(H-290) Identification and characterization of ECM-derived peptides for cardiac regeneration and repair.

Introduction:: While the use of organ specific ECM has led to significant improvements in tissue engineering/ regenerative medicine based strategies for cardiac repair, much of this work has utilized ECM from the adult organ, even though it is known that cardiac growth and regeneration potential are highest at earlier stages in development. While the effects of solubilized cardiac ECM are well described, the specific mechanisms and specific peptides in these proteolytically digested materials that led to the described effects is unclear. The identification of specific matrikines that drive the beneficial responses of treatment with solubilized ECM in cardiac repair could lead to a more robust, streamlined and personalized response that is faster, cheaper and more consistent to produce than whole ECM digests.

Materials and Methods:: In order to determine specific matrikines (signaling peptides derived from the ECM) that may play a role in cardiac regeneration, adult and fetal Sprague Dawley rat hearts were decellularized (Fig. 1A), samples were fractionated according to molecular weight using SDS-PAGE. Half of the gel was transferred to a PVDF membrane and cardiomyocytes (CMs) were cultured in monolayer directly on the PVDF membrane (Fig. 1B). The other half of the gel was saved for subsequent analysis of composition of the cECM fraction via LC/MS-MS proteomics. Following identification of potential matrikines from LC-MS/MS data, candidate peptides were synthesized and coated onto tissue culture plastic to assess their potential for promoting cardiomyocyte proliferation. Scrambled versions of the peptides were used as controls and the role of the structure of the peptides was assessed by linearizing them via cysteine alkylation. Matrikines were also tested using iPS-derived CMs.


Results, Conclusions, and Discussions:: We have previously demonstrated that neonatal rat cardiomyocytes exhibited increased proliferation on cardiac ECM (cECM) derived from fetal rat hearts¹. In addition, we subsequently demonstrated that partially digested adult cECM can replicate this proliferative response if the adult cECM is digested for shorter periods (1 and 3 hours)². To assess whether specific peptides were involved in these responses, we fractionated solubilized cECM by molecular weight. CMs cultured directly on the PVDF membranes with fractionated cECM (5 days in serum-free media) revealed specific bands of cECM with enhanced proliferation (Fig. 1C), and corresponding proteomics analysis of these molecular weight regions in the stored gel fragments implicated several peptides near the N-terminus of fibrillin-1 (Fig. 1D). The first of these regions, denoted the “F1R1” peptide (residues 55-86), was fabricated and adsorbed onto TCPS. CMs cultured in serum free conditions on F1R1 demonstrated significantly enhanced proliferation over both positive (TCPS/serum media) and negative (PLL, serum-free media) controls (p< 0.05) at conditions of 6 mg/ml and 10 mg/ml (Fig. 1E). Scrambled F1R1 (S) and linearized F1R1 (A) did not elicit CM proliferation. This effect was also seen with human iPS-CMs. These data suggest that the N-terminus of fibrillin-1 may play a role in CM plasticity in the developing myocardium and could have utility as a novel therapeutic for cardiac re-generation. Ongoing studies are assessing the ability of fetal cECM to promote a better healing response following myocardial infarction. Future work includes evaluating the effect of the F1R1 peptide on adult CMs and cardiac progenitor cells, as well as tests of in vivo efficacy.


The use of cardiac ECM derived from earlier developmental ages has the potential to enhance regeneration in tissue engineering strategies for the heart.


Acknowledgements (Optional): :

References (Optional): : 1. Williams, C. et al., Acta. Biomater., 10(1):194-204, 2013.

2. Williams, C. et al., Adv. Healthc. Mater., 4(10):1545-54, 2015.