The Mechanism By Which Adipocyte Fatty Acid-binding Protein (FABP4) Mediating The Contractility Of Cardiomyocytes

Objective:It was reported that FABP3 and FABP4 are able to suppress contraction of cardiomyocytes.Previously our group has demonstrated that the mechanism by which FABP3 regulating contractility was through calcium handling.However,the mechanism by which FABP4 regulating contractility is still undetermined.Our data showed that although the structure of FABP4 is very similar to that of FABP3,the inhibitory effect of FABP4 on contractility of cardiomcyotes was almost independent of calcium regulation.It was also reported that the a small peptide consist of the first 20 amino acids from N terminus of FABP4（FABP4-P20）retains the inhibitory effect of FABP4,our data demonstrated that the mechanism could be completely different from that of FABP4.Additionally,E15K mutation of FABP4-P20 reduced the inhibitory effect by more than 50%,suggesting E15 is key to the inhibitory effect of FABP4-P20.Methods:1.Male C57BL/6 mice 12-14 weeks of age provided by Hebei Experimental Animal Center were used in this study.2.To study contractile function of cardiomyocytes by IonOptix video-based edge-detection method.3.Calcium imaging was used to study the dynamic changes of calcium transients in mouse left ventricular cardiomyocytes.4.Statistical analysis and related analysis.The data was represented by MEAN±SE.SPSS 13.0software was used for statistical analysis.The test was significant at P<0.05.Results:1 Characteristics of FABP4 inhibition on excitation-contraction coupling in mouse ventricular myocytes.1.1 FABP4 inhibited cell shortening of mouse ventricular myocytes in a dose-dependent manner.The curve fitting data suggested that FABP4 had two different affinities for the inhibition of cardiomyocyte contraction.The low affinity mechanism had an EC₅₀ of 0.120 nmol/L and the high affinity mechanism had an EC₅₀ of approximately 0.010 pmol/L.1.2 FABP4 inhibited calcium transient in mouse ventricular myocytes in a dose dependent manner.1.2.1 FABP4 inhibited calcium transient amplitude in mouse ventricular Myoc-ytes with an EC₅₀ of 0.412 nmol/L（extremely high,thus it’s unlikely that）.1.2.2 FABP4 inhibited calcium removal in mouse ventricular myocytes but only at extremely high concentration.with an EC₅₀ of 1.312 nmol/L.2 Characteristics of inhibition on contractility of cardiomyocytes by FABP4derived peptide FABP4-P20.2.1 FABP4-P20 inhibited cell shortening in a dose-dependent manner.The curve fitting data suggested that the inhibition of cardiomyocyte contraction by FABP4-P20 retained only a low affinity mechanism with an EC₅₀ of 0.110nmol/L.2.2 FABP4-P20 inhibited the calcium transient amplitude in mouse ventricular myocytes in a dose-dependent manner,and the maximal inhibition was about3-fold higher than FABP4,and the EC₅₀ was 1.860 nmol/L.2.3 FABP4-P20 inhibited calcium recovery in mouse ventricular myocytes in a dose-dependent manner,with a maximum inhibition of about 7-fold compared to FABP4 and an EC₅₀ of 1.564 nmol/L.2.4 E15K mutation weakened the inhibition of FABP4-P20 on cell shortening by approximately 50%,suggested glutamate was critical to this inhibitory function of FABP4-P20.Conclusions:1.FABP4 inhibited the contraction of cardiomyocytes mainly through a calcium-independent pathway,and had two mechanisms of action with different affinities.2.The FABP4-derived small peptide FABP4-P20 retained only the low affinity inhibition of FABP4,but showed a similar effect of inhibiting calcium regulation as FABP3.3.The 15th glutamic acid at the amino terminus was a key site for FABP4-P20 contractile inhibition.