Partially Digested Adult Cardiac Extracellular Matrix Promotes Cardiomyocyte Proliferation In Vitro

Abstract

Stimulating or maintaining the proliferative capacity of postnatal mammalian cardiomyocytes is a major challenge to cardiac regeneration. Previously, we found that fetal cardiac extracellular matrix (ECM) could promote neonatal rat cardiomyocyte proliferation in vitro better than neonatal or adult ECM. We hypothesized that partial digestion of adult ECM (PD-ECM), would liberate less crosslinked components that promote cardiomyocyte proliferation, similar to fetal ECM. Neonatal rat cardiac cells were seeded onto substrates coated with adult rat cardiac ECM that had been solubilized in pepsin-HCl for 1, 3, 6, 12, 24, or 48 hr. Cardiomyocyte proliferation and fold-change in numbers from 1 to 5 days were highest on 1hr and 3hr PD-ECM compared to other conditions. Sarcomeres tended to mature on 24hr and 48hr PD-ECM where low proliferation was observed. 3hr PD-ECM was primarily composed of Fibrillin-1, Fibrinogen, and Laminins while 48hr PD-ECM was dominated by Collagen I. Our results suggest that adult ECM retains regenerative cues that may be masked by more abundant, mature ECM components. PD-ECM provides a simple yet powerful approach to promoting cardiomyocyte proliferation.

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

“…A study comparing the bioactivity of solubilized decellularized cartilage and tendon ECM prepared through urea extraction versus pepsin digestion found that only the urea‐extracted samples could promote the lineage‐specific differentiation of human bone‐marrow‐derived MSCs . Another study demonstrated that reducing pepsin digestion times enhanced the capacity of decellularized cardiac ECM coatings to promote cardiomyocyte proliferation, which was postulated to be due to changes in the composition of the substrates . Building from this, we hypothesize that the enhanced proliferation on the α‐amylase‐digested DAT was due to the fibrous ultrastructure and more complex tissue‐specific ECM composition of these substrates.…”

“…[16] Another study demonstrated that reducing pepsin digestion times enhanced the capacity of decellularized cardiac ECM coatings to promote cardiomyocyte proliferation, which was postulated to be due to changes in the composition of the substrates. [14] Building from this, we hypothesize that the enhanced proliferation on the -amylase-digested DAT was due to the fibrous ultrastructure and more complex tissue-specific ECM composition of these substrates. Our findings are supported by other studies that have indicated that ECM coatings derived from other tissue sources can enhance cell attachment, survival, and proliferation in a tissue-specific manner.…”

“…[9][10][11][12][13] To generate coatings, decellularized tissues are typically digested using the proteolytic enzyme pepsin. [14][15][16][17] However, pepsin digestion is highly nonspecific, with digestion times and enzyme activity influencing the profile of resulting peptides. [14] Further, it remains unclear whether pepsin digestion is the best approach for preserving ECM bioactivity.…”

“…[14][15][16][17] However, pepsin digestion is highly nonspecific, with digestion times and enzyme activity influencing the profile of resulting peptides. [14] Further, it remains unclear whether pepsin digestion is the best approach for preserving ECM bioactivity. Some studies indicate that pepsin-digested ECM coatings promote cell proliferation and/or differentiation compared to collagen type I, [17] gelatin, [15] Matrigel, [18] and uncoated substrates.…”

Scarring vs. functional healing: Matrix-based strategies to regulate tissue repair

“…Degradation products of UBM injected at the site of digit amputation caused an accumulation of multipotent progenitors expressing Sox2, Sca1, and Rex1 [126] and these effects were later attributed in part to a cryptic peptide from the C-terminal region of collagen III [127]. Bioactive components of degraded fetal cardiac ECM [128] and partially degraded adult cardiac ECM [129] have also been shown to promote the expansion of cardiomyocytes. Most frequently, these studies were conducted using a complex mix of degraded ECM materials which makes it difficult identifying contributions and interpreting mechanism of action.…”