Activation of extracellular signal-regulated kinase 5 reduces cardiac apoptosis and dysfunction via inhibition of a phosphodiesterase 3A/inducible cAMP early repressor feedback loop

Abstract

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Figure 1

IGF-1 stimulated both ERK5 kinase and transcriptional activity. A, Cardiomyocytes were stimulated with IGF-1 (20 ng/mL), and phosphorylated (top) and total (bottom) ERK5 were measured by Western blot analysis at the indicated times. B, ERK5 transcriptional activity was measured by mammalian 1-hybrid assay with Gal4-ERK5 construct transfection, as we described previously.

Figure 2

Role of ERK5 activation on IGF-1–mediated inhibition of PDE3A reduction and ICER induction (PDE3A/ICER feedback loop). A, Schematic diagram showing experimental protocol. Cardiomyocytes were transduced with Ad-LacZ, Ad-DN-ERK5, or Ad-DN-MEK1 at 50 multiplicities of infection for 24 hours (B) or ERK5 or control siRNA for 72 hours (C), followed by treatment with or without IGF-1 (20 ng/mL) for 24 hours and then stimulated with vehicle or ISO for 24 to 48 hours as indicated. Expression of PDE3A, ICER, and ERK5 activation were detected by Western blotting. B through D, Critical role of ERK5 activation on IGF-1–mediated inhibition on PDE3A/ICER feedback loop. B and C, Cardiomyocytes were transduced with Ad-LacZ or Ad-DN-ERK5, or ERK5 or control siRNA, followed by IGF-1 and ISO treatment, as described for A. D, Cardiomyocytes were transduced with Ad-LacZ or Ad-CA-MEK5α, followed by ISO treatment for 24 hours. E, Role of ERK5 activation on IGF-1–mediated inhibition on ISO-induced apoptosis. Cardiomyocytes were transduced with Ad-LacZ, Ad-CA-MEK5α, or Ad-DN-ERK5 at 50 multiplicities of infection for 24 hours, followed by treatment with or without IGF-1 (20 ng/mL) for 24 hours, and then stimulated with vehicle or ISO for 48 hours. IGF-1–induced ERK5 activation was critical for inhibiting ISO-mediated apoptosis. Data represent mean of 3 repeats (mean±SD). Similar results were obtained from at least 2 independent experiments. IB indicates immunoblot.

Figure 3

Critical role of MEF2 activation on IGF-1–mediated inhibition on PDE3A/ICER feedback loop. Cardiomyocytes were transduced with Ad-LacZ, Ad-DN-MEF2 (A, B), or Ad-CA-MEK5α (B), followed by treatment with or without IGF-1 (20 ng/mL) for 24 hours (A), as described in Figure 2A, and then stimulated with vehicle or ISO for 24 to 48 hours as indicated. Expression of PDE3A, ICER, and ERK5 activation were detected by Western blotting. IB indicates immunoblot.

Figure 4

Role of ERK5 activation on ICER stability and PDE3A/ICER feedback loop regulation. A, Schematic diagram showing experimental protocol. Cardiomyocytes were transduced with Ad-LacZ or Ad-CA-MEK5α for 24 hours and then transduced with Ad-ICER. Twelve hours after Ad-ICER transduction, cells were pretreated with cycloheximide, then stimulated with forskolin (10 μmol/L) or vehicle for 16 hours. B, Western blotting showing ICER and cAMP-responsive element modulator (CREM) protein expression in cardiomyocytes treated with the above protocol. C, The intensity of the band representing ICER was normalized to the intensity of Ad-LacZ and vehicle-treated control samples, which was designated as 1. Data represent mean±SD of 3 samples. *P<0.05. D, Effect of ERK5 activation on ICER-mediated apoptosis. Cardiomyocytes were transduced with Ad-LacZ, Ad-CA-MEK5α, or Ad-ICER at 50 multiplicities of infection for 24 hours. ERK5 activation could not reverse ICER-mediated apoptosis. Data represent mean of 3 repeats (mean±SD). Similar results were obtained from at least 2 independent experiments. E, Cardiomyocytes were transduced with Ad-LacZ, Ad-ICER, or Ad-CA-MEK5α. After 24 hours of transduction, expression of PDE3A, ICER, and ERK5 activation were detected by Western blotting. IB indicates immunoblot.

Figure 5

Pressure overload and Dox-mediated PDE3A reduction and ICER induction in NLC and CA-MEK5α-Tg mice. A, Western blot showing PDE3A and ICER expression and ERK5, Akt, and ERK1/2 kinase activity in sham and 8-week TAC mouse hearts in NLC and CA-MEK5α-Tg mice. B, Western blot showing PDE3A and ICER expression and ERK5, Akt, and ERK1/2 kinase activity in vehicle and Dox-treated (5 days) hearts in NLC and CA-MEK5α-Tg mice. IB indicates immunoblot.

Figure 6

Pressure overload– and Dox-induced myocardial apoptosis in NLC and CA-MEK5-Tg mice. A, Pressure overload–mediated (left) or Dox-mediated (right) percentage of TUNEL-positive nuclei in NLC and CA-MEK5α-Tg mice. B, Pressure overload–mediated (left) or Dox-mediated (right) cleaved caspase-3 expression in NLC and CA-MEK5α-Tg mice. The intensity of the band representing cleaved caspase-3 was normalized to the intensity of 1 control samples that was designated as 1. Data represent mean±SD of 3 samples.** P<0.01 compared with NLC sham (left) or vehicle treatment (right).

Figure 7

Cardioprotective role of ERK5 activation in Dox- and TAC-mediated heart failure model. A, Representative M-mode echocardiogram of NLC and CA-MEK5α-Tg mice treated with vehicle or Dox. Dd indicates diameter of diastole; Ds, diameter of systolic. B, Percentage of fractional shortening (FS) in NLC and CA-MEK5α-Tg mice after vehicle or Dox treatment. C and D, Hemodynamic measurements in NLC and CA-MEK5a-Tg mice after Dox treatment (C) or TAC (D). All data are expressed as mean±SD. **P<0.01.

Figure 8

Scheme of ERK5/MEF2 activation and PDE3A/ICER feedback loop. ISO induced sustained downregulation of PDE3A expression and upregulation of ICER, which is regulated by PDE3A/ICER-positive feedback loop. IGF-1–induced ERK5 activation leads to PDE3A expression caused by MEF2-dependent PDE3A promoter activation and inhibits induction of PDE3A/ICER autoregulatory positive-feedback loop, which plays a key role in cardiomyocyte apoptosis.