Is the heart a pump or an excitatory tissue? It is both and more of course, but the fundamental function of the adult heart is to contract its internal volume in order to pump blood. Moreover, the only way that a solid-walled tissue such as the heart can contract in volume is if the wall of the heart is elastically deformable. What has been less clear is whether the elasticity of the heart wall impacts the function of beating cardiomyocytes at the single-cell level. The issue is both important and timely because there are many groups that aim to generate—from embryonic stem cells, induced pluripotent stem cells, and cardiac stem cells, among others (Sachinidis et al. 2003
; Zwi et al. 2009
; Segers and Lee 2008
)—cardiomyocytes that will repair adult hearts after a heart attack or other injury. Ultimately, heart is a muscle that does repetitive work on a load, and tissue elasticity Etissue
at the scale of a cell is a significant part of that load in contributing to remodeling at a basic molecular level.
Several recent studies have sought to physically quantify in culture the effects of matrix elasticity Em
on late embryonic and neonatal cardiomyocytes. Engler et al. (2008)
first made measurements of tissue elasticity Em
with atomic force microscopy (AFM) of chick heart at embryonic days 4, 7, 10 (E4,7,10) and then isolated cardiomyocytes from E7 embryos and characterized the morphological and functional effects of substrate stiffness on cells. Collagen-I-coated polyacrylamide gels provide a tunable matrix to which these embryonic cardiomyocytes attach firmly and beat spontaneously (). Beating of cardiomyocytes, which, as in the human heart, occurs at approximately 1 Hz, applies periodic strains to the matrix that can be estimated from the displacement of beads embedded near the gel surface. The cells thus do an amount of work on the substrates that can be estimated by multiplying the square of the mean matrix strain under the cell, εout
, by matrix elasticity, Em
. The estimated strains were relatively constant up to about the mean elasticity for heart of ~10 kPa as measured by AFM, and then the strains decrease at higher Em
. The latter decrease reflects the fact that there must be some rigidity beyond which the cells simply cannot contract; it turns out that the limiting rigidity is close to the physiological tissue stiffness of ~10 kPa. Work done on the substrate goes as
multiplied by a prefactor with units of volume that depends on the geometry of the system (Friedrich and Safran 2012
) and can thus be neglected. Engler et al. (2008)
estimate this work as 1/2
, which exhibits an optimum. Below about 10 kPa, the cells do little work on soft matrix (low Em
), and above about 10 kPa, the cells cannot strain the stiff matrix (low εout
). As pointed out, rigid matrix also arises in scarring after a myocardial infarction in adults, which is well known to impede contractile function of the heart. Regardless, the optimal substrate stiffness aligns remarkably well with the micro-elasticity measurements for E7 myocardium.
Fig. 1 Isolated cardiomyocytes plated on gels of various stiffnesses. a
Engler et al. (2008) characterized the morphological and functional effects of substrate stiffness on embryonic cardiomyocytes isolated from E7 chick embryos cultured on collagen-I-coated (more ...)
Using a similar system of gel substrates, Jacot et al. (2008)
cultured neonatal rat ventricular myocytes (NRVM) and made careful measurements of both the rhythmic forces in beating as well as the much smaller ‘resting’ forces that are sustainably applied to a substrate due to a basal muscle tone in the cardiomyocytes (). NRVM do not beat spontaneously and need to be electro-stimulated, whereas the embryonic cardiomyocytes studied by Engler et al. (2008)
beat spontaneously. Importantly, calcium spike dynamics measured by Jacot et al. (2008)
showed that 10 kPa matrix maximized both contractile force and the amplitude of calcium dynamics. Excitation–contraction coupling (ECC) is a classic phenomenon in muscle physiology (Bers 2001
), and these results are consistent with ECC, but highlight the key role of matrix elasticity as a load on cardiomyocytes. The results are clear for individual cells with no confounding impact of cell-to-cell electrical communication, which suggests that one needs to consider Excitation–contraction–Matrix
C) in order to understand heart development and pathophysiology.
Evidence in both studies above showed that the main protein motor in cardiomyocyte contraction, cardiac myosin, maintained a relatively constant level of expression. However, Jacot et al. (2008)
used a pharmacological inhibitor of the nonmuscle myosin pathway, namely a drug that blocks Rho-associated kinases (ROCKs), and the results showed that this inhibitor suppresses the decrease in force exerted by NRVM on stiff gels (). Such findings could lend insight into how and why the same drug protects against heart injury in animal models (Cadete et al. 2010
Co-cultures of NRVM and matrix-secreting fibroblasts derived from the same hearts were grown on PA gels for 5 days by Bhana et al. (2009)
, who reported that—in their dense culture systems—cardiomyocyte function and cardiomyocyte numbers relative to fibroblasts appeared optimal at substrate stiffness in the range of Em
= 22–50 kPa (). The higher optimum in substrate stiffness is thought to match the mechanics of adult rat cardiac tissue, which the same group measured by a pipette aspiration method. It should be noted that cardiomyocytes beat synchronously when in direct contact with each other. Synchronously beating cardiomyocyte aggregates produce more force than individual cells (Liu et al. 2012
), which may explain why these cells functioned so well on somewhat stiffer substrates than in the other studies and the neonatal tissue from which they were derived.
Bajaj et al. (2010)
looked at similar dense co-cultures of E8 chick-derived cardiomyocytes and fibroblasts on PA gels of 1, 18, and 50 kPa and on tissue culture plates for 1–5 days. They found that the cells initially beat with frequencies modulated by substrate stiffness, with the fastest beating on the 18 kPa. However, after 5 days, as the cells proliferated and came into contact with each other, the beat frequencies became more uniform within each culture and across culture conditions, and the fastest beating occurred in the 50 kPa gel cultures. This is likely due to the cells in contact with each other beating in synchrony. Immunofluorescent imaging of focal adhesion (FA) formation and growth in the different culture conditions revealed increased FA area and number on stiff substrates over time and decreased FA area and number on the softest gels over time. Interestingly, this decrease in FA number and size corresponds to a less organized sarcomeric cytoskeleton on soft substrates relative to the well-developed and aligned myofibrils observed on stiff substrates.
Using a very different type of substrate, Rodriguez et al. (2011)
cultured NRVM on fibronectin-coated elastic micropilli arrays with effective shear moduli estimated to range from 3 to 20 kPa. The twitch force, work, and power generated by single cells once again increased with substrate stiffness (). In addition, calcium activity increased in the NRVM on stiffer substrates. The authors also made direct comparisons of forces produced by neonatal myofibrils to adult myofibrils, showing that neonatal myofibrils generate only about one-third the power of adult myofibrils. The results underscore the importance of developmental stage and age of the cells studied.
In addition to functional characterizations of the effects of substrate mechanics on force and work output of cardiomyocytes, several of the studies above also attempted to uncover some of the molecular changes that underlie measurable functional changes. Engler et al. (2008)
imaged alpha-actinin and noted that 1 day cultures on ~10 kPa matrix exhibited a maximum fraction of cells with sarcomeric striations. They also applied a novel method of labeling proteins within cells to expose differences in molecular structure or activity (cysteine shotgun mass spectrometry; Johnson et al. 2007
), and the analysis indeed identified substrate stiffness–dependent differences in myosin and other cytoskeletal proteins as well as one metabolic protein, the muscle-specific pyruvate kinase M1. The latter is intriguing because the studies of ROCK inhibition of the heart cited above also identified drug-dependent difference in several metabolic proteins (Cadete et al. 2010
). On the other hand, such results are very sensible because differential force-generation by muscle places differential demands on metabolism. Moreover, in the drug studies of Jacot et al. (2008)
, imaging of alpha-actinin in untreated cells revealed a tendency for reorganization of striated sarcomeres into stress fibers, whereas drug treatment blocked this reorganization. As mentioned above, Bajaj et al. (2010)
noted that disorganized and unaligned myofibrils in cardiomyocytes grown on soft substrates corresponds to decreased FA area and number relative to the those of cells grown on stiffer substrates, which had well-aligned myofibrils. Rodriguez et al. (2011)
quantified the striations of cardiomyocytes on their microposts through measurements of sarcomere spacing and z-disk width. They reported that sarcomere spacing, a sign of myofibril maturity and an indicator of likely force output, fell within accepted values for mature myofibrils on all substrates, and increased with increasing stiffness. Z-disk width, which indicates increased coupling of sarcomeres within a myofibril, also increased with E
. Increased sarcomere spacing is associated with increased force production because it allows for a greater number of cross-bridges to form during contraction. Increased z-disk width, in turn, maintains registry of sarcomeres within a myofibril, minimizing myofibril buckling during contraction and maximizing contraction velocity.