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. 2015 Apr 1:184:350-363.
doi: 10.1016/j.ijcard.2015.01.091. Epub 2015 Feb 25.

Cardiac tissue inhibitor of matrix metalloprotease 4 dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to therapy

Pankaj Chaturvedi  1 , Anuradha Kalani  2 , Anastasia Familtseva  2 , Pradip Kumar Kamat  2 , Naira Metreveli  2 , Suresh C Tyagi  2
Affiliations

Cardiac tissue inhibitor of matrix metalloprotease 4 dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to therapy

Pankaj Chaturvedi et al. Int J Cardiol. .

Abstract

Background: TIMP4 (Tissue Inhibitors of Matrix Metalloprotease 4), goes down in failing hearts and mice lacking TIMP4 show poor regeneration capacity after myocardial infarction (MI). This study is based on our previous observation that administration of cardiac inhibitor of metalloproteinase (~TIMP4) attenuates oxidative stress and remodeling in failing hearts. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by augmenting contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes.

Methods: To validate this hypothesis, we transfected mouse cardiomyocytes with TIMP4 and TIMP4-siRNA and performed contractility studies in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. We evaluated the calcium channel gene serca2a (sarcoplasmic reticulum calcium ATPase2a) and mir122a which tightly regulates serca2a to explain the changes in contractility. We treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody) to examine the effect of TIMP4 on differentiation of cardiac progenitor cells.

Results: Contractility was augmented in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. There was elevated expression of serca2a in the TIMP4 transformed myocytes and down regulation of mir122a. The cells treated with cardiac extract containing TIMP4 showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression.

Conclusion: This is a novel report suggesting that TIMP4 augments contractility and induces differentiation of progenitor cells into cardiac phenotype. In view of the failure of MMP9 inhibitors for cardiac therapy, TIMP4 provides an alternative approach, being an indigenous molecule and a natural inhibitor of MMP9.

Keywords: Cardiomyocytes; Contractility; MicroRNA; Stem cells; Tissue inhibitor of matrix metalloprotease (TIMP).

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Figures

Fig. 1
Fig. 1
A) Cardiomyocytes were isolated from mouse (C57BL/6) heart and represented 90% rod shaped cells. B) Schematic representation of cloning of TIMP4 in pIRES2DsRed2 Vector. TIMP4 was cloned at the multiple cloning site between EcoR1 and Sac II and confirmed by sequencing.
Fig. 2
Fig. 2
Transfection of TIMP4 and GFP in mouse cardiomyocytes: GFP and TIMP4 were transfected into mouse cardiomyocytes using lipofectamine 2000 (Invitrogen) and imaged by confocal microscopy. A) The TIMP4 transfected cardiomyocytes appeared red in color due to the DeRed2 fluorescent tag and the GFP transfected cardiomyocytes appeared green in color (arrows). B). TIMP4 expression in cardiomyocytes was confirmed by RT-PCR and Western blots. In cardiomyocytes transfected with TIMP4-siRNA, there was negligible expression of TIMP4. The expression of TIMP4 was observed at different time points: 0 h, 6 h and 12 h and there was increase in the expression with time (UT — untransformed; *p = 0.01 as compared to untransformed or 0 h-Student's t-test; n = 6). C). Real time expression of TIMP4 in the transformed cardiomyocytes and in untransformed, at 6 h and 12 h. The real time PCR showed increase in the expression at different time points which confirmed the Western results. *p = 0.001 as compared to untransformed, Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
The contractility studies in the transformed cardiomyocytes by ION OPTIX: We performed the contractility studies in the cardiomyocytes which were transformed with TIMP4 and TIMP4-siRNA after 6 h and 12 h of transfection. There was increase in contractility in the TIMP4 transformed cardiomyocytes as depicted from the graphs generated by the ION OPTIX measurements (for contractility measurements, n = 20). A). Representative length vs time graphs (as generated by ION OPTIX) showing contractility in the untransformed, TIMP4 transformed and TIMP4-siRNA transformed cardiomyocytes. The data shows the average of such 20 recordings in different cardiomyocytes. B). The representative length vs time graphs and the amplitude graphs of the untransformed, TIMP4 transformed and TIMP4-siRNA transformed cardiomyocytes. n = 20 and for significance *p = 0.01 as compared to untransformed, Student's t-test.
Fig. 4
Fig. 4
The expression of serca2a in TIMP4 transformed cardiomyocytes and mir122a: Serca2a or sarcoplasmic reticulum calcium ATPase2a is a calcium channel in myocytes and it regulates contractility by regulating the levels of calcium. We examined the effect of TIMP4 overexpression on serca2a and found increase in the expression seerca2a with TIMP4. A). RT-PCR showed increase in the expression of serca2a in the TIMP4 transformed cardiomyocytes while in the TIMP4-siRNA transformed myocytes, the serca2a levels were decreased. B). Western blots showed an increase in the expression of serca2a with TIMP4 C). Real time PCR confirmed an increase in the expression of serca2a in the TIMP4 transformed cardiomyocytes as compared to TIMP4-siRNA transformed myocytes. D). The expression of serca2a is tightly regulated by mir122a so we looked into the expression of mir122a in the TIMP4 transformed myocytes. There was a decrease in the expression of mir122a in the cells transformed with TIMP4 as compared to the TIMP4-siRNA transfected cells. #, *p = 0.004 as compared to untransformed; n = 6.
Fig. 5
Fig. 5
Schematic presentation of growth and differentiation of mouse embryonic stem cells to cardiomyocytes. A). The mouse embryonic stem cells were cultured on mouse embryonic fibroblasts in a similar manner as described earlier [47]. To determine whether TIMP4 helps in the differentiation of embryonic stem cells to cardiomyocytes, we treated the embryoid bodies with cardiac extract with and without TIMP4. We used monoclonal antibody for TIMP4 to block the effect of TIMP4 in the cardiac extract. We observed that cells treated with cardiac extract showed distinct phenotypes of cardiomyocytes (the extreme right panel shows pictures obtained from UV–visible microscope-LEICA at 40×). EB-embryoid bodies, MEF-mouse embryonic fibroblasts, mESC-mouse embryonic stem cells.
Fig. 6
Fig. 6
Flow cytometry analysis for the determination of Ckit and Oct4 positive cells. To determine the pluripotency of stem cells, we used anti-Oct 4 antibody (rabbit-1:100) along with negative control (rabbit IgG). We used anti-Ckit for determining the differentiation of stem cells into cardiomyocytes. A). There is a decrease in the number of cells expressing Oct 4 after 15 days of differentiation (arrow). B). The cells that were treated with cardiac extract showed increased expression of Ckit as compared to those treated with cardiac extract minus TIMP4 (arrows). The cells stained in green show Ckit-FITC. C). Graph presenting the number of cells expressing Oct 4 and Ckit as determined by Flow cytometry. *p = 0.01 as compared to 0 day treatment.
Fig. 7
Fig. 7
Expression of cardiac specific genes. The expression of cardiac specific genes and transcription factors was checked by RT-PCR at days 0, 5, 10 and 15. There was an increase in the expression of GATA4, Nkx2.5, myosin light chain (MYL7), Troponin T and connexin 43 genes. *, #, §, € p < 0.05 as compared to 0 day treatment.
Fig. 8
Fig. 8
Expression of α-actinin and MYL7 (myosin light chain) as determined by immunocytochemistry. The differentiated stem cells showed the expression of α-actinin and MYL7 (myosin light chain) which is the specific property of cardiomyocytes. A) All panels show the cells treated with cardiac extract that differentiate into cardiomyocytes and develop actin and myosin fibers (arrows). B). The first row shows untreated cells and the second row shows cells treated with cardiac extract minus TIMP4 that do not develop actin–myosin fibers.
Fig. 9
Fig. 9
Expression of cardiac specific transcription factors in the differentiated stem cells A). Western blot showing CKit expression B). Western blot showing the expression of GATA4 which is a cardiac specific transcription factor in cells treated with cardiac extract. C). Western blots showing the expression of NKx2.5 which is also a cardiac specific transcription factor similar to GATA-4. *p < 0.05 as compared to untreated cells; #p < 0.05 as compared to cells treated with cardiac extract.
Fig. 10
Fig. 10
Overall picture of how TIMP4 can be effective in cardiac regeneration by alleviating contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes.
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