Physiological stress improves stem cell modeling of dystrophic cardiomyopathy.

Heart failure contributes to Duchenne muscular dystrophy (DMD), which arises from mutations that ablate dystrophin, rendering the plasma membrane prone to disruption. Cardiomyocyte membrane breakdown in patients with DMD yields a serum injury profile similar to other types of myocardial injury with the release of creatine kinase and troponin isoforms. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly useful but can be improved. We generated hiPSC-CMs from a patient with DMD and subjected these cells to equibiaxial mechanical strain to mimic in vivo stress. Compared to healthy cells, DMD hiPSC-CMs demonstrated greater susceptibility to equibiaxial strain after 2 h at 10% strain. We generated an aptamer-based profile of proteins released from hiPSC-CMs both at rest and subjected to strain and identified a strong correlation in the mechanical stress-induced proteome from hiPSC-CMs and serum from patients with DMD. We exposed hiPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced biomarker release in DMD and control hiPSC-CMs subjected to strain. Thus, the application of mechanical strain to hiPSC-CMs produces a model that reflects an in vivo injury profile, providing a platform to assess pharmacologic intervention.

Simultaneous electromechanical monitoring in engineered heart tissues using a mesoscale framework.

Engineered heart tissues (EHTs) generated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent powerful platforms for human cardiac research, especially in drug testing and disease modeling. Here, we report a flexible, three-dimensional electronic framework that enables real-time, spatiotemporal analysis of electrophysiologic and mechanical signals in EHTs under physiological loading conditions for dynamic, noninvasive, longer-term assessments. These electromechanically monitored EHTs support multisite measurements throughout the tissue under baseline conditions and in response to stimuli. Demonstrations include uses in tracking physiological responses to pharmacologically active agents and in capturing electrophysiological characteristics of reentrant arrhythmias. This platform facilitates precise analysis of signal location and conduction velocity in human cardiomyocyte tissues, as the basis for a broad range of advanced cardiovascular studies.

Susceptibility to innate immune activation in genetically mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, DSP-/- EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-κB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.

Aqueous humor in primary open-angle glaucoma contains an increased level of CD44S.

PURPOSE: To determine whether the cell adhesion molecule CD44, the principal receptor of hyaluronan, is altered in the aqueous humor and the anterior segment of patients with primary open-angle glaucoma (POAG). METHODS: The trabecular meshwork (TM), iris, ciliary body, and sclera of POAG and age-matched control eyes preserved in ethanol were microdissected and subjected to 1% Triton X-100 solubilization at 4 degrees C. Western blot analysis was performed using monoclonal antibodies that recognize either CD44H (hematopoietic; extracellular domain) or CD44S (soluble ectodomain). The concentration of soluble CD44S in aqueous and microdissected tissues was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: ELISA of soluble CD44S of aqueous from eyes of patients with POAG indicated that the concentration of soluble CD44S is increased in comparison with that of aqueous from normal eyes (P < 0.0003). Western blot analysis and densitometry of POAG iris and ciliary body revealed a statistically significant increase in the Triton X-100 extraction of CD44H. The predominant increases were in the 180-kDa (P < 0.001) and the 85-kDa (P < 0.001) forms. ELISA of soluble CD44S indicated that the concentration is statistically decreased in iris (P < 0.05), ciliary body (P < 0.001), and TM (P < 0.005) of POAG eyes. CONCLUSIONS: Increased amounts of soluble CD44S in POAG aqueous and Triton X-100-solubilized CD44H characterized POAG in the iris and ciliary body. These soluble CD44 isoforms may influence the activity of the transmembrane CD44H by acting as inhibitors of CD44H and, thereby, adversely influence the cell survival of TM and retinal ganglion cells in POAG.

Ursodeoxycholic acid suppresses Cox-2 expression in colon cancer: roles of Ras, p38, and CCAAT/enhancer-binding protein.

In the azoxymethane (AOM) model of experimental rodent colon cancer, cholic acid and its colonic metabolite deoxycholic acid (DCA) strongly promote tumorigenesis. In contrast, we showed that ursodeoxycholic acid (UDCA), a low abundance bile acid, inhibited AOM tumorigenesis. Dietary UDCA also blocked the development of tumors with activated Ras and suppressed cyclooxygenase-2 (Cox-2) upregulation in AOM tumors. In this study, we compared the effect of dietary supplementation with tumor-promoting cholic acid to chemopreventive UDCA on Cox-2 expression in AOM tumors. Cholic acid enhanced Cox-2 upregulation in AOM tumors, whereas UDCA inhibited this increase and concomitantly decreased CCAAT/enhancer binding protein beta (C/EBPbeta), a transcriptional regulator of Cox-2. In HCA-7 colon cancer cells, DCA activated Ras and increased C/EBPbeta and Cox-2 by a mechanism requiring the mitogen-activated protein kinase p38. UDCA inhibited DCA-induced p38 activation and decreased C/EBPbeta and Cox-2 upregulation. Using transient transfections, UDCA inhibited Cox-2 promoter and C/EBP reporter activation by DCA. Transfection with dominant-negative (17)N-Ras abolished DCA-induced p38 activation and C/EBPbeta and Cox-2 upregulation. Taken together, these studies have identified a transcriptional pathway regulating Cox-2 expression involving Ras, p38, and C/EBPbeta that is inhibited by UDCA. These signal transducers are novel targets of UDCA's chemopreventive actions.

Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.

Hypoxia regulates PDGF-B interactions between glomerular capillary endothelial and mesangial cells.

BACKGROUND: Platelet-derived growth factor (PDGF)-B regulates mesangial cell and vessel development during embryogenesis, and contributes to the pathogenesis of adult renal and vascular diseases. Endothelial cell PDGF-B exerts paracrine effects on mesangial cells, but its regulation is not well defined. We examined the impact of hypoxia on PDGF-B-mediated interactions between glomerular endothelial and mesangial cells, a condition of potential relevance in developing, and diseased adult, kidneys. METHODS: Glomerular endothelial or mesangial cells were subjected to hypoxia and responses compared to normoxic cells. Endothelial PDGF-B was studied by Northern and Western analysis. Mesangial proliferative responses to PDGF-B were assessed by (3)H-thymidine incorporation, and migration by a modified Boyden chamber assay. Hypoxia-induced changes in receptor specific binding capacity were studied by saturation binding assays. RESULTS: Hypoxia stimulated increases in endothelial PDGF-B mRNA and protein. In normoxic mesangial cells, PDGF-B stimulated dose-dependent proliferation, but the proliferative response of hypoxic cells was two to three times greater. Exogenous PDGF-B also caused prompter migration in hypoxic mesangial cells. Mesangial cells were treated with endothelial cell-conditioned medium. More cells migrated when hypoxic cells were stimulated with hypoxic conditioned medium, than when normoxic cells were stimulated with normoxic conditioned medium. Preincubating conditioned medium with PDGF-B neutralizing antibody greatly decreased the chemoattractant activity. Binding studies demonstrated increased specific binding capacity in hypoxic cells. CONCLUSION: Hypoxia enhances PDGF-B paracrine interactions between glomerular endothelial and mesangial cells. These hypoxia-regulated interactions may be important during glomerulogenesis in fetal life and during the pathogenesis of adult glomerular disease.