Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes

Figures

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  Fig. 1. Study design and treatment protocol. Cardiomyocytes were treated with hypertrophic growth stimuli (indicated in green) for 24 h in order to determine MMP expression under these stimuli. In another set of experiments, cardiomyocytes were treated with MMP inhibitors (indicated in red) for 24 h in order to determine their influence on hypertrophic growth. In addition, inhibition of MMP activity was proved in zymography assays. Finally, phosphorylation of kinases, typically related to hypertrophic growth (ERK, Akt, and GSK3β), was determined under MMP inhibition with TAPI-0 within 5 min to 2 h, and the influence of ERK or PI3K inhibition on TAPI-0 induced hypertrophic growth was determined

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  Fig. 2. MMP mRNA expression in cardiomyocytes. (A) Total RNA was isolated from ventricular cardiomyocytes, RT-PCR performed, and amplification products were loaded on agarose gels (n = 4 independent culture preparations). mRNA expression of MMP types1, 2, 3, 9, and 14 was detected (indicated by arrows). (B) To determine the changes in mRNA expression under hypertrophic growth stimulation, cardiomyocytes were incubated for 24 h with phenylephrine (PE, 10 μM), TGFβ1 (1 ng/ml), or angiotensin II (Ang, 100 nM). mRNA expression of MMP types was analyzed by real-time RT-PCR (n = 7 – 9, *p < 0.05 vs. controls)

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  Fig. 3. MMP protein expression under hypertrophic growth stimulation. Cardiomyocytes were incubated for 24 h with phenylephrine (PE, 10 μM), TGFβ1 (1 ng/ml), or angiotensin II (Ang, 100 nM). Protein expression of MMP-2, MMP-9, and MMP-14 could be detected in (A) Western blots, and (B) bands were densiometrically evaluated. Vinculin expression served as loading control. (n = 7 – 11, *p < 0.05 vs. controls)

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  Fig. 4. MMP mRNA expression in SHR rats. Total RNA was isolated from the hearts of pre-hypertensive 1.5-month-old and hypertensive 7.5-month-old SHR rats, as well as from age-matched normotensive Wistar rats. mRNA expression of MMP-9 was analyzed by real-time RT-PCR (n = 5 – 6, *p < 0.05 vs. normotensive Wistar rats)

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  Fig. 5. Pharmacologic inhibition of MMP activity. Cardiomyocytes were incubated with phenylephrine (PE, 10 μM), ARP-100 (50 nM), or TAPI-0 (50 μM) for 24 h. Then, MMP activity was determined by zymography. (A) Representative zymogram. (B) Densiometric evaluation of MMP activity (n = 6 for PE, n = 11 for ARP-100, n = 6 for TAPI-0, p < 0.05 vs. control)

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  Fig. 6. MMP inhibitors enhance hypertrophic growth. Cardiomyocytes were incubated with the MMP inhibitors TAPI-0 (50 μM), TIMP2 (24 mM), and ARP-100 (50 nM) for 24 h. For detection of hypertrophic growth, incorporation of 14C-phenylalanine during 24 h, or cross-sectional area, was determined after 24 h. Data are expressed as percent increase relative to untreated controls and are means ± SE of 4–17 independent culture preparations. *Differences from unstimulated controls with p < 0.05

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  Fig. 7. Hypertrophic growth under MMP inhibition is mediated by ERK. Cardiomyocytes were stimulated with TAPI-0 (50 μM) for 24 h. For inhibition of ERK, PD98059 (PD, 10 μM) was added 30 min before TAPI-0. (A) For detection of phosphorylated and, thus, activated ERK, cardiomyocytes were stimulated with TAPI-0 (50 ng/ml) for 5 min up to 2 h. Total protein extracts were prepared, separated on 10% SDS gels. ERK activation was determined in Western blots by determination of p-ERK/ERK levels. Data are expressed as percent increase relative to untreated controls and are means ± SE of seven independent culture preparations. *Differences from unstimulated controls with p < 0.05. (B) For detection of hypertrophic growth, an increase in cross-sectional area during 24 h was determined. Data are expressed as percent increase relative to untreated controls and are means ± SE of six independent culture preparations. *Differences from unstimulated controls with p < 0.05. #Differences from TAPI-0 stimulated cells with p < 0.05

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  Fig. 8. Hypertrophic growth under MMP inhibition is mediated by PI3K. Cardiomyocytes were stimulated with TAPI-0 (50 μM) for 24 h. For inhibition of PI3K, LY290042 (LY, 10 μM) was added 30 min before TAPI-0. (A) For detection of phosphorylated and thus activated PI3K, cardiomyocytes were stimulated with TAPI-0 (50 ng/ml) for 5 min up to 2 h. Total cell extracts were prepared and separated on 10% SDS-gels. PI3K activation was determined in Western blots by determination of p-PI3K. (B) For detection of hypertrophic growth, an increase in cross-sectional area during 24 h was determined. Data are expressed as percent increase relative to untreated controls and are means ± SE of n = 4–10 independent culture preparations. *Differences from unstimulated controls with p < 0.05. #Differences from TAPI-0 stimulated cells with p < 0.05

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  Fig. 9. MMP inhibition reduces GSK3β phosphorylation and activity. (A) Cardiomyocytes were stimulated with TAPI-0 (50 ng/ml) for 5 min up to 2 h. Total cell extracts were prepared and separated on 10% SDS gels. GSK3β activation was determined in Western blots using antibodies specific for P-serine9-GSK3β. Data are expressed as percent increase relative to untreated controls and are means ± SE of nine independent culture preparations. *Differences from unstimulated controls with p < 0.05. (B) Cardiomyocytes were stimulated with TAPI-0 (50 ng/ml) for 1 h. Total cell extracts were prepared, GSK3β was immunoprecipitated, and phosphorylation of GSK3β substrate peptide was determined in vitro. Controls for GSK3β specificity were done either by addition of the GSK3β inhibitor SB 415286 (SB) to the kinase reaction or by omission of GSK3β antibodies (AB) during immunoprecipitation. Data are expressed as percent increase relative to untreated controls and are means ± SE of three independent culture preparations. *Differences from unstimulated controls with p < 0.05. **Differences from unstimulated controls with p < 0.01

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  Fig. 10. Proposed signaling pathway under MMP inhibition. Inhibitors used for dissection of the pathway are indicated in red rectangles. The pathway via Akt and GSK3 beta is assumed due to the activation/inhibition of these molecules under MMP inhibition combined with the knowledge that PI3K can signal via these pathways, as outlined in the discussion