograms recorded by telemetry showed similar basal HR in both sham and HF mice. However, the HR variability was lower in HF mice, as well as the intrinsic HR under autonomous nervous system (ANS) blockade by intraperitoneal injection of atropine and propranolol (2mg/kg each). These results show pacemaker dysfunction in HF mice.In order to analyze the underlying mechanism, we dissected the SAN and loaded it with a fluorescent Ca2+ indicator, fluo-4 and viewed it by confocal microscopy. The [Ca2+]i transient rate was slower in HF and its decay significantly longer suggesting alteration in SERCA and/or NCX function. Indeed, Western-Blot assays showed depressed expression of NCX and increased expression of Phospholamban (PLB, a natural SERCA inhibitor when not phosphorylated) in HF group. Moreover, PLB was relatively less phosphorylated at the site phosphorylated by CaMKII (T17). Taken together, the longer decay time could be the result of less NCX expression and more SERCA inhibition. The decreasing of the SERCA function could decrease the SR Ca2+ load, as was found by rapid caffeine perfusion in HF SAN cells.In order to assess the RyR2activity, we analyzed Ca2+ sparks, elementary events produced by activation of a RyR2s cluster. Ca2+ sparks frequency was significantly smaller in HF group, along with reduced peak amplitude and duration. Thus, the calcium released during diastole through Ca2+ sparks were smaller in HF group, indicating impairment of the Ca2+ clock.In order to further investigate the change in RyR2 at the molecular level, we performed Western-Blot. The expression level of RyR2 in HF SAN was significantly decreased, and the relative phosphorylation at S2814 site (CaMKII site) was significantly decreased. Together with the reduced PLB phosphorylation at T17 suggested that the activation level of CaMKII in HF SAN might be altered. However, the CaMKII expression was similar in the two groups, although its phosphorylation level was significantly decreased, indicating less activation. To test functional significance, we perfused a CaMKII inhibitor, KN93 on the SAN tissue and analysed Ca2+ movements. KN93 significantly decreased transient rate in both groups, indicating some basal activation, but the effect was proportionally less in HF SAN, making the rate even faster in HF SAN.To conclude, we have found that CaMKII is less activated in SAN from HF mice. As a consequence, PLB and RyR2 are less phosphorylated, slowing the Ca2+ clock. Moreover, the expression level of RyR2 and NCX, two main factors of the Ca2+ clock are decreased, further impairing the sinus node function in HF.
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Submitted on : Monday, February 22, 2021 - 3:36:30 PM
Last modification on : Wednesday, February 24, 2021 - 3:28:19 AM

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Jianbin Xue. Changes in Calcium Cycling and Its Mechanisms in Sinoatrial-node underlying the TAC-induced Heart Failure in Mouse. Cardiology and cardiovascular system. Université Paris-Saclay, 2020. English. ⟨NNT : 2020UPASQ009⟩. ⟨tel-03148869⟩

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