Adipocyte fatty acid-binding protein 4 suppresses contraction of mouse ventricular myocytes via a calcium-independent pathway

Figures

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  FIGURE 1. FABP4 expression in mouse myocardial tissue and cardiomyocytes. (A,B) Representative immunoblots demonstrating FABP4 protein expression in whole myocardial tissue and isolated ventricular myocytes. (C) Agarose gel electrophoresis of qRT-PCR products for FABP4. (D) Relative mRNA expression of FABP4 shows no significant difference between whole myocardial tissue (Tissue) and isolated ventricular myocytes (Cell). (E) Comparison of FABP3 and FABP4 mRNA expression reveals significantly higher levels of FABP3 in both tissue and cells. Data are mean ± SEM. (N = 4 animals, ***P < 0.001). Statistical significance was determined using a Student’s t-test.

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  FIGURE 2. Dose-dependent inhibition of cardiomyocyte contraction by FABP4. (A–C) Representative traces of cardiomyocyte contraction before (NT, Normal Tyrode) and after perfusion with 0.5 nM FABP4. (D) Contraction amplitude (ΔSL%) is significantly inhibited by 0.5 nM FABP4. (E) Maximal rate of contraction (Max -dL/dt) is reduced. (F) Time to peak contraction (TTP) is shortened. (G) Time required for maximal contraction rate is unchanged. For D-G, CON: n = 117 cells from N = 8 animals; FABP4: n = 47 cells from N = 5 animals. Statistical significance was determined using an unpaired Student’s t-test (*P < 0.05, ***P < 0.001). (H) Bar chart showing the effect of increasing FABP4 concentrations on contractile amplitude. For H, the bars represent control and incubations with 0.05 nM, 0.5 nM, 5 nM, and 50 nM FABP4, respectively. (I) Dose-response curve of FABP4-induced inhibition of contractile amplitude. Curve fitting with a two-site model yielded a high-affinity EC50 of 0.010 pM and a low-affinity EC50 of 0.120 nM. Data points represent mean ± SEM from at least N = 4 animals per concentration.

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  FIGURE 3. Effect of FABP4 on calcium transients in mouse ventricular myocytes. (A) Representative line-scan images of Ca2+ transients. (B) Ca2+ transient amplitude (F/F0) is slightly but significantly reduced by 0.5 nM FABP4. (C) The rate of Ca2+ recovery (τ) and (D) normalized time to peak (TTP) are not significantly altered. (E) Superimposed average Ca2+ transient traces for control (black) and FABP4-treated (red) cells. For B-E, CON: n = 103 cells from N = 7 animals; FABP4: n = 60 cells from N = 5 animals. Statistical significance was determined using an unpaired Student’s t-test (*P < 0.05). (F,G) Dose-response curves for FABP4 inhibition of Ca2+ transient amplitude (EC50 = 0.412 nM) and Ca2+ removal rate (EC50 = 1.312 nM). (H) Comparison of the dose-dependent inhibition of contractile amplitude (ΔSL%) versus Ca2+ transient amplitude (ΔF/F0), highlighting the mechanistic dissociation. Data are mean ± SEM from N = 4-5 animals per concentration.

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  FIGURE 4. FABP4 does not affect L-type Ca2+ current. Current-voltage (I–V) relationship for L-type Ca2+ current (ICa,L) in cardiomyocytes before (Control) and after exposure to 5 nM FABP4. No significant difference was observed. Data are mean ± SEM (Control: n = 15 cells from N = 4 animals; FABP4: n = 26 cells from N = 5 animals). Comparisons at each voltage step were made using a Student’s t-test.

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  FIGURE 5. The N-terminal peptide FABP4aa1-20 inhibits cardiomyocyte contractile function. (A) Representative traces of myocyte contraction showing inhibition by 0.5 nM FABP4aa1-20. (B–E) FABP4aa1-20 significantly reduces contractile amplitude (ΔSL%), maximal rate of contraction (Max -dL/dt), and time to peak (TTP). CON: n = 134 cells from N = 8 animals; FABP4aa1-20: n = 62 cells from N = 5 animals. Statistical significance was determined using an unpaired Student’s t-test (***P < 0.001). (F,G) Dose-response curve for FABP4aa1-20 inhibition of contraction, yielding a monophasic fit with an EC50 of 0.110 nM. Data are mean ± SEM from N = 4-5 animals per concentration.

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  FIGURE 6. FABP4aa1-20 inhibits calcium transients in a dose-dependent manner. (A,B) Application of 0.5 nM FABP4aa1-20 significantly reduces Ca2+ transient amplitude (F/F0) and slows the Ca2+ recovery rate (τ). CON: n = 239 cells from N = 10 animals; FABP4aa1-20: n = 61 cells from N = 5 animals. Statistical significance was determined using an unpaired Student’s t-test (**P < 0.01, ***P < 0.001). (C,D) Dose-response curves for FABP4aa1-20 inhibition of Ca2+ transient amplitude (EC50 = 1.860 nM) and Ca2+ removal rate (EC50 = 1.564 nM). Data are mean ± SEM from N = 4-5 animals per concentration.

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  FIGURE 7. Glutamate at position 15 is a key determinant of FABP4aa1-20 inhibitory activity. (A) Comparison of contractile inhibition by 0.5 nM FABP3aa1-20 and FABP4aa1-20. Statistical significance was determined using a Student’s t-test (***P < 0.001). (B) Dose-dependent comparison showing greater potency of FABP4aa1-20. (C) Amino acid sequences of the N-terminal peptides, highlighting the key difference at position 15 (K vs. (E) and the engineered mutants (K15E and E15K). (D,E) Concentration-dependent effects of a gain-of-function mutant (K15E) and a loss-of-function mutant (E15K). (F) The E15K mutation significantly attenuates the inhibitory effect of FABP4aa1-20 on contraction compared to the wild-type peptide at 0.5 nM. Statistical significance was determined using a Student’s t-test (**P < 0.01). Data are mean ± SEM from N = 5-6 animals for each group.

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  FIGURE 8. Proposed mechanism of FABP4-induced inhibition of cardiomyocyte contraction. Full-length FABP4 primarily suppresses contraction via a Ca2+-independent pathway, likely by decreasing the Ca2+ sensitivity of the myofilaments. This effect is independent of changes in L-type Ca2+ channels (ICa,L) or sarcoplasmic reticulum (SR) Ca2+ release and reuptake. In contrast, the isolated N-terminal peptide (FABP4aa1-20) inhibits contraction via a Ca2+-dependent mechanism, suppressing SR Ca2+ release.