Contractile and electrophysiologic characterization of optimized self-organizing engineered heart tissue.

BACKGROUND: Engineered heart tissue (EHT) is being developed for clinical implantation in heart failure or congenital heart disease and therefore requires a comprehensive functional characterization and scale-up of EHT. Here we explored the effects of scale-up of self-organizing EHT and present detailed electrophysiologic and contractile functional characterization. METHODS: Fibers from EHT were generated from self-organizing neonatal rat cardiac cells (0.5×10(6) to 3×10(6)/fiber) on fibrin. We characterized contractile patterns and measured contractile function using a force transducer, and assessed force-length relationship, maximal force generation, and rate of force generation. Action potential and conduction velocity of EHT were measured with optical mapping, and transcript levels of myosin heavy chain beta were measured by reverse transcriptase-polymerase chain reaction. RESULTS: Increasing the cell number per construct resulted in an increase in fiber volume. The force-length relationship was negatively impacted by increasing cell number. Maximal force generation and rate of force generation were also abrogated with increasing cell number. This decrease was not likely attributable to a selective expansion of noncontractile cells as myosin heavy chain beta levels were stable. Irregular contractile behavior was more prevalent in constructs with more cells. Engineered heart tissue (1×10(6)/construct) had an action potential duration of 140.2 milliseconds and a conduction velocity of 23.2 cm/s. CONCLUSIONS: Engineered heart tissue displays physiologically relevant features shared with native myocardium. Engineered heart tissue scale-up by increasing cell number abrogates contractile function, possibly as a result of suboptimal cardiomyocyte performance in the absence of vasculature. Finally, conduction velocity approaches that of native myocardium without any electrical or mechanical conditioning, suggesting that the self-organizing method may be superior to other rigid scaffold-based EHT.

Computational analysis of contractility in engineered heart tissue.

Engineered heart tissue (EHT) is a potential therapy for heart failure and the basis of functional in vitro assays of novel cardiovascular treatments. Self-organizing EHT can be generated in fiber form, which makes the assessment of contractile function convenient with a force transducer. Contractile function is a key parameter of EHT performance. Analysis of EHT force data is often performed manually; however, this approach is time consuming, incomplete and subjective. Therefore, the purpose of this study was to develop a computer algorithm to efficiently and objectively analyze EHT force data. This algorithm incorporates data filtering, individual contraction detection and validation, inter/intracontractile analysis and intersample analysis. We found the algorithm to be accurate in contraction detection, validation and magnitude measurement as compared to human operators. The algorithm was efficient in processing hundreds of data acquisitions and was able to determine force-length curves, force-frequency relationships and compare various contractile parameters such as peak systolic force generation. We conclude that this computer algorithm is a key adjunct to the objective and efficient assessment of EHT contractile function.

A novel SULF1 splice variant inhibits Wnt signalling but enhances angiogenesis by opposing SULF1 activity.

The importance of SULF1 in modulating the activities of multiple signalling molecules is now well established. Several studies, however, reported little or no effect of Sulf1 null mutations, questioning the relevance of this gene to in vivo development. The failure of SULF1 deletion to influence development may be predicted if one considers the involvement of a naturally occurring SULF1 antagonist, generated by alternative splicing of the same gene. We demonstrate that while the previously described SULF1 (SULF1A) enhances Wnt signalling, the novel shorter isoform (SULF1B) inhibits Wnt signalling. Our studies show developmental stage specific changes in the proportions of SULF1A and SULF1B isoforms at both the mRNA and protein levels in many developing tissues, with particularly pronounced changes in developing and adult blood vessels. Unlike SULF1A, SULF1B promotes angiogenesis and is highly expressed in endothelial cells during early blood vessel development while SULF1A predominates in mature endothelial cells. We propose that the balance of two naturally occurring SULF1 variants, with opposing functional activities, may regulate the overall net activities of multiple secreted factors and the associated signalling cascades essential for normal development and maintenance of most tissues.