Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. A new methodology has been developed to measure O consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses.

Cardiovascular diseases are a leading cause of death, in part due to limitations of existing models of the myocardium. Myocardium consists of aligned, contractile cardiac myocytes interspersed with fibroblasts that synthesize extracellular matrix (ECM). The cellular demographics and biochemical and mechanical properties of the ECM remodel in many different cardiac diseases. However, the impact of diverse cellular and extracellular remodeling on the contractile output of the myocardium are poorly understood. To address this, we micropatterned 13 kPa and 90 kPa polyacrylamide gels with aligned squares of fibronectin (FN) or laminin (LN). We seeded gels with two concentrations of primary neonatal rat ventricular myocytes, which naturally contain fibroblasts. Cells assembled into aligned "μMyocardia" with fibroblast : myocyte ratios dependent on initial seeding concentration. Using traction force microscopy (TFM), we found that the peak systolic longitudinal cross-sectional force was similar across conditions, but the peak systolic work was significantly lower on 90 kPa gels. This indicates that ECM elasticity dominates over ECM ligand and cell demographics in regulating contractile output. Because our platform provides independent control over cell-cell and cell-matrix interactions, it has many applications for cardiac disease modeling.