Allogeneic mesenchymal stromal cells overexpressing mutant human Hypoxia-inducible factor 1-α (HIF1-α) in an ovine model of acute myocardial infarction

Background-Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. Methods and Results-Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P < 0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P < 0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF. Conclusions-Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.Fil: Hnatiuk, Anna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Ong, Sang-Ging. Stanford University School of Medicine; Estados UnidosFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Riegler, Johannes. Stanford University School of Medicine; Estados UnidosFil: Lee, Won Hee. Stanford University School of Medicine; Estados UnidosFil: Jen, Cheng Hao. University of London; Reino UnidoFil: De Lorenzi, Andrea. Fundación Favaloro; ArgentinaFil: Giménez, Carlos Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Laguens, Rubén. Universidad Favaloro; ArgentinaFil: Wu, Joseph C.. Stanford University School of Medicine; Estados UnidosFil: Crottogini, Alberto José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentin

Spatial and temporal variations in hemodynamic forces initiate cardiac trabeculation

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function

Longitudinal morphological and functional characterization of human heart organoids using optical coherence tomography

Organoids play an increasingly important role as in vitro models for studying organ development, disease mechanisms, and drug discovery. Organoids are self-organizing, organ-like three-dimensional (3D) cell cultures developing organ-specific cell types and functions. Recently, three groups independently developed self-assembling human heart organoids (hHOs) from human pluripotent stem cells (hPSCs). In this study, we utilized a customized spectral-domain optical coherence tomography (SD-OCT) system to characterize the growth of hHOs. Development of chamber structures and beating patterns of the hHOs were observed via OCT and calcium imaging. We demonstrated the capability of OCT to produce 3D images in a fast, label-free, and non-destructive manner. The hHOs formed cavities of various sizes, and complex interconnections were observed as early as on day 4 of differentiation. The hHOs models and the OCT imaging system showed promising insights as an in vitro platform for investigating heart development and disease mechanisms

Genetic and environmental determinants of diastolic heart function

Diastole is the sequence of physiological events that occur in the heart during ventricular filling and principally depends on myocardial relaxation and chamber stiffness. Abnormal diastolic function is related to many cardiovascular disease processes and is predictive of health outcomes, but its genetic architecture is largely unknown. Here, we use machine learning cardiac motion analysis to measure diastolic functional traits in 39,559 participants of the UK Biobank and perform a genome-wide association study. We identified 9 significant, independent loci near genes that are associated with maintaining sarcomeric function under biomechanical stress and genes implicated in the development of cardiomyopathy. Age, sex and diabetes were independent predictors of diastolic function and we found a causal relationship between genetically-determined ventricular stiffness and incident heart failure. Our results provide insights into the genetic and environmental factors influencing diastolic function that are relevant for identifying causal relationships and potential tractable targets

On the averaging of cardiac diffusion tensor MRI data: the effect of distance function selection

Diffusion tensor magnetic resonance imaging (DT-MRI) allows a unique insight into the microstructure of highly-directional tissues. The selection of the most proper distance function for the space of diffusion tensors is crucial in enhancing the clinical application of this imaging modality. Both linear and nonlinear metrics have been proposed in the literature over the years. The debate on the most appropriate DT-MRI distance function is still ongoing. In this paper, we presented a framework to compare the Euclidean, affine-invariant Riemannian and log-Euclidean metrics using actual high-resolution DT-MRI rat heart data. We employed temporal averaging at the diffusion tensor level of three consecutive and identically-acquired DT-MRI datasets from each of five rat hearts as a means to rectify the background noise-induced loss of myocyte directional regularity. This procedure is applied here for the first time in the context of tensor distance function selection. When compared with previous studies that used a different concrete application to juxtapose the various DT-MRI distance functions, this work is unique in that it combined the following: (i) Metrics were judged by quantitative - rather than qualitative – criteria, (ii) the comparison tools were non-biased, (iii) a longitudinal comparison operation was used on a same-voxel basis. The statistical analyses of the comparison showed that the three DT-MRI distance functions tend to provide equivalent results. Hence, we came to the conclusion that the tensor manifold for cardiac DT-MRI studies is a curved space of almost zero curvature. The signal to noise ratio dependence of the operations was investigated through simulations. Finally, the “swelling effect” occurrence following Euclidean averaging was found to be too unimportant to be worth consideration

MRI Evaluation of Injectable Hyaluronic Acid Hydrogel Therapy to Attenuate Myocardial Infarct Remodeling

Left ventricular (LV) remodeling following myocardial infarction (MI) leads to maladaptive processes that often progress to heart failure. Injectable biomaterials can alter the mechanical signaling post-MI to limit this progression. To design optimal therapies, noninvasive techniques are needed to elucidate the reciprocal interaction between the injected material and the surrounding myocardial tissue. Towards this goal, the general hypothesis of this dissertation was that magnetic resonance imaging (MRI) can be used to characterize the properties of injectable materials once delivered to the myocardium and evaluate the temporal effects of injectable materials on myocardial tissue properties post-MI. To test this hypothesis, injectable hyaluronic acid (HA) hydrogels were developed with a range of gelation, degradation and mechanical properties by altering the initiator concentration, macromer modification, and macromer concentration, respectively. Non-contrast MRI was then used to characterize the properties (e.g., distribution, chemical composition) of injectable HA hydrogels in myocardial explants. Altering hydrogel gelation led to differences in distribution in myocardial tissue, as quantified by T2-weighted MRI. As an alternative to conventional (i.e.T2-weighted) MRI where contrast depends on differences in MR properties and thus, is non-specific for the material, chemical exchange saturation transfer (CEST) MRI was used to specifically image hydrogels based on their functional (i.e. exchangeable proton) groups. CEST contrast correlated with changes in material properties, specifically macromer concentration. Furthermore, CEST MRI was shown to simultaneously visualize and discriminate between different injectable materials based on their unique chemistry. Finally, the effect of injectable HA hydrogels on myocardial tissue properties was temporally evaluated in a porcine infarct model up to 12 weeks post-MI. Outcome assessment using MRI (e.g. cine, late-gadolinium enhancement, and spatial modulation of magnetization MRI) and finite element (FE) modeling demonstrated that hydrogel therapy led to improved global LV structure and function, increased wall thickness, preserved borderzone contractility, and increased infarct stiffness, respectively. This work demonstrates that MRI can be used to simultaneously study hydrogel properties after injection into the myocardium and evaluate the ability of injectable hydrogels to alter myocardial tissue properties to ultimately improve cardiac outcomes and enable future optimization of biomaterial therapies to attenuate adverse remodeling post-MI

Remote refocusing light-sheet fluorescence microscopy for high-speed 2D and 3D imaging of calcium dynamics in cardiomyocytes

The high prevalence and poor prognosis of heart failure are two key drivers for research into cardiac electrophysiology and regeneration. Dyssynchrony in calcium release and loss of structural organization within individual cardiomyocytes (CM) has been linked to reduced contractile strength and arrhythmia. Correlating calcium dynamics and cell microstructure requires multidimensional imaging with high spatiotemporal resolution. In light-sheet fluorescence microscopy (LSFM), selective plane illumination enables fast optically sectioned imaging with lower phototoxicity, making it suitable for imaging subcellular dynamics. In this work, a custom remote refocusing LSFM system is applied to studying calcium dynamics in isolated CM, cardiac cell cultures and tissue slices. The spatial resolution of the LSFM system was modelled and experimentally characterized. Simulation of the illumination path in Zemax was used to estimate the light-sheet beam waist and confocal parameter. Automated MATLAB-based image analysis was used to quantify the optical sectioning and the 3D point spread function using Gaussian fitting of bead image intensity distributions. The results demonstrated improved and more uniform axial resolution and optical sectioning with the tighter focused beam used for axially swept light-sheet microscopy. High-speed dual-channel LSFM was used for 2D imaging of calcium dynamics in correlation with the t-tubule structure in left and right ventricle cardiomyocytes at 395 fps. The high spatio-temporal resolution enabled the characterization of calcium sparks. The use of para-nitro-blebbistatin (NBleb), a non-phototoxic, low fluorescence contraction uncoupler, allowed 2D-mapping of the spatial dyssynchrony of calcium transient development across the cell. Finally, aberration-free remote refocusing was used for high-speed volumetric imaging of calcium dynamics in human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM) and their co-culture with adult-CM. 3D-imaging at up to 8 Hz demonstrated the synchronization of calcium transients in co-culture, with increased coupling with longer co-culture duration, uninhibited by motion uncoupling with NBleb.Open Acces

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity. Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 μm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell. Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules. Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure

Insight Into Myocardial Microstructure of Athletes and Hypertrophic Cardiomyopathy Patients Using Diffusion Tensor Imaging

Background Hypertrophic cardiomyopathy (HCM) remains the commonest cause of sudden cardiac death among young athletes. Differentiating between physiologically adaptive left ventricular (LV) hypertrophy observed in athletes' hearts and pathological HCM remains challenging. By quantifying the diffusion of water molecules, diffusion tensor imaging (DTI) MRI allows voxelwise characterization of myocardial microstructure. Purpose To explore microstructural differences between healthy volunteers, athletes, and HCM patients using DTI. Study Type Prospective cohort. Population Twenty healthy volunteers, 20 athletes, and 20 HCM patients. Field Strength/Sequence 3T/DTI spin echo. Assessment In‐house MatLab software was used to derive mean diffusivity (MD) and fractional anisotropy (FA) as markers of amplitude and anisotropy of the diffusion of water molecules, and secondary eigenvector angles (E2A)—reflecting the orientations of laminar sheetlets. Statistical Tests Independent samples t‐tests were used to detect statistical significance between any two cohorts. Analysis of variance was utilized for detecting the statistical difference between the three cohorts. Statistical tests were two‐tailed. A result was considered statistically significant at P ≤ 0.05. Results DTI markers were significantly different between HCM, athletes, and volunteers. HCM patients had significantly higher global MD and E2A, and significantly lower FA than athletes and volunteers. (MDHCM = 1.52 ± 0.06 × 10−3 mm2/s, MDAthletes = 1.49 ± 0.03 × 10−3 mm2/s, MDvolunteers = 1.47 ± 0.02 × 10−3 mm2/s, P < 0.05; E2AHCM = 58.8 ± 4°, E2Aathletes = 47 ± 5°, E2Avolunteers = 38.5 ± 7°, P < 0.05; FAHCM = 0.30 ± 0.02, FAAthletes = 0.35 ± 0.02, FAvolunteers = 0.36 ± 0.03, P < 0.05). HCM patients had significantly higher E2A in their thickest segments compared to the remote (E2Athickest = 66.8 ± 7, E2Aremote = 51.2 ± 9, P < 0.05). Data Conclusion DTI depicts an increase in amplitude and isotropy of diffusion in the myocardium of HCM compared to athletes and volunteers as reflected by increased MD and decreased FA values. While significantly higher E2A values in HCM and athletes reflect steeper configurations of the myocardial sheetlets than in volunteers, HCM patients demonstrated an eccentric rise in E2A in their thickest segments, while athletes demonstrated a concentric rise. Further studies are required to determine the diagnostic capabilities of DTI. Evidence Level 1 Technical Efficacy Stage

Computational modelling of diffusion magnetic resonance imaging based on cardiac histology

The exact relationship between changes in myocardial microstructure as a result of heart disease and the signal measured using diffusion tensor cardiovascular magnetic resonance (DT-CMR) is currently not well understood. Computational modelling of diffusion in combination with realistic numerical phantoms offers the unique opportunity to study effects of pathologies or the efficacy of improvements to acquisition protocols in a controlled in-silico environment. In this work, Monte Carlo random walk (MCRW) methods are used to simulate diffusion in a histology-based 3D model of the myocardium. Sensitivity of typical DT-CMR sequences to changes in tissue properties is assessed. First, myocardial tissue is analysed to identify important geometric features and diffusion parameters. A two-compartment model is considered where intra-cellular compartments with a reduced bulk diffusion coefficient are separated from extra-cellular space by permeable membranes. Secondary structures like groups of cardiomyocyte (sheetlets) must also be included, and different methods are developed to automatically generate realistic histology-based substrates. Next, in-silico simulation of DT-CMR is reviewed and a tool to generate idealised versions of common pulse sequences is discussed. An efficient GPU-based numerical scheme for obtaining a continuum solution to the Bloch--Torrey equations is presented and applied to domains directly extracted from histology images. In order to verify the numerical methods used throughout this work, an analytical solution to the diffusion equation in 1D is described. It relies on spectral analysis of the diffusion operator and requires that all roots of a complex transcendental equation are found. To facilitate a fast and reliable solution, a novel root finding algorithm based on Chebyshev polynomial interpolation is proposed. To simulate realistic 3D geometries MCRW methods are employed. A parallel simulator for both grid-based and surface mesh--based geometries is presented. The presence of permeable membranes requires special treatment. For this, a commonly used transit model is analysed. Finally, the methods above are applied to study the effect of various model and sequence parameters on DT-CMR results. Simulations with impermeable membranes reveal sequence-specific sensitivity to extra-cellular volume fraction and diffusion coefficients. By including membrane permeability, DT-CMR results further approach values expected in vivo.Open Acces