From Stem Cells to Cardiomyocytes: The Role of Forces in Cardiac Maturation, Aging, and Disease

Abstract

Stem cell differentiation into a variety of lineages is known to involve signaling from the extracellular niche, including from the physical properties of that environment. What regulates stem cell responses to these cues is there ability to activate different mechanotransductive pathways. Here, we will review the structures and pathways that regulate stem cell commitment to a cardiomyocyte lineage, specifically examining proteins within muscle sarcomeres, costameres, and intercalated discs. Proteins within these structures stretch, inducing a change in their phosphorylated state or in their localization to initiate different signals. We will also put these changes in the context of stem cell differentiation into cardiomyocytes, their subsequent formation of the chambered heart, and explore negative signaling that occurs during disease.

1. INTRODUCTION

Mechanics play an essential role in developing or maintaining function at every stage of the heart’s lifespan, from differentiation and maturation to regulation of cardiac structure with advancing age. Cells not only respond to external stresses but are also capable of affecting them through internal and external restructuring. This remarkable ability is due to the abundance of mechanosensitive molecules and mechanisms populating the cardiac tissue, which form a closed feedback loop in which mechanics regulate mechanics.

Mechanotransduction, the process by which cells sense external forces and translate them into biochemical signals that can change cell function, is regulated in the heart by a diverse array of factors operating at different length scales. Externally, arterial blood pressure, valve compliance, passive stiffness and adhesivity of the cellular niche, and ventricular wall stress have all been shown to impact form and function of the heart. Through intracellular mechanosensitive pathways, cardiac cells can sense these changes and remodel themselves and their surroundings in order to achieve and maintain a level of function that meets physiological demand.¹ Evidence also suggests that “inside-out” mechanical signaling is crucial for tissue morphogenesis, maintenance of homeostasis, and prolonging function over decades of life.^2–4

In this chapter, we will first describe the establishment of cardiac fate from stem cells and subsequent morphogenesis of the heart. Then, we will highlight several major mechanosensitive subcompartments of the heart, noting the way in which they engage in mechanical and biochemical cross talk. Throughout the chapter, we will also discuss how mechanical signaling helps establish cardiac fate, construct the contractile apparatus, shape cardiac morphogenesis, regulate force transmission between myocytes and their niche, and underline multiscale remodeling during aging and altered mechanical loads. We will also argue that establishment and long-term heart maintenance are highly dependent upon the cardiomyocyte’s ability to remodel its intracellular structure in order to adapt to changing mechanical loads and physiological demand. By dissecting the effector and affected pathways of cardiac mechanotransduction, we hope that the reader will appreciate how mechanics regulates cardiac differentiation and how physical parameters help engineer the function of adult cardiac myocytes in addition to developing a better understanding of the pathophysiology of genetic and age-related cardiomyopathies.

2. CARDIAC MORPHOGENESIS DURING THE LIFESPAN OF THE HEART

2.1. Specification, differentiation, and heart morphogenesis

The cells that eventually become the myocardium are derived from the mesoderm within the primitive streak.^5,6 Early cardiogenesis is driven by time-dependent biochemical signaling, such as bone morphogenic protein (BMP) and suppression of wingless-related integration site (WNT) signaling.^7,8 At this stage, cardiac progenitors begin to migrate and form two populations of cells, one of which will eventually become the early, beating heart tube and the other the outflow tract and portions of the right heart.^5,9 It is shortly after formation of the heart tube that contractions begin and underline further growth and remodeling to loop and subdivide into a primitive four-chambered heart. Morphogenesis can continue in embryonic mice hearts ex vivo¹⁰ as it is guided by an internal mechanism: the forces created by interactions of myosin and actin.¹¹

2.2. Cell maturation and maintenance

For the remainder of embryogenesis, growth is caused by hyperplasia or proliferation of the early cardiomyocyte population.^12,13 Postnatal cardiomyocytes continue remodeling in a process dubbed maturation, which includes both hyperplasia and hypertrophy that are considered adaptive as the growth contributes to improved function.¹³ Postmaturation myocardial remodeling, either through concentric or through eccentric hypertrophy, is underlined by the addition of sarcomeres, remodeling of cortical ultrastructure, protein expression, and altered cell morphology, and is associated with age-related dysfunction such as impaired fractional shortening.^1,14,15 While primarily composed of terminally differentiated adult cardiomyocytes, cardiac stem cells have also been recently identified^16–18; despite the presence of these progenitor cells, however, the adult heart is still thought to have limited regenerative potential compared to other tissue systems given that the heart does not repair itself like other muscles. Therefore, adult cardiomyocytes must be extremely responsive to these changing mechanical environments (e.g., elevated arterial pressure, fibrosis) to maintain function over several decades, and mechanosensitive molecules provide a convenient feedback mechanism to maintain cardiac function.

3. MECHANOSENSITIVE COMPARTMENTS IN CARDIOMYOCYTES

Cardiomyocytes are composed of several subcompartments involved in mechanotransduction, including the contractile sarcomeres, the cytoskeletal filament networks, transmembrane cell–cell and cell–matrix junctions, stretch-sensitive membrane structures, and calcium-handling machinery as shown in Fig. 9.1. Each of these is involved in providing structural integrity as well as generation, sensation, transmission, and/or modulation of forces in the heart. In the following sections, we will summarize the basic structure and highlight known and hypothesized functions of each of these subcompartments.

Figure 9.1.

Mechanosignaling between subcellular compartments. Illustration highlights the mechanical and chemical interplay of indicated intracellular, extracellular, and membrane components. Black lines indicate mechanic and/or structural interaction between elements, whereas arrows indicate the direction of signaling pathways. Note the following abbreviations: intercalated disc, ICD; sarcoplasmic reticulum, SR; extracellular matrix, ECM.

4. THE SARCOMERE

The primary function of the heart is to pump blood throughout the circulatory system, dispersing oxygen, nutrients, and biochemical factors to both distal organs as well as itself.¹⁹ This contraction is contingent upon the sarcomere, a dynamic protein complex that serves as the basic mechanical unit of muscle. A sarcomere is composed of actin-based thin filaments and myosin-based thick filaments as illustrated in Fig. 9.1; these filaments slide past one another to create contraction. These structures are cross-linked together by Z-disks. Each of these sarcomeric components is critical in regulating how muscle performs mechanical work, maintains passive tension,²⁰ undergoes biochemical signaling,²¹ and facilitates mechanotransduction.²² In vertebrates, the expression of these molecules during development is highly coordinated²³ and mutations or alternative splicing of genes encoding these proteins are associated with a variety of congenital defects and cardiomyopathies²⁴ that manifest in aberrant molecular, cellular, or organ-level structure.

4.1. Cardiac structure and mechanosignaling

Sarcomeric contraction and subsequent force production begins with an action potential. Depolarization of the cardiac membrane results in calcium influx from the extracellular space via L-type calcium channels located in structural membrane invaginations known as the transverse-tubules.²⁵ Local increases in calcium concentration are detected by proximal ryanodine receptors in the sarcoplasmic reticulum (SR), causing the latter to release additional calcium ions into the cytosolic space. This event is known as a “calcium spark”^26,27 and is part of a calcium-induced calcium-release mechanism central to cardiac excitation–contraction coupling. Though each spark is spatiotemporally constrained, it can result in a significant elevation of global cytoplasmic calcium concentration when summed (~>10⁴ sparks). This elevation is transient, as sarco/endoplasmic reticulum ATP-ase pump and the sodium–calcium exchanger will transport calcium back into the SR and interstitium, respectively. This rise and fall of calcium is referred to as the calcium transient.

Myosin thick filaments bind to actin-based thin filaments transiently, and as they bind and unbind, they undergo a power stroke where the myosin head moves forward relative to the actin filament. Myosin’s cyclic binding is known as cross-bridge cycling, and this process creates the net contraction of the sarcomere. Binding can be blocked in the absence of calcium by the troponin–tropomyosin complex²⁸ where troponin C can cover up myosin’s binding site on the thin filament. The probability of acto-myosin binding is therefore associated with increased intracellular calcium concentration.²⁹ Changes in the calcium transient directly affect contraction and relaxation dynamics and short- and long-term power output of the heart, making it a potent regulator of transient and long-term cardiac mechanotransduction.³⁰

In addition to perturbations in calcium handling, sarcomeric function is also modulated by changes in absolute gene expression,³¹ alternative splicing,^24,32 impaired protein homeostasis,^33,34 decreased protein quality,³⁵ and altered ultrastructure of its subcomponents. Biochemical and mechanical cell stress can affect the quantity, quality, and integrity of the sarcomeres. In kind, molecules in the sarcomere are also capable of acting as stress sensors that are capable of nuclear signaling, allowing the myocyte to respond.^36,37

The Z-disc was once thought to be a static architectural support for the myofilaments, responsible for anchoring and transversely cross-linking adjacent thin filaments. However in recent years, an intricate complex of force sensing and signaling molecules have been identified within Z-discs, and many of these proteins have critical roles in development and disease.³⁰ To briefly outline the contents of the Z-disc, it is primarily composed of α-actinin, a spectrin-family protein required for actin-filament anchoring to the Z-disc.^38,39 Nonsarcomeric clustering of α-actinin is a marker of sarcomere degeneration and is associated age-related dilated cardiomyopathy.⁴⁰

In addition to α-actinin, Z-discs also contain a significant amount of titin, which helps maintain resting passive tension and longitudinal stiffness in the cell. Titin also acts as a molecular spring which spans the length of the Z-disk to the M-line.^41–43 Embryonic titin is considerably shorter than adult titin, suggesting that transverse elasticity interplays with the changes in cardiac morphology and mechanics during development.⁴⁴ On the other hand, maladaptive cross-linking and differential splicing of titin have been shown to modulate resting sarcomere length and perturb sarcomere force production.⁴⁵ In addition to its mechanical properties, titin has several binding sites for ankyrin-repeat proteins (ARPs).⁴⁶ ARP signaling proteins are capable of translocating to the nucleus, where they presumably alter the transcriptome of the cell.⁴⁶ In addition to binding α-actinin at its N-terminus, titin forms a complex with muscle LIM protein (MLP), which has been hypothesized to act as a stress and/or length tensor.^36,47 MLP-null myocytes experience decreased longitudinal passive stiffness and muted response to stretch in the form of BMP expression^48,49 as well as decreased power output.⁵⁰ MLP mutations are also associated with diastolic dysfunction followed by dilated cardiomyopathy, suggesting time-dependent remodeling.⁵¹ Other LIM-domain-containing proteins, including myopodin⁵² and zyxin,⁵³ localize at the Z-disc and further suggest a critical sensory role for these types of proteins. During myoblast differentiation, myopodin is localized to the nucleus but translocates to the nascent Z-disk as sarcomerogenesis progresses.⁵⁴ Acute, short-term heat shock of differentiated myotubes results in reverse translocation of myopodin from mature Z-discs to the nucleus, suggesting that myopodin plays a role in stress-response signaling.^52,54 Nebulin, a hypothesized sarcomeric “ruler,”^55–57 is associated with nebulin-related anchoring protein (N-RAP), another well-known LIM protein and member of the Z-disk-associated mechanosensitive network.^58,59 Myomesin is another sarcomeric mechanosensor that is thought to bind titin near its C-terminal and act as a molecular spring.^60,61 In addition to its contributions to mature sarcomere stability, it is associated with myosin during the earliest stages of sarcomerogenesis, suggesting that it is crucial for performing mechanical work given the few proteins present in these early contractile structures.⁶²

4.2. Sarcomere mutations, microenvironmental changes, and their impact

Mutations in vital myofilament proteins are associated with congenital defects and adult-onset cardiomyopathies and can arise due to increased or decreased sensitivity to calcium.^36,63–73 These changes reflect a potential role of the myofilaments in cardiogenesis and maintenance of structure. A missense mutation in α-MHC can result in maladaptive hypertrophy, myofibrillar disarray, and fibrosis due to impaired calcium homeostasis in the SR.^74,75 This process can be ameliorated by inhibition of L-type calcium channel activity upstream⁷⁶ and potentially other negative-inotropic agents, suggesting that the mutation results in a maladaptive gain of function. Certain mutations in cardiac troponins are associated with restrictive cardiomyopathy,^68,69,77 a condition in which diastolic and systolic dimensions dramatically decline. Recent studies show that such mutations in critical regions can expose the myosin-binding site on thin filaments without the need for calcium influx, resulting in cross-bridge formation independent of calcium release, elevated diastolic tension, cortical stiffening, and impaired fractional shortening.^68,71,78 A mutation in troponin C resulting in calcium-independent sarcomeric perturbation has been shown to be rescued by an engineered molecule.⁷⁹

Sarcomeres are also known to respond to alterations in the mechanical forces being presented to the heart, i.e., “outside-in” mechanotransduction. For example, application of stretch on cardiomyocytes in vitro results in increased sarcomerogenesis and hypertrophic signaling.^80–82 The addition of new sarcomeres appears to occur at the intercalated disc, suggesting synergy between the two compartments in this phenomena.⁸³ Developing embryonic chick cardiomyocytes has increased sarcomerogenesis from when plated on time-dependent, stiffening hydrogels as compared to static elastic substrates.^84,85 The rate of MHC and thin filament actin turnover is stretch sensitive.^86,87 Thus, sarcomerogenesis and maintenance of sarcomeric protein quality may depend upon the ability of the sarcomeres to act as a stress/strain sensor; perturbations of this structure could impair the resultant signaling.

5. OTHER INTRACELLULAR MECHANOSENSITIVE STRUCTURES

In order for sarcomeric shortening to translate into cellular contraction, and for the contractile apparatus to sense changes in external loads, they require mechanical coupling to the membrane. This is achieved via the sarcolemma, which consists of highly ordered junctions and a cortical cyto-skeletal network. Cardiomyocytes are coupled longitudinally by an electromechanically active junctional complex known as the intercalated disc (ICD) and transversely at the Z-disc by special focal adhesion plaques known as costameres.¹² Sensation and application of external loads by the sarcomeres are enabled by these transmembrane contacts and their associated cytoskeletal networks, making them the primary responders to forces leading to and from the contractile apparatus. Here, we will discuss these structures, their influence on downstream signaling, and its impact on contractile function.

5.1. Actin-associated intercalated disc and costameric proteins

Recent work has identified the intercalated disc and costameres, shown in Fig. 9.1 at the cell membrane facilitating cell–cell and cell–extracellular matrix (ECM) connections, as being remarkably mechanosensitive, i.e., capable of responding to and producing forces and enabling biochemical signaling through a variety of sensing molecules.^88–93 Most of these studies have focused on in vitro systems⁸⁹ or noncardiac cells,^88,92 which have the same components but are organized differently. For example, Le Duc and coworkers applied cyclic stress to E-cadherin-coated magnetic beads bonded to endothelial cells and measured changes in bead deflection as the cell cytoskeleton responded.⁹³ They found that cadherin-mediated binding is mechanosensitive and results in local stiffening, potentially driven by reinforcement actin of the cortical cytoskeleton. The adhesions eventually asymptote to a new stiffness, the rate and magnitude of which depends on the presence of the actin-binding, mechanosensitive molecule vinculin.^90,93 In this way, the actin cytoskeleton downstream of cadherin–cadherin bonds reacts similarly to those proximal to integrin–ECM bonds.^91,94 It is reasonable to assume that cadherin contacts perform similarly in cardiomyocytes as all the critical machinery exists and cell–cell contacts in cultured myocytes are known to remodel during cyclic and static stretch.⁹⁵ However, the mechanosensing machinery is not always present or highly organized. In early development, cadherins are expressed ubiquitously throughout the membrane.¹² It is only as sarcomerogenesis advances and cells elongate and hypertrophy that cadherins become polarized and constrained to the longitudinal ends. Once it is fully formed, the intercalated disc does not remain static; advancing age and dilation associates with convolution of the ICD ultrastructure and enrichment with mechanotransductive molecules.^96,97 Remodeling of actin-binding mechanosensors is hypothesized to result in cortical or transverse stiffening during aging,³⁸ though the ultimate impact of this event on cardiac function remains unclear.

Beyond normal changes with development and age, studies suggest that cortical remodeling from disease or mutation may play a functional role in modulating sarcomeric function. For example, Tangney and coworkers showed that vinculin-null neonatal mouse cardiomyocytes experience decreased cortical stiffness and increased interfilament spacing, the distance between thin and thick filaments in the myofilament lattice.⁹⁸ Interfilament spacing impacts the calcium sensitivity and power output of the sarcomeres,^99–101 perhaps due to a lack of cortical transverse compression upon the myofilaments. Cortical compression through osmotic loading in vitro has been shown to affect contractile function in rat myocardium.^101,102

Lastly, it is important to consider mechanotransduction at focal adhesions, which play a leading role in contractility^103,104 and mechanical induction of myogenic differentiation.⁸⁸ In cardiomyocytes specifically, integrin expression and signaling, as well as focal adhesion and integrin-linked kinases, have been the focus of understanding age-related hypertrophic signaling.^105–111 Integrin expression is regulated by both passive and active mechanical forces as well as the ligands and ligand density presented to the cell.¹¹² Integrin clustering and isoform expression depend upon the ECM proteins and their arrangement.^113–115 Another crucial component in lateral coupling to the membrane is the actin-binding dystrophin molecule, which is thought to stabilize the membrane and provide additional mechanical coupling to the cortical actin cytoskeleton.^116–118 Dystrophin knockout or expression of a mutated isoform results in impaired sarcomeric force transmission to the membrane. Furthermore, without dystrophin as a structural support, conformational changes in stretch-activated channels are thought to occur, resulting in pathological intracellular leakage of calcium and eventual cell death.^119–121

5.2. Intermediate filament and microtubule networks

While actin plays a dominant role in regulating cardiomyocyte development and function, both the intermediate filament (IF) and microtubule networks play important supporting roles. For example, IFs are extremely deformable, capable of being stretched to several times their slack length.^122–124 One of these IFs, desmin, provides scaffolding around the Z-disk and links them laterally, binds to desmosomes at the intercalated disc, and bridges the nucleus to adjacent sarcomeres.¹²⁵ Desmin is also believed to localize to costameres, perhaps conferring additional coupling between sarcomeres and the ECM.¹¹⁸ It is an early marker of cardiogenesis.^126,127 Desminopathies result from mutations in the desmin gene and subsequent cytoplasmic aggregation of desmin and impaired myofibrillar assembly.^128–130 Patients with arrhythmogenic right ventricular cardiomyopathy have been shown to have desmin mutations, suggesting that it may play a crucial role in EC coupling via longitudinal load bearing along the sarcomeres or by stabilizing gap junctions at the intercalated disc.^131–133

Another IF protein prevalent in cardiomyocytes is lamin, which, as with desmin, is also extremely compliant. For example, during lineage specification of stem cells, lamin expression in stem cells scales with substrate stiffness¹³⁴ and is a determinant of nuclear cytoskeleton deformability.^135,136 Moreover, it may act as a universal transducer of mechanical signals into the nucleus to regulate gene expression.¹³⁷ Thus, defects in lamin A/C can adversely affect signal transduction, making them less efficient. Defects in Lamin are associated with advanced aging,¹³⁸ presumably due to genetic instability and accumulated DNA damage from external stressors,¹³⁹ and dilated cardiomyopathy.^15,140,141 The existence of mechanical couplings between the nucleus and the cytoskeletal network implies that some form of communication is required during development.¹⁴²

Note that there exist structural overlaps in the actin and IF networks; ICD-localizing vinculin is also thought to stabilize gap junctions,¹³⁴ and per turbations of actin-binding nesprins, found at the nucleus, are also known to mute biomechanical signaling and induce cardiovascular dysfunction.^143,144 These findings suggest functional overlap in both cytoskeletal networks.

On the other hand, microtubules are crucial in guiding cytokinesis¹⁴⁵ and directing vesicular transport. The microtubule network also serves several load bearing and signaling functions which impact cardiac function.^146–148 Much focus has been placed on how signal transduction is altered following application of colchicine, a pharmacological agent that induces deconstruction of microtubule filaments, as well as how microtubule dysregulation occurs during cardiac hypertrophy.^149–152 While it is unclear whether mechanical perturbation, impaired vesicular transport, or both are responsible for alterations in function, what is known is that micro-tubules contribute to passive stiffness²⁰ and are remodeled during age³⁸; both of these observations point to microtubules as being important regulators of cardiomyocyte function.

5.3. The cardiomyocyte membrane

In addition to intracellular and transmembrane mechanosensitive compartments, the cardiac myocyte membrane is also enriched with stretch-sensitive structures.^153–157 Stretch-activated channels have been implicated in modulation of calcium handling and rhythmicity, although the strains required to observed stretch activation are often superphysiological.¹⁵⁸ The membrane is partially buffered from stretch via caveolins, cytoskeletally regulated invaginations which can add additional material to the membrane through rapid disassembly under osmotic loads¹⁵⁹ or stretch.^160–163 Caveolin is known to alter its expression with age.^164,165 Deletion of Cav3^166,167 is associated with progressive dilated cardiomyopathy, while Cav1 appears to play a greater role in endothelial function.^168,169

6. ECM AND MECHANOSENSING

The cardiac interstitium provides cardiomyocytes with avenues of mechanical and biochemical communication with their environment.^{22,115,170–176} In particular, the insoluble ECM is secreted by cardiac fibroblasts,^170,171 which are the most abundant cells in the heart by number.¹⁷⁷ This ECM plays a crucial role in providing architectural support for cardiomyocytes and allowing for efficient transmission of forces during contraction. ECM organization is known to direct integrin assembly and signaling and vice versa¹⁷⁸ which can influence cell morphology and sarcomere alignment.^1,179,180 This interstitium, like the cardiomyocytes, remodels during development¹² and disease.^170,171,181 Deposition of fibrillar ECM proteins, such as collagens, laminins, and fibronectin,^182,183 is vital for the initiation of cardiogenesis and wound healing but is also associated with mal-adaptive hypertrophic growth. In addition to ECM deposition and paracrine signaling, cardiac fibroblasts are known to directly communicate with cardiomyocytes via connexins^184,185 and N-cadherin,^186,187 allowing for electromechanical coupling and modulation of action potential propagation.¹⁸⁷ Cardiac fibroblasts also respond to external forces by altering their internal cytoskeletal expression,¹⁸⁸ extracellular biochemical signaling,^{187,189–191} and increasing matrix production, which can lead to the induction of a smooth muscle phenotype.¹⁹² These “myofibroblasts” (Fig. 9.1, top) experience calcium-dependent contractility,¹⁹³ elevated ECM deposition,^171,194 and influence cardiac conduction.^186,195 Increased substrate stiffness can also increase differentiation of fibroblasts into myofibroblasts,^196–198 suggesting a positive feedback loop following ECM deposition.

7. THE INFLUENCE OF MECHANOTRANSDUCTION ON APPLICATIONS OF CARDIAC REGENERATION

In this chapter, we have broadly discussed how maintenance of cardiac structure and function is underlined by spatiotemporal patterning of biochemical and mechanical signaling. We have seen cases in which mutation, alternative splicing, maladaptive posttranslational modifications, and altered cytoskeletal and ECM assembly can dramatically alter morphology and function from the cell to the organ. One final consideration to note is how these changes, induced by development or disease, guide or can be used to guide regeneration.

The numerous heart shapes and sizes in the animal kingdom suggest that biology knows of many ways to build a heart. However, the sensitivity of the developing and aging human heart to genetic perturbations and external stresses further suggests that there is a narrow window to form a human tissue that is competently functional. Minor alterations in calcium handling or cytoskeletal ultrastructure and organization appear to have broad impacts on both basal state and response to mechanical and biochemical or pharmacological stress. For example, the use of time-dependent soluble cues appears to be sufficient for initial differentiation of cardiac lineage but insufficient for maturation.^7,199,200 The latter can be assisted by the application of external loads which in turn promote intracellular remodeling.^14,201–203 These mechanical cues may need to be dynamic; the limitations of static mechanical cues have become apparent in recent years. Attempts to revive cardiac function postinfarct via stem cell injection into the stiffened, infarcted niche, for example, result in differentiation into an osteogenic lineage instead of cardiac.²⁰⁴ Bulk hydrogels do little to provide mechanical support and may introduce arrhythmogenic defects by disrupting electrical coupling.²⁰⁵ Adult-like phenotype can be induced in developing cells through micro-patterning of ECM in 2D.^206,207 In this way, a symmetry-breaking event can be used to guide integrin clustering and sarcomerogenesis downstream. However, this kind of ECM-mediated “boundary condition” guidance in cardiomyocytes is also dynamic in vivo, suggesting that the same will be required in 3D cultures of mature cardiac tissue, which cannot be created through current micropatterning technologies. All of these concerns are ameliorated if it is shown that, for a given concern, the response and function of engineered tissues are similar to those observed in vivo. Such criteria have held for the use of simpler animal models with otherwise limited homology to human structure and function.^78,208–211

8. CONCLUSION

What is most evident from a review of current literature is that our understanding of precisely how the cardiomyocyte closes its mechanical feedback loop remains unknown; what are the basic signals that induce hypertrophy and self-assembly into a mature organ and what pathways signal the heart to cease or undergo aberrant growth? In the coming years, additional mechanosensitive molecules in cardiomyocytes will likely highlight cross talk between subcompartments, such as cell–cell and cell–matrix cooperation or myocyte–fibroblast communication. What is likely more important, however, is improving our understanding of the precise timing of mechanotransduction and its downstream pathways during the lifespan of the heart. Improved understanding of cardiac differentiation from stem cells and the mechanotransductive signaling that enables this may reveal indirect therapeutic targets and/or enable better direct engineering of cells and tissue for repair and regeneration.