Toward improved myocardial maturity in an organ-on-chip platform with immature cardiac myocytes.

In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the ß-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.

Gene expression analysis and urinary biomarker assays reveal activation of tubulointerstitial injury pathways in a rodent model of chronic proteinuria (Doxorubicin nephropathy).

BACKGROUND: Tubular atrophy and interstitial fibrosis are well-recognized sequelae of chronic proteinuria; however, little is known regarding the molecular pathways activated within tubulointerstitium in chronic proteinuric nephropathies. METHODS: To investigate the molecular mechanisms of proteinuria-associated tubulointerstitial (TI) disease, doxorubicin nephropathy was induced in rats. Progression of disease was monitored with weekly urinary biomarker assays. Because histopathology revealed multifocal TI injury, immunodirected laser capture microdissection was used to identify and isolate injured proximal tubules, as indicated by kidney injury molecule-1 immunolabeling. Adjacent interstitial cells were harvested separately. Gene expression microarray, manual annotation of gene lists, and Gene Set Enrichment Analysis were performed. A subset of the regulated transcripts was validated by quantitative PCR and immunohistochemistry. RESULTS: Severe proteinuria preceded tubular injury biomarkers by 1 week. Histology revealed multifocal, mild TI damage at 3 weeks, which progressed in severity at 5 weeks. Affymetrix microarray analysis revealed tissue-specific regulation of gene expression. Manual annotation of gene lists, gene set enrichment analysis, and urinary biomarker assays revealed similarities to pathways activated in direct TI injuries. This suggests commonalities amongst the molecular mechanisms of TI injury secondary to proteinuria, ischemia-reperfusion, and nephrotoxicity. © 2013 S. Karger AG, Basel.

Integrated and translational nonclinical in vivo cardiovascular risk assessment: gaps and opportunities.

Cardiovascular (CV) safety concerns are a significant source of drug development attrition in the pharmaceutical industry today. Though current nonclinical testing paradigms have largely prevented catastrophic CV events in Phase I studies, many challenges relating to the inability of current nonclinical safety testing strategies to model patient outcomes persist. Contemporary approaches include a spectrum of evaluations of CV structure and function in a variety of laboratory animal species. These approaches might be improved with a more holistic integration of these evaluations in repeat-dose studies, addition of novel endpoints with greater sensitivity and translational application, and use of more relevant animal models. Particular opportunities present with advances in imaging capabilities applicable to rodent and non-rodent species, technical capabilities for measuring CV function in repeat-dose animal studies, detection and quantitation of microRNAs and wider use of alternative animal models. Strategic application of these novel opportunities considering putative CV risk associated with the molecular drug target as well as inherent risks present in the target patient population could tailor or 'personalize' nonclinical safety assessment as a more translational evaluation. This paper is a call to action for the clinical and nonclinical drug safety communities to assess these opportunities to determine their utility in filling potential gaps in our current cardiovascular safety testing paradigms.

An initial characterization of N-terminal-proatrial natriuretic peptide in serum of Sprague Dawley rats.

In the clinical setting, natriuretic peptides (NPs) have proven to be reliable noninvasive markers for diagnostic, prognostic, and therapeutic monitoring of heart failure. Given their proven utility in humans, NPs are potential candidates for translational biomarkers during drug development to detect drug-induced hemodynamic stress resulting in cardiac hypertrophy in preclinical species. We evaluated the intra- and interassay precision and the stability of serum N-terminal-proatrial natriuretic peptide (NT-proANP) using a commercially available enzyme-linked immunoassay (EIA). We then measured NT-proANP concentrations in 532 serum samples from 337 male Crl:CD(SD) rats with or without pressure-induced cardiac hypertrophy. Additionally, we established a reference range using samples from control animals across multiple studies. The data demonstrate that the NT-proANP EIA is a robust and reproducible assay for the measurement of NT-proANP. The noninvasive translational utility, minimal sample volume requirement, and the lack of existing hypertrophic biomarkers in the male rat make NT-proANP an excellent candidate for further interrogation as a biomarker of cardiac hypertrophy in preclinical toxicology investigations.

Time course gene expression using laser capture microscopy-extracted bile ducts, but not hepatic parenchyma, reveals acute alpha-naphthylisothiocyanate toxicity.

Acute toxic responses to a 50-mg/kg oral dose of 1-naphthylisothiocyanate (ANIT) were evaluated by microarray analysis of laser capture-microdissected rat biliary epithelium or hepatic parenchyma obtained 2 and 6 hours postdose. Distinct differences in gene expression patterns between biliary epithelium and hepatic parenchyma were noted at the 2-hour postdose time point, where 375 genes were altered in biliary epithelium but only 38 genes were altered in hepatic parenchyma. Endoplasmic reticulum stress genes were uniquely expressed in biliary epithelial cells at 2 hours postdose. By 6 hours postdose, 620 genes were altered in biliary epithelium, but only 32 genes were altered in hepatic parenchyma. In biliary epithelium, expression of genes involved in the unfolded protein response had decreased compared with the 2-hour time point, while expression of genes involved in protein degradation such as proteasome-ubquination pathways and cell death pathways had increased. At this same time, hepatic parenchymal gene expression changed little. Within 6 hours following oral exposure to ANIT, prior to morphologic changes, specific biliary epithelial gene expression changes, indicative of a vigorous unfolded protein response with protein destruction and cell death pathway activation were noted, in contrast to minor changes in the hepatic parenchyma.

What do we need to know prior to thinking about incorporating an epigenetic evaluation into safety assessments?

The International Life Sciences Institute, Health and Environmental Sciences Institute sponsored a workshop entitled "State of the Science: Evaluating Epigenetic Changes," hosted by the National Institute of Environmental Health Sciences, Research Triangle Park, NC, 28-30 October 2009. The goal was to evaluate and enhance the scientific knowledge base regarding epigenetics and its role in disease, including potential relationships between epigenetic changes and transgenerational effects. A distinguishing aspect of the workshop was the highly interactive discussion session on the final morning. Meeting participants formed breakout groups (with representation from academia, industry, and government in each group) and were tasked with integrating their previous knowledge of epigenetics with what was learned during the workshop. The participants addressed the issue of what needs to be known prior to thinking about incorporating an epigenetic evaluation into safety assessment. To this end, the breakout groups were asked to address the following questions: (1) What model systems might be employed to evaluate the ability of a chemical to produce an epigenetic change (affecting the F1 and/or F3 generation); (2) What end points/targets might be evaluated; (3) What techniques might be employed; and (4) Regulatory Perspective: When is it appropriate to incorporate "new" science, in this case epigenetics, into the regulatory process? What does one need to know, what are the pitfalls and how might these be overcome/avoided? The basis of this paper is a synopsis of these discussions. The workshop highlighted the fact that the field of epigenetics is evolving at a very rapid pace and indicated that a great deal needs to be learned prior to being able to rationally incorporate an epigenetic evaluation into safety assessment. The value of the workshop is that it called attention to key data/knowledge gaps that should serve to focus attention on the areas where research and new thinking are needed to better understand epigenetics and its relationship to safety assessment.

PPAR alpha, more than PPAR delta, mediates the hepatic and skeletal muscle alterations induced by the PPAR agonist GW0742.

Therapeutic use of certain peroxisome proliferator-activated receptor (PPAR) alpha agonists (fibrates) for the treatment of dyslipidemia has infrequently been associated with the untoward side effect of myopathy. With interest in PPAR-delta as a therapeutic target, this study assessed whether a PPAR-delta agonist induced similar hepatic and skeletal muscle alterations as noted with some fibrates. PPAR-alpha null (KO) and corresponding wild-type (WT) mice were administered toxicological dosages of a potent PPAR-delta agonist tool ligand (GW0742; which also has weak PPAR-alpha agonist activity) or a potent PPAR-alpha agonist (WY-14,643) for 10 days. Increases in liver weights and clinical chemistry indicators of skeletal muscle damage and/or liver injury were more pronounced in WT mice compared with KO mice administered the PPAR-delta agonist. Likewise, the incidence and severity of skeletal myopathy were greater in WT mice given GW0742 compared with KO mice. Ultrastructural and immunohistochemical analyses revealed significant peroxisome proliferation in muscle and liver of WT mice treated with each agonist; however, KO animals showed little or no evidence of hepatic and muscle peroxisome proliferation. PMP-70 protein expression in liver was consistent with these results. The hepatomegaly, hepatic and skeletal muscle peroxisome proliferation, and skeletal myopathy induced by this PPAR-delta ligand was predominantly mediated by its cross-activation of PPAR-alpha, though PPAR-delta agonism contributed slightly to these effects.