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PKG and PKC Are Down-Regulated during Cardiomyocyte Differentiation from Embryonic Stem Cells: Manipulation of These Pathways Enhances Cardiomyocyte Production.

Mobley S, Shookhof JM, Foshay K, Park M, Gallicano GI - Stem Cells Int (2010)

Bottom Line: Subjecting nascent ESC-derived cardiomyocytes to a proteomics assay, we found that cardiomyogenesis is influenced by up- and down-regulation of a number of kinases, one of which, cGMP-dependent protein kinase (PKG), is markedly down-regulated during differentiation.Delving further, we found that manipulating the PKG pathway using PKG-specific inhibitors produced significantly more cardiomyocytes from ESCs when compared to ESCs left to differentiate without inhibitors.In addition, we found combinatorial effects when culturing ESCs in inhibitors to PKG and PKC isotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Med/Dent Building NE205, 3900 Reservoir Road NW, Washington, DC 20057-1455, USA.

ABSTRACT
Understanding signal transduction mechanisms that drive differentiation of adult or embryonic stem cells (ESCs) is imperative if they are to be used to cure disease. While the list of signaling pathways regulating stem cell differentiation is growing, it is far from complete. Indentifying regulatory mechanisms and timecourse commitment to cell lineages is needed for generating pure populations terminally differentiated cell types, and in ESCs, suppression of teratoma formation. To this end, we investigated specific signaling mechanisms involved in cardiomyogenesis, followed by manipulation of these pathways to enhance differentiation of ESCs into cardiomyocytes. Subjecting nascent ESC-derived cardiomyocytes to a proteomics assay, we found that cardiomyogenesis is influenced by up- and down-regulation of a number of kinases, one of which, cGMP-dependent protein kinase (PKG), is markedly down-regulated during differentiation. Delving further, we found that manipulating the PKG pathway using PKG-specific inhibitors produced significantly more cardiomyocytes from ESCs when compared to ESCs left to differentiate without inhibitors. In addition, we found combinatorial effects when culturing ESCs in inhibitors to PKG and PKC isotypes. Consequently, we have generated a novel hypothesis: Down-regulation of PKG and specific PKC pathways are necessary for cardiomyogenesis, and when manipulated, these pathways produce significantly more cardiomyocytes than untreated ESCs.

No MeSH data available.


Related in: MedlinePlus

Gene expression was measured by semiquantitative RT-PCR using PKG1-specific primers. (a) Beating and nonbeating areas of similar sizes were dissected apart as described in [4, 10], followed by RNA isolation and quantification. Control GAPDH RT-PCR amplification revealed that 35 cycles were in the linear range and nucleotides in the PCR reaction were not exhausted. (b) After normalization to GAPDH levels, PKG1 mRNA levels were lower by approximately one-half in beating areas when compared to nonbeating areas (pixel intensity = 70 ± 11.0 to 139 ± 19.1, resp.). A representative ratio of PKG mRNA signal revealed 1.98X more in nonbeating areas when compared to beating areas (inset in (b)). (c) Inhibiting PKG1 resulted in dramatic and significant (P < .005) increase in the size of beating areas within EBs; however, the total size of EBs was not changed by PKG inhibition (d).
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fig2: Gene expression was measured by semiquantitative RT-PCR using PKG1-specific primers. (a) Beating and nonbeating areas of similar sizes were dissected apart as described in [4, 10], followed by RNA isolation and quantification. Control GAPDH RT-PCR amplification revealed that 35 cycles were in the linear range and nucleotides in the PCR reaction were not exhausted. (b) After normalization to GAPDH levels, PKG1 mRNA levels were lower by approximately one-half in beating areas when compared to nonbeating areas (pixel intensity = 70 ± 11.0 to 139 ± 19.1, resp.). A representative ratio of PKG mRNA signal revealed 1.98X more in nonbeating areas when compared to beating areas (inset in (b)). (c) Inhibiting PKG1 resulted in dramatic and significant (P < .005) increase in the size of beating areas within EBs; however, the total size of EBs was not changed by PKG inhibition (d).

Mentions: The raw data from the kinase expression screen indicated a dramatic decrease of PKG1 in beating areas when compared to nonbeating control areas (Figures 1(a)–1(c)). Western blot analyses by two independent laboratories verified PKG1 downregulation in cardiomyocytes (Figure 1(d)). After normalizing intensity of protein levels to tubulin, nonbeating areas were determined to have 1.7X (69%) more PKG1 protein than beating areas. Confocal microscopy corroborated the proteomics screen (Figures 1(e)–1(g)). Newly differentiated, beating cardiomyocytes identified by cardiac troponin T-(Tnnt2-) positive staining (Figure 1(f)) showed lower levels of PKG1 in beating areas when compared to Tnnt2-negative, nonbeating areas (Figures 1(e)–1(g)). Semi-quantitative RT-PCR revealed PKG1 mRNA levels lower by approximately one-half in beating areas when compared to nonbeating areas confirming Western blot analyses (Figures 2(a) and 2(b)).


PKG and PKC Are Down-Regulated during Cardiomyocyte Differentiation from Embryonic Stem Cells: Manipulation of These Pathways Enhances Cardiomyocyte Production.

Mobley S, Shookhof JM, Foshay K, Park M, Gallicano GI - Stem Cells Int (2010)

Gene expression was measured by semiquantitative RT-PCR using PKG1-specific primers. (a) Beating and nonbeating areas of similar sizes were dissected apart as described in [4, 10], followed by RNA isolation and quantification. Control GAPDH RT-PCR amplification revealed that 35 cycles were in the linear range and nucleotides in the PCR reaction were not exhausted. (b) After normalization to GAPDH levels, PKG1 mRNA levels were lower by approximately one-half in beating areas when compared to nonbeating areas (pixel intensity = 70 ± 11.0 to 139 ± 19.1, resp.). A representative ratio of PKG mRNA signal revealed 1.98X more in nonbeating areas when compared to beating areas (inset in (b)). (c) Inhibiting PKG1 resulted in dramatic and significant (P < .005) increase in the size of beating areas within EBs; however, the total size of EBs was not changed by PKG inhibition (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2963170&req=5

fig2: Gene expression was measured by semiquantitative RT-PCR using PKG1-specific primers. (a) Beating and nonbeating areas of similar sizes were dissected apart as described in [4, 10], followed by RNA isolation and quantification. Control GAPDH RT-PCR amplification revealed that 35 cycles were in the linear range and nucleotides in the PCR reaction were not exhausted. (b) After normalization to GAPDH levels, PKG1 mRNA levels were lower by approximately one-half in beating areas when compared to nonbeating areas (pixel intensity = 70 ± 11.0 to 139 ± 19.1, resp.). A representative ratio of PKG mRNA signal revealed 1.98X more in nonbeating areas when compared to beating areas (inset in (b)). (c) Inhibiting PKG1 resulted in dramatic and significant (P < .005) increase in the size of beating areas within EBs; however, the total size of EBs was not changed by PKG inhibition (d).
Mentions: The raw data from the kinase expression screen indicated a dramatic decrease of PKG1 in beating areas when compared to nonbeating control areas (Figures 1(a)–1(c)). Western blot analyses by two independent laboratories verified PKG1 downregulation in cardiomyocytes (Figure 1(d)). After normalizing intensity of protein levels to tubulin, nonbeating areas were determined to have 1.7X (69%) more PKG1 protein than beating areas. Confocal microscopy corroborated the proteomics screen (Figures 1(e)–1(g)). Newly differentiated, beating cardiomyocytes identified by cardiac troponin T-(Tnnt2-) positive staining (Figure 1(f)) showed lower levels of PKG1 in beating areas when compared to Tnnt2-negative, nonbeating areas (Figures 1(e)–1(g)). Semi-quantitative RT-PCR revealed PKG1 mRNA levels lower by approximately one-half in beating areas when compared to nonbeating areas confirming Western blot analyses (Figures 2(a) and 2(b)).

Bottom Line: Subjecting nascent ESC-derived cardiomyocytes to a proteomics assay, we found that cardiomyogenesis is influenced by up- and down-regulation of a number of kinases, one of which, cGMP-dependent protein kinase (PKG), is markedly down-regulated during differentiation.Delving further, we found that manipulating the PKG pathway using PKG-specific inhibitors produced significantly more cardiomyocytes from ESCs when compared to ESCs left to differentiate without inhibitors.In addition, we found combinatorial effects when culturing ESCs in inhibitors to PKG and PKC isotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Med/Dent Building NE205, 3900 Reservoir Road NW, Washington, DC 20057-1455, USA.

ABSTRACT
Understanding signal transduction mechanisms that drive differentiation of adult or embryonic stem cells (ESCs) is imperative if they are to be used to cure disease. While the list of signaling pathways regulating stem cell differentiation is growing, it is far from complete. Indentifying regulatory mechanisms and timecourse commitment to cell lineages is needed for generating pure populations terminally differentiated cell types, and in ESCs, suppression of teratoma formation. To this end, we investigated specific signaling mechanisms involved in cardiomyogenesis, followed by manipulation of these pathways to enhance differentiation of ESCs into cardiomyocytes. Subjecting nascent ESC-derived cardiomyocytes to a proteomics assay, we found that cardiomyogenesis is influenced by up- and down-regulation of a number of kinases, one of which, cGMP-dependent protein kinase (PKG), is markedly down-regulated during differentiation. Delving further, we found that manipulating the PKG pathway using PKG-specific inhibitors produced significantly more cardiomyocytes from ESCs when compared to ESCs left to differentiate without inhibitors. In addition, we found combinatorial effects when culturing ESCs in inhibitors to PKG and PKC isotypes. Consequently, we have generated a novel hypothesis: Down-regulation of PKG and specific PKC pathways are necessary for cardiomyogenesis, and when manipulated, these pathways produce significantly more cardiomyocytes than untreated ESCs.

No MeSH data available.


Related in: MedlinePlus