Introduction: The prevalence of obesity has tripled over the past 40 years and is continuing to increase rapidly. Cardiovascular diseases were reported to be the primary cause of death associated with a high body mass index and to be responsible for two-thirds of fatalities caused by obesity. Epicardial adipose tissue (EAT) is the visceral fat deposit that directly covers the surface of the myocardium without any fascial structure. While typically supporting cardiac health, in caloric surplus, it undergoes excessive adipogenesis and becomes hypertrophic, secreting proinflammatory adipokines. Furthermore, the absence of a fascial structure between EAT and myocardium can result in fat infiltration into the myocardium, which compromises the cell-cell contact of cardiomyocytes. This results in irregular beating patterns called arrhythmia. Atrial fibrillation (AF) is reported to be the most prevalent type of arrhythmia affecting more than 60 million people globally. Obesity was reported to be associated with a 50% increase in the risk of AF development. Recent imaging techniques were able to assess the EAT thickness, volume, and density to predict the arrhythmogenic risk in obese individuals. These studies showed that increased EAT thickness and infiltration, mainly associated with adipocyte hypertrophy, is associated with worse atrial function, higher hospitalization, and mortality rate. Current 3D cardiac models lack the crucial adipose tissue component, preventing the assessment and mitigation of adipogenic hypertrophy effects on cardiomyocytes. Thus, there is an urgent need for biomimetic models to better understand the underlying pathology of adipose tissue expansion and to develop new therapies to battle obesity.
Materials and
Methods: Primary adipose-derived stem cells (ADSCs) were incorporated into bioink made of collagen type 1 (6 mg/ml), 20% GelMA and Irgacure2959 photoinitiator solution and bioprinted in hollow cylinder shape employing CELLINK BioX6 Bioprinter utilizing a custom-made G-Code (Figure 1a). After bioprinting, ADSCs were differentiated into adipocytes using adipogenic induction media, with an experimental group undergoing obese-like induction (OLI) using BSAPalmitate (Figure 1b). Viability of the constructs were analyzed using Live-Dead staining (n=3) (Figure 2) and lipid droplet sizes were compared between groups using Nile Red staining. Insulin sensitivity was assessed by immunostaining phosphorylated-insulin receptors (p-INSR) (n=3) and quantified using western blot (n=3). Secreatome of lean and obese adipocyte constructs were characterized in terms of adiponectin and leptin concentrations were measured using ELISA (n=2). The DiPS 1016 SevA hiPSC line was cultured and differentiated into atrial cardiomyocytes (a-iCMs), characterized by MLC2A, cardiac Troponin T (TnT), and Sarcomeric alpha-actinin (SAA) markers. After differentiation, atrial cardiomyocytes were incorporated into the mentioned GelMA-collagen hybrid bioink and bioprinted into the middle of the lean and obese adipocyte constructs. Constructs were stained using Nile Red and Troponin T to colocalize adipocytes and iCMs in the constructs (data not shown). After a week in culture, atrial cardiomyocytes in both cultures were compared in terms of beating utilizing bright field video analysis (MATLAB), active insulin receptor (Figure 4b) and gap junction Connexin43 concentration (Figure 4a) utilizing immunostaining (n=3) and western blot (n=3).
Results, Conclusions, and Discussions: All bioprinted constructs have high viability (>93%) (Figure 2) and the obese-like adipocyte group exhibited larger droplets (Figure 3b and 3c). Under normal conditions, the leptin to adiponectin ratio is expected to be lower than 0.5. However, in obesity, this ratio is reported to increase beyond that threshold and become a CVD risk factor. Obese-like adipocyte constructs had higher leptin to adiponectin concentration ratio, with OL group exceeding 0.5 (Figure 3d). After a week in culture, atrial cardiomyocytes cultured with obese-like adipocytes had a significant decrease in beating frequency compared to control (atrial cardiomyocyte only) and beating irregularities such as palpitations were recorded (data not shown). According to the immunostaining (Figure 4b) and western blot analysis (data not shown), atrial cardiomyocytes cultured with obese-like adipocytes had a significantly lower gap junction expression and pINSR to INSR ratio (Figure 4). Thus far, our results indicated that the 3D hypertrophic adipocyte model were showing hallmarks of obese adipocytes and atrial cardiomyocytes incorporated into these models were simulating the in-vivo effects of obesity-related cardiac damage. Research conducted by The American Obesity Association foresees that half of adults will be affected by obesity by the year 2030. Using human tissues to develop effective therapeutics is not feasible due to the lack of a systemic immune response and the majority of laboratory animals (mice, rats, rodents..) lack EAT. Therefore, engineering an obese heart under laboratory conditions is important to understand underlying pathology and come up with new therapies to treat obesity-related cardiac pathophysiology.