| Fatty acid binding proteins(FABPs) are a family of cytoplasmic protein that are approximately 14~15 KDa, which can interact with the long chain fatty acids to facilitate the transportation of the long chain fatty acids. FABPs are thought to act as lipid chaperones, which promote the fatty acids tracfficking into mitochondria for oxidation, into lipid droplets for storage, into endoplasmic reticulum for membrane synthesis, and into the nucleus for regulating lipid-modulated transcription factors. Fatty acid-binding protein 3, FABP3, also known as a heart type of FABP(H-FABP), is highly expressed in cardiac muscle and involved in lipid and glucose metabolism to maintain the energy supply of the heart as well as other parts of the body. Under normal physiological conditions, FABP3 promotes the long chain fatty acids transport into cardiomyocyte mitochondria for ATP synthesis through β-oxidation system. The overexpression of FABP3 contributes to a significant decline in cytosolic ATP concentration. During the myocardial ischemic injury, FABP3 is released into the circulation, leading to a elevated concentration of plasma FABP3. According to researches, FABP3 can inhibit the myocardial contractility of rat heart, but the specific mechanism remains to be determined.Generally, patients with diabetes mellitus have a greater chance to get the left ventricular systolic dysfunction complication. Intracellular calcium is a main regulator of myocardial contractility, defects in regulating intracellular calcium homeostasis may cause cardiac dysfunction. Insufficient SR calcium release usually leads to the reduced myocardial contractility, which can be a resu It of multiple causes. The classical theory suggests the reduced SR calcium load resulted from decreased SERCA activity is a major cuase of heart failure. The decreased SERCA activity directly results in decreasing the ability of the SR to re-take up calcium, which cause SR calcium store reduced in the cardiomyocyte. Alternatively, increasing Ry R2-mediated SR calcium leak could be another major reason for the underachieved SR Ca content. Chronic Ry R2-mediated SR calcium leak may deplete SR calcium content, eventually causing heart failure.ATP content directly affects the activity of SERCA. Inorder to maintain ATP production, the heart needs to proceed oxidative metabolism highly and continuously. In a healthy heart, approximately 70% ATP is generated through the oxidation of fatty acids. The rate of oxidative phosphorylation is closely related to the rate of ATP hydrolysis, which makes ATP content remain constant in the heart. The glucose utilization is depressed in diabetic heart, in which synthesis of total ATP almost completely depends on mitochondrial β-oxidation. However, excessive intake of fatty acids may be detrimental to the mitochondria by effecting oxidative phosphorylation. It has been suggested that ATP/oxygen ratios is decreased in the diabetic heart because of mitochondrial uncoupling. Oxidizing more fatty acids needs to consumpte more oxygen, which makes the oxygen that the heart can use is reduced. Thus, the increased demand of oxygen for oxidizing fatty acids may cause contractile dysfunction in the diabetic heart.So, what’s the role of FABP3 in the progression of diabetic cardiomyopathy? Whether FABP3 is associated with cardiac dysfunction or alterations in FABP3 are associated with the changes in mitochondrial function in the cardiomyocytes? In the present study, we used diabetic mouse as a model and isolated left ventricular cardiomyocytes to explore the aforementioned questions. First, the FABP3 expression level was assessed in cardiac tissues. Then we analyzed the correlation between the FABP3 expression level and cardiac contractility, the relation between FABP3 protein and the function of mitochondria. Second, exogenous FABP3 protein was used to stimulate cardiomyocyte in mice to investigate that FABP3 can regulat cardiomyocyte contractility, SR Ca2+ store, calcium transients and calcium spark in cardiomyocytes. Our data demonstrated that FABP3 contributes to depress SR Ca2+ store and the activity of SERCA, alter calcium transient and inhibited cardiomyocytes contractility. Finally, FABP3 reduces the content of ATP, then further affects the SERCA activity and inhibit cardiomyocytes contractility. This study aims to provide new insights into mechanisms of the development of diabetic cardiomyopathy. Part 1 FABP3 Expression was changed in a model of diabetes mice, and the effect on cardiac functionObjective: To detect the expression and subcellular localization of FABP3, the fatty acid and glucose oxidation genes expression. Study the correlation between FABP3 and cardiac function injury. Analyze the relationship between FABP3 and ATP content.Methods:1 A diabetic mice model was generate by injecting streptozocin.2 To analyze body weight, blood glucose level, blood pressure, blood lipid level and cardiac function of mice.3 To evaluate FABP3 protein expression by Western blotting.4 To investigate the subcellular localization of FABP3 by immunofluorescence.5 The correlation analysis between FABP3 and EF% by spearman correlation test.6 Detection of mitochondria structure of DM mice by scanning electron microscopy(SEM).7 Detection of the m RNA expression of fatty acid and glucose oxidation related genes by Real time PCR.Results:1 The establishment of diabetic mouse model.As determination of physiological indexes in mice after STZ injection, we observed increased blood glucose(>16.7 mmol/L), weight loss and symptoms of polyuria, which indicated that type 1 diabetes model was successfully generated. The evaluation of cardiac function showed a significant reduction in the ejection fraction in DM mice. The above mentioned results demonstrated that the DM mice developed diabetic cardiomyopathy successfully.2 ATP content was reduced in DMComparison of ATP content in CON and DM mouse, ATP content was reduced in DM(117 ± 0.8 μM vs. 62 ± 0.6 μM, P<0.05).3 The expression of FABP3 was increasedWestern blotting data showed that in DM mice, the expression level of FABP3 was increased by 2.7-fold(P<0.05) and m RNA was increased by 3-fold compared with CON mice(P<0.05).4 The negatively correlation between the expression of FABP3 and ATP content, EF%The analysis of correlation between the protein expression of FABP3 and ATP content in the heart of mice showed that expression level of FABP3 was negatively correlated with ATP content(r2=0.5143, P<0.01).The analysis of correlation between the protein expression of FABP3 and ejection fraction(EF%) in the heart of mice showed that expression level of FABP3 was negatively correlated with EF%(r2=0.6913, P<0.01).5 Altered subcellular localization of FABP35.1 In DM mice, immunofluorescence staining demonstrated that FABP3 in the nucleus was 1.89 ± 0.05-fold more than that in CON mice and the difference was statistically significant(P<0.01).5.2 Colocalization of FABP3 and mitochondria displayed in DM mice was more than that in CON mice(91.518 ± 2.6% vs. 71.17 ± 5.8%, P<0.05) and the difference was statistically significant.6 Changed expression of fatty acid and glucose oxidation-related geneRT-PCR data showed that m RNA level of MCAD, CPT-1α, PEPCK genes that are related to mitochondrial β-oxidation does not altered significantly; whereas the m RNA level of THLA, DBP, AOX1 genes related to peroxisomal β-oxidation was significantly increased; expression of GLUT4 and G6PC3 gene related to glucose oxidation was depressed; expression of ATP synthase and Cyt C gene related to mitochondrial respiratory chain did not change; expression of uncoupling protein UCP2 and UCP3 was increased in the heart tissue of DM mice.7 Disrupted structure of myocardial mitochondrial in DM miceOur electron microscropic data showed that myocardial mitocho-ndri al structure was severely disrupted, about 63% of mitochondria was fuse d.Summary:In diabetic cardiomyocytes, the expression level of FABP3 is increased and is correlated with depressed cardiac contractility. Part II Inhibitory effect of FABP3 on myocardial contractionObjective: To determine the effect of FABP3 on cardiac contractility and calcium handling.Methods:1 To study contractile function of cardiomyocytes by Ion Optix video-based edge-detection method.2 To study calcium spark, calcium transient and SR calcium load by confocal calcium imaging techniques.3 To study the effects of FABP3 on cardiomyocytes contraction and Calcium signal.Results:1 Measurement of cardiac contractility in control and DM miceThe level of cardiomyocyte contraction was significantly reduced in cardiomyocyte of DM mice compared with CON mice(4.38 ± 0.81 μm vs. 2.57 ± 0.26 μm, P<0.05).2 Calcium regulation disorders of cardiomyocytes in DM mice2.1 Calcium transient changed of cardiomyocyte in DM mice.Calcium transient amplitude was decreased in DM mice(2.15 ± 0.09 F/F0 vs. 1.88 ± 0.08 F/F0, P<0.05). Time constants of calcium recovery are prolonged in DM mice(0.068 ± 0.006 vs. 0.094 ± 0.008 s, P<0.05), Time to Peak are increased in DM mice(25.92 ± 1.99 ms vs. 35.93 ± 2.82 ms, P<0.05). Calcium transient synchronization decreased in DM mice.2.2 Sarcoplasmic reticulum calcium content was markedly reduced in DM mice.Compared with CON mice, calcium uptake was markedly reduced in DM mice(2.38 ± 0.74 F/F0 vs. 1.98 ± 0.37 F/F0, P<0.05).2.3 Increased Calcium Spark frequency in DM cardiomyocytesCalcium Spark frequency was markedly increased in DM cardiomyocytes(0.90 ± 0.44 Sparks/100 μm/s vs. 2.63 ± 0.52 sparks/100 μm/s, P<0.05).3 FABP3 acutely inhibits cardiomyocyte contractionAt a concentration of 50 nmol/L, FABP3 significantly decreased the shortening amplitude from 4.38 ± 0.31 to 1.49 ± 0.16%, whereas has no significant effect on time to peak.4 Calcium metabolism are destructed by FABP3 of cardiomyocyte in DM mice.4.1 FABP3 depress calcium transient change of cardiomyocyteIn the presence of FABP3, calcium transient change was significantly reduced(2.15 ± 0.094 vs. 1.69 ± 0.118, P<0.05) and tau was significantly prolonged(0.068 ± 0.006 vs. 0.146 ± 0.175 s, P<0.001). Time to Peak was unaltered by applying FABP3. FABP3 make calcium transient dynamics change in left ventricular of cardiomyocytes in mice. FABP3 affected the activity of SERCA.4.2 FABP3 reduced SR calcium load. In the presence of FABP3, the SR calcium load of cardiomyocytes was significantly decreased(2.39 ± 0.74 F/F0 vs. 1.27 ± 0.08 F/F0, P<0.001).4.3 Calcium sparks was changed by FABP3 in mouse cardiomyocytesCalcium Spark frequency was increased in DM cardiomyocytes(0.74 ± 0.28 Sparks/100 μm/s vs. 1.20 ± 0.29 Sparks/100 μm/s, P<0.05). Calcium Spark amplitude was decreased by FABP3(0.84 ± 0.04 F/F0 vs. 0.64 ± 0.01 F/F0, P<0.001)Calcium Spark diameter was decreased by FABP3(26.90 ± 1.26 vs. 20.27 ± 1.47, P<0.01)Summary:FABP3 can decrease cardiomyocytes contraction; FABP3 can reduce the activity of SERCA and SERCA in the state of diabetes. Part III Mechanism of FABP3 regulating sarcoplasmic reticulum calcium contentObjective:In this part, we investigated that the effect of FABP3 on SERCA and Ry R2, further illustrated the role of FABP3 in myocardial contraction.Methods:1 Deal with the cells in the form of rapid perfusion of FABP3.2 Detection of ATP content by ATP Assay Kit.3 Analysis of colocalization of FABP3 with SERCA and Ry R2 by Immunofluorescence.4 Detection of the level of ROS in cell by DCFH-DA fluorescent probe.Results:1 ATP content was down-regulated by FABP3We detected ATP content in the cardiomyocytes by ATP Assay Kit.The results showed ATP content was decreased in the cardiomyocytes after FABP3 incubation. Therefore, we speculated that FABP3 may depress the activity of SERCA by down-regulating ATP.2 The colocalization of FABP3 and SERCA was decreasedThe decline of colocalization FABP3 and SERCA, indicating that more FABP3 left SERCA, which may affect the SERCA activity in DM mice(80.22 ± 3.55% vs. 62.52 ± 2.88%, P<0.01).3 Colocalization of FABP3 and Ry R2 was decreasedThe decline of colocalization FABP3 and Ry R2, indicating that more FABP3 left Ry R2(96.50 ± 1.27% vs. 79.38 ± 3.70%, P<0.01).4 The effects of FABP3 on ROSWe treated cardiomyocytes with 0.5 n M FABP3 in the form of rapid perfusion. The results of ROS changes before and after perfusion showed that ROS fluorescence was abated after FABP3 perfusion. The level of ROS was not altered by FABP3. Therefore, we speculated the regulation of FABP3 on SERCA activity was not related to oxidative stress.Summary:FABP3 may depress the activity of SERCA by down-regulating ATP content and further inhibit cardiomyocytes contraction. Colocalization of FABP3 may affect the active of SERCA and Ry R2.Conclusions:1 The expression of FABP3 was increased in the heart of diabetes, which was related to heart fuction and ATP content, further indicating correlation between FABP3 and mitochondria.2 FABP3 may contribute to decrease the active of SERCA and Ry R2, SR Ca2+ content and cardiomyocytes contraction in diabetes.3 FABP3 may influence the activity of SERCA by down-regulating ATP in diabetes. |
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