Macrophage migration inhibitory factor plays a permissive role in the maintenance of cardiac contractile function under starvation through regulation of autophagy - PubMed (original) (raw)

Macrophage migration inhibitory factor plays a permissive role in the maintenance of cardiac contractile function under starvation through regulation of autophagy

Xihui Xu et al. Cardiovasc Res. 2013.

Abstract

Aims: The cytokine macrophage migration inhibitory factor (MIF) protects the heart through AMPK activation. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, helps preserve and recycle energy and nutrients for cells to survive under starvation. This study was designed to examine the role of MIF in cardiac homeostasis and autophagy regulation following an acute starvation challenge.

Methods and results: Wild-type (WT) and MIF knockout mice were starved for 48 h. Echocardiographic data revealed little effect of starvation on cardiac geometry, contractile and intracellular Ca²⁺ properties. MIF deficiency unmasked an increase in left ventricular end-systolic diameter, a drop in fractional shortening associated with cardiomyocyte contractile and intracellular Ca²⁺ anomalies following starvation. Interestingly, the unfavourable effect of MIF deficiency was associated with interruption of starvation-induced autophagy. Furthermore, restoration of autophagy using rapamycin partially protected against starvation-induced cardiomyocyte contractile defects. In our in vitro model of starvation, neonatal mouse cardiomyocytes from WT and MIF-/- mice and H9C2 cells were treated with serum free-glucose free DMEM for 2 h. MIF depletion dramatically attenuated starvation-induced autophagic vacuole formation in neonatal mouse cardiomyocytes and exacerbated starvation-induced cell death in H9C2 cells.

Conclusion: In summary, these results indicate that MIF plays a permissive role in the maintenance of cardiac contractile function under starvation by regulation of autophagy.

Keywords: AMPK; Autophagy; MIF; Rapamycin; Starvation.

PubMed Disclaimer

Figures

Figure 1

Echocardiographic parameters in WT and MIF−/− mice normally fed or starved (ST) for 48 h. (A) Wall thickness; (B) septal thickness; (C) peak shortening (PS, normalized to resting cell length); (D) LV end-systolic diameter (LVESD); and (E) LV end-diastolic diameter (LVEDD). Mean ± SEM, n = 8–9 mice per group, *P < 0.05 vs. FED group, #P < 0.05 vs. WT ST group.

Figure 2

Contractile function and intracellular Ca2+ handling properties of cardiomyocytes isolated from WT and MIF−/− mice normally fed or starved (ST) for 48 h with or without rmMIF reconstitution. (A) Resting cell length; (B) maximal velocity of shortening (+d_L_/d_t_); (C) maximal velocity of relengthening (−d_L_/d_t_); (D) peak shortening (PS, normalized to resting cell length); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90); (G) resting fura-2 fluorescence intensity (FFI); (H) electrically stimulated rise in FFI (ΔFFI); and (I) single exponential intracellular Ca2+ decay rate. Mean ± SEM, n = 100–130 cells, *P < 0.05 vs. FED group, #P < 0.05 vs. WT ST group, $P < 0.05 vs.MIF knockout ST group.

Figure 3

TUNEL staining of frozen cardiac sections from WT and MIF−/− mice normally fed or starved for 48 h. (A) Triple immunofluorescence for nuclei (blue), TUNEL (green), and cardiomyocytes (red). (B) Quantitative analysis of the percentage of TUNEL-positive nuclei measurement of ∼100 nuclei; (C) representative gel blots depicting levels of the apoptotic proteins Bax and Bcl-2 and GAPDH (loading control) using respective specific antibodies; (D) Bax; and (E) Bcl-2 levels. All expressions were normalized to GAPDH. Mean ± SEM, n = 100 cells for TUNEL analysis, n = 5–6 mice per group for western blot analysis, *P < 0.05 vs. FED group, #P < 0.05 vs. WT ST group.

Figure 4

Expression of autophagy signalling molecules in hearts from WT and MIF−/− mice normally fed or starved for 48 h. (A) Representative gel blots depicting levels of autophagic markers including LC3BI/II, p62, Beclin1, Atg5, AMPKα phosphorylation at Thr172, AMPKα, mTOR phosphorylation at Ser2448, mTOR, p70 S6kinase phosphorylation at Thr389, and p70 S6K. GAPDH was used as the loading control; (B) LC3B I expression; (C) LC3B II expression; (D) LC3B II-to-LC3B I ratio; (E) p62 expression; (F) Beclin1 expression; (G) Atg5 expression; (H) AMPKα phosphorylation at Thr172 (pAMPKα-to-AMPKα ratio); (I) pan AMPKα expression; (J) mTOR phosphorylation at Ser2448 (pmTOR-to-mTOR ratio); (K) total mTOR expression; (L) p70 S6kinase phosphorylation at Thr389 (pS6K-to-S6K ratio); and (M) total S6K. Mean ± SEM, n = 5–6 mice per group, *P < 0.05 vs. FED group, #P < 0.05 vs. WT ST group.

Figure 5

The effect of rapamycin (Rapa) on contractile function of cardiomyocytes isolated from WT and MIF−/− mice normally fed or starved for 48 h. (A) Resting cell length; (B) maximal velocity of shortening (+d_L_/d_t_); (C) maximal velocity of relengthening (−d_L_/d_t_); (D) peak shortening (PS, normalized to resting cell length); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 100–130 cells, *P < 0.05 vs. FED group, #P < 0.05 vs. WT ST group, $P < 0.05 vs. MIF knockout ST group.

Figure 6

The effect of rapamycin (Rapa), 3-MA on starvation-induced autophagosome formation in neonatal cardiomyocyte (NCM) isolated from WT and MIF−/− mice. (A) NCM isolated from WT mice in normal culture medium with glucose and serum; (B) NCM isolated from WT mice in glucose and serum-free culture medium; (C) NCM isolated from WT mice in normal culture medium with rapamycin; (D) starved NCM isolated from WT mice treated with rapamycin; (E) starved NCM isolated from WT mice rapamycin and 3-MA; (F) NCM isolated from MIF−/− mice in normal culture medium; (G) NCM isolated from MIF−/− mice cultured in glucose and serum-free culture medium; (H) NCM isolated from MIF−/− mice treated with rapamycin in normal culture medium; (I) starved NCMs isolated from MIF−/− mice were treated with rapamycin; (J) NCMs isolated from MIF−/− mice were treated with rapamycin and 3-MA; and (K) percentage of cells with autophagosomes. Cells with 10 or more punctuate spots were scored as positive for autophagosomes. Mean ± SEM, n = 300–400 cells, *P < 0.05 vs. control (CONT) group, #P < 0.05 vs. starvation (ST) group; $P < 0.05 vs. MIFko +starvation (ST) group; &P < 0.05 vs. MIFko + starvation (ST) + rapamycin (Rapa) group.

References

1. Kanamori H, Takemura G, Maruyama R, Goto K, Tsujimoto A, Ogino A, et al. Functional significance and morphological characterization of starvation-induced autophagy in the adult heart. Am J Pathol. 2009;174:1705–1714. doi:10.2353/ajpath.2009.080875. - DOI - PMC - PubMed
1. Hariharan N, Maejima Y, Nakae J, Paik J, Depinho RA, Sadoshima J. Deacetylation of FoxO by Sirt1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes. Circ Res. 2010;107:1470–1482. doi:10.1161/CIRCRESAHA.110.227371. - DOI - PMC - PubMed
1. Rose M, Greene RM. Cardiovascular complications during prolonged starvation. West J Med. 1979;130:170–177. - PMC - PubMed
1. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004;15:1101–1111. doi:10.1091/mbc.E03-09-0704. - DOI - PMC - PubMed
1. Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr. 2007;27:19–40. doi:10.1146/annurev.nutr.27.061406.093749. - DOI - PubMed

Macrophage migration inhibitory factor plays a permissive role in the maintenance of cardiac contractile function under starvation through regulation of autophagy - PubMed (original) (raw)