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. 2013 Sep;381(1-2):69-83.
doi: 10.1007/s11010-013-1689-4. Epub 2013 May 28.

The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction

Evangelos Mavrommatis  1 , Krystyna M Shioura, Tamara Los, Paul H Goldspink
Affiliations

The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction

Evangelos Mavrommatis et al. Mol Cell Biochem. 2013 Sep.

Abstract

Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.

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Figures

Fig. 1
Fig. 1
a Endogenous MGF isoform mRNA expression following exposure to 0.3 M sorbitol in H9c2 cells (*P < 0.05 vs. control, n = 3). b Immunostaining with MGF E-domain-specific antibody in H9c2 cells following 24 h sorbitol treatment (Red is E-domain specific antibody, blue is DAPI). c Quantification of FITC-conjugated MGF E-domain peptide uptake in H9c2 cells with various concentrations. d Competition assay with excess unlabeled MGF E-domain peptide with 10 min incubation, FITC alone and incubation at 4 °C (*P < 0.05 vs. control, n = 5). e Immunostaining of cells treated with the E-domain peptide (30 nM) for 15 min with an MGF E-domain-specific antibody and counter stained with DAPI. f Immunoblot of H9c2 cell lysates treated with MGF E-domain peptide, IGF-1 or MGF E-domain peptide + IGF-1 for 15 min. Anti-pIGF-1R (Tyr980), anti-pAkt (Ser473) to examine IGF-1 receptor and pathway activation. Anti-actin shows equal loading among samples. (Color figure online)
Fig. 2
Fig. 2
MGF E-peptide exerts anti-apoptotic actions in H9c2 cells. a Mitochondrial membrane potential in response to 0.3 M sorbitol and MGF E-domain peptide treatment for various times (*P < 0.05 vs. sorbitol + MGF E-domain). b Caspase-3 activity in response to sorbitol treatment (12 h), with MGF E-domain peptide and MGF E-domain peptide containing an amino acid substitution (S/A), (*P < 0.05 vs. control, # P < 0.05 vs. IGF-1, n = 5)
Fig. 3
Fig. 3
Expression and quantification of IGF-1 isoform expression in the mouse heart following MI. a, b Quantification of the 3′-splice variants using isoform specific primers at various times post-MI (*P < 0.05, n = 4). c Analysis of 5′-splice variants of the MGF transcript (rodent IGF-1Eb), with class/isoform specific primers 24 h post-MI (*P < 0.05). d Western blot of myocyte protein extracts isolated from control and 24 h post-MI hearts (duplicate samples). Protein extracts from the atria of a PKCε transgenic mouse run as positive controls (arrow indicates migration of fragment <15 kDa). The synthetic E-domain peptide was also run under similar conditions and probed with an MGF E-domain-specific antibody
Fig. 4
Fig. 4
Cardiac contractility based on P–V loop measurements collected during transient occlusion of thoracic vena cava in 2 week post-MI mice with and without MGF E-peptide treatment. a ESPVR-end systolic pressure volume relationship. b EDPVR-end diastolic pressure volume relationship. c PRSW-preload recruitable stroke work. d dP/dtmax versus EDVmax-maximal dP/dt versus maximal end diastolic volume. e E max-time-varying maximal elastance. f The relationship between A–V coupling ratio (E a/E s) and cardiac contractility efficiency (CCE) in all 2 week post-MI mice (*P < 0.05 vs. control and † P < 0.05 vs. 2 week MI, n = 6)
Fig. 5
Fig. 5
Quantification of cardiac mass and gene expression in 2 week post-MI mice with and without MGF E-peptide treatment. a Heart weight to body weight ratios. b α-myosin heavy chain isoform expression. c β-myosin heavy chain isoform expression. d ANF expression. e Metalloproteinase expression MMP2 (*P < 0.05, # P < 0.01 vs. control, n = 5)
Fig. 6
Fig. 6
Quantification of cell death in different regions and infarct size in 2 week post-MI mice. a The percentage of TUNEL positive nuclei in the border zone and viable myocardium 2 weeks post-MI with E-domain peptide treatment (*P < 0.05 vs. 2 week MI). b Infarct size in MI and MI + E-peptide-treated mice (n = 5)
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