Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Song-Jung Kim; Maha Abdellatif; Sharat Koul; George J Crystal

doi:10.1152/ajpheart.00298.2008

. 2008 May 2;295(1):H130–H135. doi: 10.1152/ajpheart.00298.2008

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Song-Jung Kim ^1,3, Maha Abdellatif ⁴, Sharat Koul ¹, George J Crystal ^1,2,3

PMCID: PMC2494754 PMID: 18456736

Abstract

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10⁻⁶ M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 ± 0.3% vs. 4.5 ± 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 ± 4 vs. 106 ± 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca²⁺ transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 ± 0.02 vs. Con, 0.25 ± 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10⁻⁹ M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca²⁺ handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.

Keywords: myocardial contractility, sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a

insulin-like growth factor I (IGF-I) is a single-chain polypeptide that is structurally and functionally similar to insulin. IGF-I is an autocrine and a paracrine hormone, the synthesis of which is under the control of the growth hormone (GH) (9, 31, 33, 38). IGF-I is produced in various cell types, including myocytes, where it is known to stimulate protein synthesis and exert metabolic and antiapoptoic effects (9, 33, 38). Various lines of evidence have suggested that the GH/IGF-I axis may also be an important physiological regulator of the inotropic state of the heart. Patients with GH/IGF-I deficiency show impaired cardiac function, which could be reversed with a chronic administration of GH (3, 26). Similar beneficial effects were observed by treating patients with congestive heart failure with recombinant human GH or IGF-I (7, 8, 10, 29). Furthermore, IGF-I administration improved cardiac function in experimental animal models with diabetes, IGF-I deficiency, heart failure, or myocardial infarction (18, 32–34).

Recent studies have indicated that IGF-I activates multiple signaling pathways, which involve the sequential activation of the phosphatidylinositol (PI)3-kinase and Akt (33). It is well established that the PI3-kinase-Akt cascade modulates diverse cellular functions, including antiapoptotic signaling and cell survival pathways. A recent study has demonstrated that acute treatment with IGF-I of myocardium from end-stage human failing hearts caused a positive inotropic effect (accompanied by an increase in the Ca²⁺ transients), which could almost be abolished with wortmannin, a PI3-kinase inhibitor (37). This suggested that Akt may also play a role in the IGF-I-induced increases in cardiac contractile function.

The objective of the present study was to investigate the effect of chronic treatment with IGF-I on intrinsic myocyte function and to clarify systematically the role of the PI3-kinase-Akt-sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a signaling cascade in these responses. Myocytes isolated from rat hearts were used to avoid the reduction in afterload secondary to an endothelium-dependent, nitric oxide-mediated vasodilating effect of IGF-I associated with in vivo administrations (6, 8). This simplified the interpretation of the findings. The chronic treatment of the myocytes with IGF was employed to make the findings more clinically relevant.

In our initial studies, we assessed the effect of IGF-I on intrinsic myocyte function, along with the associated changes in the intracellular Ca²⁺ transients (fura-2), and the levels of the Ca²⁺-handling proteins (SERCA2a, phospholamban, and calsequestrin) by Western blot analysis. Our findings demonstrated that IGF-I caused increases in myocyte contraction and relaxation function, increases in intracellular Ca²⁺ transients, and an upregulation of SERCA2a; there was no change in phospholamban or calsequestrin. We next evaluated whether the IGF-I-induced upregulation in the SERCA2a protein was mediated by Akt. This was accomplished by culturing the myocytes, transfecting them with an adenovirus harboring either Akt or dominant negative Akt (dnAkt), and then assessing both myocyte function and SERCA2a expression. Additional mechanistic insights were obtained using a PI3-kinase inhibitor (LY-249002) and a SERCA2a inhibitor (thapsigargin).

MATERIALS AND METHODS

Animals were used in this study in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, Revised 1996). The study was conducted after the approval from the Institutional Animal Care Committee.

Preparation of adult cardiac myocyte culture and adenovirus transfection.

Cardiac myocytes were isolated and cultured from the left ventricle of 23 adult Sprague-Dawley rats (200–250 g and 1 to 2 mo old) as described in detail previously (15, 17). In brief, the heart was rapidly excised and perfused with a digesting solution composed of MEM (Joklik's modification, Cat. No. M0519; Sigma), 5 mM taurine, 2 mM creatine, 5 mM HEPES, 5 mM NaHCO₃, 20 units insulin, and 1% penicillin-streptomycin containing 75 U/ml each of collagenase 1 and 2 (Worthington Biochemical, Freehold, NJ) at 37°C. The solution was continuously bubbled with 95% O₂-5% CO₂ at 37°C. The digested heart was then minced and poured into an Erlenmeyer flask containing the enzyme solution for a second digestion. This second digestion took place in a warm (37°C) shaking water bath. After the minced tissue was shaken for 15 min, it was filtered into a 100-μm diameter Nylon cell strainer. The supernatant was removed, and MEM solution containing 0.3 mM CaCl₂ and 6% BSA was added stepwise with CaCl₂ concentrations (0.5 and 1.0 mM).

The Ca²⁺-tolerant myocytes were isolated and plated on laminin-coated petri dishes. IGF-I was added to the experimental groups to yield a final concentration of 10⁻⁶ M. Other groups without IGF-I were used as controls. Myocytes were incubated in 1 mM Ca²⁺ basic media solution at 37°C for 48 h. The myocyte function was then assessed, and Western blot analysis procedures were performed. In selected studies, adenovirus harboring either constitutively active Akt (multiplicity of infection, 15) or dnAkt (multiplicity of infection, 15) was applied 2 h after myocyte isolation. The methods for adenovirus transduction have been described in detail (27).

Measurement of myocyte function and Ca²⁺ transients.

Myocytes were field stimulated at 1 Hz, and contraction was measured using a video motion edge detector (VED103; Crescent Electronics). Intracellular Ca²⁺ transients were measured with 5 μM of fura-2 AM (Sigma) using the Photoscan dual-beam spectrofluorophotometer (Photon Technology), as described previously (15–17). The external solution contained 1 mM Ca²⁺. Myocyte function was also assessed in the presence of a PI3-kinase inhibitor (LY-249002; 10⁻⁹ M; Sigma) and a SERCA2a inhibitor (thapsigargin; 10⁻¹⁰ M; Sigma).

Western blotting.

A lysis buffer containing 150 mmol/l NaCl, 50 mmol/l Tris (pH 7.5), 0.1 mmol/l Na₃VO₄, 1 mmol/l NaF, 0.5 mmol/l 4-(2-aminoethyl)benzenesulfonyl fluoride, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), and 0.5% deoxycholic acid and a protease inhibitor were used to collect the cardiac myocytes from the culture petri dishes. Protein concentration was measured using a BSA protein assay. The samples were obtained in equal amounts of protein (40 μg) and run in an 8% SDS-PAGE using the Bio-Rad Mini-gel system. The gels were transferred to a nitrocellulose membrane using a wet transfer apparatus (Bio-Rad) with 20% methanol, 25 mmol/l Tris, and 19 mmol/l glycine buffer. The expression of Akt, SERCA2a, calsequestrin, and phospholamban was analyzed in both the control and IGF-I-treated groups. Blots for Akt were incubated overnight at 4°C with a 1:500 dilution in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Blots for SERCA2a and phospholamban were incubated overnight at 4°C with a 1:5,000 dilution of rabbit anti-SERCA2a polyclonal antibody (a generous gift from Dr. Frank Wuytack, Leuven, Belgium) and 1:5,000 mouse anti-PLB monoclonal Ab (Affinity BioReagents, Golden, CO) in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Blots for calsequestrin were incubated overnight at 4°C with a 1:1,000 dilution of rabbit anti-canine cardiac calsequestrin polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) in the same buffer. The intensities of the bands were evaluated by densitometric scanning using a Personal Densitometer SI with ImageQuaNT software (Amersham Biosciences) and normalized for protein loading. All Western blot exposures were in the liner range of detection, and the intensities of the resulting bands were quantified by densitometry (Corel Photo).

Statistical analysis.

Data are reported as means ± SE. Comparisons between the groups were made by the Student's t-test. Differences between means were considered statistically significant if the probability of their occurring by chance was <5% (P < 0.05).

RESULTS

Myocyte function and Ca²⁺ transients in IGF-I-treated myocytes.

Table 1 summarizes the effect of the chronic treatment of myocytes with IGF-I on baseline contraction and relaxation along with the associated effect on intracellular Ca²⁺. Myocytes treated with IGF-I caused 70% increases in contractile function (both percent contraction and the rate of contraction). This enhanced contractile function was associated with a proportional increase in Ca²⁺ amplitude (control, 0.25 ± 0.03 vs. IGF-I, 0.42 ± 0.02; P < 0.05). Treatment with IGF-I also increased myocyte relaxation function; the rate of relaxation (+dL/dt) was increased by 109% (control, 77 ± 6 vs. IGF-I, 161 ± 13 μm/s; P < 0.05), and the time required for 70% relengthening (TR70%) was accelerated by 22% (control, 104 ± 7 vs. IGF-I, 81 ± 7 ms; P < 0.05). The time required for 70% Ca²⁺ reuptake by the sarcoplasmic reticulum (SR) was also accelerated by 14% (control, 385 ± 14 vs. IGF-I, 331 ± 10 ms; P < 0.05). The specific PI3-kinase inhibitor LY-249002 abolished the IGF-I-induced increases in myocyte contraction and relaxation function, as well as in intracellular Ca²⁺ (Fig. 1, A and B).

Table 1.

Myocyte contractile and relaxation function in response to 48 h chronic treatment with IGF-I

	Control	IGF-I, 10⁻⁶ M
Diastolic length, μm	124±3	122±4
Contractile function
Contraction, %	4.5±0.4	7.7±0.3^*
−dL/dt, μm/s	100±5	171±13^*
Relaxation function
+dL/dt, μm/s	77±6	161±13^*
TR70%, ms	104±7	81±7^*
Number of animals (cells)	6 (28)	6 (27)
Intracellular Ca²⁺
Diastolic, 340/380 nm	0.91±0.02	0.90±0.02
Amplitude, 340/380 nm	0.25±0.03	0.42±0.02^*
TRC70%, ms	385±14	331±10^*
Number of animals (cells)	5 (26)	5 (25)

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Values are means ± SE. −dL/dt, rate of contraction; +dL/dt, rate of relaxation; TR70%, time for 70% relengthening; TRC70%, time for 70% Ca²⁺ reuptake.

P < 0.05 vs. control.

Fig. 1. — Myocyte contraction (A) and intracellular Ca²⁺ (B) changes in response to LY-249002 (10⁻⁹ M) in the untreated (control) and insulin-like growth factor I (IGF-I)-treated (10⁻⁶ M) myocytes. IGF-I enhances contractile function, which was associated with the increase in Ca²⁺ transients. The enhanced function and Ca²⁺ transients were abolished in the presence of LY-249002 (n = 6 rats; total number of cells = 27). *P < 0.05 vs. respective baseline; †P < 0.05 vs. IGF-I baseline. TRC70%, time for 70% Ca²⁺ reuptake; −dL/dt, rate of contraction.

Akt, SERCA2a, phospholamban, and calsequestrin protein expression in IGF-I-treated myocytes.

The protein expressions of Akt and SERCA2a were markedly increased in myocytes treated with IGF-I compared with untreated myocytes (2.5- and 4.8-fold increases, respectively); the levels of phospholamban and calsequestrin were not changed (Fig. 2). Figure 3 shows that the ability of IGF-I to increase the level of SERCA2a was absent in myocytes transfected by adenovirus harboring dnAkt. It also shows that neither the culture medium nor the reporter gene that codes for β-galactosidase (LacZ) was evident.

Fig. 2. — Western blots of Akt, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a, phospholamban (PLB), and calsequestrin (CQN) from adult cultured myocytes chronically treated with IGF-I (10⁻⁶ M; n = 4 rats) for 48 h compared with control myocytes. *Top*: representative gel pictures. *Bottom*: the intensity of each band is expressed in arbitrary units. Chronic treatment of IGF-I significantly increased the levels of Akt and SERCA2a but not of PLB and CQN. *P < 0.01 vs. control.

Fig. 3. — Western blots of SERCA2a (*top*) and Akt (*bottom*) from adult cultured myocytes. From *left*: culture medium [control (CT)], LacZ, IGF-I (10⁻⁶ M), and IGF-I transfected with dominant negative Akt (dnAkt). Noteworthy is that chronic treatment with IGF-I upregulated the expression of SERCA2a in normal myocytes but not in myocytes transfected with dnAkt, suggesting that IGF-I-induced upregulation of SERCA2a is mediated by Akt.

Myocyte function and Ca²⁺ transients in Akt-transfected myocytes.

Studies were performed to determine whether the overexpression of Akt could mimic the changes in myocyte function, intracellular Ca²⁺, and the SERCA2a level caused by chronic treatment with IGF-I and whether these changes could be attenuated with the SERCA2a inhibitor thapsigargin. Figure 4A shows a representative immunoblotting of SERCA2a 2 days after Akt transfection compared with control virus transfected (LacZ). The protein expression of SERCA2a was increased 3.4-fold in Akt- compared with LacZ-treated myocytes (Fig. 4A, bottom). Figure 4B presents representative recordings of myocyte contraction (Fig. 4B, left) and Ca²⁺ transient (Fig. 4B, right) from Akt- and LacZ-transfected myocytes. The percent contraction (Fig. 4B, left) and Ca²⁺ amplitude (Fig. 4B, right) in Akt-transfected myocytes were significantly (P < 0.05) increased by 47% and 108%, respectively, compared with the values from LacZ-transfected myocytes (Fig. 4C). These changes were abolished by thapsigargin.

Fig. 4. — A: Western blot of SERCA2a and Akt from myocytes transfected with LacZ and Akt (Ad.Akt; n = 4 rats). A, *top*: representative gel pictures. A, *bottom*: the intensity of each band is expressed in arbitrary units. B: representative contraction and intracellular Ca²⁺ transients recordings. C: summarized results of percent contraction and Ca²⁺ amplitude and in response to thapsigargin (10⁻¹⁰ M). Myocyte contractile function (n = 4 rats; total number of cells = 14) and Ca²⁺ amplitude (n = 4; total number of cells = 26) were significantly increased in Akt-transfected compared with LacZ myocytes. The enhanced responses were abolished by thapsigargin (Ad.Akt/Thp). *P < 0.05 vs. LacZ.

DISCUSSION

The main findings from this study were as follows. 1) The chronic treatment of normal rat myocytes with IGF-I increased their contraction and relaxation, Ca²⁺ transients, and the expression of Akt and SERCA2a. These changes were abolished by the specific PI3-kinase inhibitor LY-2490022. 2) The myocytes overexpressing Akt demonstrated the same changes in function, Ca²⁺ transients, and the SERCA2a level found during the chronic treatment with IGF-I. 3) Transfection with an adenovirus harboring dnAkt negated SERCA2a overexpression. These effects were also prevented with the SERCA2a inhibitor thapsigargin. Taken together, our findings indicate that the enhanced myocyte contraction and relaxation functions following chronic treatment with IGF-I are mediated by the PI3-kinase-Akt-SERCA2a signaling cascade.

Various lines of evidence have demonstrated that treatment with GH/IGF-I has a beneficial effect on cardiac function in vivo. For example, animal and clinical studies have shown that GH/IGF-I was an effective treatment for congestive heart failure and ischemic cardiomyopathy (9, 31, 33, 38). Furthermore, chronic GH replacement with recombinant human GH for 3 to 9 mo has been demonstrated to improve cardiac function in patients with GH deficiency or dilated cardiomyopathy (3, 8, 10, 26). Finally, a recent study found that transgenic mice with IGF-I overexpression showed attenuated aging-associated contractile dysfunction, which was linked to an increase in SERCA2a protein expression (24).

The objective of the present study was to investigate the effect of chronic treatment with IGF-I on intrinsic myocyte function and to clarify systematically the role of the PI3-kinase-Akt-SERCA2a signaling cascade in these responses. To accomplish this objective, it was necessary to employ an experimental model and design that allowed us to rule out the effects of other potential mechanisms for improved cardiac function by GH/IGF-I. This entailed the use of isolated rat myocytes subjected to a chronic, albeit limited, exposure to IGF-I. This approach rendered irrelevant the following mechanisms: 1) myocardial hypertrophy reported during chronic treatment with GH, leading to an increased wall thickness and a reduction in wall stress (10, 26, 36). This mechanism was not applicable to the present study because of an insufficient duration of treatment. As proof, we found that the treated myocytes were similar in size and geometry to the controls (Table 1); 2) an antiapoptotic effect of GH/IGF-I (20, 23). This effect did not pertain to the present study since we only examined contracting myocytes. Thus neighboring nonfunctional, apoptotic cells could not have contributed to the observed changes in cardiac function; and 3) an endothelium-dependent and nitric oxide-mediated mechanism resulting in peripheral vasodilation and a reduction in afterload (6). Since only isolated myocytes were treated and studied, i.e., no systemic administrations were used, the secondary changes in cardiac function due to the peripheral vasodilating effects of IGF-I were excluded.

Previous studies using isolated cardiac myocytes have reported direct, acute inotropic effects by IGF-I (4, 12, 19, 34, 37). The preponderance of evidence suggests that these effects were attributable to an increase in Ca²⁺ availability to the myofilaments (4, 18, 34). Although some studies have shown increases in myofilament sensitivity during IGF-I (4, 30), others have shown decreases (35). If enhanced myofilament sensitivity had played a major role in increasing myocyte contraction in response to IGF-I in our study, we would have found no increase in intracellular Ca²⁺ transients, if any, and a prolonged relaxation. The increased Ca²⁺ transients and acceleration of relaxation time that we observed are consistent with a minimal influence of an increased myofilament sensitivity in the inotropic effect of chronic treatment with IGF-I.

The present study is the first to demonstrate that chronic exposure to IGF-I increases myocyte contractility associated with an increase in Ca²⁺ availability, i.e., Ca²⁺ transient amplitude. We also provide the first evidence that IGF-I can enhance the relaxation function (both TR70% and +dL/dt). The latter effect was not found during acute treatments of myocytes with IGF-I (12, 19). Our functional findings in the myocytes pointed to potential changes in the intracellular Ca²⁺-handling proteins, such as SERCA2a, phospholamban, and calsequestrin (21, 22, 25), during the chronic exposures to IGF-I. However, the Western blot analysis findings demonstrated an upregulation of SERCA2a, although phospholamban and calsequestrin remained unchanged (Fig. 2).

Although the expression of phospholamban was not affected by IGF-I, changes in its level of phosphorylation cannot be ruled out. Previous studies have demonstrated that IGF-I can stimulate protein kinase C (PKC) (33), PKC can cause the phosphorylation of phospholamban (28), and phosphorylated phospholamban can increase Ca⁺² uptake into the SR (14). These findings describe a mechanism that may have contributed to the increases in Ca⁺² uptake and myocyte contractility observed during chronic IGF-I in the present study.

IGF-I binds to IGF-I receptors, which are most abundant in myocardium, or to insulin receptors and activates PI3-kinase (11). The activated PI3-kinase phosphorylates various downstream targets and modulates diverse cellular functions, including cell survival and apoptosis, protein synthesis, and glucose metabolism (1, 2, 5, 13). In the present study, the increase in myocyte contractility caused by chronic IGF-I treatment was abolished by the PI3-kinase inhibitor LY-249002, which provided evidence for a role of PI3-kinase. Furthermore, it was accompanied by an increased expression of Akt, as well as of SERCA2 (as stated above), implying that an upregulation of Akt was also involved. The findings that SERCA2a protein expression was upregulated in adenovirus-mediated Akt-transfected myocytes and abolished in the presence of dnAkt provided further evidence for the Akt-SERCA2a pathway in the observed IGF-I-induced increases in myocyte contractility. The present findings are consistent with a previous study from our laboratory (17) demonstrating that transgenic mice with cardiac-specific overexpression of Akt and hypertrophic hearts had an enhanced left ventricular function, associated with an increased expression of SERCA2a. They are also in accord with the work of Von Lewinski et al. (37), which suggested that Akt made a contribution to the acute inotropic effect of IGF-I in myocytes from human failing hearts.

In summary, the chronic stimulation of IGF-I enhanced myocyte contraction and relaxation function through the acceleration of intracellular Ca²⁺ transients. The underlying cellular mechanism for this response was an upregulation of the SERCA2a protein, which is mediated by the PI3-kinase-Akt-SERCA2a signaling cascade.

GRANTS

This work was supported, in part, by the National Heart, Lung, and Blood Institute Grant HL-62442 and the American Heart Association Grant 0030125N and by the Joan and Norman Chapman Family Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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PERMALINK

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Song-Jung Kim

Maha Abdellatif

Sharat Koul

George J Crystal

Abstract