Study On Structural Remodeling And Ca～(2+)-CaMKⅡ-CREB Signaling Pathway In Atrial Myocardium Of Pathway In Atrial Myocardium Of Patients With Chronic Atrial Fibrillation

Background Atrial fibrillation (AF) is the most frequently encountered arrhythmia in the clinical setting. It is also a progressive disease, experimental and clinical studies point at two major mechanisms involved in the intrinsically progressive nature of AF. Firstly, AF is associated with elaborate adaptive and maladaptive changes in atrial tissue and cellular architecture. By parallel, this type of change was denominated structural remodeling. Furthermore, it also consists of a change in the electrical properties of the atrium, notably a shortening of the AERP and a loss of rate adaptation,and hence was named electrophysiological remodeling. Together, these mechanisms will increase the probability of generating multiple atrial wavelets by enabling rapid atrial activation and dispersion of refractoriness and promote the long-term maintenance of AF. Studies believed that intracellular Ca²⁺ overload played an important role in atrial structural and electrophysiological remodeling during AF. Studies on animal model of AF indicated that chronic AF led to marked structural changes in the atrium, and some of these findings were characteristics of intracellular Ca²⁺ overload. On the molecular level, Ca2+ overload resulting from the increased activation rate in AF in myocytes seems to trigger electrophysiological changes leading to the reduction in L-type Ca²⁺ channel function and the reduction in the intensity of L-type calcium current. The reduced L-type Ca2+ channel protein levels were associated with short AERP and poor rate adaptation. The short of AERP underlies the occurrence and maintenance of AF. But studies on structural remodeling and Ca²⁺-CaMKII-CREB signaling pathway of atrial myocardium in patients with chronic AF is not adequate. In this paper, the characteristics of structural remodeling of atrial myocardium, the intracellular free calcium concentration of atrial myocytes and the Ca²⁺-CaMKII-CREB signaling pathway of atrial remodeling in patients with chronic AF were investigated. For further study the Ca2+-CaMKII-CREB signaling pathway of atrial remodeling in celluar model of AF, a recombinant adenoviral vector with CREB-EGFP were constructed. Methods Part I Study on structural remodeling of atrial myocardium in patients with chronic AF. Light microscopy and electron microscopy were used to observe the characteristics of structural remodeling in atrial myocardium of patients with chronic AF. TUNEL and immunohistochemistry were used to study cell apoptosis and expression of CASP-3,bcl-2,bax and calpain I in atrial myocardium of patients with chronic AF. Part II The observation of intracellular free calcium concentration of atrial myocytes in patients with chronic AF. The intracellular free calcium concentration in fresh isolated atrial myocytes was measured by laser scanning cofocal microscopy technique using Fluo-4/AM as the fluorecent Ca2+ indicator. Part III Study of Ca2+-CaMKII-CREB signal pathway of atrial remodeling in patients with chronic AF. The expression of CaM,CaMKII,CREB,p-CREB of atrial myocardium in patients with chronic AF and with normal sinus rhythm was measured by Western blotting respectively. Part IV Construction of recombinant adenoviral vector with CREB-EGFP gene. Results Part I Characteristics of structural remodeling of atrial myocardium in patients with chronic AF 1. Light microscopy of atrial myocardium stained with periodic acid Schiff (PAS) shows that PAS positive material (glycogen) is scarce within the sections of atrial from patients with sinus rhythm and sections of atrial from patients with chronic AF show glycogen accumulation (PAS positive). 2. Electron microscopic shows the ultrastructure of atrial myocardium of patients with chronic AF include depletion of sarcomeres, loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and size, fragmentation of the SR, and accumulation of secondary lysosome. And atrial myocardium from patients with sinus rhythm show nearly regularly structured.3. Immunohistochemistry analysis of the expression of CASP-3 and calpain I revealed that the expression of both proteases in sections of atrial from patients with chronic AF was enhanced (p<0.05), and both of the expression of the proapoptotic bax and the antiapoptotic bcl-2 proteins were also enhance in the sections of atrial from patients with with chronic AF(p<0.05). 4. In situ detection of DNA cleavage using the TUNEL method showed that a high percentage of myocytes had nuclei TUNEL positive (48.2 ±5.2%) in Sections of atrial from patients with chronic AF, whereas almost none of the atrial myocytes of control tissue (sinus rhythm) contained TUNEL-positive nuclei. Part II The intracellular Ca2+ concentration of atrial myocytes from patients with chronic AF was significantly higher than that of control group (276.38±38.12 vs 122.28±45.63nmol/L, p <0.05). Part III Western blotting analysis of atrial samples showed that CaM and CaMKII expression was enhanced in chronic AF group(p<0.05). The analysis of CREB,p-CREB by western blotting showed that both proteins were expressed in AF group and normal sinus rhythm group, whereas CREB expression was not significantly different among the different samples studied(p>0.05), the expression of p-CREB was enhanced in AF group (p<0.05). Part IV Construction of recombinant adenovirus vector Ad-CREB-EGFP 1. The CREB-EGFP gene was cloned into shuttle plasmid pDC315 to construct shuttle plasmid pDC315-CREB-EGFP. By sequencing the recombinant plasmid pDC315-CREB-EGFP, it was proved that the CREB-EGFP gene had correct base pair sequence and the read frame work was right. 2. Recombinant adenovirus vector Ad-CREB-EGFP was rescued in 293 packaging cells by co-transfection of shuttle plasmid pDC315-CREB-EGFP and adenovirus genomic plasmid pBHGloxdelta1, 3Cre. Conclusion 1. Structural changes in atrial myocardium of patients with chronic AF include loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and size, fragmentation of the SR, and accumulation of secondary lysosome and apoptosis. The expression of calpain I was enhanced in AF group indicate increased proteolytic activity, and also indicate calciumoverload in atrial myocytes of patients with chronic AF. 2. Calcium overload was found directly in atrial myocytes of patients with chronic AF. Calcium overload may contribute to atrial structural and electrophysiological remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia. 3. Western blotting analysis of atrial samples showed that CaM, CaMKII and p-CREB expression was enhanced during chronic AF indicate Ca2+-CaMKII-CREB signal pathway may play an important role in AF induced atrial remodeling. Understanding the signal pathway of AF induced atrial remodeling has the potential to improve our appreciation of the pathophysiology of clinical AF and to allow for the development of useful new therapeutic approaches. 4. For further study the Ca2+-CaMKII-CREB signaling pathway of atrial remodeling in cellular model of AF, recombinant adenoviral vectors Ad-CREB-EGFP was constructed successfully, and thus provide valuable tool for the future study.