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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03185

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HDAC activity regulates entry of mesoderm cells into the cardiac muscle lineage

¹ Department of Biochemistry, Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada
² Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada

View larger version (136K): [in a new window]   Fig. 1. TSA induces cardiomyogenesis. P19 cells were aggregated in the (A,B) absence of DMSO, (C,D) with 0.8% DMSO or (E,F) with 5 nM TSA. After 6 days, cells were stained with an anti-myosin heavy-chain antibody (anti-MyHC) and examined by immunofluorescence (B,D,F). Hoechst dye 33258 was used to visualize nuclei (A,C,E). Magnification 400x.

View larger version (44K): [in a new window]   Fig. 2. TSA treatment of P19 cells induces the expression of cardiac -actin, Gata4, Bmp4, Mef2c and Nkx2-5. P19 cells were aggregated and treated with 0, 2 nM, 5 nM or 10 nM TSA, or DMSO each day (day 0 to day 4). Total RNA was harvested on days 0, 2, 3, 4 and 6. (I) Northern blot analysis was used to detect the transcripts for cardiac -actin, Gata4, Bmp4 and Brachyury T from 12 µg of total RNA on the days indicated. (II) RT-PCR was performed on the RNA harvested on days 0, 2, 3, 4 and 6, and Southern blot analysis was used to detect for the expression of Nkx2-5 and Mef2c. Lane 17 (+) shows a positive control with RNA from DMSO-induced cardiomyocytes on day 6. RT-PCR controls include the positive control without reverse transcriptase (lane 18), with control RNA (lane 19) and with PCR-H2O (lane 20). (III, IV) Densitometric quantification was performed for the expression of (III) Gata4 and cardiac -actin by northern blot analysis and (IV) Nxk2-5 and Mef2c by RT-PCR for cells treated with 5 nM TSA. Results are shown as the fold enhancement of transcript levels in TSA-treated cells compared with untreated cells on days 2, 3, 4 and 6. Results are also shown for the fold enhancement of Gata4 and cardiac -actin transcripts on day 6 in cells treated with DMSO compared with untreated cells. Error bars represent the standard error (+ s.e.) of three separate experiments.

View larger version (42K): [in a new window]   Fig. 3. Overexpression of HDAC4 inhibits cardiomyogenesis. P19(control) and P19(HDAC4) cell lines were aggregated in the presence of DMSO and total RNA was harvested on days 0, 3 and 6. (I) Northern blot analyses of 12 µg of total RNA from P19(control) and P19(HDAC4) cells probed as indicated on the right. (II) Total RNA was reverse-transcribed and PCR products for Nkx2-5 were detected by Southern blot analysis. Lanes were spliced from the same autoradiogram.

View larger version (30K): [in a new window]   Fig. 4. Inhibition of CaMK activity diminishes the specification of P19 cells to cardiomyoblasts. P19 cells were aggregated with and without DMSO in the presence or absence of the CaMK inhibitor KN-93 or its non-functional analogue KN-92. (I) Total RNA was harvested on day 0 (lane 1); day 3 without DMSO (lane 2), day 3 with DMSO (lane 3), day 3 with DMSO and in the presence of KN-92 at 10 µM or 7.5 µM (lanes 4 or 5, respectively), and day 3 with DMSO and in the presence of KN-93 at 10 µM or 7.5 µM (lanes 6 or 7, respectively). Northern blot analysis was performed with 12 µg of total RNA to detect expression of the transcripts indicated on the right. (II) P19 cells were treated with 2.5 µM, 5.0 µM, 7.5µM and 10 µM of KN-93 or KN-92 and the level of Gata4 expression was quantified by densitometry. Bars represent the densitometric measurement of Gata4 transcripts from one representative experiment relative to rabbit 18S cDNA.

View larger version (19K): [in a new window]   Fig. 5. CaMKIV* enhances cardiomyogenesis in P19 cells grown in monolayer. P19 cells were transfected with or without CaMKIV* (1.6 µg). (I) Total RNA was isolated from transfected cells after 1 day of transfection and reverse transcribed. PCR products from the amplification of Nkx2-5, Mef2c, Gata4 and ß-actin were detected using Southern blot analysis. (II) P19 cells were fixed after 4 days of transfection, stained with anti-myosin heavy-chain (MyHC) antibody, and examined by immunofluorescence. The number of MyHC-positive cells under each condition was counted. Error bars represent the standard error (+ s.e.); n=3.

View larger version (14K): [in a new window]   Fig. 6. CaMKIV* alone can activate a MEF2-responsive promoter, in a fashion that depends on the MEF2 sites. P19 cells were co-transfected with 0.8 µg of HDAC4, MEF2C and CaMKIV* expression vectors individually and in combination, and with either a wild-type or mutated MEF2-responsive promoter (1.4 µg) as indicated. Cells were harvested 24 hours after transfection and CAT assays were used to measure the activity from the MEF2-responsive promoter. Error bars represent the standard error (+ s.e.); n=2-13.

View larger version (46K): [in a new window]   Fig. 7. Overexpression of dominant-negative MEF2 (MEF2C/EnR) enhances the development of cardiac muscle. P19(MEF2C/EnR) and P19(control) cells were differentiated in the presence of DMSO. (I), (A) P19(control) and (B) P19 (MEF2C/EnR) cells were fixed and immunostained on day 6 of differentiation with anti-MyHC antibody to detect cardiomyocytes (magnification 400x). (II) RNA was harvested on days 0, 6 and 9. Northern blots containing 6 µg of total RNA were probed as indicated on the right. (III) RNA from day 6 and 9 were reverse-transcribed and Nkx2-5 PCR products were detected using Southern blot analysis. (IV) 10T1/2 fibroblasts were transfected with and without MyoD and MEF2C/EnR. After 6 days in differentiation medium, MyHC-positive cells were counted and normalized for transfection efficiency with GFP and standardized to MyoD conversion levels. Error bars represent the standard error (+ s.e.); n=3.

View larger version (10K): [in a new window]   Fig. 8. Mechanistic model. HDAC activity negatively regulates the entry of mesoderm cells into the cardiac-muscle lineage. Relief of HDAC inhibition by TSA, CaMK or, possibly, DMSO enhances the differentiation of P19 stem cells into cardiomyocytes, whereas the overexpression of HDAC4 inhibits differentiation (bold, black). Thus, HDAC activity inhibits mesoderm cells from entering the cardiac lineage and upregulating the expression of Nkx2-5, Gata4 and Mef2c. Furthermore, the overexpression of the dominant-negative MEF2C/EnR enhances cardiomyogenensis, by possibly disrupting HDAC activity, allowing more cells into the cardiac lineage. Similarly, MEF2C/EnR expressed in cardiomyoblasts displays an initial enhancement of the cardiac factors; however, MEF2C/EnR in these cells (asterisk) results in the subsequent downregulation of this positive loop and inhibition of cardiomyogenesis (Karamboulas et al., 2006).

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