The differential expression of the insulin-like growth factor 1 receptor in the developing postnatal and adult heart suggests opposing biological functions

Objectives: The insulin-like growth factor 1 (IGF1) receptor (IGF1R) is a transmembrane protein tyrosine kinase that is activated by IGF1, IGF2 and, to a much lesser extent, insulin. IGF1R exerts potent anabolic effects and mice lacking this receptor have significantly lower body weight and die postnatally. Since IGF1 exerts hypertrophic and proliferative effects, we sought to determine whether IGFR1 expression is associated with cell proliferation, hypertrophy, or both in the developing postnatal heart.

Methods: Tissues and cell isolates were analysed by Western blot (WB) and microscopy (phase contrast and fluorescence). Significance of p<0.05 versus reference/con is indicated by P*.

Mouse hearts (n=4-7) were isolated from 3 days (3d), 7d, 14d, 4weeks (w) and 8w (adult) old animals. 3d old mice served as reference (100%). Differentiation status was assessed by non-muscle α-actin (Acta2) expression, α-actinin 1 & 4 expression. Since IGF1 and insulin are potent activators by phosphorylation of the MAPK (mitogen-activated protein kinase) master gatekeeper MEK1/2 (pMEK), the expression of their receptors and pMEK was evaluated by WB. Proliferative response was assessed by the determination of Mad2, P-H3 (Ser10) and PCNA. Cardiomyocyte growth was determined by myofibrillogenesis (myomesin and sarcomeric α-actinin) and the expression of the muscle O₂ carrier myoglobin and the mitochondrial electron carrier cytochrome C.

For comparison cultures of 2-day-old neonatal (NRC) and 8-week-old adult cardiomyocytes (ARC) were pretreated with 2% serum and then stimulated with 50ng/ml albumin (control, con) or IGF-1 (n=3-6). Cardiomyocyte surface area was determined after 2d (NRC) and 7d (ARC). Oxygen consumption (con = 100%) of NRC and ARC was monitored with the Seahorse after 36h and 7d, respectively.

Results: In mouse hearts, InsR expression decreased moderately postnatally to 47% (8w), but IGF1R was dramatically reduced from day 7 to less than 30% at 14d and <10% at 8w (P*). Similar to IGF1R, pMEK decreased to 29% and 16%, respectively. Cardiomyocyte differentiation was evidenced by more densely packed myofibrils and complete loss of Acta2 and α-actinin 1&4 expression in cardiomyocytes from 8w old animals (P*). The proliferative response was strongly downregulated, as Mad2, P-H3 (Ser10) and PCNA were barely detectable after day 7 (P*), indicating that cardiomyocyte proliferation is limited to the first week postnatally. The increase in cytochrome C and myoglobin expression more than doubles from day 7, indicating a shift from glycolysis to oxidative phosphorylation.

In cultured NRC, the rate of O₂ consumption after IGF1 stimulation showed almost no change despite an elevation in surface area of more than 150% (P*), whereas O₂ consumption in cultured adult cardiomyocytes increased to 140% (P*), consistent with the IGF-1-induced increases in sarcomerogenesis in ARC. In addition, IGF-1 was able to reverse the dedifferentiation of ARC pretreated with dedifferentiation factors.

Conclusions: Our data suggest that during the first 7 days, IGF1R plays a major role in glycolysis-dependent cell proliferation, whereas from day 14 on, other growth factors such as FGFs might play a more dominant function in the regulation of cardiac growth. Conversely, in the stressed or diseased adult heart, IGF1R may mediate primarily O₂-dependent hypertrophy, but little proliferative effects.