The Macrophage Landscape Across the Lifespan of a Human Cardiac Allograft.

BACKGROUND: Much of our knowledge of organ rejection after transplantation is derived from rodent models. METHODS: We used single-nucleus RNA sequencing to investigate the inflammatory myocardial microenvironment in human pediatric cardiac allografts at different stages after transplantation. We distinguished donor- from recipient-derived cells using naturally occurring genetic variants embedded in single-nucleus RNA sequencing data. RESULTS: Donor-derived tissue resident macrophages, which accompany the allograft into the recipient, are lost over time after transplantation. In contrast, monocyte-derived macrophages from the recipient populate the heart within days after transplantation and form 2 macrophage populations: recipient MP1 and recipient MP2. Recipient MP2s have cell signatures similar to donor-derived resident macrophages; however, they lack signatures of pro-reparative phagocytic activity typical of donor-derived resident macrophages and instead express profibrotic genes. In contrast, recipient MP1s express genes consistent with hallmarks of cellular rejection. Our data suggest that recipient MP1s activate a subset of natural killer cells, turning them into a cytotoxic cell population through feed-forward signaling between recipient MP1s and natural killer cells. CONCLUSIONS: Our findings reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft that contributes to allograft failure.

Activated fibroblasts drive cellular interactions in end-stage pediatric hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a relatively rare but debilitating diagnosis in the pediatric population and patients with end-stage HCM require heart transplantation. In this study, we performed single-nucleus RNA sequencing on pediatric HCM and control myocardium. We identified distinct underling cellular processes in pediatric, end-stage HCM in cardiomyocytes, fibroblasts, endothelial cells, and myeloid cells, compared to controls. Pediatric HCM was enriched in cardiomyocytes exhibiting "stressed" myocardium gene signatures and underlying pathways associated with cardiac hypertrophy. Cardiac fibroblasts exhibited clear activation signatures and heightened downstream processes associated with fibrosis, more so than adult counterparts. There was notable depletion of tissue-resident macrophages, and increased vascular remodeling in endothelial cells. Our analysis provides the first single nuclei analysis focused on end-stage pediatric HCM.

Single nucleus transcriptome of a "Super RV" shows increased insulin and angiogenesis signaling.

The right ventricle (RV) is one of the four pumping chambers of the heart, pumping blood to the lungs. In severe forms of congenital heart disease and pulmonary hypertension, the RV is made to pump into the systemic circulation. Such systemic RVs typically display early failure due to pressure overload. In rare cases a systemic RV persists into later decades of life - colloquially called a 'Super RV'. Here we present the single-nucleus transcriptome of a systemic RV from a 60-year-old with congenitally corrected transposition of great arteries (ccTGA). Our data shows two specific signaling pathways enriched in the ccTGA RV myocardium. First, we show increased insulin like growth factor (IGF1) signaling within the systemic RV myocardium: there is increased expression of the main receptor IGFR1 within the cardiomyocytes, and IGF1 ligands within the cardiofibroblasts and macrophages. Second, we find increased VEGF and Wnt9 ligand expression in cardiomyocytes and increased VEGF1R and Wnt9 receptors in endothelial cells, which are implicated in angiogenesis. We show that increased insulin and angiogenesis signaling are potentially beneficial RV adaptations to increased pressure overload. This study of an adult systemic RV provides an important framework for understanding RV remodeling to systemic pressures in congenital heart disease and pulmonary hypertension.

Endothelial cells adopt a pro-reparative immune responsive signature during cardiac injury.

Modulation of the heart's immune microenvironment is crucial for recovery after ischemic events such as myocardial infarction (MI). Endothelial cells (ECs) can have immune regulatory functions; however, interactions between ECs and the immune environment in the heart after MI remain poorly understood. We identified an EC-specific IFN responsive and immune regulatory gene signature in adult and pediatric heart failure (HF) tissues. Single-cell transcriptomic analysis of murine hearts subjected to MI uncovered an EC population (IFN-ECs) with immunologic gene signatures similar to those in human HF. IFN-ECs were enriched in regenerative-stage mouse hearts and expressed genes encoding immune responsive transcription factors (Irf7, Batf2, and Stat1). Single-cell chromatin accessibility studies revealed an enrichment of these TF motifs at IFN-EC signature genes. Expression of immune regulatory ligand genes by IFN-ECs suggests bidirectional signaling between IFN-ECs and macrophages in regenerative-stage hearts. Our data suggest that ECs may adopt immune regulatory signatures after cardiac injury to accompany the reparative response. The presence of these signatures in human HF and murine MI models suggests a potential role for EC-mediated immune regulation in responding to stress induced by acute injury in MI and chronic adverse remodeling in HF.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Single-Cell Determination of Cardiac Microtissue Structure and Function Using Light Sheet Microscopy.

Native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models composed of stem cell-derived cardiomyocytes (CMs) require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal CMs and cardiac microtissues have to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact three-dimensional (3D) tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy (LSFM) overcomes these constraints to enable single-cell resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that CMs and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of CM calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that LSFM can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact statement The ability to achieve single-cell resolution by advanced three-dimensional light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.

Functional organization of glomerular maps in the mouse accessory olfactory bulb.

The mammalian accessory olfactory system extracts information about species, sex and individual identity from social odors, but its functional organization remains unclear. We imaged presynaptic Ca(2+) signals in vomeronasal inputs to the accessory olfactory bulb (AOB) during peripheral stimulation using light sheet microscopy. Urine- and steroid-responsive glomeruli densely innervated the anterior AOB. Glomerular activity maps for sexually mature female mouse urine overlapped maps for juvenile and/or gonadectomized urine of both sexes, whereas maps for sexually mature male urine were highly distinct. Further spatial analysis revealed a complicated organization involving selective juxtaposition and dispersal of functionally grouped glomerular classes. Glomeruli that were similarly tuned to urines were often closely associated, whereas more disparately tuned glomeruli were selectively dispersed. Maps to a panel of sulfated steroid odorants identified tightly juxtaposed groups that were disparately tuned and dispersed groups that were similarly tuned. These results reveal a modular, nonchemotopic spatial organization in the AOB.

Aberrations and their correction in light-sheet microscopy: a low-dimensional parametrization.

Light sheet microscopy allows rapid imaging of three-dimensional fluorescent samples, using illumination and detection axes that are orthogonal. For imaging large samples, this often forces the objective to be tilted relative to the sample's surface; for samples that are not precisely matched to the immersion medium index, this tilt introduces aberrations. Here we calculate the nature of these aberrations for a simple tissue model, and show that a low-dimensional parametrization of these aberrations facilitates online correction via a deformable mirror without introduction of beads or other fiducial markers. We use this approach to demonstrate improved image quality in living tissue.

Organization of vomeronasal sensory coding revealed by fast volumetric calcium imaging.

A long-standing goal in neuroscience is to perform exhaustive recording of each neuron in a functional local circuit. To achieve this goal, one promising approach is optical imaging of fluorescent calcium indicators, but typically the tens or hundreds of cells imaged simultaneously comprise only a tiny percentage of the neurons in an intact circuit. Here, we show that a recent innovation, objective-coupled planar illumination (OCPI) microscopy, permits simultaneous recording from three-dimensional volumes containing many thousand neurons. We used OCPI microscopy to record chemosensory responses in the mouse vomeronasal epithelium, for which expression of hundreds of receptor types implies high functional diversity. The implications of this diversity for sensory coding were examined using several classes of previously reported vomeronasal ligands, including sulfated steroids. A collection of just 12 sulfated steroids activated more than a quarter of the neurons in the apical vomeronasal epithelium; unexpectedly, responses were functionally organized into a modest number of classes with characteristic spatial distribution. Recording from a whole sensory system thus revealed new organizational principles.

Image-based calibration of a deformable mirror in wide-field microscopy.

Optical aberrations limit resolution in biological tissues, and their influence is particularly large for promising techniques such as light-sheet microscopy. In principle, image quality might be improved by adaptive optics (AO), in which aberrations are corrected by using a deformable mirror (DM). To implement AO in microscopy, one requires a method to measure wavefront aberrations, but the most commonly used methods have limitations for samples lacking point-source emitters. Here we implement an image-based wavefront-sensing technique, a variant of generalized phase-diverse imaging called multiframe blind deconvolution, and exploit it to calibrate a DM in a light-sheet microscope. We describe two methods of parameterizing the influence of the DM on aberrations: a traditional Zernike expansion requiring 1040 parameters, and a direct physical model of the DM requiring just 8 or 110 parameters. By randomizing voltages on all actuators, we show that the Zernike expansion successfully predicts wavefronts to an accuracy of approximately 30 nm (rms) even for large aberrations. We thus show that image-based wavefront sensing, which requires no additional optical equipment, allows a simple but powerful method to calibrate a deformable optical element in a microscope setting.

Miniaturization and defocus correction for objective-coupled planar illumination microscopy.

Recently, a light sheet-based technique called objective-coupled planar illumination (OCPI) microscopy [Holekamp et al., Neuron 57, 661 (2008)] was shown to permit low-phototoxicity, high-speed, three-dimensional fluorescence imaging of extended tissue samples. Here, we introduce two major improvements in OCPI microscopy. First, we miniaturize the objective coupler by using a uniaxial gradient-index lens to produce the light sheet. Second, we demonstrate theoretically and experimentally that refractive index mismatch at the fluid/tissue interface causes a significant defocus aberration. By introducing the ability to tune the angle of the light sheet, we show that defocus correction in a miniaturized OCPI microscope leads to a significant improvement in image sharpness deeper into tissue.

Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy.

Unraveling the functions of the diverse neural types in any local circuit ultimately requires methods to record from most or all of its cells simultaneously. One promising approach to this goal is fluorescence imaging, but existing methods using laser-scanning microscopy (LSM) are severely limited in their ability to resolve rapid phenomena, like neuronal action potentials, over wide fields. Here we present a microscope that rapidly sections a three-dimensional volume using a thin illumination sheet whose position is rigidly coupled to the objective and aligned with its focal plane. We demonstrate that this approach allows exceptionally low-noise imaging of large neuronal populations at pixel rates at least 100-fold higher than with LSM. Using this microscope, we studied the pheromone-sensing neurons of the mouse vomeronasal organ and found that responses to dilute urine are largely or exclusively restricted to cells in the apical layer, the location of V1r-family-expressing neurons.