Ribonucleosides from tRNA in hyperglycemic mammalian cells and diabetic murine cardiac models.

AIMS: Cardiomyopathy is a diabetic comorbidity with few molecular targets. To address this, we evaluated transfer RNA (tRNA) modifications in the diabetic heart because tRNA modifications have been implicated in diabetic etiologies. MAIN METHODS: tRNA was isolated from aorta, apex, and atrial tissue of healthy and diabetic murine hearts and related hyperglycemic cell models. tRNA modifications and canonical ribonucleosides were quantified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using stable isotope dilution. Correlations between ribonucleosides and diabetic comorbidity pathology were assessed using statistical analyses. KEY FINDINGS: Total tRNA ribonucleoside levels were analyzed from cell types and healthy and diabetic murine heart tissue. Each heart structure had characteristic ribonucleoside profiles and quantities. Several ribonucleosides were observed as significantly different in hyperglycemic cells and diabetic tissues. In hyperglycemic models, ribonucleosides N4-acetylcytidine (ac4C), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methylcytidine (m5C), and N1-methylguanosine (m1G) were anomalous. Specific tRNA modifications known to be on murine tRNAIni(CAU) were higher in diabetic heart tissue which suggests that tRNA modifications could be regulating translation in diabetes. SIGNIFICANCE: We identified tRNA ribonucleosides and tRNA species associated with hyperglycemia and diabetic etiology.

Injectable Extracellular Matrix Microparticles Promote Heart Regeneration in Mice with Post-ischemic Heart Injury.

Ischemic heart injury causes permanent cardiomyocyte loss and fibrosis impairing cardiac function. Tissue derived biomaterials have shown promise as an injectable treatment for the post-ischemic heart. Specifically, decellularized extracellular matrix (dECM) is a protein rich suspension that forms a therapeutic hydrogel once injected and improves the heart post-injury response in rodents and pig models. Current dECM-derived biomaterials are delivered to the heart as a liquid dECM hydrogel precursor or colloidal suspension, which gels over several minutes. To increase the functionality of the dECM therapy, an injectable solid dECM microparticle formulation derived from heart tissue to control sizing and extend stability in aqueous conditions is developed. When delivered into the infarcted mouse heart, these dECM microparticles protect cardiac function promote vessel density and reduce left ventricular remodeling by sustained delivery of biomolecules. Longer retention, higher stiffness, and slower protein release of dECM microparticles are noted compared to liquid dECM hydrogel precursor. In addition, the dECM microparticle can be developed as a platform for macromolecule delivery. Together, the results suggest that dECM microparticles can be developed as a modular therapy for heart injury.

Microenvironment Stiffness Amplifies Post-ischemia Heart Regeneration in Response to Exogenous Extracellular Matrix Proteins in Neonatal Mice.

The cardiogenesis of the fetal heart is absent in juveniles and adults. Cross-transplantation of decellularized extracellular matrix (dECM) can stimulate regeneration in myocardial infarct (MI) models. We have previously shown that dECM and tissue stiffness have cooperative regulation of heart regeneration in transiently regenerative day 1 neonatal mice. To investigate underlying mechanisms of mechano-signaling and dECM, we pharmacologically altered heart stiffness and administered dECM hydrogels in non-regenerative mice after MI. The dECM combined with softening exhibits preserved cardiac function, LV geometry, increased cardiomyocyte mitosis and lowered fibrosis while stiffening further aggravated ischemic damage. Transcriptome analysis identified a protein in cardiomyocytes, CLCA2, confirmed to be upregulated after MI and downregulated by dECM in a mechanosensitive manner. Synthetic knock-down of CLCA2 expression induced mitosis in primary rat cardiomyocytes in the dish. Together, our results indicate that therapeutic efficacy of extracellular molecules for heart regeneration can be modulated by heart microenvironment stiffness in vivo.

Accounting for Material Changes in Decellularized Tissue with Underutilized Methodologies.

Tissue decellularization has rapidly developed to be a practical approach in tissue engineering research; biological tissue is cleared of cells resulting in a protein-rich husk as a natural scaffold for growing transplanted cells as a donor organ therapy. Minimally processed, acellular extracellular matrix reproduces natural interactions with cells in vitro and for tissue engineering applications in animal models. There are many decellularization techniques that achieve preservation of molecular profile (proteins and sugars), microstructure features such as organization of ECM layers (interstitial matrix and basement membrane) and organ level macrofeatures (vasculature and tissue compartments). While structural and molecular cues receive attention, mechanical and material properties of decellularized tissues are not often discussed. The effects of decellularization on an organ depend on the tissue properties, clearing mechanism, chemical interactions, solubility, temperature, and treatment duration. Physical characterization by a few labs including work from the authors provides evidence that decellularization protocols should be tailored to specific research questions. Physical characterization beyond histology and immunohistochemistry of the decellularized matrix (dECM) extends evaluation of retained functional features of the original tissue. We direct our attention to current technologies that can be employed for structure function analysis of dECM using underutilized tools such as atomic force microscopy (AFM), cryogenic electron microscopy (cryo-EM), dynamic mechanical analysis (DMA), Fourier-transform infrared spectroscopy (FTIR), mass spectrometry, and rheometry. Structural imaging and mechanical functional testing combined with high-throughput molecular analyses opens a new approach for a deeper appreciation of how cellular behavior is influenced by the isolated microenvironment (specifically dECM). Additionally, the impact of these features with different decellularization techniques and generation of synthetic material scaffolds with desired attributes are informed. Ultimately, this mechanical profiling provides a new dimension to our understanding of decellularized matrix and its role in new applications.

Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG.

Controlling fibrosis is an essential part of regenerating the post-ischemic heart. In the post-ischemic heart, fibroblasts differentiate to myofibroblasts that produce collagen-rich matrix to physically stabilize the infarct area. Infarct models in adult mice result in permanent scarring unlike newborn animals which fully regenerate. Decellularized extracellular matrix (dECM) hydrogels derived from early-aged hearts have been shown to be a transplantable therapy that preserves heart function and stimulates cardiomyocyte proliferation and vascularization. In this study, we investigate the anti-fibrotic effects of injectable dECM hydrogels in a cardiac explant model in the context of age-associated tissue compliance. Treatments with adult and fetal dECM hydrogels were tested for molecular effects on cardiac fibroblast activation and fibrosis. Altered sensitivity of fibroblasts to the mechanosignaling of the remodeling microenvironment was evaluated by manipulating the native extracellular matrix in explants and also with elastomeric substrates in the presence of dECM hydrogels. The injectable fetal dECM hydrogel treatment decreases fibroblast activation and contractility and lowers the stiffness-mediated increases in fibroblast activation observed in stiffened explants. The anti-fibrotic effect of dECM hydrogel is most observable at highest stiffness. Experiments with primary cells on elastomeric substrates with dECM treatment support this phenomenon. Transcriptome analysis indicated that dECM hydrogels affect cytoskeleton related signaling including Macrophage capping protein (CAPG) and Leupaxin (LPXN). CAPG was down-regulated by the fetal dECM hydrogel. LPXN expression was decreased by stiffening the explants; however, this effect was reversed by dECM hydrogel treatment. Pharmacological disruption of cytoskeleton polymerization lowered fibroblast activation and CAPG levels. Knocking down CAPG expression with siRNA inhibited fibroblast activation and collagen deposition. Collectively, fibroblast activation is dependent on cooperative action of extracellular molecular signals and mechanosignaling by cytoskeletal integrity.