Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment

Abstract

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.

Cardiac Cell-Derived Matrices Impart Age-Specific Functional Properties to Human Cardiomyocytes

“…3C). The enrichment of these factors in adult CDMs is consistent with adult cardiac biology, which exhibits increased ECM crosslinking 56 and organization 23 and is associated with greater CM maturity 22,57 .…”

“…CM function is also strongly influenced by morphology 22 , with cells increasing in size as they mature, and high aspect-ratio CMs achieving the most efficient contractions 16 . CMs cultured on fetal CDMs exhibited greater than a two-fold increase in morphological aspect ratio than those cultured on Matrigel, and CMs on both fetal and adult CDMs adopted narrower cell widths than those on Matrigel (Fig.…”

“…CDMs are particularly amenable to modeling and interrogating cell-ECM interactions at a structural and functional level, making them well suited to study cardiac biology. Cardiomyocyte (CM) behavior relies heavily on morphology and signaling events [15][16][17][18][19][20] , which are both influenced and reinforced by the local ECM microenvironment 6,21,22 . Specifically, the fetal cardiac ECM is loosely organized 15, 23 and enriched with growth factors and GAGs 24,25 to support the developing myocardium and smaller, proliferative fetal CMs 19,[26][27][28] , whereas the adult ECM is a more fibrillar 23 and collagen-rich 29 environment intended to main homeostasis of highly aligned, energetically-demanding adult CMs that constantly experience significant contractile strain [30][31][32] .…”

Sevoflurane Pre-conditioning Ameliorates Diabetic Myocardial Ischemia/Reperfusion Injury Via Differential Regulation of p38 and ERK

“…Seahorse Bioscience XFe96 analyzer, as previously described 37 . Briefly, after cardiac function measurements (hemodynamic evaluation), heart tissue (400 mg) was harvested and homogenized in a prepared mitochondrial buffer (10 ml of 0.1 M Tris-MOPS and 1 ml of 0.1 M EGTA/Tris in 20 ml of 1 M sucrose).…”

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors

“…There is little data in the literature related to whether ECM stiffness affects mitochondrial structure and function. Recently, it was reported that in cardiac myocytes, baseline metabolism is influenced by ECM stiffness and, even more interestingly, the ability of cells to adapt to metabolic stress is regulated by both ECM stiffness and fiber alignment [ 92 ]. To complement these findings, below we present preliminary data that suggest a weak dependence of mitochondrial structure and function on ECM stiffness in VSMCs in culture, in qualitative agreement with the result in cardiac myocytes.…”