| Background and Purposes: Heart failure is a major cause of morbidity and mortality worldwide, which is characteristic of function disturbance of myocardial pumping. Cytosolic free calcium plays a critical role in the regulatory of cardiomyocytes function and metabolism. The disturbance of cytosolic calcium is not only the central link of myocardium hypertrophy development stimulated by outside factors, but also the core of abnormal excitation contraction couple. FK506 binding proteins (FKBPs) is a group of receptor protein family combined with FK506 and rapamycin. Research indicates that FKBPs is the accessory protein of ryanodine receptors (RyRs), calcium release channel of sarcoplasmic reticulum, and can stabilize RyRs in close state. Further researches show that FKBP12/12.6 participates in heart development, and plays an important role in the cardiac morphogenesis and cardiac excitation contraction couple.The basic mechanism of heart failure is myocardium remodeling, which is induced by a series of complicated molecular and cellular factors. The changes of myocardium remodeling including cardiomyocytes hypertrophy and apoptosis, embryo genes and protein expressions, and the changes of extracellular matrix, of which cardiomyocytes hypertrophy and apoptosis is the distinctive alteration. In the many cell signal pathways participating myocardium hypertrophy, calcineurin (CaN) and mitogen activated protein kinase (MAPK) pathways show critical importance. Bax/bcl-2 are regulatory genes of cell apoptosis, the later can anti-apoptosis and promote cell survival, and the former has the opposite effects.Then, how is the relationship between FKBP12/12.6 and human heart function and development? What is the effects of FKBP12/12.6 to myocardium remodeling and the associated molecular mechanism? In the present study, the expressions of FKBP12/12.6 and RyR2 in clinical myocardial tissue examples were measured to initially show the relationship between FKBP12/12.6 and heart diseases. The effects of FKBP12/12.6 and rapamycin and FK506 on to the growth of cardiomyocytes, and the pathophysiological role of CaN, ERK1/2 and bax/bcl-2 signal pathways were all investigated. The influence ofFKBP12/12.6 on the calcium signal and the possible mechanism were also discussed. Another purpose of this study is to find experimental data for the mechanism of cardiac structural development and a new method of prevention and treatment of heart failure. Methods:1. Clinical study: The study was conducted in 11 chronic congestive heart failure (CHF) patients receiving valve replacement opertations because of valvular heart diseases and 6 control subjects, and divided into 4 groups according to NYHA. 24 patients with different congenital heart diseases (CHD) were also studied. The expression of protein and mRNA of FKBP12/12.6 and RyR2 in myocardium tissue were measured by Western blot and RT-PCR respectively.2. Experimental study: Wild and mutant FKBP12.6 (FKBP12.6M) and wild FKBP12 recombine adenovirus were constructed and used to infect cultured neonatal cardiomyocytes, and rapamycin and FK506 were chosen as the interventional drugs. (1) Celluar diameter and area were measured by Motic Image Advanced 3.0 analysis system. (2) Expression of 3 -MHC mRNA was assessed by RT-PCR.(3) Western blotting was used for detecting signal moleculars such as CaN> ERK1/2 and bax/bcl-2, calcium channels — transient receptor potential channel (TRPC3) and RyR2 as well. (4) DNA fragmentation was used to reflect apoptosis by agarose gel electrophoresis. (5) The cytosolic calcium was measured by PTI ratio fluorescence spectrometers.Results: Clinical study:(1) With the decrease of heart function, the expressions of FKBP12/12.6 and RyR2 protein or mRNA were declined, and with significant difference between the different grades of heart function.(2) Compared with control, the mRNA and protein expressions of FKBP12 and RyR2 in myocardium tissue of atrial septal defect (ASD), interventricular septal defect (ISD) and multiple CHD were reduced, with no significant difference between different CHD. However, the FKBP12.6 mRNA level of CHD was upregulated compared with control, and the expression level of it in myocardium tissue of multiple CHD was higher than that of ASD.Experimental study:(1) The recombined Ad-FKBP12.6, Ad-FKBP12.6M and Ad-FKBP12 vectors were successfully constructed and could effectively infect cardiomyocytes. At the third day after infected with the adenovirus, the mRNA expressions of target genes of cardiomyocytes were increased significantly, and which could sustain at least one week.(2) Overexpression of FKBP12.6M and FKBP12 reduced the area, perimeter and the expression of P - MHC of cardiomyocytes, while overexpression of FKBP12.6 increased the area, perimeter and P - MHC, and which was associated with the inhibition and activation of CaN and ERK1/2 signal pathways.(3) Myocytes infected with adenovirus carrying only GFP (Control) had no bax and bcl-2 protein expression. Myocytes overexpressed FKBP12.6M, FKBP12.6 and FKBP12 had all bax and bcl-2 protein expressions, so had DNA fragmentation.(4) Rapamycin and FK506 could not only reverse the hypertrophy, P - MHC expression of cardiomyocytes infected with Ad-FKBP12.6, but also downregulate the CaN and ERK1/2 signal proteins expressions, and apoptosis signal moleculars bax and bcl-2 as well.(5) Overexpression of FKBP12.6M and FKBP12 reduced the basal cytosolic calcium of cardiomyocytes, while overexpression of FKBP12.6 increased it. Activation of RyR2 by ryanodine at a concentration of 1 u M caused a rapid rise of cytosolic calcium followed by a plateau and repetitive calcium spikes on the plateau. To the transient rise and amplitude of vibration stimulated by ryanodine, FKBP12.6M and FKBP12 groups had a tendency of declining, but FKBP12.6 group increasing.(6) Overexpression of FKBP12.6M reduced the RyR2 and TRPC3 proteins expression of cardiomyocytes. While overexpression of FKBP12.6 significantly increased the TRPC3 protein expression, which could be reversed by rapamycin.Conclusions:1. The expressions of FKBP12/12.6 changed in the myocardial tissue of CHF and CHD, which indicated that FKBP12/12.6 may paticipate in the cardiac development and maintaining the normal heart function. FKBP12/12.6 may be the susceptive genes of cardiac structural development.2. Overexpression of FKBP12.6 promoted the hypertrophy of cardiomyocytes, whileoverexpression of FKBP12.6M and FKBP12 inhibited the growth of cardiomyocytes, which was associated with the activation or inhibition of CaN and ERK1/2 signal pathways. Overexpression of FKBP12.6M, FKBP12.6 and FKBP12 could all induce apoptosis of cardiomyocytes by causing bax and bcl-2 expressions. Rapamycin and FK506 could inhibit myocardium remodeling by downregulating the CaN and ERK1/2 signals, and apoptosis signal moleculars bax and bcl-2 as well.3. Through regulating RyR2 and TRPC3 channels expression, not only the basal cytosolic calcium, but also the transient rise and amplitude of vibration stimulated by ryanodine of cardiomyocytes overexpressed FKBP12.6 had increasing tendency, while cardiomyocytes overexpressed FKBP12.6M and FKBP12 had a lower cellular calcium reactivity. |
