The Hippo signal transduction network for exercise physiologists

Abstract

The ubiquitous transcriptional coactivators Yap (gene symbol Yap1) and Taz (gene symbol Wwtr1) regulate gene expression mainly by coactivating the Tead transcription factors. Being at the center of the Hippo signaling network, Yap and Taz are regulated by the Hippo kinase cassette and additionally by a plethora of exercise-associated signals and signaling modules. These include mechanotransduction, the AKT-mTORC1 network, the SMAD transcription factors, hypoxia, glucose homeostasis, AMPK, adrenaline/epinephrine and angiotensin II through G protein-coupled receptors, and IL-6. Consequently, exercise should alter Hippo signaling in several organs to mediate at least some aspects of the organ-specific adaptations to exercise. Indeed, Tead1 overexpression in muscle fibers has been shown to promote a fast-to-slow fiber type switch, whereas Yap in muscle fibers and cardiomyocytes promotes skeletal muscle hypertrophy and cardiomyocyte adaptations, respectively. Finally, genome-wide association studies in humans have linked the Hippo pathway members LATS2, TEAD1, YAP1, VGLL2, VGLL3, and VGLL4 to body height, which is a key factor in sports.

key discoveries especially during the last decade have led to the characterization of the mammalian Hippo signal transduction pathway or network (51, 141, 159). The Hippo signal transduction network is relevant for exercise physiologists because many exercise-associated signals and signaling molecules affect Hippo signaling. Additionally, Hippo effectors regulate several exercise-related genes and adaptations. Starting with Booth in the mid-1990s (19), exercise physiologists have sporadically studied Hippo pathway members in an exercise context. However, to date only a few studies on Hippo in an exercise context have been published. In this review, we will first introduce the Hippo pathway to exercise physiologists. We will then discuss evidence showing that exercise-associated signals and signaling modules cross-talk to the key Hippo effectors YAP and TAZ. Next, we will review studies that implicate Hippo signaling in the regulation of exercise adaptations. Finally, we will discuss the emerging genetic link between Hippo and body height, a key variable linked to performance in several sports.

Hippo Signal Transduction Pathway and Network

The discovery of the Hippo pathway is based on two strands of research. First, since the early 20th century, researchers have used the fruit fly (Drosophila melanogaster) to identify genes whose knockout results in cancer-like overgrowth (40). Since 1995, this line of research has led to the discovery of the several growth-inhibiting genes (68, 156) that together form the core Hippo pathway (56). In the fly, the mutation of one kinase resulted in an overgrown head that reminded the researchers of the skin of a Hippopotamus. Consequently, this kinase was named hippo by Georg Halder group of researchers (134). Subsequently “Hippo” was adopted as the name for the pathway in both the fly and mammals. The Hippo pathway is highly evolutionarily conserved (60). In mammals (see Fig. 1 for a schematic of the mammalian Hippo pathway), two homologues of the fly hippo gene exist; namely, the upstream kinases Mst1 (Stk4) and Mst2 (Stk3). With the help of scaffolding proteins, Mst1 and Mst2 activate the downstream kinases Lats1 and Lats2. Recently, Map4k4/6/7 isoforms were identified as alternative kinases capable of phosphorylating Lats1 and Lats2 (82, 97, 165). Phosphorylated Lats1 and Lats2 then inhibit the transcriptional cofactors Yap and Taz by phosphorylating multiple serine residues (86, 162).

Fig. 1.

Schematic representation of the Hippo signal transduction network and its links to exercise-associated signals and signalling modules. A: MST1, MST2, LATS1, and LATS2 form the core kinase cassette of the Hippo pathway. SAV1 and MOB1 act as scaffolding proteins. The MAP4K 4, 6, and 7 kinase isoforms can independently regulate LATS1/2 (82, 97, 165). Active LATS1 and LATS2 inhibits YAP and TAZ through the phosphorylation of multiple HXRXXS motifs (where “S” within HXRXXS indicates the phosphorylated serine) (162). Phosphorylation of YAP on serine127 generates a binding site for 14-3-3 proteins, which sequester YAP and TAZ in the cytoplasm. Phosphorylation of serine381 results in the ubiquitination and degradation of YAP (162). Similar regulatory events also affect TAZ. B: the classical model stipulates that active unphosphorylated YAP and TAZ are nuclear and coactivate the TEAD transcription factors, whereas VGLL4 acts as a repressor. YAP/TAZ-TEAD complexes bind the CATTCC/GGAATG (MCAT or GTIIC) motifs found especially in enhancers that loop to the promoters of genes even though they are located several base pairs away from the promoter itself [see (39) and the text for more information]. C: resistance (strength) exercise and muscle growth-associated signals that are linked to YAP/TAZ (see text for more information). D: endurance exercise-associated signals that are linked to YAP/TAZ (see text for more information).

The second strand of research started with the identification of CATTCC DNA motifs, termed muscle CAT (MCAT) (90) or GTIIC (24) motifs. Such CATTCC DNA motifs and their reverse-strand GGAATG complement form a DNA-binding site for the Tead (TEA domain) transcription factors (named transcription enhancer factors, or Tefs in earlier papers) (6, 24). Teads repress their target genes, especially when bound by their corepressor, Vgll4 (66, 76). Teads become activated only when they are bound by the transcriptional cofactor Yap (Yes1-associated protein; gene symbol Yap1), which was discovered by Sudol et al. (122, 123, 138). In contrast to the fly, mammals possess a Yap paralogue termed Taz [transcriptional coactivator with PDZ-binding motif (70)].

The two origins of Hippo research were merged when the Pan group of researchers (63) demonstrated that the Hippo kinase cascade inhibited Yorkie, the fly homologue of Yap and Taz. Hippo research then developed exponentially especially after studies by the Pan group (29) and Camargo from the Jaenisch group (18) both found that expression of a constitutively active YAP1 S127A in mouse livers resulted in a fourfold increase in liver size. These landmark findings confirmed that Yap also functions as a highly potent organ size regulator in mammals.

However, the Hippo kinase cascade (i.e., the Mst1/2-Lats1/2) is only one of many signaling modules that regulate the activity of Yap and Taz. For this reason, it seems most appropriate to refer to the wider signaling system as the Hippo signal transduction network. Importantly, numerous exercise-related signals also cross-talk to Yap and Taz, as illustrated in Figure 1 and discussed in the next section.

Cross-Talk Between Exercise-Related Signaling Molecules and Hippo Mechanotransduction

Mechanical loading, in the form of resistance exercise or synergist ablation, stimulates skeletal muscle growth (42). However, the molecular mechanosensor that triggers growth processes in response to mechanical loading has long remained elusive. Additionally, the stiffness of the cellular environment or niche is an additional mechanical signal that influences, for example, the differentiation of mesenchymal stem cells into muscle and other cell types (34, 35). Moreover, mechanical cues also influence the fate of resident stem cells in skeletal muscle, named satellite cells (41). To identify signaling molecules that regulate gene expression in response to the mechanical signal triggered by substrate stiffness, the Piccolo group of researchers (31) cultured mammary epithelial cells on soft and stiff substrates. They found that Hippo-related genes showed the most important changes between the two conditions in terms of expression levels. Subsequent experiments confirmed that stiffer substrates led to increased Yap/Taz activity in a cytoskeleton-dependent manner (31). Later, it was shown that increased cell-cell contact reduces the mechanical loading of cells, which explained the previously observed (163) deactivation of Yap and Taz in response to cell-cell contact at high cell density (8). Although this is intriguing and relevant for processes such as myoblast differentiation, it is unclear whether mechanical changes of the extracellular matrix during exercise regulate transcriptional responses through Yap and Taz in muscle fibers or satellite cells.

In skeletal muscle, the Höhfeld group (135) has identified a specific Hippo and autophagy-regulating mechanosensor complex located at the Z disc. In this complex, the protein Bag3 senses mechanical unfolding of the actin-crosslinking protein filamin. Importantly, Bag3 contains a WW domain, a rare protein domain frequently found in Hippo members [WW indicates two tryptophan residues; reviewed in (124)]. The location of Bag3 in the Z disc is an ideal position for a mechanosensor. Indeed, unlike proteins that lie in parallel to the force-generating sarcomeres, such as integrins, Z-disc proteins directly experience contractile force. One Hippo-independent function of Bag3 is to mediate tension-induced autophagy, which might contribute to the increased protein breakdown observed during resistance exercise (127). Additionally, Bag3 regulates Yap and Taz activity by binding to Yap and Taz binding partners, such as the Hippo kinase Lats1, Amotl1, and Amotl2 [reviewed in (102)]. Given that increased Yap activity can promote muscle hypertrophy (46, 148), Bag3-Hippo mechanosensing might be one of several mechanisms regulating muscle growth in response to mechanical signals. The importance of Bag3 for skeletal muscle is further demonstrated by the finding that a loss of function of Bag3 causes severe myopathy symptoms in mice and humans (62, 117). Moreover, the phosphorylation of human BAG3 on Thr285 and Ser289 decreases in response to endurance exercise (61). Also, BAG3 expression decreases after acute, high-intensity resistance exercise, but increases together with force-bearing cytoskeleton proteins (136). Therefore, the Hippo-dependent and -independent functions of Bag3 are of potentially great interest to exercise physiologists. Currently, it is unclear whether Bag3 is the major mediator of mechanical loading-induced hypertrophy or whether it mainly senses the changes in the stiffness of a cell's niche; for example, satellite cells, and accordingly regulates their behavior (34).

Cross-Talk with Akt-Tsc-mTOR Signaling

The mechanistic target of rapamycin (mTOR) pathway was first linked to overload-induced muscle growth by Baar and Esser (11). They demonstrated that the phosphorylation of the mTOR-related kinase p70 S6k correlated with increased muscle mass in a rat electrical muscle stimulation model. Since then, many studies have confirmed the key role of mTOR signaling for resistance exercise-induced muscle hypertrophy. This includes a study in humans showing that the mTORC1 inhibitor rapamycin prevented the increased muscle protein synthesis triggered by resistance exercise (28).

Given that both the Hippo and mTOR networks regulate organ size, it is intuitive to assume that cross-talk exists between the mTOR and Hippo signaling pathways. This is indeed the case. Akt was initially shown to phosphorylate Yap on Ser127 (13), but this finding was not confirmed by subsequent studies. In another study, the Hippo kinase Mst1 (gene symbol Stk4) was shown to bind and inhibit Akt1 (also known as Pkb) (22). Conversely, Akt phosphorylates Mst1 on Thr387 (65) and Thr120 (161), suggesting that Akt1 and Mst1 regulate each others activity. Additionally, the Hippo effector Yap de-represses mTOR by inhibiting the expression of the phosphatase Pten via the Pten-targeting miRNA miR-29 (133). Cross-talk also exists between Tsc1 and Tsc2 and the Hippo effector Yap because Tsc1/2-deficient cells have higher Yap levels due to less Yap degradation via the autophagosome system (83). YAP/TAZ also increase the expression of genes that encode the leucine transporter LAT1 (52), which is significant because leucine is a potent stimulator of mTORC1 signaling (10). Collectively, these findings demonstrate that Hippo and mTOR-mediated growth signals are closely coupled by multiple mechanisms. However, a caveat of this research from an exercise physiology standpoint is that most of the above studies have been conducted in models that are not related to exercise. Therefore, molecular exercise physiologists now need to test whether these mechanisms also function in an exercise context.

Cross-Talk with Myostatin-Smad Signaling

TGFβ and BMPs are two classes of small extracellular molecules that bind to activin receptors. Bound activin receptors then either phosphorylate the receptor-regulated Smad2/3 or Smad1/5/8 proteins, respectively. These two classes of receptor-regulated Smads compete for the common mediator Smad4 to form either transcriptionally active Smad2/3-4 complexes that promote muscle loss or Smad1/3/5-4 complexes that have recently been proposed to promote muscle growth (114). In skeletal muscle, the knockout of myostatin (gene symbol Gdf8, a TGFβ-related ligand) or its “natural” loss-of-function mutation resulted in a doubling of muscle size in mice and cattle, respectively (48, 69, 95, 96). The link between myostatin and muscle mass was confirmed in humans by showing that a toddler with a high muscle mass was homozygous for a knockout mutation in the first intron of the human myostatin-encoding GDF8 gene (116). Furthermore, dogs with a heterozygous loss of Gdf8 show increased racing performance (105), linking myostatin not only with muscle mass but also with actual athletic performance. If the loss of myostatin is combined with the overexpression of a follistatin transgene, then muscle mass quadruples, suggesting that myostatin is not the only muscle mass regulator in the TGFβ-Smad system (79).

Several studies have shown that Yap and Taz coregulate not only Teads, but also several other transcription factors, including Smads (143). In line with this, overexpression of Yap in mouse tibialis anterior muscle reduces the activity of a Smad-binding element (SBE) reporter by ∼85% (46). In contrast, overexpression of Yap in myoblasts, which are activated satellite cells, potently increases expression of the Smad regulator Bmp4 (67). This is intriguing because both Yap (67) and Bmp4 (109) stimulate satellite cell proliferation while inhibiting differentiation into myotubes. Several mechanisms have been proposed that can explain how Yap or Taz can interact with Smads. These include the binding of Yap to the inhibitory Smad7 (7, 49), the promotion of Smad1 transcriptional action by Yap binding (3), and an effect of Yap and Taz on Smad2/3 localization (137). For molecular exercise physiologists, the challenge is to determine whether Hippo-Smad cross-talk regulates exercise phenomena and especially where it is involved in skeletal muscle mass regulation.

Cross-Talk with AMPK and Glucose Signaling

In the sections above, we discussed the mechanisms connecting the Hippo network to resistance exercise and organ growth signals and signaling modules. Below, we will discuss the cross-talk between Hippo signaling and endurance exercise.

During the transition from rest to exercise, ATP turnover can rise potentially more than 200-fold (98). To maintain homeostasis during such a large step change in ATP hydrolysis, systems have evolved to sense energy levels and initiate the signaling processes that regulate both short- and long-term adaptations of energy metabolism. In this system, glucose, glycogen, AMP, and ADP are sensed principally by the heterotrimeric AMPK complex (53, 55). Indeed, exercise increases the concentrations of AMP and ADP in contracting skeletal and cardiac muscle, and exercise depletes glycogen, especially in muscle (45). Therefore, it comes as no surprise that AMPK is a key mediator of the adaptation to endurance exercise, particularly in skeletal muscle (54). Several recent studies have demonstrated that AMPK is a regulator of YAP, linking a key exercise kinase to Hippo signaling.

Both glucose starvation (36, 100, 145) and AMPK activators (26, 100, 145) inhibit Yap in different cell types. This suggests that Yap-dependent growth is inhibited when cellular energy levels are low. Further research led to identification of the molecular mechanisms mediating this effect. These include the phosphorylation of the Yap regulator Amotl1 on Ser293 by AMPK (26) and the direct phosphorylation of YAP on Ser61/94, which is key for the interaction between Yap and Teads (100, 145). In another study, the Dupont group (36) showed that the glycolytic enzyme phosphofructokinase (PFK1) directly binds to and regulates YAP and TAZ. Finally, two studies (101, 108) have identified the AMPK-kinase LKB1 (gene symbol STK11) as an AMPK-independent YAP regulator. Collectively, these studies demonstrate that glucose starvation and energy stress inhibit YAP via both AMPK-dependent and -independent mechanisms in multiple cell types.

The fact that energy stress and the key exercise kinase AMPK regulate Yap suggest that Yap should be affected by exercise and diet. This now needs to be demonstrated in an exercise model. Also, because Hippo signaling responds to glucose and regulates the expression of glucose transporters (145), it should be studied whether Hippo signaling mediates the augmented adaptations in response to endurance training under low carbohydrate supply (12), or mediates some of the antidiabetic effects of exercise.

Cross-Talk with Hypoxia Signaling

The rise in atmospheric oxygen more than 2 billion years ago was followed by the evolution of oxidative phosphorylation by mitochondria, which use oxygen as their main electron acceptor. The emergence of oxygen-related metabolism drove the evolution of oxygen-sensing systems, as oxygen became critical for survival. Oxygen-sensing systems allow cells and organisms to adapt to low oxygen levels (i.e., hypoxia), especially through the transcription factor hypoxia-inducible factor (Hif1). Hypoxic conditions lead to an increase in expression levels of the Hif1α isoform by blocking its degradation. The hypoxia-induced increase in Hif1α then induces multiple adaptations through gene expression (126). During exercise, hypoxia-induced signaling is also at work. For example, HIF1α levels increase in response to normoxic endurance exercise (5). Moreover, altitude training is often used to stimulate the molecular adaptations to hypoxia, including the erythropoeitin (EPO)-mediated hematopoiesis that increases the athlete's oxygen transport capacity (118).

Hypoxia and Hippo signaling also interact. For example, hypoxia activates the E3 ligase Siah2, which leads to the degradation of Lats2. This results in a decreased level of Yap phosphorylation, thereby increasing the activity of Yap in the nucleus (88). Additionally, Yap directly interacts with and stabilizes Hif1α (88). Hif1α also promotes expression of Taz, and Taz transactivates Hif1α, highlighting a mechanism by which Taz and Hif1α are acting as reciprocal coactivators (153). It is unknown whether hypoxia-Hippo mechanisms function during normoxic endurance exercise (5) and mediate adaptations to high-altitude or low-intensity occlusion training. Direct evidence in an exercise or altitude model is required.

Sensing of Catecholamines and Other G Protein-Coupled Receptor Ligands by Hippo

Catecholamines such as adrenaline (i.e., epinephrine in the United States) and noradrenaline (norepinephrine in the US), mediate the “fight-or-flight” responses. Catecholamine concentrations generally increase with the intensity and duration of exercise and drive the systemic responses to exercise via the α- and β-adrenergic receptors. These receptors, in turn, signal through G protein-coupled receptors (GPCRs) to trigger exercise adaptations such as increasing the heart rate and muscle contractility. Moreover, β2 agonists such as clenbuterol promote skeletal muscle hypertrophy, suggesting an involvement of this system in the control of skeletal muscle growth (89). In the renin-angiotensin system (RAS), the angiotensin receptors are also coupled to protein G. The RAS was related to exercise when the ACE I/D polymorphism was associated with exercise-related traits such as strength and endurance (111). Also, angiogensin II contributes to an adaptation to overload-induced skeletal muscle hypertrophy (47) and stretch-induced cardiac hypertrophy (112). In accordance with this, the ACE I/D polymorphism was associated with left ventricular mass changes occurring in response to endurance training (103).

Multiple studies have linked GPCRs to Hippo signaling. Adrenaline/epinephrine represses YAP/TAZ through Gα_S-coupled GPCRs and protein kinase A (PKA) (74, 158). In contrast, angiotensin II and other ligands signal through the Gα_12/13 and Gα_q/11 GPCRs to activate YAP/TAZ (150, 160). Given that Yap has been shown to mediate skeletal muscle hypertrophy (46, 148) and can promote cardiac hypertrophy [reviewed in (141)], it will be key to test whether GPCR-Hippo signaling is involved in mediating such adaptations.

Interleukin-6 and Hippo

Interleukin-6 is a myokine (i.e., a circulating signaling molecule) that is produced by contracting muscle but whose functions are incompletely understood (106, 110). Recently, IL-6 has been shown to activate Yap through the gp130 coreceptor in the intestine (125). However, it remains unclear whether this mechanism explains some of the effects of exercise-generated IL-6.

Hippo and Exercise-Related Phenomena

In the text above we have shown that many resistance and endurance exercise-associated signals can cross-talk to the Hippo signal transduction network (see Fig. 1). However, much of this evidence was obtained in the context of cancer or nonexercise contexts. In the following section, we will review a small number of studies that provide evidence of a role for Hippo signaling in the adaptation to exercise. In relation to this, some key findings are summarized in Table 1. Additionally, we review emerging evidence that body height is associated with single nucleotide polymorphisms (SNPs) in the vicinity of Hippo genes.

Table 1.

Key experiments in which the perturbation of Hippo members affects skeletal and cardiac muscle in a way that is relevant to exercise physiology

Key Protein, Experiment	Effects of Intervention vs. Control	Reference
MST1: STK4 (protein MST1) knockout vs. wild-type mice, denervation-induced atrophy in vivo	Attenuation of atrophy: skeletal muscle atrophy after denervation ↓; expression of atrophy mediators ↓.	(149)
YAP: injection of rAAV vector to express the main YAP1 isoform vs. control into mouse tibialis anterior in vivo	Hypertrophy: skeletal muscle mass per body weight ↑; fiber cross-sectional area ↑; protein synthesis ↑ (no evidence for mTOR involvement).	(148)
YAP: electroporation of YAP1 vs. control constructs into mouse tibialis anterior in vivo	Hypertrophy: fiber cross-sectional area ↑ (mTORC1 independent); MyoD reporter ↑; c-Myc reporter ↑, MurRF1 reporter ↓; Smad reporter ↓.	(46)
YAP: overexpression of YAP1 S127A, wild-type YAP or empty vector in satellite cells or cultured muscle fibers in vivo	Satellite cell proliferation ↑; differentiation ↓.	(67)
TAZ: injection of TAZ activator IBS008738 (specificity unclear) vs. vehicle into mouse tibialis anterior after cardiotoxin-induced injury or dexamethasone-induced atrophy in vivo	Regeneration, atrophy prevention: IBS008738 injections accelerated skeletal muscle regeneration after injury and reduced atrophy after dexamethasone-induced atrophy.	(157)
TEAD1: muscle creatine kinase promoter-driven expression of TEAD1 in mouse muscle fibers and heart in vivo	Fast-to-slow muscle phenotype shift but cardiomyopathy: extensor digitorum longus shortening velocity ↓; peak power ↓ by approximately 40%; fast-to-slow shift in myosin heavy chains; cardiomyopathy and heart failure.	(130, 132)
YAP: transduction of neonatal rat cardiomyocytes with Yap1 or control adenovirus in vitro	Cardiomyocyte hypertrophy: Cardiomyocyte size ↑ (Akt-independent) and survival ↑ (Akt-dependent).	(25)
YAP: inducible, Tnnt2-promoter-driven expression of YAP1 S127A in fetal and postnatal cardiomyocytes in vivo	Cardiac proliferation: cardiomyocyte proliferation ↑; relative heart weight ↑; regulation of cell cycle-related genes.	(139)
Salvador: inducible, Myh6-driven knock-out of Wwtr1 (Salvador), Lats 1 and Lats2 in adult cardiomyocytes in mice in vivo; apex resection or myocardial infarction	Cardiac proliferation, regeneration: cardiomyocyte proliferation ↑, regeneration of injured hearts.	(58)
YAP: α myosin heavy chain-driven expression of Yap1 S112A in the mouse heart in vivo (homolog of human S127A mutation); myocardial infarction	Cardiac proliferation, regeneration: Cardiomyocyte proliferation ↑; relative heart weight ↑. After myocardial infarction: cardiac function ↑, cardiomyocyte proliferation ↑, fibrosis ↓.	(154)
YAP: inducible expression of α myosin heavy chain-driven expression of Yap1 S127A in the adult mouse heart in vivo; myocardial infarction	Cardiac proliferation, regeneration: Cardiomyocyte proliferation ↑; relative heart weight unchanged. After myocardial infarction: cardiac function ↑, cardiomyocyte proliferation ↑, scar size ↓.	(85)

rAAV, recombinant adeno-associated virus; Nkx2.5 and Tnnt2 are promoters used to drive the specific expression of genes in cardiomyocytes.

Hippo and Adaptive Changes in Skeletal Muscle Fiber Phenotypes

Gollnick and Saltin (44) were the first to demonstrate a higher percentage of slow type-1 muscle fibers and a higher oxidative activity in the muscles of endurance athletes compared with controls and other athletes. They also observed a nonsignificant increase from 32% to 36% in the frequency of slow type-1 fibers in response to endurance training (43). Subsequent research has shown that chronic exercise training programs mainly induce type 2X-to-2A interconversions (151).

In the early 2000s, the Tsika group of researchers (71, 131, 140) investigated the role of MCAT elements and Tead1 transcription factors in the regulation of muscle fiber type-specific gene expression. The functional relevance of their work was demonstrated in vivo using a creatine kinase muscle (CKM) promoter to overexpress Tead1 in mouse skeletal muscle fibers, which caused an increased in slow-muscle-specific gene expression in vivo [Table 1, (132)]. Functionally, CKM-driven Tead1 overexpression reduced the shortening velocity (V_max) and increased the contraction and relaxation times of extensor digitalis longus muscles (132). This suggests that Hippo signaling affects muscle fiber type-specific gene expression and fiber type percentages.

Hippo and Skeletal Muscle Hypertrophy

Muscle hypertrophy is a key response to resistance exercise. After resistance exercise, protein synthesis and protein breakdown both increase. In the fed state, protein synthesis is higher than breakdown, resulting in protein accretion and hypertrophy (128). However, the effect of resistance exercise on muscle hypertrophy and strength differs greatly among the human population (64). A key mediator of muscle protein synthesis is mTOR signaling, as shown for example, by the inhibitory effect of rapamycin on the increase in muscle protein synthesis after resistance exercise in human muscles (30). The key effect of resistance exercise on muscles is mechanical loading, which was discussed in the first part of this review along with the extensive links between mechanosensing and Hippo signaling [reviewed in (50, 87)], including the Z-disc-located Bag3 mechanosensor in skeletal muscle (135, 136).

Several studies support a link between Hippo signaling and resistance exercise and muscle fiber size. In the first study on Hippo signaling in relation to exercise, the Booth group of researchers (19) used chronic stretch overload to induce hypertrophy of the anterior latissimus dorsi muscle and found an increased expression of the skeletal α-actin gene. They showed that stretch overload activated a CATTCC (MCAT) motif-containing luciferase reporter, suggesting that Tead transcription factor activity was present during mechanical overload of skeletal muscle (19). In a human study, eight men performed 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion (75). The researchers found that the mRNA of Hippo marker genes such as cysteine-rich angiogenic protein 61 (CYR61) and connective tissue growth factor (CTGF) (77, 164), increased 14- and 2.5-fold 30 min after exercise, respectively. Additionally, CYR61 protein levels were approximately twofold higher at both 30 min and 48 h after the exercise compared with resting control levels. This suggests that some forms of mechanical loading can induce expression of Hippo marker genes. However, it is unclear whether increases in CYR61 and CTGF expression are a direct consequence of altered Hippo signaling.

Two recent studies have linked Yap activity directly to muscle fiber hypertrophy. Watt and colleagues (148) used an adeno-associated viral vector (rAAV6)-mediated shRNA knockdown strategy to reduce Yap levels in mouse limb muscles. They found a decreased muscle fiber size and reduced protein synthesis. Additionally, they used the same rAAV6 system to overexpress the predominant YAP isoform in muscle and found increases in muscle mass, cross-sectional area, and protein synthesis [Table 1, (148)]. Intriguingly, despite the extensive evidence of cross-talk between Hippo and mTOR signaling discussed in the first part of this review, their YAP interventions did not seem to affect mTOR activity. In another study, the Hornberger group of researchers (46) reported that YAP expression increases up to approximately 4.5-fold in the hypertrophying plantaris muscle days synergist ablation, a model commonly used to induce skeletal muscle hypertrophy. They then used electroporation to overexpress YAP in the tibialis anterior muscles and analyzed the muscles 7 days later. The fibers that overexpressed YAP were larger than control fibers, demonstrating that elevated YAP activity could cause hypertrophy (Table 1). Additionally, they found that YAP induced MyoD and Myc reporters, while inhibiting a Smad-binding element (CAGA)-containing reporter (46). Reductions in myostatin produce a similar effect on a Smad-binding element (CAGA) reporter (166), and a myostatin knockout similarly induces muscle hypertrophy (95). In summary, Hippo members can affect fiber type proportions, and increased levels of Yap can induce skeletal muscle hypertrophy. Additionally, Hippo marker genes increase after resistance exercise in human skeletal muscle.

Hippo and Satellite Cells

Satellite cells were discovered by Mauro (92) using electron microscopy and are now well recognized as the resident stem cells of skeletal muscle (115). The key tool that allowed Mauro to characterize their function in vivo was the Pax7-DTA knockout mouse line, which is used specifically to deplete the satellite cell pool in mouse muscles. Studies with the mice showed that satellite cells are essential for the regeneration of skeletal muscle after injury (2, 81, 107), suggesting that in a sports context, satellite cells are needed to regenerate the muscles damaged by eccentric exercises, such as marathon running (59). In contrast, satellite cell-depleted muscles show a normal hypertrophic response to overload in the short term (93). However, satellite cell-depleted muscle cannot maintain its initial hypertrophy after more than 8 wk (38), suggesting that satellite cells are essential for muscle regeneration after injury and are required to maintain the size of hypertrophied muscles in the long term.

Hippo members are key mediators of proliferation and differentiation in satellite cells and myoblasts (mononuclear muscle cells). Yap is active in proliferating C2C12 myoblasts, and high Yap activity promotes their proliferation but inhibits myogenic differentiation (147). In satellite cells, Yap protein levels are low in the quiescent state, but they increase when satellite cells become activated and develop into MyoD-expressing myoblasts. Again, high Yap activity resulting from expression of the constitutively active YAP1 S127A mutant promotes proliferation but inhibits differentiation (67). Conversely, knocking down Yap in satellite cell-derived myoblasts reduces proliferation by ∼40% (67). Overexpression of YAP1 S127A in satellite cell-derived myoblasts also increases the expression of proliferation-associated genes and known satellite cell regulators such as BMP4 (109) and CD34 (4), while reducing the expression of differentiation markers and the myogenic differentiation regulator Mrf4 (67). Collectively, these results indicate that Yap promotes myoblast and satellite cell proliferation but inhibits differentiation into myotubes and muscle fibers. This suggests that Yap might be an important regulator of muscle development (myogenesis) and satellite cell-derived myoblast proliferation after injury and in response to hypertrophy. The requirement of Yap and other Hippo members such as Taz during the response to exercise by satellite cells remains to be formally demonstrated.

Hippo and the Athlete's Heart

Maximal oxygen uptake (V̇o_{2 max}) is the key determinant of an individual's endurance capacity (27) and is also associated with longevity (15). Early physiological studies in humans demonstrated that one of the key predictors of an individual's V̇o_{2 max} is the blood flow generated by the heart, termed cardiac output (99), which determines the efficiency of oxygen transport to the exercising musculature. The V̇o_{2 max} and cardiac output parameters respond to exercise training and detraining, as demonstrated by Saltin and colleagues (113). They showed that 20 days of bed rest reduces resting heart volume by ∼10% and exercise cardiac output by 15%, resulting in a significantly reduced V̇o_{2 max} (113). Conversely, 55 days of endurance exercise training following the bed rest increased cardiac output above prebed rest levels. This partially explains the restoration of V̇o_{2 max} (142).

The increase in cardiac output can be attributed to the development of an athlete's heart in response to endurance training. Indeed, electrocardiographic studies show that athletes have enlarged hearts (9), which was later confirmed by comparative echocardiography on endurance athletes (91). Generally, the main cellular mechanism underlying the athlete's heart is cardiomyocyte hypertrophy. For example, endurance running training for 8 wk increases cardiomyocyte size by 17–32% in mice (73). Until several years ago, researchers thought that adult cardiomyocytes were unable to proliferate and regenerate the heart. However, this view is now changing (33). By determining the levels of ¹⁴C DNA integration from nuclear bomb tests, Bergmann et al. (14) estimated that between 0.45 and 1% of human cardiomyocytes renew per annum. Furthermore, they showed that by the age of 50, 40% of cardiomyocytes had emerged after birth (14). Moreover, swim endurance training for 2 wk results in increased expression of proliferation markers in mouse cardiomyocytes (16), suggesting that exercise promotes cardiomyocyte proliferation, at least in mice. There is emerging evidence supporting the existence of a cardiac stem cell population that may engage in some limited regeneration (32) in addition to a low renewal rate of preexisting cardiomyocytes (119). Two recent studies found that both cardiac stem cells and cardiomyocytes proliferate in response to endurance exercise in rodents (80, 146). Collectively, these data demonstrate that endurance exercise increases left ventricular volume, thickness, and pumping performance and suggest that these effects occur mainly through cardiomyocyte hypertrophy with a limited contribution of cardiac stem cells and cardiomyocyte proliferation. Consequently, this results in the development of the athlete's heart, which in turn increases the individual's V̇o_{2 max} and aerobic exercise capacity.

The heart can respond to increased loads in two different ways, depending on the nature of the load (94): 1) physiological hypertrophy (i.e., athlete's heart) can occur as a response to endurance exercise or pregnancy (volumetric hypertrophy) or resistance exercise (nonvolumetric hypertrophy); or 2) pathological hypertrophy can occur after cardiac injury or in individuals with high blood pressure or defective valves.

Physiological hypertrophy, which is associated with exercise or pregnancy, differs from pathological hypertrophy by the nature of the stimuli, the structural response, the absence of fibrosis, and the molecular drivers leading to the adaptation (94). Generally, physiological hypertrophy does not progress into cardiac dysfunction. In contrast, pathological hypertrophy often decompensates, reducing cardiac function and resulting in end-stage heart failure (72). Both types of hypertrophy differ at the molecular level in their response the divergent stimuli (1).

Currently, whether and how Hippo signaling contributes to the different forms of cardiac hypertrophy remains incompletely understood. Several studies show that Yap loss- or gain-of-function in the embryonic heart is frequently lethal [reviewed in (84, 141, 155)]. This suggests that normal Yap function is essential for normal cardiac development. Presumably, Yap is active only in certain cell populations during specific periods, which could explain why permanent Yap gain- or loss-of-function has such a detrimental effect. There is some evidence that Hippo signaling is perturbed during pathological cardiac hypertrophy. Yap is expressed at higher levels and dephosphorylated (activated) in samples obtained from pathologically hypertrophied human hearts (144). Furthermore, Yap is activated in hearts stressed by pathological pressure overload (144) and in the area bordering a myocardial infarction in mice (25).

Are Hippo members contributing to the formation of the athlete's heart in response to exercise? Although this question has not been addressed directly, published data suggest possible roles for Hippo members in mediating the athlete's heart (see Table 1). First, Yap1 overexpression in cultured neonatal rat cardiomyocytes promotes hypertrophy and survival compared with control cardiomyocytes (25). In contrast, two other teams reported no cardiomyocyte hypertrophy upon Yap activation in postnatal mouse hearts in vivo (139, 154). The reasons behind these contrasting results are unknown, and so it is unclear whether Hippo signaling contributes to cardiomyocyte hypertrophy in response to exercise (73).

Another response of the heart to endurance exercise in rodents is the limited proliferation of cardiomyocytes and cardiac stem cells (16, 80, 146). Although it has not been tested whether Hippo members promote cardiomyocyte or cardiac stem cell proliferation in response to endurance exercise, evidence supporting that notion that Hippo members regulate the proliferation of adult cardiomyocytes and can enhance regeneration after cardiac injury has been reported (Table 1). Knocking out the Hippo members Sav1 or Lats1/2 in adult mouse cardiomyocytes increases proliferation, promotes regeneration after myocardial infarction, and reduces scar tissue formation (58). Similarly, Yap1 S112A overexpression in cardiomyocytes improves cardiac regeneration after myocardial infarction, both in neonatal and adult mice, with evidence for increased cardiomyocyte proliferation compared with controls (154). Finally, in a mouse model of myocardial infarction, forcing human YAP expression in the heart using adeno-associated virus delivery results in increased cardiomyocyte proliferation and improved cardiac function and survival (85).

In summary, the normal function of Yap and Hippo signaling is essential for normal cardiac development. Currently, it is unknown whether Hippo members and Yap in cardiomyocytes and cardiac stem cells respond to exercise and contribute to the development of an athlete's heart. Available studies suggest that Yap can promote cardiomyocyte hypertrophy in some contexts, while in other contexts, Yap appears to promote cardiomyocyte proliferation and enhance cardiac regeneration after injury.

Hippo and Body Height

Body height is a key factor in sports, which is most striking in NBA basketball players. Body height is approximately 70–90% inherited (121) and depends on hundreds, if not thousands, of common DNA sequence variations with a small effect size each (152). In rare cases, body height can be affected by single, rare mutations with a large effect size. Examples for the latter are dwarfism caused by FGFR3 mutations (37), and acromegaly resulting from AIP gene mutations (20).

Given that a core function of Hippo signaling is to control cell numbers, one would expect links between Hippo gene DNA sequence variations and body height. Interestingly, genome-wide association studies (GWAS) involving analysis of data from up to 250,000 individuals (152) show that single nucleotide polymorphisms (SNPs) in several Hippo genes are associated with body height (23, 78, 152). Indeed, SNPs associated with genes encoding for LATS2, TEAD1, YAP1, VGLL2, VGLL3, and VGLL4 are associated with body height (23, 57, 78, 152). In the largest meta-analysis study using data obtained from 253,288 individuals of recent European ancestry (152), body height-associated SNPs in LATS2 (rs1199734), TEAD1 (rs6485978, rs2099745), VGLL2 (rs1405212), and VGLL4 (rs13078528) were identified. In 2010, Lango Allen et al. (78) identified an association between SNPs in TEAD1 (rs7926971) and VGLL2 (rs961764) with body height in 133,653 individuals of recent European ancestry. Also, SNPs in YAP1 (rs11225148) and VGLL3 (rs7628864) were individually associated with a shorter stature during pubertal growth in a longitudinal meta-analysis involving 18,737 European individuals (23). Interestingly, the SNP in VGLL3 was significantly associated with the trait only in women. So far, no sex-related differences have been reported for the Hippo pathway functions, but this association could suggest that such differences might actually exist in some contexts. Finally, an SNP in VGLL4 (rs6772112) was associated with height in 36,227 subjects with East Asian ancestry (57). Another interesting association of the study by Cousminer et al. (23) is the identification of a female-specific SNP in LIN28B associated with late pubertal growth. The LIN28/LET-7 pathway, which has recently emerged as a potent regulator of organismal development and cellular metabolism (120), has been functionally linked with the Hippo pathway (21, 104). In summary, common DNA sequence variants in several Hippo genes influence body height, but the effect of each variant on height is small, presumably because de novo DNA sequence variants with a large effect size either become fixed or lost relatively quickly (17).

Summary and Future Research

In this review we have listed mainly indirect evidence suggesting that Hippo signaling may mediate some of the physiological adaptations to exercise and that SNPs, especially in the Hippo transcriptional regulators, are associated with body height as a measure of whole body cell numbers. The task for molecular exercise physiologists is now to directly show that these mechanisms mediate adaptation to exercise in exercise models and that Hippo gene variants are associated with sport and exercise-related traits. We end with three questions:

Because resistance and endurance exercise trigger different adaptations in skeletal muscle, how can it be explained that the activity of Hippo members is both affected by both resistance and endurance exercise-associated signals?

Given that Hippo signaling affects amino acid (52) and glucose transporter expression (145), can this be used to develop strategies to alter the responsiveness to nutrients? For example, can we target through Hippo modulation the leucine transporter LAT1 (52) to make muscles and other organs more sensitive to protein intake? Could such a strategy be beneficial for strength athletes or in cases of muscle weakness and wasting, for example, in elderly individuals or patients with cancer and sarcopenia?

Given that the Hippo pathway is involved in regulating the fate of many stem cells (129), can this be exploited to develop interventions aimed at improving the repair of muscle, tendons, and cartilage after sports injury or in degenerative muscle diseases?

GRANTS

B.M. Gabriel is supported by a Wenner-Gren Foundation Postdoctoral Fellowship, a European Foundation for the Study of Diabetes Albert Renold Travel Fellowship, and a Novo Nordisk Foundation Challenge Grant. Work in the Aberdeen Hippo laboratory is funded by Medical Research Council Grant 99477, Sarcoma UK, and Friends of Anchor. A.M. Tremblay is recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.M.G., D.L.H., A.M.T., and H.W. prepared figures; B.M.G., D.L.H., A.M.T., and H.W. drafted manuscript; B.M.G., D.L.H., A.M.T., and H.W. edited and revised manuscript; B.M.G., D.L.H., A.M.T., and H.W. approved final version of manuscript.