RESUMO
Aging induces cardiac remodeling, resulting in an increase in the risk of suffering heart diseases, including heart failure. Collagen deposition increases with age and, together with sarcomeric changes in cardiomyocytes, may lead to ventricular stiffness. Multiphoton (MP) microscopy is a useful technique to visualize and detect variations in cardiac structures in a label free fashion. Here, we propose a method based on MP imaging (both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) modalities) to explore and objectively quantify age-related structural differences in various components of cardiac tissues. Results in transmural porcine left ventricle (LV) sections reveal significant differences when comparing samples from young and old animals. Collagen and myosin SHG signals in old specimens are respectively 3.8x and >6-fold larger than in young ones. Differences in TPEF signals from cardiomyocyte were â¼3x. Moreover, the increased amount of collagen in old specimens results in a more organized pattern when compared to young LV tissues. Since changes in collagen and myosin are associated with cardiac dysfunction, the technique used herein might be a useful tool to accurately predict and measure changes associated with age-related myocardium fibrosis, tissue remodeling and sarcomeric alterations, with potential implications in preventing heart disease.
RESUMO
Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.