Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes.

Neonatal rat ventricular cardiomyocytes were used to investigate mechanisms underlying transient changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by pulsed infrared radiation (IR, 1862 nm). Fluorescence confocal microscopy revealed IR-evoked [Ca2+]i events with each IR pulse (3-4 ms pulse⁻¹, 9.1-11.6 J cm⁻² pulse⁻¹). IR-evoked [Ca2+]i events were distinct from the relatively large spontaneous [Ca2+]i transients, with IR-evoked events exhibiting smaller amplitudes (0.88 ΔF/F0 vs. 1.99 ΔF/F0) and shorter time constants (τ =0.64 s vs. 1.19 s, respectively). Both IR-evoked [Ca2+]i events and spontaneous [Ca2+]i transients could be entrained by the IR pulse (0.2-1 pulse s⁻¹), provided the IR dose was sufficient and the radiation was applied directly to the cell. Examination of IR-evoked events during peak spontaneous [Ca2+]i periods revealed a rapid drop in [Ca2+]i, often restoring the baseline [Ca2+]i concentration, followed by a transient increase in [Ca2+]i.Cardiomyocytes were challenged with pharmacological agents to examine potential contributors to the IR-evoked [Ca2+]i events. Three compounds proved to be the most potent, reversible inhibitors: (1) CGP-37157 (20 µM, n =12), an inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX), (2) Ruthenium Red (40 µM, n =13), an inhibitor of the mitochondrial Ca2+ uniporter (mCU), and (3) 2-aminoethoxydiphenylborane (10 µM, n =6), an IP3 channel antagonist. Ryanodine blocked the spontaneous [Ca2+]i transients but did not alter the IR-evoked events in the same cells. This pharmacological array implicates mitochondria as the major intracellular store of Ca2+ involved in IR-evoked responses reported here. Results support the hypothesis that 1862 nm pulsed IR modulates mitochondrial Ca2+ transport primarily through actions on mCU and mNCX.

Defining and evaluating wrapping surfaces for MRI-derived spinal muscle paths.

Muscle paths can be approximated in biomechanical models by wrapping the path around geometric objects; however, the process for selecting and evaluating wrapping surface parameters is not well defined, especially for spinal muscles. In this study, we defined objective methods to select the shape, orientation, size and location of wrapping surfaces and evaluated the wrapping surfaces using an error metric based on the distance between the modeled muscle path and the centroid path from magnetic resonance imaging (MRI). We applied these methods and the error metric to a model of the neck musculature, where our specific goals were (1) to optimize the vertebral level at which to place a single wrapping surface per muscle; and (2) to define wrapping surface parameters in the neutral posture and evaluate them in other postures. Detailed results are provided for the sternocleidomastoid and the semispinalis capitis muscles. For the sternocleidomastoid, the level where the wrapping surface was placed did not significantly affect the error between the modeled path and the centroid path; use of wrapping surfaces defined from the neutral posture improved the representation of the muscle path compared to a straight line in all postures except contralateral rotation. For the semispinalis capitis, wrapping surfaces placed at C3 or C4 resulted in lower error compared to other levels; and the use of wrapping surfaces significantly improved the muscle path representation in all postures. These methods will be used to improve the estimates of muscle length, moment arm and moment-generating capacity in biomechanical models.

Quantitative analysis of cardiac tissue including fibroblasts using three-dimensional confocal microscopy and image reconstruction: towards a basis for electrophysiological modeling.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.

Electrical stimulation directs engineered cardiac tissue to an age-matched native phenotype.

Quantifying structural features of native myocardium in engineered tissue is essential for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. We applied three-dimensional confocal imaging and image analysis to quantitatively describe the features of native and engineered cardiac tissue. Quantitative analysis methods were developed and applied to test the hypothesis that environmental cues direct engineered tissue toward a phenotype resembling that of age-matched native myocardium. The analytical approach was applied to engineered cardiac tissue with and without the application of electrical stimulation as well as to age-matched and adult native tissue. Individual myocytes were segmented from confocal image stacks and assigned a coordinate system from which measures of cell geometry and connexin-43 spatial distribution were calculated. The data were collected from 9 nonstimulated and 12 electrically stimulated engineered tissue constructs and 5 postnatal day 12 and 7 adult hearts. The myocyte volume fraction was nearly double in stimulated engineered tissue compared to nonstimulated engineered tissue (0.34 ± 0.14 vs 0.18 ± 0.06) but less than half of the native postnatal day 12 (0.90 ± 0.06) and adult (0.91 ± 0.04) myocardium. The myocytes under electrical stimulation were more elongated compared to nonstimulated myocytes and exhibited similar lengths, widths, and heights as in age-matched myocardium. Furthermore, the percentage of connexin-43-positive membrane staining was similar in the electrically stimulated, postnatal day 12, and adult myocytes, whereas it was significantly lower in the nonstimulated myocytes. Connexin-43 was found to be primarily located at cell ends for adult myocytes and irregularly but densely clustered over the membranes of nonstimulated, stimulated, and postnatal day 12 myocytes. These findings support our hypothesis and reveal that the application of environmental cues produces tissue with structural features more representative of age-matched native myocardium than adult myocardium. We suggest that the presented approach can be applied to quantitatively characterize developmental processes and mechanisms in engineered tissue.

Three-dimensional modeling and quantitative analysis of gap junction distributions in cardiac tissue.

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

The mechanically enhanced phase separation of sprayed polyurethane scaffolds and their effect on the alignment of fibroblasts.

This paper reports a method to fabricate anisotropic scaffolds of tunable porosity and mechanical properties. Scaffolds were fabricated using a computer controlled sprayed phase separation technique. Following fabrication, the sheets were elongated 0, 35 or 70% of their original length to induce varying degrees of scaffold alignment and anisotropy. The nonsolvent used in the phase separation was shown to affect porosity and the elastic modulus. Mouse embryo NIH-3T3 fibroblasts were cultured on the scaffolds to investigate cell response to the anisotropy of the scaffold. A 2D FFT method was used to quantify cellular alignment. Cells were shown to align themselves with the scaffold. This sheet-like scaffold material can be used in single plys or can be laminated to form porous 3D composite scaffolds.

Design and characterization of a modified T-flask bioreactor for continuous monitoring of engineered tissue stiffness.

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force-displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T-flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T-flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R(2) > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF-beta, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development.

Towards modeling of cardiac micro-structure with catheter-based confocal microscopy: a novel approach for dye delivery and tissue characterization.

This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901 +/- 1713 and 10 299 +/-3598 mum (3) (mean+/-sd), respectively. Atrial and ventricular myocyte volume fractions were 72.4 +/-4.7% and 79.7 +/- 2.9% (mean +/-sd), respectively. Atrial and ventricular myocyte density was 165 571 +/- 55 836 and 86 957 +/- 32 280 cells/mm (3) (mean+/-sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.