IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

Abstract

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.

Fig 1

Cardiomyocyte contractile properties in cells from sham- or AAC-treated C57 and LID mouse hearts. (A) Representative cell shortening traces from C57 mice with sham or AAC treatment. (B) Representative cell shortening traces from LID mice with sham or AAC treatment. (C) Resting cell length. (D) PS (normalized to cell length). (E) Maximal velocity of shortening (+dL/dt). (F) Maximal velocity of relengthening (−dL/dt). (G) TPS and (H) TR₉₀. Mean ± S.E.M., n = 94–103 cells per group, *P < 0.05 versus C57-sham group, ^#P < 0.05 versus C57-AAC group.

Fig 2

PS amplitude-stimulus frequency and intracellular Ca²⁺ transient responses in cardiomyocytes from sham- or AAC-treated mouse hearts. (A) PS amplitude at various stimulus frequencies (0.1–5.0 Hz). (B) Representative intracellular Ca²⁺ transients from sham- or AAC-treated C57 and LID mice. (C) Resting FFI. (D) Electrically stimulated rise in FFI (ΔFFI). (E) Single exponential intracellular Ca²⁺ decay and (F) Bi-exponential intracellular Ca²⁺ decay. Mean ± S.E.M., n = 22 (A) and 80–95 (C–F) cells per group.

Fig 3

Whole heart and cardiomyocyte diameter in sham- or AAC-treated C57 and LID mice. (A) Representative hearts from sham and AAC-treated C57 and LID mice. (B) Pooled cardiomyocyte diameter (longitudinal) from haematoxylin and eosin staining images (400X and (C)–(F): haematoxylin and eosin staining from C57-sham, C57-AAC, LID-sham and LID-AAC groups, respectively. Mean ± S.E.M., n = 12 fields from three mice (B), *P < 0.05 versus C57-sham group, ^#P < 0.05 versus C57-AAC group.

Fig 4

Western blot analysis of the hyper-trophic markers ANF and GATA4 from C57 and LID mice hearts with sham or AAC treatment. (A) Representative gel blots depicting expression of ANF, GATA4 and α-tubulin (loading control) using specific antibodies; (B) ANF and (C) GATA4. Mean ± S.E.M., n = 6/group, *P < 0.05 versus C57-sham group.

Fig 5

Western blot analysis of pan and phosphorylated Akt, GSK-3β, and mTOR in myocardium from C57 and LID mice with either sham or AAC treatment. (A) Representative gel blots depicting pan and phosphorylated Akt, GSK-3β and mTOR (α-tubulin as loading control) using specific antibodies; (B) pAkt-to-Akt ratio; (C) pGSK-3β-to-GSK-3β ratio and (D) pmTOR-to-mTOR ratio; mean ± S.E.M., n = 6 hearts/group, *P < 0.05 versus C57-sham group, ^#P < 0.05 versus C57-AAC group.

Fig 6

Western blot analysis of phosphory-lated AS160 (pAS160), GLUT4, PP2AA, PP2AB and PP2Cα in myocardium from C57 and LID mice with sham or AAC treatment. (A) Representative gel blots depicting pAS160, GLUT4, PP2AA, PP2AB, PP2Cα and α-tubulin (loading control) using specific antibodies; (B) pAS160; (C) GLUT4; (D) PP2AA; (E) PP2AB and (F) PP2Cα. Mean ± S.E.M., n = 6 hearts per group, *P < 0.05 versus C57-sham group.

Fig 7

Quantitative real time-PCR analysis of miRNA-1, miRNA-133a and miRNA-208 in ventricles from C57 and LID mice with sham or AAC treatment. (A) miRNA-1; (B) miRNA-133a and (C) miRNA-208a; mean ± S.E.M., n = 6 hearts per group, *P < 0.05 versus C57-sham group, ^#P < 0.05 versus C57-AAC group.

Fig 8

Effect of overexpression of miRNA-1 and miRNA-133a on IGF-1-induced hypertrophic response in NRCMs. (A) Intracellular localization of miRNA (Cy3 fluorescence) detected by a Nikon Eclipse TE300 microscope (left panel). Middle panel displays image of cells (FITC fluorescence) with the right panel exhibiting the overlay depicting Cy3 fluorescence in one NRCM. (B) Representative images of NRCMs transfected with negative control miRNA, miRNA-1 and miRNA-133a for 48 hrs in the absence of IGF-1 (unstimulated). (C) Representative images of NRCMs transfected with negative control miRNA, miRNA-1 and miRNA-133a in the presence of IGF-1 (30 ng/ml) for 48 hrs. Cells were stained with the anti-α-actin antibody to confirm cardiomyocyte purity. (D) Cell surface area of NRCMs overexpressing negative control miRNA, miRNA-1 and miRNA-133a treated with or without IGF-1 (30 ng/ml) for 48 hrs and (E) ³H-Leucine incorporation in NRCMs overexpressing negative control miRNA, miRNA-1 or miRNA-133a treated with or without IGF-1 (30 ng/ml) for 48 hrs; mean ± S.E.M., n = 6 independent experiments per group, *P < 0.05 versus negative control group in the absence of IGF-1, ^#P < .05 versus corresponding IGF-1-stimulated negative control group.