The effects of rosiglitazone on the neonatal rat cardiomyocyte transcriptome: a temporal analysis.

Aim: The objective was to determine via high-throughput RNA sequencing the temporal effects of rosiglitazone (Avandia®) on the neonatal rat ventricular myocyte transcriptome. Materials & methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to rosiglitazone in vitro. Meta analyses utilized temporal comparisons of 0.5 h control versus 0.5 h treatment, 0.5 h treatment versus 24 h treatment and 24 h treatment versus 48 h treatment. Results: Time dependent responses were observed. At 0.5 h, the PI3K-AKT signaling pathway was impacted. At 24 h endoplasmic reticulum activity and protein degradation were altered. At 48 h, oxytocin signaling was perturbed. Conclusion: The effects of rosiglitazone occured early and increased in magnitude over time. A protective molecular response was triggered at 24 h and maintained until 48 h. In parallel, a response that can cause cardiac damage was activated. Our findings suggest that rosiglitazone has deleterious effects.

Developmental and extracellular matrix-remodeling processes in rosiglitazone-exposed neonatal rat cardiomyocytes.

OBJECTIVE: The objective of this study was to investigate the effects of rosiglitazone (Avandia(®)) on gene expression in neonatal rat ventricular myocytes. MATERIALS & METHODS: Myocytes were exposed to rosiglitazone ex vivo. The two factors examined in the experiment were drug exposure (rosiglitazone and dimethyl sulfoxide vs dimethyl sulfoxide), and length of exposure to drug (½ h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h and 48 h). RESULTS: Transcripts that were consistently expressed in response to the drug were identified. Cardiovascular system development, extracellular matrix and immune response are represented prominently among the significantly modified gene ontology terms. CONCLUSION: Hmgcs2, Angptl4, Cpt1a, Cyp1b1, Ech1 and Nqo1 mRNAs were strongly upregulated in cells exposed to rosiglitazone. Enrichment of transcripts involved in cardiac muscle cell differentiation and the extracellular matrix provides a panel of biomarkers for further analysis in the context of adverse cardiac outcomes in humans. Original submitted 15 November 2013; Revision submitted 14 February 2014.

Contractility assessment in enzymatically isolated cardiomyocytes.

The use of enzymatically isolated cardiac myocytes is ubiquitous in modern cardiovascular research. Parallels established between cardiomyocyte shortening responses and those of intact tissue make the cardiomyocyte an invaluable experimental model of cardiac function. Much of our understanding regarding the fundamental processes underlying heart function is owed to our increasing capabilities in single-cell stimulation and direct or indirect observation, as well as quantitative analysis of such cells. Of the many important mechanisms and functions that can be readily assessed in cardiomyocytes at all stages of development, contractility is the most representative and one of the most revealing. The purpose of this review is to provide a survey of various methodological approaches in the literature used to assess adult and neonatal cardiomyocyte contractility. The various methods employed to evaluate the contractile behavior of enzymatically isolated mammalian cardiac myocytes can be conveniently divided into two general categories-those employing optical (image)-based systems and those that use transducer-based technologies. This survey is by no means complete, but we have made an effort to include the most popular methods in terms of reliability and accessibility. These techniques are in constant evolution and hold great promise for the next generation of breakthrough studies in cell biology for the prevention, treatment, and cure of cardiovascular diseases.

Image processing techniques for assessing contractility in isolated neonatal cardiac myocytes.

We describe a computational framework for the quantitative assessment of contractile responses of isolated neonatal cardiac myocytes. To the best of our knowledge, this is the first report on a practical and accessible method for the assessment of contractility in neonatal cardiocytes. The proposed methodology is comprised of digital video recording of the contracting cell, signal preparation, representation by polar Fourier descriptors, and contractility assessment. The different processing stages are variants of mathematically sound and computationally robust algorithms very well established in the scientific community. The described computational approach provides a comprehensive assessment of the neonatal cardiac myocyte contraction without the need of elaborate instrumentation. The versatility of the methodology allows it to be employed in determining myocyte contractility almost simultaneously with the acquisition of the Ca(2+) transient and other correlates of cell contraction. The proposed methodology can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease models, transgeneity, or other common applications of neonatal cardiocytes.

Introduction of non-linear elasticity models for characterization of shape and deformation statistics: application to contractility assessment of isolated adult cardiocytes.

BACKGROUND: We are exploring the viability of a novel approach to cardiocyte contractility assessment based on biomechanical properties of the cardiac cells, energy conservation principles, and information content measures. We define our measure of cell contraction as being the distance between the shapes of the contracting cell, assessed by the minimum total energy of the domain deformation (warping) of one cell shape into another. To guarantee a meaningful vis-à-vis correspondence between the two shapes, we employ both a data fidelity term and a regularization term. The data fidelity term is based on nonlinear features of the shapes while the regularization term enforces the compatibility between the shape deformations and that of a hyper-elastic material. RESULTS: We tested the proposed approach by assessing the contractile responses in isolated adult rat cardiocytes and contrasted these measurements against two different methods for contractility assessment in the literature. Our results show good qualitative and quantitative agreements with these methods as far as frequency, pacing, and overall behavior of the contractions are concerned. CONCLUSIONS: We hypothesize that the proposed methodology, once appropriately developed and customized, can provide a framework for computational cardiac cell biomechanics that can be used to integrate both theory and experiment. For example, besides giving a good assessment of contractile response of the cardiocyte, since the excitation process of the cell is a closed system, this methodology can be employed in an attempt to infer statistically significant model parameters for the constitutive equations of the cardiocytes.

Image processing techniques for assessing contractility in isolated adult cardiac myocytes.

We describe a computational framework for the comprehensive assessment of contractile responses of enzymatically dissociated adult cardiac myocytes. The proposed methodology comprises the following stages: digital video recording of the contracting cell, edge preserving total variation-based image smoothing, segmentation of the smoothed images, contour extraction from the segmented images, shape representation by Fourier descriptors, and contractility assessment. The different stages are variants of mathematically sound and computationally robust algorithms very well established in the image processing community. The physiologic application of the methodology is evaluated by assessing overall contraction in enzymatically dissociated adult rat cardiocytes. Our results demonstrate the effectiveness of the proposed approach in characterizing the true, two-dimensional, "shortening" in the contraction process of adult cardiocytes. We compare the performance of the proposed method to that of a popular edge detection system in the literature. The proposed method not only provides a more comprehensive assessment of the myocyte contraction process but also can potentially eliminate historical concerns and sources of errors caused by myocyte rotation or translation during contraction. Furthermore, the versatility of the image processing techniques makes the method suitable for determining myocyte shortening in cells that usually bend or move during contraction. The proposed method can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease modeling, transgeneity, or other common applications to mammalian cardiocytes.

The antidiabetic agent rosiglitazone upregulates SERCA2 and enhances TNF-alpha- and LPS-induced NF-kappaB-dependent transcription and TNF-alpha-induced IL-6 secretion in ventricular myocytes.

Positive hemodynamic effects of the antidiabetic agent rosiglitazone on perfused whole hearts have recently been described, but the mechanisms regulating these effects are not well understood. This study reports the effects of rosiglitazone on calcium regulation in isolated neonatal rat ventricular myocytes by measurement of Ca2+ transient decay rates and SERCA2 gene expression, and shows that rosiglitazone enhances known cardioprotective signaling pathways. Myocyte treatment with 10 micromol/L rosiglitazone accelerated Ca2+ transient decay rates by approximately 30%, enhanced SERCA2 mRNA levels by approximately 1.5-fold and SERCA2 production by approximately 3-fold. Rosiglitazone treatment (1, 5, and 10 micromol/L) also led to a dose-dependent increase (approximately 1.2-1.5-fold) in SERCA2 promoter activity. Comparable levels of cardiac SERCA overexpression have been associated with physiologically relevant and compensatory effects in vivo. These data link thiazolidinedione-induced improvement in cardiac myocyte function to an upregulation of SERCA2 gene expression. Since NF-kappaB-dependent pathways, including the upregulation of IL-6 secretion, were shown to protect neonatal rat ventricular myocytes from apoptosis upon TNFalpha stimulation, additional experiments were designed to determine whether rosiglitazone enhances TNFalpha-induced NF-kappaB-dependent transcription and IL-6 secretion. Because the endotoxin stress response in ventricular myocytes involves the upregulation of TNFalpha, and the activation of NF-kappaB, the effects of rosiglitazone on lipopolysaccharide-induced NF-kappaB-dependent transcription were also investigated. Treatment of neonatal rat ventricular myocytes with 10 micromol/L rosiglitazone enhanced TNF-alpha- and lipopolysaccharide-induced NF-kappaB-dependent transcription by approximately 1.8- and approximately 1.4-fold respectively, and TNF-alpha-induced IL-6 secretion by n1.5-fold. Rosiglitazone had no significant effects on basal levels of NF-kappaB-dependent transcription and IL-6 secretion. Thus, cardioprotective effects of rosiglitazone may be partly mediated by NF-kappaB.