Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart

Abstract

Background: IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1-regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo.

Methods and results: IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1-mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1-induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury.

Conclusions: These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.

Figure 1

IGF-1 mediates cardiomyocyte survival in a wortmannin-sensitive manner. A, Time course of cell survival effects of IGF-1 on cardiac myocytes. Cardiac myocytes were incubated with (●) or without (○) 50 ng/mL of IGF-1 under serum-deprivation conditions for the indicated times, and surviving cells were counted by the trypan blue exclusion assay. B, IGF-1 has no effects on the viability of nonmyocyte cells under serum-depleted conditions. Serum-deprived non–cardiac myocyte culture was incubated in the presence (+) or absence (−) of IGF-1 (50 ng/mL) for 72 hours, and viable cells were counted by trypan blue exclusion assay. C, IGF-1 promotes myocyte survival in a dose-dependent manner. Cardiac myocytes were cultured with the indicated concentrations of IGF-1 in the serum-deprivation medium for 72 hours, and cell viability was determined by trypan blue exclusion assay. D, IGF-1–mediated myocyte survival is inhibited by wortmannin. Cardiac myocytes were cultured in presence of indicated concentrations of wortmannin (Wort) with or without IGF-1 (50 ng/mL) for 72 hours, and surviving cell number was analyzed by trypan blue methods. All assays were performed in quadruplicate, and data are shown as mean±SEM.

Figure 2

IGF-1 activates Akt in a wortmannin-sensitive manner. A, IGF-1 activates Akt kinase activity. After serum starvation, cardiomyocytes were stimulated with IGF-1 (50 ng/mL) for 15 minutes. Cell lysates were prepared and immunoprecipitated with an antibody specific for Akt in absence or presence of competitor peptides (Comp). Immunoprecipitated kinase activity was measured with histone H2B as a substrate. B, Activation of Akt by IGF-1 is blocked by wortmannin. Cells were pretreated with wortmannin (Wort, 200 nmol/L) and stimulated with IGF-1 (50 ng/mL) for 15 minutes. Akt kinase activity was measured as described in A.

Figure 3

Adenovirus constructs expressing Akt molecules. A, Structures of replication-defective adenovirus vectors expressing wild-type (wtAkt), dominant-negative (dnAkt), or constitutively active (myrAkt) Akt fused to HA. B, Adenoviral constructs express comparable levels of transgene proteins. Cardiomyocytes were transfected with adenovirus vector at MOI 25. Cell lysates were prepared and protein concentrations determined. Proteins (10 μg) were immunoblotted with anti-HA antibody. Adenoviral vector encoding β-galactosidase (β-gal) was used as a negative control. C, Kinase activities of adenovirally encoded Akt proteins. Cardiac myocytes were infected with adenovirus vectors and stimulated with IGF-1 (50 ng/mL) for 15 minutes. Cell lysates were immunoprecipitated with anti-HA antibody and assayed for kinase activities.

Figure 4

Akt is necessary and sufficient for IGF-1–mediated myocyte survival. A, Dominant-negative Akt abrogates IGF-1–mediated cell survival. Cells were mock-infected (△) or infected with adenovirus vector expressing dnAkt (●) or β-gal (▲). Cells were cultured in indicated concentrations of IGF-1 for 72 hours, and cell viability was analyzed by trypan blue exclusion assay. Assays were performed in quadruplicate, and data are demonstrated as mean±SEM (*P<0.05). B, Overexpression of wild-type Akt facilitates myocyte survival in response to IGF-1. Cells were mock-infected (solid columns) or infected with β-gal (shaded columns) or wtAkt (open columns). Parallel cultures were incubated in serum-free medium in presence or absence of 12.5 ng/mL IGF-1 for 72 hours. Viable cell number was determined by trypan blue exclusion assays. Assays were performed in quadruplicate, and data are shown as mean±SEM (*P<0.05). C, Constitutively active Akt protects cardiomyocytes from apoptosis. Cardiac myocytes were transfected with β-gal or constitutively active Akt (myrAkt). Cells were cultured in serum-deprived condition for 48 hours, and DNA fragmentation was analyzed. M indicates marker of 123-bp DNA ladder.

Figure 5

Akt is necessary and sufficient for IGF-1–mediated myocyte survival. A, Representative image of TUNEL (TNL)-positive cardiac myocytes in culture. Cardiac myocytes were cultured in absence of serum for 48 hours. Cells were stained with Hoechst, TUNEL, and anti–α-sarcomeric actin antibody. B, Effects of Akt-expressing adenovirus vectors on cardiac myocyte survival. Cultured cells were infected with mock (no virus) or adenovirus vectors expressing β-galactosidase, wild-type, dominant-negative, or constitutively active Akt (β-gal, wtAkt, dnAkt, and myrAkt, respectively). After infection, cells were cultured in presence of indicated concentrations of IGF-1 for 48 hours. Myocyte viability was assessed by staining with Hoechst and TUNEL. Cultured cells were identified as cardiomyocytes by staining with anti–α-sarcomeric actin antibody. Data are shown as mean±SEM (n=4).

Figure 6

Adenovirus-mediated transfer of constitutively active Akt gene confers resistance to apoptosis in myocytes subjected to ischemia-reperfusion injury in vivo. A, Schematic of virus injection site relative to site of left coronary artery (LCA) occlusion and area at risk. B, Transverse heart section in which area at risk is demarcated by absence of Evans blue staining. Dye was injected retrogradely through aorta after 4 hours 45 minutes of LCA occlusion. Note that area at risk is restricted to left ventricular free wall. Animals subjected to sham operation or to 45 minutes of ischemia followed by 4 hours of reperfusion exhibited uniform Evans blue staining. Blue sutures are apparent. Virus injection site is indicated by arrow. C, Section from a heart injected with Adeno-βgal and stained for LacZ activity illustrating that myocytes adjacent to needle track (note tissue damage) can be successfully transduced. Similar results were obtained with hearts injected with Adeno-myrAkt as indicated by HA staining. D and E, Adeno-myrAkt protects myocytes from ischemia-reperfusion injury. Representative section from a heart injected with Adeno-myrAkt 20 hours before being subjected to 45 minutes of ischemia and 4 hours of reperfusion in vivo. Myr-Akt–positive cells are indicated by red staining (E). Apoptotic nuclei are shown by TUNEL (TNL) staining (green in D and E). Total nuclei were demarcated by Hoechst 33342 (blue in E). Arrowheads in E indicate TUNEL-positive nuclei seen in D. F, Quantitative analysis of cardioprotective effects of myrAkt. Percentage of TUNEL-positive nuclei in transgene-positive (+) and adjacent transgene-negative (−) myocytes were compared in randomly chosen fields that contained ≈200 myocytes each. Myocytes that expressed myrAkt (HA-positive) exhibited significantly fewer TUNEL-positive nuclei than those that did not express myrAkt (HA-negative). In contrast, percentages of TUNEL-positive myocyte nuclei were similar in myocytes that were β-gal–positive or β-gal–negative. Myocyte identity was assessed by staining with anti–α-sarcomeric actin antibody (*P<0.05; ND indicates no TUNEL-positive cells detected).