Decoding the Impact of the Hippo Pathway on Different Cell Types in Heart Failure

Abstract

The evolutionarily conserved Hippo pathway plays a pivotal role in governing a variety of biological processes. Heart failure (HF) is a major global health problem with a significant risk of mortality. This review provides a contemporary understanding of the Hippo pathway in regulating different cell types during HF. Through a systematic analysis of each component’s regulatory mechanisms within the Hippo pathway, we elucidate their specific effects on cardiomyocytes, fibroblasts, endothelial cells, and macrophages in response to various cardiac injuries. Insights gleaned from both in vitro and in vivo studies highlight the therapeutic promise of targeting the Hippo pathway to address cardiovascular diseases, particularly HF.

Heart disease is a leading cause of death worldwide and carries a high risk of progression to heart failure (HF). According to the Heart Disease and Stroke Statistics 2023, from 2017 to 2020, an estimated 6.7 million US adults over the age of 20 years had HF.¹ The total HF population is expected to increase from 2.4% in 2012 to 3.0% in 2030.¹ Hence, investigating the underlying mechanisms of HF is essential for identifying innovative therapeutic targets and improving cardiac function. In the exploration of the regulatory mechanisms of cardiac function, the Hippo pathway has been shown to be highly significant within the heart. The Hippo pathway plays a crucial role in governing organ growth and size, as well as tumorigenesis, exhibiting evolutionary and functional conservation across species.² Since its demonstrated importance in organ size and regeneration in Drosophila,^3,4 extensive research over the past 2 decades has revealed the pivotal role of the Hippo pathway in cardiac development, growth, disease, and tissue regeneration. The Hippo pathway regulates cardiac function by regulating multiple cell types in the heart, including cardiomyocytes, fibroblasts, macrophages, and endothelial cells. In this review, we provide a brief overview of the Hippo pathway and elucidate the importance of its individual components in regulating the activity of different cell types during HF based on current research.

Hippo Pathway

The Hippo signaling pathway is an evolutionarily conserved signaling pathway that regulates cell proliferation, differentiation, and apoptosis.^2,4,5 Increasing evidence substantiates the role of the Hippo pathway as a pivotal regulator of not only organ size, but also cell development, regeneration, and disease, including in the heart.^6–13 In mammals, the Hippo pathway consists of multiple core components, including mammalian STE20-like protein kinase 1/2 (MST1/2), the scaffold protein Salvador homologue 1 (SAV1), large tumor suppressor kinase 1/2 (LATS1/2), the scaffolding proteins MOB domain kinase activator 1A/B (MOB1A/B), and the main downstream effector proteins Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ; Figure 1). The core of the Hippo pathway involves a kinase cascade, in which activation of MST1/2 generally serves as the initial signal. Many studies have focused on exploring the upstream signals that regulate activation of the Hippo kinase cascade, and various upstream signals, including stress response and mechanical signaling, have been discovered.²

Figure 1.

Summary of the Hippo pathway in mammals. When the Hippo pathway is “ON” (Left), activated mammalian STE20-like protein kinase 1/2 (MST1/2) and the scaffold protein Salvador homologue 1 (SAV1) activate large tumor suppressor kinase 1/2 (LATS1/2) and the scaffolding proteins MOB domain kinase activator 1A/B (MOB1A/B), which, in turn, phosphorylate and promote the degradation or cytoplasmic retention of the downstream effector proteins Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). In the heart, the Hippo pathway can be activated by myocardial infarction (MI), myocardial ischemia/reperfusion (I/R), oxidative stress, chronic pressure overload (PO), and doxorubicin (DOX) treatment. In contrast, striatin-interacting phosphatase and kinase (STRIPAK) complexes negatively regulate the activity of MST1/2. When the Hippo pathway is “OFF” (Right), activated YAP and TAZ enter the nucleus and interact with various transcription factors (TFs), acting as transcriptional coactivators to regulate gene transcription. Some small molecules have demonstrated the ability to selectively inhibit the activity of individual components of the Hippo pathway, including XMU-MP-1 (targeting MST1/2), TRULI and TDI-011536 (targeting LATS1/2), and verteporfin (targeting YAP).

In the heart, the Hippo pathway can be activated by myocardial infarction (MI), myocardial ischemia/reperfusion (I/R), oxidative stress, and chronic pressure overload (PO).^14,15 For example, studies have demonstrated that mechanical signaling and oxidative stress-induced neurofibromin 2 (NF2) activates the Hippo pathway in the heart.^2,16–18 When Hippo signaling is activated or “ON”, MST1/2 or mitogen-activated protein kinase kinase kinase kinase (MAP4K)¹⁹ phosphorylate and activate LATS1/2, which subsequently phosphorylate the downstream effectors YAP and TAZ. This prevents YAP and TAZ from translocating into the nucleus and induces their degradation or retention in the cytoplasm. Conversely, the striatin-interacting phosphatase and kinase complexes (STRIPAK) negatively regulate the kinase activity of MST1/2, leading to the subsequent activation of YAP and TAZ.^2,20 When the Hippo pathway is “OFF”, the upstream kinase cascade is inactivated, and unphosphorylated YAP and TAZ translocate into the nucleus and bind to transcription factors to act as transcriptional cofactors, activating or repressing the expression of genes and thereby regulating cell growth and survival. The TEA domain transcription factor family members (TEADs) are common transcription factors with which YAP and TAZ interact.²¹ In addition, YAP and TAZ have been observed to interact with other transcription factors, including hypoxia-inducible factor-1α (HIF-1α),^22,23 forkhead box protein O1 (FoxO1),²⁴ CCAAT/enhancer-binding protein-α,²⁵ nuclear factor (NF)-κB,²⁶ and runt-related transcription factors 1 and 2 (RUNX1/2).^27,28

Hippo Pathway in Different Cell Types of the Heart

The heart comprises various types of cells, including cardiomyocytes, fibroblasts, endothelial cells (ECs), and immune cells^6,11,29,30 (Figure 2). Cardiomyocytes, responsible for the contractile function of the heart and pumping blood into the circulatory system, are highly differentiated cells that have limited renewal ability after birth.^31–33 The primary hallmark of HF is the loss of cardiomyocytes, often accompanied by scar formation and adverse cardiac remodeling.³² This may be due to adult cardiomyocytes having exited the cell cycle, making it challenging for them to regenerate naturally after injury in adult mammals, ultimately resulting in cardiac remodeling and HF.³³ Persistent HF induces qualitative changes in cardiomyocytes, including in metabolism and cardiac contraction.

Figure 2.

Impact of the Hippo pathway on various cell types in the heart. Under basal conditions or in response to cardiac injuries, each component of the Hippo pathway has been shown to modulate the function of different cell types in the heart. Mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor kinase 1/2 (LATS1/2), and Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) show positive (green “+”) and negative (red “−”) correlations with different cell activities in cardiomyocytes, fibroblasts, macrophages, and endothelial cells. SAV1, Salvador homologue 1.

Fibroblasts represent approximately 40% of the total cells in the heart,³⁰ and their primary role is to maintain the cardiac extracellular matrix and offer structural and functional support to resident cardiomyocytes.³⁴ Resting or quiescent fibroblasts can be activated and terminally differentiate to myofibroblasts, which is important for wound healing, tissue repair, and fibrosis.^35–37

ECs form the inner surface layer of blood vessels and release various auto- and paracrine agents, including nitric oxide and angiotensin II. ECs play a crucial role in regulating cardiac metabolism, growth, and contractile performance.³⁸ EC dysfunction is also associated with multiple cardiovascular diseases and HF.^39,40

Inflammation is a pivotal factor in cardiovascular disease, contributing to pathological cardiac remodeling, cardiac dysfunction, and HF. Single-cell RNA sequencing showed that 5–10% of cells in the human heart are immune cells, including macrophages.³⁰ Cardiac macrophages play a crucial role in development, homeostasis, and injury responses.^41–45 Cardiac macrophages not only clear debris from damaged cardiomyocytes after cardiac injury, but they may also contribute to pro- and anti-inflammatory effects.⁴²

Each component of the Hippo pathway has been demonstrated to regulate the function of the different cell types in the heart in response to cardiac insults such as I/R, MI, and PO. In the subsequent sections of this review, we summarize the studies conducted thus far, highlighting the importance of the Hippo pathway in different cardiovascular diseases and HF. These studies are classified according to the different cell types in the heart.

Cardiomyocytes

MST1 With the progression of research, the potential role of MST1 in promoting the development of heart disease has become increasingly evident.⁴⁶ MST1 can be activated by I/R, MI, PO, and doxorubicin (DOX)-induced cardiomyopathy. In 2003, MST1 was identified as a key to triggering apoptosis in cardiomyocytes.⁴⁷ In addition, cardiac-specific overexpression of MST1 in mice induced dilated cardiomyopathy and hindered compensatory cardiomyocyte hypertrophy despite increased wall stress.⁴⁷ Conversely, suppressing endogenous MST1 by overexpressing dominant-negative MST1 in mouse hearts prevented cardiomyocyte death in response to I/R injury.⁴⁷

MST1 also has been demonstrated to play a crucial role in mediating cardiac dilation, apoptosis, fibrosis, and cardiac dysfunction in response to MI.⁴⁸ Inhibiting MST1 improved cardiac function without impacting cardiac hypertrophy.⁴⁸ Notably, treatment with XMU-MP-1 (XMU), a novel pharmacological inhibitor of MST1/2 enzyme function, has been shown to effectively inhibit cardiomyocyte apoptosis and rescue cardiac dysfunction in response to PO stress or I/R injury.^49,50

Autophagy is an evolutionarily conserved mechanism responsible for lysosome-mediated cellular degradation, playing an important role in mediating cellular protein and organelle quality control mechanisms.¹⁰ Activated MST1 has been implicated in inhibiting autophagy in the heart after MI.^10,51 Stress induced MST1 suppression of autophagosomes in cardiomyocytes by inducing phosphorylation of Beclin1 and enhancing the interaction between Bcl-2 and Beclin1.⁵¹ In addition, oxidative stress-activated MST1 phosphorylated Bcl-xL and disrupted Bcl-xL-Bax binding in the heart; this disruption resulted in the activation of Bax and subsequently induced mitochondria-mediated apoptotic cell death in response to I/R.⁵²

DOX, an effective chemotherapeutic agent used for cancer treatment, is the most frequently reported drug in chemotherapy-induced cardiotoxicity. DOX treatment activated MST1, thereby inducing mitochondrial damage and dysfunction, oxidative stress and cardiac fibrosis.⁵³ The DOX-induced cardiac phenotype was attenuated by expressing cardiac-specific dominant-negative MST1.⁵³ Another study corroborated this discovery, indicating that MST1 contributed to DOX-induced cardiomyopathy by downregulating sirtuin 3, a deacetylase involved in mitochondrial protection.⁵⁴ Furthermore, XMU treatment significantly inhibited DOX-induced cardiac dysfunction.⁵⁴

These findings suggest that MST1 is important in promoting cardiomyocyte apoptosis in multiple cardiac diseases. In summary, suppressing the expression of MST1 in cardiomyocytes is an effective strategy to ameliorate the pathological phenotypes and cardiac dysfunction associated with cardiovascular diseases and HF. It should be noted that some of the important actions of MST1 and MST2 in the heart are mediated through direct phosphorylation of unique downstream substrates such as Beclin1⁵⁵ and Bcl-xL⁵² rather than through activation of LATS1/2 and inhibition of YAP and TAZ. Thus, one may expect that inhibition of MST1 may possess unique features not shared by stimulation of YAP in some disease conditions. It would be interesting to identify additional direct substrates of MST1 and MST2 and their subcellular localizations.

SAV1 (WW45) MST1/2 form an active complex with the WW repeat scaffolding protein SAV1, also known as WW45. In a study exploring the role of SAV1 in heart development, deleting SAV1 specifically in the mouse embryonic heart resulted in upregulation of WNT-β-catenin target genes and expression of cell survival-related genes.⁵⁶ Moreover, the absence of SAV1 led to a decrease in YAP^Ser127 phosphorylation, potentially contributing to the interaction between YAP and β-catenin in the nucleus.⁵⁶ This discovery suggests that the Hippo pathway limits the WNT pathway in developing hearts, thereby suppressing the growth and proliferation of cardiomyocytes and the size of the heart.⁵⁶

Interestingly, in the postnatal pig heart, adeno-associated virus 9 (AAV9)-mediated knockdown of SAV1 in border zone cardiomyocytes resulted in increased nuclear YAP localization and upregulated cardiomyocyte proliferation compared with the control group.⁵⁷ Administration of AAV9-Sav short hairpin (sh) RNA significantly enhanced cardiomyocyte proliferation and rescued cardiac function (14.3% improvement in ejection fraction) in the pig heart following I/R-induced MI.⁵⁷ This implies that local delivery of AAV9-Sav shRNA could potentially rescue tissue renewal and improve function, thereby holding promise for the treatment of HF.⁵⁷ In another study, cardiac-specific homozygous knockout of WW45 resulted in higher nuclear YAP expression in cardiomyocytes.⁵⁸ Under PO, mice with cardiac-specific knockout of WW45 (WW45cKO) exhibited an increase in TEAD1 target cardiomyocyte dedifferentiation genes, such as bone morphogenetic protein 2 (Bmp2), caveolin 3 (Cav3), and delta like canonical Notch ligand 1 (Dll1), and a decrease in cardiomyocyte differentiation genes like arrestin, β 2 (Arrb2), ephrin B2 (Efnb2), and E1A binding protein p300 (Ep300). This effect may be attributable to the upregulation of the YAP-TEAD1 pathway and the subsequent activation of oncostatin M (OSM) and OSM receptors.⁵⁸ Unexpectedly, the WW45cKO mice experienced exacerbated cardiac dysfunction and decreased survival rates during PO, even though apoptosis decreased and cardiomyocytes re-entered the cell cycle.⁵⁸ This suggests that although YAP activation in the heart promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and HF in the long-term presence of PO through activation of the YAP-TEAD1-OSM positive-feedback mechanism.⁵⁸ Therefore, the function of upstream Hippo components in response to different cardiac stresses may produce different results due to differential regulation of gene expression. Furthermore, interventions either maintaining or promoting the differentiation of cardiomyocytes may be helpful when loss of WW45 function is considered as a modality to induce regeneration of fully functional cardiomyocytes in the failing heart.

LATS1/2 MST1/2 form a complex with SAV1, thereby phosphorylating and activating downstream kinases LATS1/2 in the Hippo pathway. Systemic loss of LATS2 in mice (LATS2^+/−) attenuated cardiac hypertrophy and dysfunction induced by 4 weeks of transverse aortic constriction.⁵⁹ Cardiac-specific knockout of SAV1 and LATS1/2 in adult mice stimulated cardiomyocyte regeneration and improved cardiac function after MI.⁶⁰ Furthermore, intraperitoneal administration of small molecules that effectively inhibit the activity of LATS1/2, namely TRULI and TDI-011536, resulted in the induction of cardiomyocyte proliferation, accompanied by upregulated transcriptional activation of YAP target genes in the heart.^61,62 Another study demonstrated that LATS2, but not LATS1, inhibited cardiomyocyte growth and induced their apoptosis.⁶³ This finding is supported by the significant reduction in MST1-induced cardiomyocyte apoptosis observed when cardiac-specific dominant-negative LATS2 was expressed using an Myh6-Cre driver, indicating that LATS2 mediates the function of MST1 in cardiomyocytes.⁶³ Cardiac-specific overexpression of LATS2 in mice significantly reduced the size of both the left and right ventricles and decreased cardiac function. Notably, dominant-negative LATS2 enhanced cardiac hypertrophy and inhibited cardiomyocyte apoptosis induced by transverse aortic constriction.⁶³ These findings suggest the importance of LATS1/2 in regulating cardiomyocytes apoptosis, hypertrophy, and regeneration.

YAP and TAZ YAP and TAZ, as major downstream effectors of the Hippo signaling pathway, play a vital role in regulating the proliferation, survival, and renewal of cardiomyocytes under both physiological and pathophysiological conditions.^9,12,64 Loss of YAP and TAZ resulted in gene dosage-dependent cardiac phenotypes, indicating their key roles in determining the proper number of cardiomyocytes, hypertrophy, and maintaining cardiac function.⁶⁵

Cardiac-specific deletion of YAP impeded neonatal heart regeneration, adult cardiomyocyte survival, hypertrophy, and proliferation.⁶⁵ In addition, loss of YAP in the heart exacerbated cardiomyocyte apoptosis, fibrosis, and cardiac dysfunction after MI.^27,65,66 Like MST1, YAP has been demonstrated to mediate the regulation of autophagy in the heart.⁶⁷ YAP is degraded in part through lysosomes at baseline. Thus, YAP is upregulated when lysosome function is impaired in cardiomyocytes.⁶⁷ Accumulation of YAP is observed in cardiac-specific RagAB-knockout mice, a model of lysosomal storage disease, where YAP promotes cardiac hypertrophy and cell death induced by excessive accumulation of autophagosomes.⁶⁷ In addition, the expression of YAPS112A (a constitutively active form of YAP) in cardiomyocytes enhanced cardiomyocyte proliferation, improved cardiac function in injured adult hearts, reduced infarct size, and promoted survival after MI.⁶⁵ Similar findings were observed with AAV9-mediated overexpression of human YAP in adult murine myocardium; AAV9:hYAP reduced cardiomyocyte apoptosis and improved cardiac function, contributing to an increased survival rate after MI.⁶⁸ It should be noted that constitutive activation of YAP, namely overexpression of YAP(5SA), induces HF in mice.⁶⁹ This may due to the cardiomyocyte hyperplasia induced by YAP(5SA); increased left ventricular wall thickness and a significantly smaller chamber result in outflow tract obstruction.⁶⁹ Thus, the salutary actions of YAP in the heart appear to be dose dependent.

YAP, as a transcription coactivator, has been observed to interact with the transcription factor FoxO1 in cardiomyocyte nuclei, enhancing the complex’s binding to the promoters of antioxidant genes.²⁴ This interaction stimulated the transcription of antioxidant genes, ultimately promoting cardiomyocyte survival.²⁴ Inhibition of the YAP-FoxO1 complex worsened cardiac function following I/R injury.²⁴ Another study found that YAP-TEAD occupied a conserved enhancer of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit β (Pik3cβ), which contributed to upregulation of cardiomyocyte proliferation.⁷⁰ Furthermore, YAP is required and sufficient to activate the phosphatidylinositol 3-kinase-Akt pathway (PIK3-Akt pathway).⁷⁰ Another study investigated the role of miR-206 in mediating YAP-TEAD upregulation and found that it induces cardiomyocyte survival and hypertrophy and protects cardiac function during I/R injury.⁷¹

YAP also plays a protective role in PO-induced compensatory cardiac hypertrophy by promoting YAP-TEAD1-HIF-1α-dependent glucose transporter 1 expression in cardiomyocytes.²³

In addition to cardiac injury, high-fat diet (HFD) consumption also induces YAP expression in mice.⁷² However, YAP is persistently active in the diabetic heart, which contributes to dedifferentiation of cardiomyocytes through activation of TEAD1.⁷² Furthermore, HFD consumption exacerbated cardiac dysfunction in response to PO by inducing YAP and TEAD1, suggesting that YAP-TEAD is a promising therapeutic target to prevent the development of HF in diabetic patients with high blood pressure.⁷² YAP-TEAD1 has also been implicated in the suppression of the mitochondrial genes, dynamin 1-like (Dnm1l) and mitofusin 1 (Mfn1), contributing to mitochondrial dysfunction in the heart under chronic PO.⁷³ Administration of verteporfin, a drug reported to inhibit YAP signaling, rescued cardiac dysfunction in mice fed an HFD and subjected to PO⁷² and attenuated cardiac hypertrophy in the presence of chronic PO.⁷³ However, chronic treatment with verteporfin resulted in a cardiomyopathy phenotype similar to that observed with chronic DOX treatment.⁵³

Thus, although YAP may play protective roles in cardiomyocytes in the heart, sustained activation of YAP under stress may be detrimental through other mechanisms, such as induction of mitochondrial dysfunction.

Fibroblasts

MST1/2 Fibroblasts play a vital role in facilitating tissue repair and wound healing after injury; however, sustained fibroblast activation can lead to excessive cardiac fibrosis and functional decline. Due to its ability to modulate fundamental cellular functions, the importance of the Hippo pathway in the cardiac fibroblast has emerged as a focal point of recent research efforts. The tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) has been shown to be a physiological activator of MST1 in the mammalian heart.¹⁸ In contrast to its harmful effect of inducing cardiomyocyte apoptosis, the Rassf1A/MST1 pathway was unexpectedly found to play a protective role in fibroblasts by inhibiting proliferation and cardiac hypertrophy.¹⁸ This signaling module negatively regulated NF-κB and the production of tumor necrosis factor-α, a key mediator of cardiomyocyte hypertrophy, fibroblast proliferation, and cardiac dysfunction.¹⁸ This suggests that suppressing Rassf1A/MST1 in cardiac fibroblasts may have negative effects on cardiac remodeling and function, and highlights the cell type-dependent consequences of activating the Rassf1A/MST1 pathway during PO in the heart.¹⁸

LATS1/2 In 2018, it was demonstrated that LATS1/2 are required for epicardial fibroblast transition during heart development.⁷⁴ Through single-cell RNA sequencing, it was found that epicardial-specific deletion of LATS1/2 resulted in cells failing to undergo fibroblast differentiation, highlighting the crucial role of LATS1/2 in maintaining the proper extracellular environment and coronary vessel development.⁷⁴ Subsequent research into the role of LATS1/2 in adult cardiac fibroblasts revealed that, even in the absence of injury, deletion of LATS1/2 in cardiac fibroblasts initiated a self-perpetuating fibrotic response, inducing spontaneous transition into a myofibroblast cell state, which was exacerbated by MI.⁷⁵ In addition, the loss of LATS1/2 in cardiac fibroblasts elicited activation of nuclear YAP, which directly activates expression of myofibroblast genes, as well as genes encoding proinflammatory factors.⁷⁵ Thus, LATS1/2 are vital for maintaining the resting cardiac fibroblast cell state by limiting YAP-induced fibroblast activation at baseline and after MI.⁷⁵

YAP and TAZ In mice, YAP is activated in cardiac fibroblasts in response to either neuroendocrine stimulation (angiotensin II administration) or MI.^76,77 MI alters extracellular matrix proteins and causes disorganization of collagen fibers, conditions that were shown to favor YAP activation.⁷⁸ Active YAP then stimulates the expression of genes related to proliferation, the cell cycle, and myofibroblast differentiation in cardiac fibroblasts through interaction with TEAD1.⁷⁶ Importantly, selective deletion of YAP in cardiac fibroblasts, using the Tcf21^iCre driver mouse, resulted in attenuation of myocardial fibrosis and cardiac dysfunction after MI.⁷⁶ A similar approach was used to delete both YAP and TAZ in cardiac fibroblasts, using Col1a2-CreERT mice. Targeting both YAP and TAZ in this compartment led to attenuated fibrosis and inflammation, with improved function following MI.⁷⁷ More recent work genetically inhibited YAP and TAZ in heart myofibroblasts by using Postn-MCM mice.⁷⁹ Consistently, deletion of myofibroblast YAP and TAZ reduced fibrosis and afforded cardioprotection against MI.⁷⁹ These studies demonstrate the beneficial effects of cardiac fibroblast-selective inhibition of YAP and TAZ. Conversely, AAV-mediated sustained expression of YAP in cardiac fibroblasts promoted fibrosis and inflammation.⁸⁰ A comparable phenotype was observed in mice genetically engineered to express active YAP in cardiac fibroblasts, providing additional evidence that YAP activation in cardiac fibroblasts is sufficient to drive cardiac fibrosis.^77,80

Transforming growth factor (TGF)-β/SMAD signaling is a well-established core regulator of cardiac fibrosis, and it has been demonstrated that there is a relationship between YAP and TGF-β. Deletion of YAP was shown to reduce TGF-β/hypoxia-induced cardiac fibroblast proliferation in human ventricular cardiac fibroblasts.⁸¹ In addition, SKI, an endogenous TGF-β1 repressor, deactivated TAZ (but not YAP), subsequently inhibiting myofibroblast activation. SKI-induced TAZ downregulation was inhibited by simultaneous LATS2 knockdown, indicating core Hippo pathway involvement.⁸² Moreover, these findings were observed in post-MI fibrosis, indicating the pathophysiological relevance of this mechanism.^15,82

Downstream signaling mediated by YAP/TAZ activation in cardiac fibroblasts is complex and regulates distinct cellular functions, including proliferation, myofibroblast differentiation, migration, and inflammatory processes.¹¹ Studies have demonstrated that YAP-TEAD1 promotes the expression and function of myocardin-related transcription factor A, thereby promoting cardiac myofibroblast transition, enhanced contractile function, and proinflammatory signaling.⁷⁶ YAP and TAZ have also been shown to positively regulate interleukin-33 and cellular communication network factor 3 to stimulate inflammatory and fibrotic responses following ischemic injury in the heart.^77,79 Importantly, the administration of verteporfin blocked the expression of profibrotic genes⁷⁶ and reduced cardiac fibrosis in mice after MI.⁸³ Together, these studies indicate that activation of fibroblast YAP and TAZ during cardiac stress contributes to injury and suggest that inhibitory strategies may prove therapeutically viable.

Endothelial Cells

Coronary atherosclerotic heart disease, commonly referred to as coronary artery disease, is characterized by the presence of atherosclerotic lesions in the coronary arteries. Endothelial dysfunction is a typical feature associated with atherosclerosis in this context.⁴⁰ Studies have shown that inhibition of YAP and TAZ suppresses inflammation and delays atherogenesis, and that endothelium-specific YAP overexpression exacerbates plaque formation in mice.⁸⁴ Laminar flow, known for its protective effect on the endothelium, disrupts the formation of atherosclerotic plaques by inhibiting YAP activity through the induction of endothelial autophagy.⁸⁵ Notably, phosphorylation of YAP at Y357, which is mediated through phosphorylation of the cytosolic non-receptor protein kinase c-Abl and induces nuclear translocation of YAP, was markedly increased in ECs within atherosclerotic vessels of mice and in human plaques compared with normal vessels.⁸⁶

In addition, YAP and TAZ are important in regulating EC proliferation and angiogenesis.^87–89 Under metabolic stress, such as hyperglycemia and dyslipidemia during diabetes and PO in the heart, ECs play a critical role in the repair process by promoting angiogenesis. Under metabolic stress, aortic ECs activate cGAS-STING-interferon regulatory factor 3 (IRF3) signaling, which, in turn, upregulates MST1 gene transcription and suppresses angiogenesis via MST1-mediated inhibition of YAP.⁹⁰

Macrophages

MST1/2 Macrophages are the most abundant population of leukocytes found within the heart and can either maintain tissue homeostasis or contribute to disease depending on their phenotype (polarization) and/or ontology.^{43,45,91–97} It is well established that macrophages can polarize to either a proinflammatory “M1” or anti-inflammatory “M2” phenotype after insult to the heart; this polarization affects inflammation or repair, respectively, and subsequently the extent of cardiac injury and remodeling.^98–102 However, the mechanisms underlying this polarization are only recently beginning to be uncovered. Previous reports have shown that loss of MST1/2 in macrophages can be either pro- or anti-inflammatory, depending on the type of immune response activated.^103–106 On the one hand, MST1 activity directly suppressed conventional Toll-like receptor 4 and tumor necrosis factor-α-mediated NF-κB signaling in macrophages, suggesting an anti-inflammatory role;^103,106 on the other hand, MST1 has been shown to activate Type 1 interferons after lipopolysaccharide stimulation via degradation of interleukin-1 receptor-associated kinase 1, promote reactive oxygen species production via protein kinase C activation, and enhance inflammatory cell recruitment via IRF3-induced CXCL1/2 gene expression after inflammatory stimulation or infection, suggesting an inflammatory function.^{103,104,107,108} MST1/2 have been shown to play a role in regulating macrophage polarization in models of HF by suppressing inflammatory polarization. Recent studies found that myeloid cell-specific deletion of MST1/2 exacerbates cardiac dysfunction and exerts a proinflammatory effect by activating the 5-lipoxygenase-leukotriene B₄-BLT1 receptor (5-LOX-LTB4-BLT1) axis in macrophages after MI, which is attributed to a shift of macrophage phenotype from expression of MP2/3 genes toward expression of proinflammatory MP1 markers.¹⁰⁹ Surprisingly, inhibition of MST1 via XMU was also shown to stimulate M2 macrophage polarization and suppressed cardiac inflammation in mice after I/R injury.⁵⁰ Similarly, in models of lipopolysaccharide infection or kidney ischemia, MST1/2 inhibition by XMU promoted M1 macrophage polarization via YAP-mediated nuclear accumulation of NF-κB.¹¹⁰ Interestingly, the same study revealed that MST1/2 inactivation suppressed M2 macrophage polarization independently of YAP activity by reducing STAT6 signaling, demonstrating both YAP-dependent and -independent mechanisms in MST1/2-directed macrophage polarization.¹¹⁰

In conclusion, MST1/2 may play a dual role in macrophage polarization in the heart, exhibiting both proinflammatory and anti-inflammatory effects. This dual effect appears to be influenced by the nature of the cardiac injury or stress condition.

YAP and TAZ Although MST1/2 activity seems to promote an anti-inflammatory phenotype in macrophages during cardiac injury, downstream Hippo effectors YAP and TAZ appear to have a more inflammatory role in these cells.^104,105 Activated YAP and TAZ have been implicated in the modulation of inflammation in macrophages in response to MI.¹¹¹ That study revealed that YAP and TAZ promote a proinflammatory response by upregulating interleukin-6 expression and impede reparative responses by reducing arginase 1.¹¹¹ Furthermore, a deficiency of YAP and TAZ reprogrammed macrophage phenotypes, leading to improved cardiac fibrosis, hypertrophy, and cardiac function after MI.¹¹¹ In addition to its role in MI injury, YAP has been implicated in promoting a proinflammatory response in macrophages under PO in the heart. Macrophages lacking YAP exhibit increased expression of resolving and angiogenic genes.¹¹² Myeloid-specific YAP-knockout mice exhibit improved systolic function, reduced cardiac fibrosis, and attenuated pathological remodeling in response to PO stress.¹¹² Together, the current studies support the idea that YAP and TAZ activation in macrophages is associated with proinflammatory responses. Conversely, a lack of YAP and TAZ in macrophages improved cardiac function after cardiac injury such as MI or PO stress.

Summary

Collectively, the studies discussed in this review underscore the critical role of the Hippo pathway in the heart and highlight the functional regulation of the Hippo pathway in different cell types during HF. Each component of the Hippo pathway exhibits cell type specificity in the heart under normal conditions and in response to different cardiac stresses or injuries, and these responses may be detrimental or protective. Therefore, additional research on the Hippo pathway in more cell types and in different heart diseases is necessary.

Increasing lines of evidence suggest that YAP and TAZ can be regulated through non-canonical mechanisms, without direct involvement of the upstream canonical components of the Hippo pathway. It is possible that the activity of the Hippo pathway in some cell types is regulated in a non-cell autonomous manner through paracrine mechanisms. Thus, it would be helpful to understand the relevant cell-to-cell interactions using either single-cell RNA sequencing or spatial genomics.

Remarkably, novel small molecule inhibitors targeting the Hippo pathway have demonstrated exciting efficacy in cardiomyocyte proliferation and in treating a variety of cardiac injuries in animal models. Examples include XMU (targeting MST1/2), TRULI and TDI-011536 (targeting LATS1/2), and verteporfin (targeting YAP). Continued exploration will deepen our understanding of the regulatory mechanisms of HF, and may further support the Hippo pathway as a promising therapeutic target for the treatment of cardiovascular disease and HF.

Acknowledgments

The authors thank Daniela K. Zablocki for critical reading of the manuscript.

Sources of Funding

This work was supported, in part, by U.S. Public Health Service Grants HL67724, HL91469, HL102738, HL112330, HL138720, HL144626, HL150881, and AG27211; the Fondation Leducq Transatlantic Network of Excellence (15CBD04 to J.S.); American Heart Association Merit Award 20 (MERIT35120374 to J.S.); and a American Heart Association Predoctoral Fellowship (916222 to C.H.).

Disclosures

J.S. is a member of Circulation Journal’s Editorial Team. The remaining authors have no conflicts of interest to declare.

Author Contributions

Conceptualization and writing: C.H., J.F., D.P.D.R., J.S.; Creating the graphical abstract and writing: C.H., J.S.; Supervision: J.S.

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