GROWTH AND CHARACTERIZATION OF A TISSUE-ENGINEERED CONSTRUCT FROM HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS.

OBJECTIVE: To optimize a bioengineered «I-Wire¼ platform to grow tissue-engineered constructs (TCs) derived from coronary artery smooth muscle cells and characterize the mechano-elastic properties of the grown TCs. MATERIALS AND METHODS: A fibrinogen-based cell mixture was pipetted in a casting mold having two parallel titanium anchoring wires inserted in the grooves on opposite ends of the mold to support the TC. The casting mold was 3 mm in depth, 2 mm in width and 12 mm in length. To measure TC deformation, a flexible probe with a diameter of 365 mk and a length of 42 mm was utilized. The deflection of the probe tip at various tensile forces applied to the TC was recorded using an inverted microscope optical recording system. The elasticity modulus was calculated based on a stretch-stress diagram reconstructed for each TC. The mechano-elastic properties of control TCs and TCs under the influence of isoproterenol (Iso), acetylcholine (ACh), blebbistatin (Bb) and cytochalasin D (Cyto-D) were evaluated. Immunohistochemical staining of smooth muscle α-actin, desmin and the cell nucleus was implemented for the structural characterization of the TCs. RESULTS: The TCs formed on day 5-6 of incubation. Subsequent measurements during the following 7 days did not reveal significant changes in elasticity. Values of the elastic modulus were 7.4 ± 1.5 kPa at the first day, 7.9 ± 1.4 kPa on the third day, and 7.8 ± 1.9 kPa on the seventh day of culturing after TC formation. Changes in the mechano-elastic properties of the TCs in response to the subsequent application of Bb and Cyto-D had a two-phase pattern, indicating a possible separation of active and passive elements of the TC elasticity. The application of 1 µM of Iso led to an increase in the value of the elastic modulus from 7.9 ± 1.5 kPa to 10.2 ± 2.1 kPa (p<0.05, n = 6). ACh did not cause a significant change in elasticity. CONCLUSION: The system allows quantification of the mechano-elastic properties of TCs in response to pharmacological stimuli and can be useful to model pathological changes in vascular smooth muscle cells.

Mechanistic analysis of challenge-response experiments.

We present an application of mechanistic modeling and nonlinear longitudinal regression in the context of biomedical response-to-challenge experiments, a field where these methods are underutilized. In this type of experiment, a system is studied by imposing an experimental challenge, and then observing its response. The combination of mechanistic modeling and nonlinear longitudinal regression has brought new insight, and revealed an unexpected opportunity for optimal design. Specifically, the mechanistic aspect of our approach enables the optimal design of experimental challenge characteristics (e.g., intensity, duration). This article lays some groundwork for this approach. We consider a series of experiments wherein an isolated rabbit heart is challenged with intermittent anoxia. The heart responds to the challenge onset, and recovers when the challenge ends. The mean response is modeled by a system of differential equations that describe a candidate mechanism for cardiac response to anoxia challenge. The cardiac system behaves more variably when challenged than when at rest. Hence, observations arising from this experiment exhibit complex heteroscedasticity and sharp changes in central tendency. We present evidence that an asymptotic statistical inference strategy may fail to adequately account for statistical uncertainty. Two alternative methods are critiqued qualitatively (i.e., for utility in the current context), and quantitatively using an innovative Monte-Carlo method. We conclude with a discussion of the exciting opportunities in optimal design of response-to-challenge experiments.

Measurements of transmembrane potential and magnetic field at the apex of the heart.

We studied the transmembrane potential and magnetic fields from electrical activity at the apex of the isolated rabbit heart experimentally using optical mapping and superconducting quantum interference device microscopy, and theoretically using monodomain and bidomain models. The cardiac apex has a complex spiral fiber architecture that plays an important role in the development and propagation of action currents during stimulation at the apex. This spiral fiber orientation contains both radial electric currents that contribute to the electrocardiogram and electrically silent circular currents that cannot be detected by the electrocardiogram but are detectable by their magnetic field, B(z). In our experiments, the transmembrane potential, V(m), was first measured optically and then B(z) was measured with a superconducting quantum interference device microscope. Based on a simple model of the spiral structure of the apex, V(m) was expected to exhibit circular wave front patterns and B(z) to reflect the circular component of the action currents. Although the circular V(m) wave fronts were detected, the B(z) maps were not as simple as expected. However, we observed a pattern consistent with a tilted axis for the apex spiral fiber geometry. We were able to simulate similar patterns in both a monodomain model of a tilted stack of rings of dipole current and a bidomain model of a tilted stack of spiraled cardiac tissue that was stimulated at the apex. The fact that the spatial pattern of the magnetic data was more complex than the simple circles observed for V(m) suggests that the magnetic data contain information that cannot be found electrically.

Polarity reversal lowers activation time during diastolic field stimulation of the rabbit ventricles: insights into mechanisms.

To fully characterize the mechanisms of defibrillation, it is necessary to understand the response, within the three-dimensional (3D) volume of the ventricles, to shocks given in diastole. Studies that have examined diastolic responses conducted measurements on the epicardium or on a transmural surface of the left ventricular (LV) wall only. The goal of this study was to use optical imaging experiments and 3D bidomain simulations, including a model of optical mapping, to ascertain the shock-induced virtual electrode and activation patterns throughout the rabbit ventricles following diastolic shocks. We tested the hypothesis that the locations of shock-induced regions of hyperpolarization govern the different diastolic activation patterns for shocks of reversed polarity. In model and experiment, uniform-field monophasic shocks of reversed polarities (cathode over the right ventricle is RV-, reverse polarity is LV-) were applied to the ventricles in diastole. Experiments and simulations revealed that RV- shocks resulted in longer activation times compared with LV- shocks of the same strength. 3D simulations demonstrated that RV- shocks induced a greater volume of hyperpolarization at shock end compared with LV- shocks; most of these hyperpolarized regions were located in the LV. The results of this study indicate that ventricular geometry plays an important role in both the location and size of the shock-induced virtual anodes that determine activation delay during the shock and subsequently affect shock-induced propagation. If regions of hyperpolarization that develop during the shock are sufficiently large, activation delay may persist until shock end.