Limits...
mTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel.

Huang TQ, Zou MX, Pasek DA, Meissner G - J Receptor Ligand Channel Res (2015)

Bottom Line: Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased.However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204.Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2(ADA/ADA) mice but did not extend longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA.

ABSTRACT

Simultaneous substitution of three amino acid residues in the calmodulin binding domain (W3587A/L3591D/F3603A, ADA) of the cardiac ryanodine receptor ion channel (RyR2) impairs calmodulin inhibition of RyR2 and causes cardiac hypertrophy and early death of Ryr2(ADA/ADA) mice. To determine the physiological significance of growth promoting signaling molecules, the protein and phosphorylation levels of Ser/Thr kinase mTOR and upstream and downstream signaling molecules were determined in hearts of wild-type and Ryr2(ADA/ADA) mice. Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased. However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204. To confirm a role for mTOR signaling in the development of cardiac hypertrophy, rapamycin, an inhibitor of mTOR, was injected into wild-type and mutant mice. Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2(ADA/ADA) mice but did not extend longevity. Taken together, the results link a dysfunctional RyR2 to an altered activity of signaling molecules that regulate cardiac growth and function.

No MeSH data available.


Related in: MedlinePlus

Protein and phosphorylation levels of mTOR and downstream targets.Notes: (A and B) Immunoblots of protein and phosphorylation levels of heart homogenates from two individual 1-day- and 10-day-old W and H mice. GAPDH was the loading control. (C–F) Protein levels and phosphorylation/protein ratios normalized to W. Data are the mean ± standard error of six mTOR, five to nine p70S6K, six to nine S6, and eight to nine 4E-BP1 determinations. *P<0.05 compared with W using two-way analysis of variance.Abbreviations: H, Ryr2ADA/ADA; W, wild-type; mTOR, mammalian target of rapamycin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4547478&req=5

Figure 1: Protein and phosphorylation levels of mTOR and downstream targets.Notes: (A and B) Immunoblots of protein and phosphorylation levels of heart homogenates from two individual 1-day- and 10-day-old W and H mice. GAPDH was the loading control. (C–F) Protein levels and phosphorylation/protein ratios normalized to W. Data are the mean ± standard error of six mTOR, five to nine p70S6K, six to nine S6, and eight to nine 4E-BP1 determinations. *P<0.05 compared with W using two-way analysis of variance.Abbreviations: H, Ryr2ADA/ADA; W, wild-type; mTOR, mammalian target of rapamycin.

Mentions: Mice impaired in CaM regulation of the cardiac muscle RyR2 ion channel (Ryr2ADA/ADA mice) develop cardiac hypertrophy and reduced cardiac function by postnatal day 1, and die 2–3 weeks after birth.9 To determine the role of mTOR signaling, hearts were collected from 1-day- and 10-day-old Ryr2ADA/ADA mice. Protein and phosphorylation levels of mTOR and downstream targets were determined by immunoblot analysis (Figure 1A and B). p-mTOR-Ser-2448/mTOR protein ratios increased 1.4- and 1.5-fold in 1-day- and 10-day-old Ryr2ADA/ADA hearts compared to wild-type hearts, respectively (Figure 1C and D), without significant changes in mTOR protein levels (Figure 1E and F).


mTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel.

Huang TQ, Zou MX, Pasek DA, Meissner G - J Receptor Ligand Channel Res (2015)

Protein and phosphorylation levels of mTOR and downstream targets.Notes: (A and B) Immunoblots of protein and phosphorylation levels of heart homogenates from two individual 1-day- and 10-day-old W and H mice. GAPDH was the loading control. (C–F) Protein levels and phosphorylation/protein ratios normalized to W. Data are the mean ± standard error of six mTOR, five to nine p70S6K, six to nine S6, and eight to nine 4E-BP1 determinations. *P<0.05 compared with W using two-way analysis of variance.Abbreviations: H, Ryr2ADA/ADA; W, wild-type; mTOR, mammalian target of rapamycin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4547478&req=5

Figure 1: Protein and phosphorylation levels of mTOR and downstream targets.Notes: (A and B) Immunoblots of protein and phosphorylation levels of heart homogenates from two individual 1-day- and 10-day-old W and H mice. GAPDH was the loading control. (C–F) Protein levels and phosphorylation/protein ratios normalized to W. Data are the mean ± standard error of six mTOR, five to nine p70S6K, six to nine S6, and eight to nine 4E-BP1 determinations. *P<0.05 compared with W using two-way analysis of variance.Abbreviations: H, Ryr2ADA/ADA; W, wild-type; mTOR, mammalian target of rapamycin.
Mentions: Mice impaired in CaM regulation of the cardiac muscle RyR2 ion channel (Ryr2ADA/ADA mice) develop cardiac hypertrophy and reduced cardiac function by postnatal day 1, and die 2–3 weeks after birth.9 To determine the role of mTOR signaling, hearts were collected from 1-day- and 10-day-old Ryr2ADA/ADA mice. Protein and phosphorylation levels of mTOR and downstream targets were determined by immunoblot analysis (Figure 1A and B). p-mTOR-Ser-2448/mTOR protein ratios increased 1.4- and 1.5-fold in 1-day- and 10-day-old Ryr2ADA/ADA hearts compared to wild-type hearts, respectively (Figure 1C and D), without significant changes in mTOR protein levels (Figure 1E and F).

Bottom Line: Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased.However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204.Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2(ADA/ADA) mice but did not extend longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA.

ABSTRACT

Simultaneous substitution of three amino acid residues in the calmodulin binding domain (W3587A/L3591D/F3603A, ADA) of the cardiac ryanodine receptor ion channel (RyR2) impairs calmodulin inhibition of RyR2 and causes cardiac hypertrophy and early death of Ryr2(ADA/ADA) mice. To determine the physiological significance of growth promoting signaling molecules, the protein and phosphorylation levels of Ser/Thr kinase mTOR and upstream and downstream signaling molecules were determined in hearts of wild-type and Ryr2(ADA/ADA) mice. Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased. However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204. To confirm a role for mTOR signaling in the development of cardiac hypertrophy, rapamycin, an inhibitor of mTOR, was injected into wild-type and mutant mice. Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2(ADA/ADA) mice but did not extend longevity. Taken together, the results link a dysfunctional RyR2 to an altered activity of signaling molecules that regulate cardiac growth and function.

No MeSH data available.


Related in: MedlinePlus