Metabolic Interventions to Prevent Hypertrophy-Induced Alterations in Contractile Properties In Vitro

Figures

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  Figure 1. Time-dependent effects of phenylephrine on hypertrophic gene expression, protein synthesis, and autophagy. (A,B) mRNA expression levels of hypertrophic markers brain natriuretic peptide (BNP) and atrial natriuretic factor (ANF) after 16 h, 24 h, and 48 h treatment with phenylephrine (50 µM), expressed in fold increase relative to control (n = 5). (C) Protein synthesis after 16 h, 24 h, and 48 h stimulation with phenylephrine (50 µM), using [14C] phenylalanine incorporation expressed in pCi/mg wet weight/hour. Data are presented as means ± standard deviation (STDEV) (n = 5). (D) Representative Western blots of the autophagy-related proteins, LC3B and p62, after 16 h, 24 h, and 48 h phenylephrine (PE) stimulation compared to control. Co-treatment with bafilomycin was added to inhibit autophagic flux, and β-actin was used as the loading control. (E,F) Quantification of LC3BII and p62 protein levels normalized to β-actin. Values are expressed as fold increase ± STDEV relative to 16 h control incubation (n = 3/n = 4). * p < 0.05 compared to control, # p < 0.05 compared to PE were considered significantly different at the same incubation time.

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  Figure 2. Time-dependent effect of phenylephrine on cellular signaling. (A) Representative Western blots showing phosphorylation levels of proteins involved in either protein synthesis (p-mTOR(ser2448), total mTOR, p-4EBP(Thr37/46), p-rS6(Ser235/236), and p-HDAC5(Ser498)) or the regulation of cellular glucose uptake (p-Akt(Ser473), p-AS160(Thr642), p-PKD1(Ser744), p-PKD1(Ser916), p-TropI(Ser23/24), p-AMPK(Thr172), total AMPK, and p-ACC(Ser79)) after 16 h, 24 h, and 48 h phenylephrine (50 µM) stimulation. (B–L) Quantification of these protein levels normalized to Caveolin 3 (CAV3). Values are expressed as fold increase ± STDEV relative to their control at the same incubation time (n = 7/n = 9). * p < 0.05 was considered significantly different compared to control at the same incubation time.

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  Figure 3. Time-dependent effect of phenylephrine on cellular substrate uptake. Glucose uptake after 16 h, 24 h, and 48 h phenylephrine (50 µM) stimulation, expressed in pmol/mg wet weight/min. Values are expressed as means ± STDEV for each incubation time (n = 7). * p < 0.05 was considered significantly different compared to control at the same incubation time.

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  Figure 4. Contractile properties of isolated cardiomyocytes after 16 h, 24 h, and 48 h phenylephrine (50 µM) treatment, at 1 Hz electric-field stimulation, and a summary of the main findings with respect to the established in vitro model of cardiac hypertrophy. (A–D) Sarcomere shortening, contraction rate, time to peak, and decay time after 16 h and 24 h phenylephrine stimulation compared to that of control. (E) Representative pictures of control and phenylephrine-stimulated cells after 48 h culture. (F) Table depicting the effects of phenylephrine treatment on glucose uptake, protein synthesis, hypertrophic gene expression, and contractile function after 16 h, 24 h, and 48 h phenylephrine stimulation. The minus symbols indicate no change. The arrows up and down indicate an increase or decrease, respectively. The hazard symbol indicates that the cells died. Values are expressed as means ± STDEV for each incubation time (n = 7; imaging of 10 cells/condition). * p < 0.05 was considered significantly different compared to control at the same incubation time.

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  Figure 5. The effect of the PAN-Akt inhibitor (5 µM) on GLUT4 translocation, glucose uptake, hypertrophic gene expression, protein synthesis, contractile function, and cellular signaling after co-treatment with PE (50 µM) for 24 h. GLUT4 translocation (scale bar 20 µm) (representative confocal images of n = 3, see Supplemental Figure S2) (A), glucose uptake (n = 5) (B), hypertrophic gene expression (n = 3) (C), and protein synthesis (n = 5) (D) are displayed in fold increase relative to control. (E–G) Contractile properties upon 1 Hz electric-field stimulation: sarcomere shortening, time to peak, and decay time. Values are expressed as means ± STDEV (n = 4). (H) Representative Western blots showing phosphorylation levels of proteins involved in the Pi3K/Akt/mTOR-pathway (p-Akt(Ser473), p-AS160(Thr642), p-mTOR(ser2448), mTOR, and p-rS6(Ser235/236)) and the PKD1-pathway (p-PKD1(ser744), p-PKD1(ser916), p-TropI(Ser23/24), and p-HDAC5(Ser498)) (H–O). Finally, phosphorylation of ERK1/2 (p-ERK1/2-thr202/tyr204) is shown (P). For quantification, protein levels were normalized to Caveolin 3 (CAV3). Values are expressed as fold increase ± STDEV relative to control (n = 7/n = 9). * p < 0.05 was…

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  Figure 6. The effect of dipyridamole (DPY) (10 µM) on GLUT4 translocation, glucose uptake, hypertrophic gene expression, protein synthesis, contractile function, and cellular signaling after 24 h stimulation with phenylephrine (50 µM). GLUT4 translocation (scale bar 20 µm) (representative confocal images of n = 3, also see Supplemental Figure S2) (A), glucose uptake (n = 5) (B), hypertrophic gene expression (n = 5) (C), and protein synthesis (n = 5) (D) are displayed in fold increase relative to control. (E–G) Contractile properties upon 1 Hz electric-field stimulation: sarcomere shortening, time to peak, and decay time. Values are expressed as means ± STDEV (n = 5). (H) Representative Western blots depicting phosphorylation levels of proteins involved in the Pi3K/Akt/mTOR-pathway (p-Akt(Ser473), p-AS160(Thr642), p-mTOR(ser2448), and p-rS6(Ser235/236)) and the PKD1-pathway (p-PKD1(ser744), p-TropI(Ser23/24), and p-HDAC5(Ser498)). (I–O) For quantification, protein levels were normalized to Caveolin 3 (CAV3). Values are expressed as fold increase ± STDEV relative to control (n = 5/n = 7). * p < 0.05 was considered significantly different compared to control.

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  Figure 7. Integration of mechanisms of the phenylephrine (PE)-induced increase in glucose uptake and hypertrophy in cardiomyocytes—a hypothetical scheme. PE induces both activation of the Akt and protein kinase D (PKD1) signaling pathways. The PE-induced phosphorylation of Akt and of PKD1 leads to increased translocation of the main glucose transporter GLUT4 from the endosomes to the cell surface, resulting in increased glucose uptake. At the same time, activated Akt and PKD1 lead to the phosphorylation and nuclear export of HDAC5, resulting in release and activation of hypertrophic transcription factors. Additionally, PE-induced Akt phosphorylation causes activation of mTOR, a major regulator of protein synthesis. Both the PE- induced increase in glucose uptake and hypertrophy contribute to contractile alterations. Treatment of cardiomyocytes with PAN-Akt inhibitor or DPY inhibit GLUT4 translocation or GLUT4-mediated glucose transport, respectively, and protect the cell from contractile alterations. Since DPY treatment does not restore the PE-induced increases in protein synthesis and hypertrophic markers, yet restores contractile properties, it follows that targeting metabolism,…