Introduction: The intercommunication between the liver and heart is established. However, the molecular factors that play a role in this communication in aging and their common age-related pathologies are understudied. Aging is an important risk factor in both liver and cardiac diseases as it leads to the accumulation of reactive oxygen species and cellular waste in the form of lipofuscin granules at the cellular level. At the organ level, an increase in the number of senescent and apoptotic cells is observed in both organs. Common age-related pathologies of the liver and heart include cirrhotic cardiomyopathy, where cirrhosis leads to dysfunction of the heart, which in most cases remains asymptomatic. Thus, a better understanding of liver-heart interactions is crucial as it could lead to the identification of molecular targets for prevention, diagnosis, and treatment of such conditions. In this study, we aimed to determine the effect of secreted factors of aged liver cells on healthy cardiomyocytes to understand the role of aging alone in liver-heart interactions. To achieve this, we developed in vitro aged liver cell cultures, introduced their secretions to healthy heart muscle cells, or cardiomyocytes (CMs), and determined the resulting age-related and functional changes in CMs.
Materials and
Methods: Induced pluripotent stem cell (iPSC) derived CMs and HepG2 cell lines were used to represent heart and liver tissues, respectively. iPSCs were differentiated into cardiomyocytes following a previously established protocol (Lian et al., 2013) and were used for experiments at 25-41 days of differentiation. To develop aged liver cells, HepG2 cells were treated with varying concentrations of hydrogen peroxide (H2O2) for different durations in 24-well plates at 1.5x105 cells per well density. The treatment conditions were 7-day duration with 200 and 300 M H2O2 (Short-term, high-exposure), 15-day duration with 50 and 100 M H2O2 (Mid-term, moderate-exposure), and 21-day duration with 25 and 50 M H2O2 (Long-term, low-exposure). Media with treatment was changed every two days. After establishing aging of liver cells, the aged liver media were introduced to CMs and incubated for three days. CMs were then fixed and characterized for lipofuscin waste accumulation, p21 expression, senescence, and beating deterioration of heart cells. Lipofuscin waste accumulation was assessed with Sudan Black B (SBB) staining. Immunostaining for p21 expression was done using p21 monoclonal antibody and Alexa FluorTM 488 Goat Anti-Rabbit SFX Kit (green), and nuclei were stained with Hoechst (blue). Senescence in CMs was determined using Senescence Cells Histochemical Staining Kit following the manufacturer’s instructions. Five images were taken per well for all stains with the Nikon-Eclipse Ti-U microscope and quantified using ImageJ software. Statistical analysis was performed using students' t-test, one-way ANOVA, and Tukey’s post hoc test with n=3 for all experiments.
Results, Conclusions, and Discussions: Among the different treatment conditions tested for aging the liver cells, 7-day 200 uM and 15-day 100 uM H2O2 treatments yielded the most aged cells (data not shown). The 15-day 100 uM H2O2 treatment led to a significantly higher percentage of cells with lipofuscin waste accumulation, p21 expression, and senescence compared to the 7-day 200 uM H2O2 treatment (Figure 1, 2). Before introducing aged liver media to CMs, we assessed the effect of different media compositions on CMs since liver cells and CMs are cultured in different media (CM+ media, for CMs, DMEM for liver cells). We also transferred media from non-aged liver cells cultured for 7 or 15 days to ensure non-aging related secretions do not induce aging in CMs. We determined that media composition or media from non-aged liver cells did not induce lipofuscin accumulation in CMs (Figure 3). Next, aged liver media was introduced to the CMs, which led to an increase in the number of cells with lipofuscin waste accumulation (Figure 4A), p21 expression (Figure 4B), and senescence (Figure 4C) in both groups compared to controls, highest values being observed with 15-day 100 uM treatment. Lastly, we observed that the beat per minute (bpm) of CMs decreased upon receiving aged liver media (bpm of 21 before treatment, bpm of 3 after treatment). The results of this study suggest that the molecular factors secreted by aged liver cells may directly be correlated with inducing age-related functional deterioration or age-related heart diseases in patients with liver pathologies. We achieved aging liver cells within 15 days with 100 uM H2O2 treatment, as shown by an increased percentage of cells with lipofuscin waste accumulation, p21 expression, and senescence. Introducing this aged liver media onto healthy CMs led to rapid aging and deterioration of their function, which suggests that direct organ-organ communication may be possible between the liver and heart, potentially playing a role in their age-related common pathologies. Future studies will explore the specific factors involved in this communication, which will help identify new targets for diagnosing, preventing, and treating such conditions.
