心衰是当今引起死亡最主要的病因之一，心肌肥厚与心力衰竭的发生发展密切相关，但是生理性心肌肥厚与病理性心肌肥厚的转归截然不同，前者很少发展成心力衰竭及病理性心脏扩张。因此，研究生理性心肌肥厚过程中有益的保护机制是有意义的。我们实验室以往的研究表明在去甲肾上腺素持续灌注成年大鼠心肌肥厚模型的基因芯片检测有14-3-3εmRNA表达上调。在此工作基础上，为了进一步探讨生理性心肌肥厚、病理性心肌肥厚及心衰小鼠心脏中14-3-3ε的表达变化及其意义，我们复制了小鼠游泳训练的生理性心肌肥厚模型和主动脉缩窄(TAC)压力负荷的病理性心肌肥厚及心衰模型。采用小动物心脏超声术、心脏称重和组织切片染色评价各种模型小鼠心脏的结构和功能变化，蛋白印迹法检测心脏组织中14-3-ε亚型(β，γ，ε，ζ)蛋白表达的变化；在心肌成纤维细胞中导入表达14-3-3抑制肽的腺病毒，观察14-3-3功能改变对心肌成纤维细胞增殖的影响。结果表明：小鼠游泳6周时出现心脏室壁厚度增加，随着游泳时间的延长，小鼠室壁厚度逐渐增加(LVPWd：0.60±0.02 ㎜ vs. 0.73±0.06㎜，p<0.05)，心率明显下降(HR： 589 ± 29.42 bpm vs. 449 ±26.16 bpm，p<0.01)。TAC小鼠2周开始即出现左心室室壁厚度增加，4周增加最显著。随着时间延长，心室室壁厚度有下降趋势；心脏收缩功能在TAC术后第 16周时明显降低(FS 33.08±1.59% vs. 20.64±0.96 %，p<0.05)，表现为心力衰竭。心脏组织切片显示，游泳训练小鼠的心肌细胞肥大，但无纤维化； TAC小鼠在心肌肥厚组和心力衰竭组均表现为心肌细胞肥大和明显的纤维化。 Western-Blot检测14-3-3ε表达水平，在游泳训练小鼠的心肌组织中14-3-3ε蛋白表达增加，在TAC术后16周的心衰小鼠心肌组织中表达减少，其它亚型(β，γ，ζ)在此三种模型中表达无明显变化。采用14-3-3抑制肽的腺病毒转染心肌成纤维细胞，当14-3-3被其抑制肽抑制时，IGF-I对小鼠心肌成纤维细胞的促增殖作用明显增强，提示14-3-3可抑制IGF-I引起的心肌成纤维细胞增殖。BrdU试验、激酶活性试验及蛋白免疫印迹试验的结果表明14-3-3可通过抑制PI3K活性，对GSK3β的磷酸化起负性调节作用。进一步通过荧光素酶一报告基因活性试验及免疫荧光试验，提示14-3-3通过抑制PI3K通路对NFAT蛋白起负性调节作用。 上述结果表明，在生理性心肌肥厚小鼠和心衰小鼠的心肌组织中，14-3-3ε蛋白表达变化相反；14-3-3蛋白可通过抑制IGF-I/PI3K/AKT/GSK3β和NFAT通路抑制心肌成纤维细胞增殖，说明14-3-3在生理性心肌肥厚中发挥重要作用，研究结果为揭示生理性心肌肥厚中的保护机制和寻找心衰治疗新靶点提供了实验依据。 关键词：14-3-3蛋白；心肌肥厚；游泳训练；主动脉弓缩窄；增殖；NFAT；磷脂酰肌醇3—激酶

 Heart failure is one of the main causes of mortality in the world. Cardiac hypertrophy, an increase in heart size, is associated with nearly all forms of heart failure. Cardiac hypertrophy is induced by pathological stimuli (e.g., pressure or volume overload) or physiological stimuli (e.g., developmental growth, exercise training). Physiological processes, such as normal developmental growth and exercise, also result in cardiac hypertrophy, but this type of hypertrophy is characterized by normal cardiac structure with a relatively normal pattern of cardiac gene expression and does not decompensate into dilated cardiomyopathy or heart failure. The elucidation of signaling cascades that play distinct roles in these two forms of hypertrophy will be critical for the development of more effective strategies to treat heart failure. Therefore, it is meaningful to study the molecular mechanism of physiological cardiac hypertrophy. In our preliminary study, by using a cDNA micro array, we found that 14-3-3ε mRNA was up-regulated in cardiac hypertrophy induced by continuous norepinephrine infusion in rats. Subsequently, to study the role of 14-3-3 effect on cardiac hypertrophy, we constructed three mouse models, physiological cardiac hypertrophy to swim training, pathological cardiac hypertrophy and chronic heart failure(CHF) to transverse aortic constriction (TAC) respectively. Furthermore, we evaluated cardiac functions and structures by echocardiography, heart/body weight and section staining, detected protein expression of 14-3-3 iso forms (β, γ, ε, ζ) by Westem-blot assay. Moreover, we constructed an adenovirus-mediated transfection of YFP-R18 peptide (AdR18), a general 14-3-3 peptide inhibitor, to disrupt 14-3-3 functions in cardiac fibroblasts. By using AdR18, we observed the influence of 14-3-3 effect on the proliferation of cardiac fibroblasts. The results showed that, left ventricular end-diastolic posterior wall thickness (LVPWd) increased after swimming 6 weeks, with time passing by, LVPWd increased more significantly(LVPWd: 0.60±0.02 ㎜ vs.0.73±0.06 ㎜, p＜0.05), whereas heart rate decreased significantly(HR: 589±29.42 bpm vs. 449±26.16 bpm, p＜0.01). In TAC mice, LVPWd increased at 2 weeks, became most significant in 4 weeks, with time passing by, LVPWd decreased gradually. Also cardiac systiolic function decrease significantly in 16 weeks compared with the sham group (fraction shortening (FS) 33.08±1.59 % vs. 20.64±0.96 %, p＜0.05). Moreover, in response to swimming training and TAC, mice showed significant increases in left ventricular diastolic posterior wall thickness (LVPWd), heart weight and normalized heart weight to body weight ratio. However, in CHF mice, LVPWd decreased, end-diastolic volume (EDV) increased and cardiac fibrosis formed significantly. Thus, overload-pressure induced decompensated heart failure and eccentric hypertrophy. Moreover, 14-3-3ε protein expression of hearts was increased in response to swimming training but decreased in CHF mice. However, other isoforms (β, γ, ζ) of 14-3-3 proteins showed no obvious change in these three models. To study mechanisms that 14-3-3 effect on physiological cardiac hypertrophy, we observed that AdR18 significantly enhanced IGF-I-induced CFs proliferation. This potentiation was due to the increased phosphoinositide 3-kinase (PI3K) and AKT (also called protein kinase B, PKB) phosphorylations, inactivated glycogen synthesis kinase-3β (GSK3β) by phosphorylation and increased transcriptional activity of the nuclear factor of activated T cells (NFAT) and its nuclear accumulation. Collectively, these results suggest that 14-3-3ε play an important role in physiological cardiac hypertrophy, 14-3-3 inhibits IGF-I-induced proliferation of cardiac fibroblasts via PI3K dependent NFAT signaling pathway. This may contribute to our understanding of the function of 14-3-3 proteins in cardiac hypertrophy. Keywords: 14-3-3 proteins; Cardiac hypertrophy; Swimming training; Transverse aortic constriction; Proliferation; NFAT; PI3K