Bastiaan J. van Nierop

Strain-time cell death threshold for skeletal muscle in a tissue-engineered model system for deep tissue injury

Deep tissue injury (DTI) is a severe pressure ulcer that results from sustained deformation of muscle tissue overlying bony prominences. In order to understand the etiology of DTI, it is essential to determine the tolerance of muscle cells to large mechanical strains. In this study, a new experimental method of determining the time-dependent critical compressive strains for necrotic cell death (E(zz)(c)(t)) in a planar tissue-engineered construct under static loading was developed. A half-spherical indentor is used to induce a non-uniform, concentric distribution of strains in the construct, and E(zz)(c)(t) is calculated from the radius of the damage region in the construct versus time. The method was employed to obtain E(zz)(c)(t) for bio-artificial muscles (BAMs) cultured from C2C12 murine cells, as a model system for DTI. Specifically, propidium iodine was used to fluorescently stain the development of necrosis in BAMs subjected to strains up to 80%. Two groups of BAMs were tested at an extracellular pH of 7.4 (n=10) and pH 6.5 (n=5). The lowest strain levels causing cell death in the BAMs were determined every 15min, during 285-min-long trials, from confocal microscopy fluorescent images of the size of the damage regions. The experimental E(zz)(c)(t) data fitted a decreasing single-step sigmoid of the Boltzmann type. Analysis of the parameters of this sigmoid function indicated a 95% likelihood that cells could tolerate engineering strains below 65% for 1h, whereas the cells could endure strains below 40% over a 285min trial period. The decrease in endurance of the cells to compressive strains occurred between 1-3h post-loading. The method developed in this paper is generic and suitable for studying E(zz)(c)(t) in virtually any planar tissue-engineered construct. The specific E(zz)(c)(t) curve obtained herein is necessary for extrapolating biological damage from muscle-strain data in biomechanical studies of pressure ulcers and DTI.

Phenotyping of Left and Right Ventricular Function in Mouse Models of Compensated Hypertrophy and Heart Failure with Cardiac MRI

Background Left ventricular (LV) and right ventricular (RV) function have an important impact on symptom occurrence, disease progression and exercise tolerance in pressure overload-induced heart failure, but particularly RV functional changes are not well described in the relevant aortic banding mouse model. Therefore, we quantified time-dependent alterations in the ventricular morphology and function in two models of hypertrophy and heart failure and we studied the relationship between RV and LV function during the transition from hypertrophy to heart failure. Methods MRI was used to quantify RV and LV function and morphology in healthy (n = 4) and sham operated (n = 3) C57BL/6 mice, and animals with a mild (n = 5) and a severe aortic constriction (n = 10). Results Mice subjected to a mild constriction showed increased LV mass (P<0.01) and depressed LV ejection fraction (EF) (P<0.05) as compared to controls, but had similar RVEF (P>0.05). Animals with a severe constriction progressively developed LV hypertrophy (P<0.001), depressed LVEF (P<0.001), followed by a declining RVEF (P<0.001) and the development of pulmonary remodeling, as compared to controls during a 10-week follow-up. Myocardial strain, as a measure for local cardiac function, decreased in mice with a severe constriction compared to controls (P<0.05). Conclusions Relevant changes in mouse RV and LV function following an aortic constriction could be quantified using MRI. The well-controlled models described here open opportunities to assess the added value of new MRI techniques for the diagnosis of heart failure and to study the impact of new therapeutic strategies on disease progression and symptom occurrence.

Quantitative first-pass perfusion MRI of the mouse myocardium.

In this article, we present a first‐pass perfusion imaging protocol to determine quantitative regional perfusion values (in mL min−1 g−1) of the mouse myocardium. Perfusion was quantified using a Fermi‐constrained deconvolution of the myocardial tissue response with the arterial input function. A dual‐bolus approach was implemented. Experimental evidence is presented for the linearity of signal intensity in the left‐ventricular lumen during the prebolus (r = 0.99, P < 0.001) and in the myocardium during the full‐bolus injection (r = 0.99, P < 0.01) as function of Gd(DTPA)2− injection concentration used. The prebolus was used to reconstruct a nonsaturated arterial input function. Regional perfusion values proved repeatable in a cohort of nine healthy C57BL/6 mice. The perfusion values over two measurements with a 1‐week interval were 7.3 ± 0.9 and 7.2 ± 0.6 mL min−1 g−1, respectively. No effects of time (P > 0.05) and myocardial region (P > 0.05) were observed. The between‐session coefficient of variation was only 6%, whereas the inter‐animal coefficient of variation was 11 and 8% for the separate experiments. We expect that the first‐pass perfusion method here presented will be useful in preclinical studies of myocardial perfusion deficits and valuable to assess the impact of pro‐angiogenic therapy after myocardial infarction. Magn Reson Med, 2013.

Assessment of Myocardial Fibrosis in Mice Using a T2*-Weighted 3D Radial Magnetic Resonance Imaging Sequence

Background Myocardial fibrosis is a common hallmark of many diseases of the heart. Late gadolinium enhanced MRI is a powerful tool to image replacement fibrosis after myocardial infarction (MI). Interstitial fibrosis can be assessed indirectly from an extracellular volume fraction measurement using contrast-enhanced T1 mapping. Detection of short T2* species resulting from fibrotic tissue may provide an attractive non-contrast-enhanced alternative to directly visualize the presence of both replacement and interstitial fibrosis. Objective To goal of this paper was to explore the use of a T2*-weighted radial sequence for the visualization of fibrosis in mouse heart. Methods C57BL/6 mice were studied with MI (n = 20, replacement fibrosis), transverse aortic constriction (TAC) (n = 18, diffuse fibrosis), and as control (n = 10). 3D center-out radial T2*-weighted images with varying TE were acquired in vivo and ex vivo (TE = 21 μs-4 ms). Ex vivo T2*-weighted signal decay with TE was analyzed using a 3-component model. Subtraction of short- and long-TE images was used to highlight fibrotic tissue with short T2*. The presence of fibrosis was validated using histology and correlated to MRI findings. Results Detailed ex vivo T2*-weighted signal analysis revealed a fast (T2*fast), slow (T2*slow) and lipid (T2*lipid) pool. T2*fast remained essentially constant. Infarct T2*slow decreased significantly, while a moderate decrease was observed in remote tissue in post-MI hearts and in TAC hearts. T2*slow correlated with the presence of diffuse fibrosis in TAC hearts (r = 0.82, P = 0.01). Ex vivo and in vivo subtraction images depicted a positive contrast in the infarct co-localizing with the scar. Infarct volumes from histology and subtraction images linearly correlated (r = 0.94, P<0.001). Region-of-interest analysis in the in vivo post-MI and TAC hearts revealed significant T2* shortening due to fibrosis, in agreement with the ex vivo results. However, in vivo contrast on subtraction images was rather poor, hampering a straightforward visual assessment of the spatial distribution of the fibrotic tissue.

In vivo mouse myocardial (31)P MRS using three-dimensional image-selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations.

31P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4 T. Cardiac 31P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright

Mimicking Cardiac Fibrosis in a Dish: Fibroblast Density Rather than Collagen Density Weakens Cardiomyocyte Function

Cardiac fibrosis is one of the most devastating effects of cardiac disease. Current in vitro models of cardiac fibrosis do not sufficiently mimic the complex in vivo environment of the cardiomyocyte. We determined the local composition and mechanical properties of the myocardium in established mouse models of genetic and acquired fibrosis and tested the effect of myocardial composition on cardiomyocyte contractility in vitro by systematically manipulating the number of fibroblasts and collagen concentration in a platform of engineered cardiac microtissues. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Despite apparent dissimilarities between native and in vitro fibrosis, the latter allows for the independent analysis of local determinants of fibrosis, which is not possible in vivo.

In vivomouse myocardial31P MRS using three-dimensional image-selectedin vivospectroscopy (3D ISIS): technical considerations and biochemical validations: IN VIVOMOUSE MYOCARDIAL31P MRS

31P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4 T. Cardiac 31P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright

In vivo mouse myocardial 31P MR spectroscopy using 3D ISIS: technical considerations and biochemical validations

31P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of 31P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized 31P MRS of the in vivo mouse heart at 9.4 T. Cardiac 31P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with 31P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo 31P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with 31P MRS under physiological conditions. Copyright

Global left ventricular function in mice during the development of heart failure

In response to pressure overload the left ventricle (LV) remodels to compensate for the increased workload. Initially this adaptation is beneficial to maintain pump function, but eventually the heart may loose its battle to cope with the increased workload resulting in heart failure (HF). In this study the evolution of global LV function was characterized in a relevant mouse model of LV pressure overload during the development of HF using MRI.

A Method for Determining the Strain-Time Endurance of Cells in Planar Tissue-Engineered Constructs Subjected to Large Compressive Deformations

The mechanical environment of cells influences their normal growth and function, and may also affect the development of diseases and chronic injuries. Accordingly, there is substantial interest in determining the endurance of cells subjected to controlled mechanical strains for given time periods. A standardized, generic experimental method for determining strain-time thresholds for cell death is so far missing in the literature. In this study, a new experimental method was developed to measure strain-time thresholds of cells in planar tissue-engineered constructs subjected to large compressive strains. The method was applied to measure a strain-time threshold for differentiated C2C12 murine skeletal muscle cells in tissue-engineered bio-artificial muscle (BAM) constructs.Copyright