Cardiomyocytes cultured on mechanically compliant substrates, but not on conventional culture devices, exhibit prominent mitochondrial dysfunction due to reactive oxygen species and insulin resistance under high glucose

Abstract

RationaleDiabetes causes cardiac dysfunction, and understanding of its mechanism is still incomplete. One reason could be limitations in modeling disease conditions by current in vitro cardiomyocyte culture. Emerging evidence suggests that the mechanical properties of the microenvironment affect cardiomyocyte function. Nevertheless, the impact of high glucose on cardiomyocytes cultured on substrates whose stiffness matches that of the heart (approximately 15 kPa) is untested.ObjectiveTo test the hypothesis that cardiomyocytes cultured in microenvironments that mimic the mechanical properties of those for cardiomyocytes in vivo may reproduce the pathophysiology characteristics of diabetic cardiomyocytes ex vivo, such as the morphological appearance, ROS accumulation, mitochondrial dysfunction, apoptosis and insulin-stimulated glucose uptake.Methods and resultsIsolated neonatal rat cardiomyocytes were seeded on 15 kPa polyacrylamide (PAA) gels, whose stiffness mimics that of heart tissues, or on glass coverslips, which represent conventional culture devices but are unphysiologically stiff. Cells were then cultured at 5 mM glucose, corresponding to the normal blood glucose level, or at high glucose levels (10 to 25 mM). Cytoskeletal disorganization, ROS accumulation, attenuated mitochondrial membrane potential and attenuated ATP level caused by high glucose and their reversal by a ROS scavenger were prominent in cells on gels, but not in cells on coverslips. The lack of response to ROS scavenging could be attributable to enhanced apoptosis in cells on glass, shown by enhanced DNA fragmentation and higher caspase 3/7 activity in cells on glass coverslips. High-glucose treatment also downregulated GLUT4 expression and attenuated insulin-stimulated glucose uptake only in cells on 15 kPa gels.ConclusionOur data suggest that a mechanically compliant microenvironment increases the susceptibility of primary cardiomyocytes to elevated glucose levels, which enables these cells to serve as an innovative model for diabetic heart research.

Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1

“…After 72h, IHG was shown to decrease the MMP upon succinate stimulation compared to both LG and NG. High glucose exposure was also reported to promote a reduction of MMP in H9c2 cells (50 mmol/l glucose during 24h) [ 21 ] and in isolated neonatal rat cardiomyocytes (15 mmol/l glucose during 24h) [ 22 ]. The decrease of MMP observed upon IHG could be either due to a reduction of the electron flux in the respiratory chain or an uncoupling of OXPHOS but our data are not consistent with this hypothesis since both conditions would promote either a decrease or an increase of the mitochondrial respiration when using the different substrates (succinate, palmitate and pyruvate), respectively; however, we only observed a low mitochondrial respiration upon succinate but not with palmitate or pyruvate in IHG compared to LG, NG and HG.…”

“…In the kidney epithelial cells MDCK (Madin-Darby Canine Kidney), both increase in ROS production and in oxidized mitochondrial proteins were detected after 96h exposure to 25 mmol/l glucose [ 25 ]. High glucose exposure was also demonstrated to increase ROS production in different cellular models including neonatal rat cardiomyocytes [ 22 ] and H9c2 cells [ 24 ].…”

Intracellular and microenvironmental regulation of mitochondrial membrane potential in cancer cells

“…Substrate stiffness has been shown to regulate the ΔΨ m of several different cell types in vitro. Pulmonary arterial endothelial and smooth muscle cells (Bertero et al, 2016), and cardiomyocytes (Morishima et al, 2018) demonstrate enhanced ΔΨ m when cultured on softer substrates. Another study on vascular smooth muscle cells found that their ΔΨ m was the highest when cultured on a substrate with stiffness close to the in vivo stiffness of the tissue of origin (Bartolák‐Suki et al, 2017), while both softer and stiffer substrates significantly reduced the ΔΨ m of these cells.…”

The Utilisation of Hydrogels for iPSC-Cardiomyocyte Research

“…The changes in CM electrophysiology based on substrate stiffness indicates PAA is also effective at mimicking the ECM. Furthermore, neonatal rat CMs cultured on 15 kPa PAA gels maintain sarcomere and F-actin structures [ 85 ]. Sarcomeric structure and myofibril alignment was also preserved in human iPSC-CMs cultured on 10 kPa polyacrylamide gels [ 67 ], thus highlighting the compatibility of PAA with several cardiac cell types.…”