Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity
Graphical abstract
Introduction
The heart is one of the most metabolically active organs in the human body, producing and consuming approximately 30 kg of ATP per day [1]. Most of this ATP is generated by the mitochondria in cardiac myocytes and used to fuel sarcomere shortening. Due to their functional importance, mitochondria and myofibrils are both prominent in cardiac myocytes, occupying approximately 30% and 60% of myocyte volume, respectively [2]. Like most organelles, the function of a single mitochondrion is highly dependent on its structure [3]. Mitochondria have an outer and inner membrane, the latter of which contains the enzymes and pumps that generate ATP through oxidative phosphorylation. Larger mitochondria are generally more efficient than smaller mitochondria, likely due to the benefits of mixing and sharing mitochondrial contents [3,4]. The architecture of the mitochondrial network itself is regulated by the balance of mitochondrial biogenesis, fusion, fission, and mitophagy [5,6]. These processes collectively shape the mitochondrial network throughout stages of physiological and pathological growth and dynamically adapt to changes in energy demands, nutrient availability, and other factors that are not completely understood. For example, mitochondria in early fetal cardiac myocytes are relatively sparse, circular, and scattered throughout the cytoplasm. As development progresses, mitochondria increase in number and densely pack into elongated structures between myofibrils [[7], [8], [9]]. This remodeling of mitochondrial architecture occurs in parallel to a switch from glycolysis to oxidative phosphorylation as the primary pathway for ATP production [10,11]. In many pathological conditions, the heart switches back to glycolysis [12] and the mitochondrial network remodels again, often towards an excessively fragmented state [13] due in part to the up-regulation of fission proteins and down-regulation of fusion proteins [[14], [15], [16], [17]]. However, although we know that mitochondrial architecture changes in development and disease, the underlying factors driving these architectural changes are not completely understood.
In addition to metabolic remodeling, many developmental and pathological events in the heart are also associated with coordinated changes in cardiac myocyte shape, myofibril architecture, and mechanical loading [18]. Embryonic cardiac myocytes are hexagonally shaped and gradually compact and elongate as their myofibrils mature, reaching an AR of approximately 7:1 in adult hearts [19]. In parallel, the mechanical load increases from embryonic to adult stages as the extracellular matrix (ECM) becomes more rigid [20,21] and hemodynamic load increases [22]. Many pathological conditions are associated with further increases in ECM rigidity [23,24] and/or hemodynamic load [25,26], which is known to trigger distinct forms of maladaptive myocyte shape remodeling. In pressure overload, myocyte aspect ratio decreases, contributing to concentric hypertrophy and a reduction in ventricular chamber volume [27,28]. In volume overload, cardiac myocytes elongate, leading to pathological eccentric hypertrophy and eventual heart failure [27,28]. Previous in vitro studies have shown that physiologically- and pathologically-relevant changes in cell shape and/or ECM rigidity alter cytoskeletal architecture [[29], [30], [31], [32]], nuclear morphology [33], contractility [20,32,[34], [35], [36]], and electrophysiology [[37], [38], [39], [40]] in cardiac myocytes. We also previously reported that tissue architecture and the physical and biochemical properties of the ECM regulate rates of oxygen consumption in cardiac myocytes [[41], [42], [43]], indicative of links between cardiac tissue microstructure, the ECM, and metabolic function. However, it has not yet been established if mitochondrial network architecture is also affected by cell architecture and/or ECM rigidity, which is highly relevant because mitochondrial structure is known to regulate mitochondrial function.
Our objective for this study was to test the hypothesis that cell AR and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To modulate AR and ECM rigidity independently, we microcontact printed single cell-sized rectangles of fibronectin of three aspect ratios (3:1, 7:1, 11:1) onto soft, moderate, and stiff surfaces. We then cultured neonatal rat ventricular myocytes on these surfaces and used confocal microscopy and image processing techniques to quantify parameters such as mitochondrial volume, surface area to volume ratio, and number. Our results suggest that mitochondria volume increases and fragmentation decreases as cardiac myocytes elongate, thereby forming a larger and more fused mitochondrial network. For some AR, we also found that mitochondrial volume decreases and fragmentation increases as ECM rigidity increases, which is consistent with a pathological state. These results suggest that extracellular cues that are prominent throughout cardiac development and disease, especially cell shape remodeling, directly affect the morphology of the mitochondrial network in cardiac myocytes. These changes in mitochondrial network architecture could contribute to both physiological and pathological changes in metabolic function, which likely has downstream implications for contractile performance and cardiac output due to the reliance of sarcomere shortening on ATP production.
Section snippets
Master wafer and PDMS stamp fabrication
Standard photolithography and soft lithography techniques were used to fabricate master wafers and PDMS stamps [29,44,45]. Briefly, in AutoCAD (Autodesk), a pattern was designed consisting of a series of rectangles with preserved surface area (~1200 μm2) and the following aspect ratios: 3:1 (60 μm × 20 μm), 7:1 (91 μm × 13 μm), and 11:1 (121 μm × 11 μm) [32,46]. This pattern was transferred to a chrome photomask. In a Class 100 cleanroom, silicon wafers were spin-coated with
Engineering cardiac myocytes with defined AR and ECM rigidity
To mimic the values of myocyte AR and ECM rigidity observed in developing, healthy, and diseased myocardium, we spin-coated glass coverslips with soft, moderate, or stiff formulations of PDMS with elastic moduli in the range of embryonic, adult, or fibrotic myocardium [20,21,23,24], respectively. We then used microcontact printing to transfer rectangles of fibronectin with surface areas of approximately 1200 μm2 and AR 3:1, 7:1, or 11:1 onto the PDMS substrates. These AR were chosen because
Discussion
Throughout stages of cardiac development and disease, the mitochondrial network in cardiac myocytes dynamically remodels in concert with several factors, including altered energy demands, nutrient availability, mechanical loading, and remodeling of many other cellular components, including myofibrils. However, the independent influence of each of these factors on mitochondrial architecture is not clearly understood. In this study, we used an in vitro approach to focus specifically on
Conclusions
As cardiac myocytes elongate, the mitochondrial network becomes larger and more fused. Increases in ECM rigidity can also selectively increase mitochondrial fragmentation. These data suggest that changes in cell shape and mechanical loading that occur during heart development and disease impact the architecture of the mitochondrial network, which likely has implications for the metabolic and therefore contractile performance of the myocardium. These results also identify mechanisms for
Disclosures
None.
Acknowledgments
This project was supported by the USC Viterbi School of Engineering, USC Graduate School, the American Heart Association Scientist Development Grant 16SDG29950005, and USC Women in Science and Engineering. We acknowledge the W.M. Keck Foundation Photonics Center Cleanroom for photolithography equipment and facilities, and the USC Translational Imaging Center for imaging software and workstations. We would like to thank Eun Sang Koo, Francesco Cutrale, and Cosimo Arnesano for assistance with
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