Spatial analysis of tissue immunity and vascularity by light sheet fluorescence microscopy.

The pathogenesis of cancer and cardiovascular diseases is subjected to spatiotemporal regulation by the tissue microenvironment. Multiplex visualization of the microenvironmental components, including immune cells, vasculature and tissue hypoxia, provides critical information underlying the disease progression and therapy resistance, which is often limited by imaging depth and resolution in large-volume tissues. To this end, light sheet fluorescence microscopy, following tissue clarification and immunostaining, may generate three-dimensional high-resolution images at a whole-organ level. Here we provide a detailed description of light sheet fluorescence microscopy imaging analysis of immune cell composition, vascularization, tissue perfusion and hypoxia in mouse normal brains and hearts, as well as brain tumors. We describe a procedure for visualizing tissue vascularization, perfusion and hypoxia with a transgenic vascular labeling system. We provide the procedures for tissue collection, tissue semi-clearing and immunostaining. We further describe standard methods for analyzing tissue immunity and vascularity. We anticipate that this method will facilitate the spatial illustration of structure and function of the tissue microenvironmental components in cancer and cardiovascular diseases. The procedure requires 1-2 weeks and can be performed by users with expertise in general molecular biology.

Subcellular analysis of nuclear and cytoplasmic redox indices differentiates breast cancer cell subtypes better than nuclear-to-cytoplasmic area ratio.

SIGNIFICANCE: Stratification of malignancy is valuable for cancer treatment. Both optical redox imaging (ORI) indices and nuclear-to-cytoplasmic volume/area ratio (N:C ratio) have been investigated to differentiate between cancers with varying aggressiveness, but these two methods have not been directly compared. The redox status in the cell nucleus has not been studied by ORI, and it remains unknown whether nuclear ORI indices add new biological information. AIM: We sought to compare the capacity of whole-cell and subcellular ORI indices and N:C ratio to differentiate between breast cancer subtypes with varying aggressiveness and between mitotic and nonmitotic cells. APPROACH: ORI indices for whole cell, cytoplasm, and nucleus as well as the N:C area ratio were generated for two triple-negative (more aggressive) and two receptor-positive (less aggressive) breast cancer cell lines by fluorescence microscopy. RESULTS: We found positive correlations between nuclear and cytoplasmic ORI indices within individual cells. On average, a nuclear redox status was found to be more oxidized than cytoplasm in triple-negative cells but not in receptor-positive cells. Whole-cell and subcellular ORI indices distinguished between the receptor statuses better than the N:C ratio. However, N:C ratio was a better differentiator between nonmitotic and mitotic triple-negative cells. CONCLUSIONS: Subcellular ORI analysis differentiates breast cancer subtypes with varying aggressiveness better than N:C area ratio.

Myocardin is required for cardiomyocyte survival and maintenance of heart function.

Despite intense investigation over the past century, the molecular mechanisms that regulate maintenance and adaptation of the heart during postnatal development are poorly understood. Myocardin is a remarkably potent transcriptional coactivator expressed exclusively in cardiac myocytes and smooth muscle cells during postnatal development. Here we show that myocardin is required for maintenance of cardiomyocyte structure and sarcomeric organization and that cell-autonomous loss of myocardin in cardiac myocytes triggers programmed cell death. Mice harboring a cardiomyocyte-restricted null mutation in the myocardin gene (Myocd) develop dilated cardiomyopathy and succumb from heart failure within a year. Remarkably, ablation of the Myocd gene in the adult heart leads to the rapid-onset of heart failure, dilated cardiomyopathy, and death within a week. Myocd gene ablation is accompanied by dissolution of sarcomeric organization, disruption of the intercalated disc, and cell-autonomous loss of cardiomyocytes via apoptosis. Expression of myocardin/serum response factor-regulated myofibrillar genes is extinguished, or profoundly attenuated, in myocardin-deficient hearts. Conversely, proapoptotic factors are induced and activated in myocardin-deficient hearts. We conclude that the transcriptional coactivator myocardin is required for maintenance of heart function and ultimately cardiomyocyte survival.

Histone-deacetylase inhibition reverses atrial arrhythmia inducibility and fibrosis in cardiac hypertrophy independent of angiotensin.

Atrial fibrosis influences the development of atrial fibrillation (AF), particularly in the setting of structural heart disease where angiotensin-inhibition is partially effective for reducing atrial fibrosis and AF. Histone-deacetylase inhibition reduces cardiac hypertrophy and fibrosis, so we sought to determine if the HDAC inhibitor trichostatin A (TSA) could reduce atrial fibrosis and arrhythmias. Mice over-expressing homeodomain-only protein (HopX(Tg)), which recruits HDAC activity to induce cardiac hypertrophy were investigated in 4 groups (aged 14-18 weeks): wild-type (WT), HopX(Tg), HopX(Tg) mice treated with TSA for 2 weeks (TSA-HopX) and wild-type mice treated with TSA for 2 weeks (TSA-WT). These groups were characterized using invasive electrophysiology, atrial fibrosis measurements, atrial connexin immunocytochemistry and myocardial angiotensin II measurements. Invasive electrophysiologic stimulation, using the same attempts in each group, induced more atrial arrhythmias in HopX(Tg) mice (48 episodes in 13 of 15 HopX(Tg) mice versus 5 episodes in 2 of 15 TSA-HopX mice, P<0.001; versus 9 episodes in 2 of 15 WT mice, P<0.001; versus no episodes in any TSA-WT mice, P<0.001). TSA reduced atrial arrhythmia duration in HopX(Tg) mice (1307+/-289 ms versus 148+/-110 ms, P<0.01) and atrial fibrosis (8.1+/-1.5% versus 3.9+/-0.4%, P<0.001). Atrial connexin40 was lower in HopX(Tg) compared to WT mice, and TSA normalized the expression and size distribution of connexin40 gap junctions. Myocardial angiotensin II levels were similar between WT and HopX(Tg) mice (76.3+/-26.0 versus 69.7+/-16.6 pg/mg protein, P=NS). Therefore, it appears HDAC-inhibition reverses atrial fibrosis, connexin40 remodeling and atrial arrhythmia vulnerability independent of angiotensin II in cardiac hypertrophy.

Tracking changes in Z-band organization during myofibrillogenesis with FRET imaging.

There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical analysis of data from FRET imaging studies yield results that are in agreement with published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our analysis has revealed changes in the arrangement of alpha-actinin and FATZ that take place during the transition as the z-bodies of premyofibrils fuse to form the Z-bands of mature myofibrils. There was no evidence for a change in the arrangement of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These FRET results from living cells support a stepwise model for the assembly of myofibrils.

How to build a myofibril.

Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) premyofibrils as precursors of myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.