Injectable OPF/graphene oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct

Abstract

After myocardial infarction (MI), the scar tissue contributes to ventricular dysfunction by electrically uncoupling viable cardiomyocytes in the infarct region. Injection of a conductive hydrogel could not only provide mechanical support to the infarcted region, but also synchronize contraction and restore ventricular function by electrically connecting isolated cardiomyocytes to intact tissue. Methods: We created a conductive hydrogel by introducing graphene oxide (GO) nanoparticles into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels. The hydrogels were characterized by AFM and electrochemistry workstation. A rat model of myocardial infarction was used to investigate the ability of OPF/GO to improve cardiac electrical propagation in the injured heart in vivo. Echocardiography (ECHO) was used to evaluate heart function 4 weeks after MI. Ca²⁺ imaging was used to visualize beating cardiomyocytes (CMs). Immunofluorescence staining was used to visualize the expression of cardiac-specific markers. Results: OPF/GO hydrogels had semiconductive properties that were lacking in pure OPF. In addition, the incorporation of GO into OPF hydrogels could improve cell attachment in vitro. Injection of OPF/GO 4 weeks after myocardial infarction in rats enhanced the Ca²⁺ signal conduction of cardiomyocytes in the infarcted region in comparison with PBS or OPF alone. Moreover, the injection of OPF/GO hydrogel into the infarct region enhanced the generation of cytoskeletal structure and intercalated disc assembly. Echocardiography analysis showed improvement in load-dependent ejection fraction/fractional shortening of heart function 4 weeks after injection. Conclusions: We prepared a conductive hydrogel (OPF/GO) that provide mechanical support and biological conduction in vitro and in vivo. We found that injected OPF/GO hydrogels can provide mechanical support and electric connection between healthy myocardium and the cardiomyocytes in the scar via activating the canonical Wnt signal pathway, thus upregulating the generation of Cx43 and gap junction associated proteins. Injection of OPF/GO hydrogel maintained better heart function after myocardial infarction than the injection of a nonconductive polymer.

Keywords: conduction; injectable biomaterials; myocardial infarction; remodeling.

Figure 1

Synthesis and characterization of OPF/GO conductive hydrogel. (A) Photographs of OPF and OPF/GO hydrogels with various concentrations. (B) Degradation behavior of OPF and OPF/GO hydrogels (n=3/group). (C) Spatial topography of OPF and OPF/GO hydrogels measured by AFM. (D) Conductivity of OPF and OPF/GO with various concentrations (n=3/group). (E) A schematic illustration of the ex vivo setup used for measuring excitation threshold of muscle tissue. One muscle was electrically stimulated while the beating or contraction of the other muscle was monitored. (F) Excitation threshold of muscle tissue connected by pure OPF and OPF/GO hybrid gels, demonstrating that OPF/GO hybrid gels gave rise to the lowest excitation threshold, which was due to the conductivity of the OPF/GO hybrid gels (± standard deviation, n=3/group, *p < 0.05, or **p < 0.01).

Figure 2

OPF/GO hydrogel supports cell growth without toxicity. (A) Representative morphological images of cardiac fibroblast cells cultured for 48 h on OPF- or OPF/GO-coated substrates. (B) Rat cardiac fibroblast cells were plated on the surfaces of wells coated with OPF or OPF/GO, grown for 48 h, and then stained with live & dead kit. (C) Immunofluorescence images of cardiac fibroblasts seeded on OPF- or OPF/GO-coated substrates (vimentin, red). (D) An MTT assay of cardiac fibroblasts cultured on the surface of pure OPF and different composite OPF/GO (0.3 mg/mL, 0.6 mg/mL, 1.0 mg/mL) (n=3, *P<0.05). MTT indicates 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide.

Figure 3

Effects of OPF and OPF/GO hydrogels on macrophage infiltration in infarcted hearts. (A) Representative fluorescence images show CD68-positive cells (left column, merged channel; middle column, CD68 (red); right column, DAPI (blue)) in PBS-, OPF-, and OPF/GO-injected hearts at 1 week post MI. (B) Quantification of CD68-positive cells of the corresponding images of (A). (C) CD68-positive signal is co-localized with the presence of OPF/GO hydrogel (CD68 (green), DAPI (blue)).

Figure 4

OPF/GO enhanced cytoskeletal structure 4 weeks after MI. (A) F-actin (green) in the infarcted areas of PBS-, OPF- and OPF/GO-treated groups at 4 weeks post MI (DAPI (blue)). (B) α-Tubulin (green) in the border zone cardiomyocytes (marked by actinin staining in red, DAPI (blue)).

Figure 5

Immunofluorescence staining and quantification of ID-related proteins in the hearts of PBS-, OPF-, OPF/GO-injected groups at 4 weeks post MI. Marker proteins including N-cadherin (NC) for adherens junctions (A), and plakoglobin (PG) (B) and desmoplakin (DP) (C) for desmosomes. (D) Western blot revealed the OPF/GO-injected group had significantly higher expression levels of NC, PG, and DP compared to the PBS- and OPF-injected groups.

Figure 6

The formation of gap junction-associated proteins in the infarcted region. (A) Immunofluorescence staining revealed higher gap junction remodeling in the infarcted region of the OPF/GO hydrogel-treated group compared to the OPF- and PBS-treated groups. The highlighted areas are presented in the bottom images. (B) qPCR analysis of the expression levels of gap junction-associated markers Cx43, Cx40, Cx37 and Cx45. (C) Western blot revealed the downstream signaling molecules of canonical Wnt signaling pathway. (D) A schematic of the possible mechanisms.

Figure 7

Masson's trichrome staining and echocardiographic measurements represent the effects of OPF/GO hydrogel on the morphology and myocardial functional recovery of infarcted hearts. (A) Masson trichrome staining of PBS-, OPF- or OPF/GO-injected heart 4 weeks post injection. Four weeks after injection, hearts were excised and left vetricle wall thickness (B) and infarct size (C) were measured. (D) Systolic ventricular function was improved after OPF/GO hydrogel injection into the border zone. Echocardiography (ECHO) was performed at 4 weeks post injection. Quantification of the parameters reflecting blood pumping function, including ejection fraction (EF) (E) and fractional shortening (FS) (F) 6 weeks after MI. Quantification of the parameters reflecting ventricular filling function, including left ventricular end-diastolic dimension (LVEDD) (G) and left ventricular end-systolic dimension (LVESD) (H). Data is presented as mean ± standard deviation; *p < 0.05, or **p < 0.01 (n=6/group).

Figure 8

Ca²⁺ transients evaluation at 4 week after MI. Spontaneous Ca²⁺ transients of cardiomyocytes isolated from the left ventricle of OPF/GO- (A), OPF- (B) or PBS- (C) injected rats four weeks after MI.