Hippo-Yap signaling in cardiac and fibrotic remodeling

Abstract

Cardiac injury initiates a tissue remodeling process in which aberrant fibrosis plays a significant part, contributing to impaired contractility of the myocardium and the progression to heart failure. Fibrotic remodeling is characterized by the activation, proliferation, and differentiation of quiescent fibroblasts to myofibroblasts, and the resulting effects on the extracellular matrix and inflammatory milieu. Molecular mechanisms underlying fibroblast fate decisions and subsequent cardiac fibrosis are complex and remain incompletely understood. Emerging evidence has implicated the Hippo-Yap signaling pathway, originally discovered as a fundamental regulator of organ size, as an important mechanism that modulates fibroblast activity and adverse remodeling in the heart, while also exerting distinct cell type-specific functions that dictate opposing outcomes on heart failure. This brief review will focus on Hippo-Yap signaling in cardiomyocytes, cardiac fibroblasts, and other non-myocytes, and present mechanisms by which it may influence the course of cardiac fibrosis and dysfunction.

Introduction

Cardiac fibrosis and adverse cardiac remodeling are present in all etiologies of heart disease. The act of remodeling the extracellular matrix (ECM) is an active process with pleiotropic effects. While it is needed to prevent wall rupture in response to an acute injury associated with extensive myocyte loss, persistent fibrosis also drives pathology, and strongly correlates with worsened cardiac outcomes, including increased risk for arrhythmias and progression to heart failure, making it a clinical concern of great importance¹. Importantly, there are currently no effective treatments for fibrosis, either to limit or reverse the process. Based on studies leveraging cell type-specific and inducible genetic mouse models to manipulate in vivo gene expression, it has become evident that the resident fibroblast is the predominant cell type maintaining cardiac ECM at baseline, and is largely responsible for coordinating and executing fibrotic remodeling in response to stress^2–6. An emerging paradigm posits that cardiac fibroblasts are more heterogeneous and transit between more states than originally thought including quiescent or resting fibroblasts, activated intermediate fibroblasts, myofibroblasts, and matrifibrocytes⁷. However, a more comprehensive understanding of how fibroblasts transition between these diverse functional phenotypes during injury and wound healing, and how fibroblasts interact with and influence other cells within the myocardium, will likely be essential to developing improved therapeutic interventions targeting pathological cardiac remodeling.

Integrative regulation of fibroblast state and activity

Until recently, resident quiescent fibroblasts were thought to become activated and terminally differentiate to myofibroblasts, the cell type largely responsible for increased production of matricellular proteins and ECM modification following stress, eventually succumbing to apoptosis and tissue clearance. However, through the use of single cell RNA sequencing, it has become clear that determination of fibroblast fate following injury such as myocardial infarction (MI) requires more nuanced classification, and is likely more fluid than originally thought⁸. In the healthy heart, quiescent fibroblasts make up roughly 15% of non-myocytes⁹, and greater than 95% of these resident fibroblasts give rise to the active myofibroblast population post injury². Evidence indicates that periostin (Postn)-expressing activated fibroblasts peak ~2–3d post-MI and are highly proliferative, whereas differentiated myofibroblasts are greatest roughly 5–7 days after MI, and are defined by enhanced expression of fibrillar collagens and α smooth muscle actin (αSMA, Acta2 gene). Approximately 2 weeks post-MI and beyond, the matrifibrocyte predominates the mature scar, and exhibits high levels of chondrocyte and osteogenic gene expression, presumably to maintain scar integrity^{4, 5, 10, 11}. Importantly, not all active fibroblasts transition to the myofibroblast state as evidenced by αSMA expression in only a subset of fibroblasts following heart injury, suggesting multifaceted regulation of fate decisions¹². Moreover, the progressive loss of identifiable myofibroblast markers in cells of fibroblast origin that persist long-term following injury suggests this status is transient and is resolved through some combination of further differentiation/maturation (i.e. matrifibrocyte)¹⁰, deactivation⁴, and/or senescence, further highlighting the complexity of fibroblast state determination.

Elucidating the underlying signaling mechanisms that modulate fibroblast activation during cardiac stress are vitally important and have been the focus of much study. Fibroblast fate is regulated by extracellular cues that activate complex signaling networks and epigenetic modulation that ultimately results in modified transcriptional responses. Soluble ligands such as transforming growth factor β (TGF-β), wingless/integrated (Wnt), or angiotensin II (AngII) signal through cognate receptors to activate downstream effectors and initiate cell responses^{1, 13}. Convincing evidence has demonstrated that mitogen activated protein kinase (MAPK) p38α is a critical node regulating fibroblast function¹⁴. p38 is activated by non-canonical TGF-β signaling, as well as mechanical stimuli, and facilitates fibroblast activation through multiple mechanisms including serum response factor (SRF)- myocardin related transcription factor A (MRTF-A) transcriptional activation¹⁵ and transient receptor potential cation channel subfamily C member 6 (TRPC6)-mediated Ca2+ signaling to promote Calcineurin/nuclear factor of activated T cells (NFAT) function¹⁶. Histone and chromatin modification also regulate fibroblast differentiation. Inhibitors of class I histone deacetylases (HDACs), as well as bromodomain and extra-terminal domain (BET) family inhibitors, can prevent transcription of key fibroblast activation genes¹⁷, suppress cardiac fibroblast proliferation, and antagonize myofibroblast differentiation^{18, 19} demonstrating the importance of epigenetic regulation in determining fibroblast state transitions²⁰. Moreover, the RNA binding protein muscleblind-like1 (MBNL1) has been shown to stabilize and alternatively splice transcripts of many previously identified mediators of myofibroblast differentiation providing an additional level of positive regulation of fibrotic signaling pathways²¹.

Hippo-Yap pathway overview

The Hippo signaling pathway is a fundamental growth regulatory mechanism that was originally discovered in Drosophila and is evolutionarily conserved to humans. It is a kinase cascade whose activation is controlled by diverse inputs including growth factors, oxidative stress, and mechanical perturbation to mediate various cellular outputs, e.g. proliferation, differentiation, and survival²². The core components of the mammalian pathway include the serine/threonine kinases mammalian STE20-like (Mst1/2) and large tumor suppressor kinases (Lats1/2), the scaffold proteins Salvador (Sav1) and Mps one binder kinase activator (MOB1A/B), and the transcriptional co-activators Yes-associate protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz). Hippo pathway activation results in enhanced kinase activity leading to increased phosphorylation and cytosolic retention/degradation of Yap/Taz. When Hippo is turned off, Yap/Taz accumulate in the nucleus and stimulate expression of genes that regulate cell cycling, survival, and cell fate, a response that is primarily mediated through interaction with the TEA domain family transcription factors (TEAD1–4), although partnering with additional transcription factors also occurs²².

There is accumulating evidence that Hippo-Yap signaling modulates fibroblast activation and differentiation, and can impact the extent of fibrosis following tissue injury in multiple organs. Increased mechanical stresses are a hallmark of fibrotic remodeling and can result from altered cell-cell contacts, changes in cellular tension, or differences in ECM stiffness. The actin cytoskeleton is highly responsive to these mechanical cues, which are transduced intracellularly by membrane spanning protein complexes (e.g. adherens junctions, focal adhesions) and stimulate increased F-actin stress fiber formation. Yap/Taz monitor and respond to altered actin dynamics through increased activation, thereby linking mechanosensation to gene expression modulation²³. For example, enhanced substrate rigidity causes increased assembly of focal adhesion complexes and stress fiber formation, which elicits Yap/Taz nuclear localization and activity²⁴. Soluble factors including TGF-β, Wnt, and AngII have also been shown to cause Yap/Taz upregulation and promote enhanced function in fibroblasts through Hippo dependent and independent mechanisms^{25, 26}. Importantly, this Yap/Taz mediated transcriptional response has been directly implicated in mechanical stress-induced fibroblast proliferation, migration, and expression of myofibroblast and contractile associated genes including pro-collagen, αSMA, brain derived neurotrophic factor (Bdnf), fibronectin, and plasminogen activator inhibitor (PAI-1)^27–29. Conversely, fibroblasts cultured on soft matrix had reduced Yap/Taz function and suppressed fibroblast-related gene induction³⁰. Additional studies have demonstrated key in vivo roles for Yap and Taz in mediating fibroblast activation and ECM remodeling in liver, kidney, and lung^{27, 28, 31}, suggesting broad implications for Yap/Taz signaling in cell fate responses to physical stress and tissue fibrosis and remodeling.

Hippo-Yap signaling in cardiomyocytes: heart development and disease

Disruption of the Hippo pathway via cardiomyocyte deletion of Sav1 (or Mst1/2, or Lats1/2) during development, led to enhanced Yap activity and hyperproliferation of cardiomyocytes, abnormal heart growth, thickened ventricles, and embryonic lethality³² (Table). Conversely, targeted cardiomyocyte deletion of Yap resulted in cardiac hypoplasia, heart failure and early embryonic death, demonstrating the importance of balanced Yap activation for proper heart development. Hippo-Yap signaling in adult cardiomyocytes also plays a critical role in modulating responses to cardiac stress^{33, 34}. Cardiac Mst1 is activated in response to ischemic and non-ischemic pressure overload (PO) stress^{35, 36}. Transgenic inhibition of cardiomyocyte Mst1 was shown to attenuate adverse remodeling and improve survival through upregulation of autophagy and inhibition of apoptosis caused by MI³⁷. Similarly, inhibition of cardiomyocyte Lats2 reduced apoptosis and cardiac dysfunction induced by PO stress³⁸. The role of Sav1 is less straightforward, as inducible myocyte deletion in the adult heart triggered Yap activation and reversed MI-induced adverse remodeling and heart failure³⁹, demonstrating a robust cardioprotective benefit. On the other hand, postnatal Sav1 deficiency led to worsened cardiac function in response to chronic PO⁴⁰. This difference in outcomes is likely due to the type of stress imposed (permanent coronary artery occlusion vs increased left ventricular afterload) and the resulting effect on cardiomyocyte loss, which would be ameliorated by enhanced cardiomyocyte proliferation in the setting of MI but not necessarily during PO, where persistent myocyte dedifferentiation impaired contractile function.

Table.

Cardiac effects of Hippo-Yap pathway modulation in mice.

Gene	Model	Intervention	Phenotype	Ref
Yap	cKO	Nkx2.5-Cre	Lethal by E10.5, reduced CM proliferation, thin myocardium.	33
Yap	cKO	Tnnt2-Cre	Lethal by E16.5, reduced ventricle size, reduced CM proliferation, hypoplastic myocardium.	34
Yap	cKO	αMHC-Cre	Die between 11–20 weeks, dilated cardiomyopathy, increased CM apoptosis, and increased fibrosis. Defective neonatal cardiac regeneration post-MI. Hemizygous mice showed attenuated adaptive growth, worsened remodeling and greater cardiac dysfunction after MI or PO stress.	41–43
Yap	cTG	αMHC-YapS112A	Increased heart size, wall thickness, CM proliferation, improved function post-MI injury.	42
Yap	cKO	Tcf21-MCM	Attenuated fibrosis and improved cardiac function after MI.	26
Yap/Taz	cKO	Col1a2-CreERT	Reduced fibrosis and inflammation with improved heart function post-MI injury.	25
Yap/Taz	cKO	LysM-Cre	Attenuated fibrotic remodeling, reduced inflammation and enhanced angiogenesis in response to MI.	50
Yap/Taz	cKO	Wt1-CreERT2	Increased inflammation, fibrosis, wall rupture, and lethality following MI stress.	55
Sav1	cKO	Nkx2.5-Cre	Increased CM proliferation, cardiomegaly, ventricular septal defect.	32
Sav1	cKO	αMHC-CreERT2	Cardioprotective after MI, reduced fibrosis, improved function	39
Sav1	cKO	αMHC-Cre	Increased CM proliferation/dedifferentiation, worsened heart function after PO stress.	40
Lats1/2cKO		Nkx2.5-Cre	Excessive heart growth, ventricular septal defect.	32
Lats1/2cKO		Wt1-CreERT2	Lethal by E15.5, defective transition of epicardial precursors, reduced mature cardiac fibroblasts, impaired coronary vessel development.	46
Lats1/2cKO		Tcf21-MCM	Spontaneous fibrosis, myofibroblast differentiation, increased inflammation, and cardiac dysfunction.	49
Lats2	cTG-Dn	αMHC-Lats2K697A	Reduced CM apoptosis and improved fun ction after PO stress	38
Mst1/2	cKO	Nkx2.5-Cre	Excessive heart growth, vent icular septal defect.	32
Mst1	cTG-Dn	αMHC-Mst1K59R	Increased autophagy, reduced scar size, and improved cardiac function after MI.	37
Rassf1a	cKO	αMHC-Cre	Reduced CM apoptosis and fibrosis, with improved cardiac function after PO stress.	36
Rassf1a	cTG-Dn	αMHC-Rassf1aL308P	Attenuated CM apoptosis and fibrosis in response to PO.	36

cKO, conditional knockout; cTG, cell type-specific transgenic; CM, cardiomyocyte; Dn, dominant-negative; MI, myocardial infarction; PO, pressure overload.

In contrast to the interventions above that upregulated Yap activity, cardiomyocyte-specific postnatal deletion of Yap caused spontaneous dilated cardiomyopathy associated with increased apoptosis and premature death between 11–20 weeks^{41, 42}. Consistently, mice with hemizygous reduction of myocyte Yap had increased cardiac fibrosis and dysfunction following either MI or PO stress^{41, 43}. Neonatal cardiac regeneration, which occurs in mice prior to P7⁴⁴, was abolished in Yap deficient mice, whereas cardiac expression of a constitutively active Yap was able to attenuate myocyte apoptosis and fibrosis, and improve heart function in response to MI⁴². Additionally, cardiomyocyte Yap was shown to promote the expression of Wntless and subsequent secretion of Wnt5a and Wnt9a, which mediated paracrine inhibitory effects on cardiac fibroblasts resulting in reduced inflammation and fibrosis, and enhanced scar resolution after MI⁴⁵. In sum, it has become increasingly clear that cardiomyocyte modulation of Hippo signaling is a critical determinant of cardiac remodeling and dysfunction in response to pathological stress, and interventions that promote Yap activation in the cardiomyocyte confer protection through enhanced survival and regenerative potential in the adult heart.

Function of Hippo-Yap/Taz in cardiac fibroblasts: homeostatic and injury responses

Studies using targeted genetic approaches in mice have demonstrated an important role for Hippo-Yap in modulating cardiac fibroblast maturation as well as myofibroblast differentiation. Epicardial cell-specific deletion of Lats1/2 kinases during embryonic development using the Wt1CreERT driver mice resulted in defective transition of epicardial precursors to mature cardiac fibroblasts, improper coronary vessel formation, and rapid lethality⁴⁶. In a series of elegant single cell RNA sequencing and lineage tracing experiments, this was shown to be driven through aberrant Yap activation, and at least partly mediated through newly identified Yap-TEAD target genes, Dpp4 and Dhrs3. Enhanced Dhrs3 expression antagonized retinoic acid signaling to prevent epicardial fibroblast formation, while upregulation of the matricellular factor Dpp4 altered ECM composition and blunted normal vascular development. These results indicate a critical role for Lats-Yap in the proper state transition of epicardial precursors to differentiated cardiac fibroblasts that occurs during cardiogenesis.

Conditional deletion of Lats1/2 in adult cardiac fibroblasts using Tcf21-MerCreMer (to target resident cardiac fibroblasts for Cre expression) was sufficient to induce spontaneous fibrosis and cardiac dysfunction within 3 weeks of tamoxifen administration²⁹. Hearts from Lats1/2 mutant mice also contained increased populations of inflammatory cardiac fibroblasts, as well as increased myofibroblast-like cells that arose from quiescent fibroblasts indicating that Lats1/2 deletion in resting cardiac fibroblasts promotes a cell state transition typically seen following injury. Enhanced Yap activity in Lats1/2 targeted cells mediated myofibroblast differentiation, and was also responsible for non-cell autonomous paracrine effects that elicited cardiac myeloid cell recruitment. Importantly, Lats1/2 deficient mice subjected to MI had augmented fibrosis, decreased survival, and an extension of the cardiac fibroblast proliferative window, a phenotype that was partly rescued by concomitant deletion of Yap and Taz. ATAC-seq analysis of global chromatin accessibility signatures indicated that Lats1/2 cardiac fibroblast deletion favored a myofibroblast-like epigenetic and transcriptional profile, likely mediated by enrichment of Yap-TEAD occupancy at active enhancer regions. These findings demonstrate a key role for Lats-Yap in modulating cardiac fibroblast state transition, whereby Lats1/2 activity restrains myofibroblast differentiation of quiescent fibroblasts and pro-inflammatory signaling between cell types.

Myocardial infarction fundamentally alters properties of the ECM and leads to heterogeneity of collagen deposition and alignment. Disturbed collagen fiber organization not only impairs heart pump function, but also signals locally to endogenous cells to influence their behavior. To this effect, recent work has elegantly demonstrated that regions of infarcted myocardium with aligned collagen were more prevalent at the border zone and exhibited myofibroblast enrichment when compared to the more disorganized collagen containing infarct core⁴⁷. Moreover, providing an aligned ECM substrate in vitro was sufficient to promote myofibroblast differentiation of adult cardiac fibroblasts, and this response was mediated by focal adhesion-dependent upregulation of a Yap-TEAD-p38 signaling complex. These findings expanded upon previous work that identified a requirement of fibroblast p38α for scar formation and myofibroblast differentiation in vivo¹⁴, and revealed a novel mechanism that links p38 to YAP posttranslational regulation and activation of myofibroblast transcriptional activity in response to collagen fiber disorganization after MI.

Several studies have examined the role of cardiac fibroblast Yap and Taz by directly modulating their expression in vivo. Yap is upregulated and activated in cardiac fibroblasts 3–5 days following MI, and selective deletion of Yap in quiescent cardiac fibroblasts using Tcf21-MerCreMer prior to infarct attenuated the extent of cardiac fibroblast proliferation and collagen deposition, and preserved heart function²⁶. Yap was shown to positively regulate expression of MRTF-A through TEAD-dependent transcription, and Yap mutant mice had reduced myocardial MRTF-A post-MI. No difference in cardiac hypertrophy was observed, although cardiomyocyte apoptosis was reduced in Yap deficient mice, indicating potential pathological paracrine Yap dependent signaling from cardiac fibroblasts to myocytes during MI. Of note, the Hippo pathway activator, Ras association domain family 1 isoform A (RASSF1A), was shown to suppress the expression and secretion of tumor necrosis factor α (TNFα) from cardiac fibroblasts thereby influencing cardiomyocyte function, however the role of Yap in mediating this response was not defined³⁶. Importantly, similar results were observed using a different Cre driver (Col1a2-CreERT) to induce deletion of both Yap and Taz in cardiac fibroblasts prior to MI²⁵. Targeted inhibition of both genes prevented cardiac dysfunction and reduced fibrosis after injury. Intriguingly, no increase in ventricular rupture was observed in Yap/Taz deficient mice after MI indicating other pathways mediate reparative processes. Double mutant mice also had attenuated macrophage recruitment and blunted pro-inflammatory gene expression. Indeed, cardiac fibroblasts deficient for Yap/Taz had reduced ability to stimulate macrophage inflammatory polarization via impaired IL-33 paracrine signaling from cardiac fibroblasts. Together these studies demonstrate the important role for cardiac fibroblast Yap/Taz in mediating fibrotic remodeling and dysfunction in response to ischemic injury, a clear contrast to their beneficial functions in cardiomyocytes (Figure).

Figure.

The role of Hippo-Yap signaling in response to cardiac stress – the cell type-specific functions of Yap/Taz activation and their influence on fibrotic remodeling. Cardiac injury resulting from myocardial infarction (MI) or pressure overload (PO) stress can modulate Hippo-Yap signaling in both positive and negative directions and is dependent on downstream signaling mechanism engagement and cell type. Activation of canonical Hippo signaling (i.e. Mst1/2 and Lats1/2) causes the inhibitory phosphorylation of Yap/Taz and their subsequent nuclear exclusion and degradation. In cardiomyocytes, this leads to increased apoptosis, suppression of cell growth and impaired heart function. Conversely, Hippo disruption and Yap activation promotes cardiomyocyte survival, growth, proliferation, and generally attenuates dysfunction. Cardiac stress can also elicit increased availability of soluble factors (e.g. TGF-β, AngII) and altered tissue stiffness, leading to increased Yap/Taz activation. In cardiac fibroblasts, Yap/Taz mediate proliferation, myofibroblast differentiation, and pro-inflammatory signaling including the secretion of cytokines and chemokines that contribute to enhanced immune cell recruitment. Following MI, Yap/Taz are activated in macrophages present in the heart. Myeloid selective deletion of Yap/Taz affords cardioprotection against MI-induced inflammation, adverse fibrotic remodeling, and cardiac dysfunction through effects on macrophage polarization, wound healing and angiogenic responses. Due to disparate outcomes mediated by Hippo-Yap signaling according to cell type, interventions aimed at modulating the pathway therapeutically must prove selective or risk unintended deleterious effects.

To directly investigate the effect of Yap gain of function in cardiac fibroblasts in vivo, an adeno-associated virus-mediated approach was employed⁴⁸. Ectopic expression of activated Yap was driven by the Tcf21 promoter in mice, and elicited myofibroblast differentiation and increased collagen deposition in the absence of stress. Markers of inflammation including macrophage abundance and pro-inflammatory cytokine and chemokine expression were also observed in Yap transduced myocardium. CC motif chemokine ligand 2 (CCL2), which is required for the tissue recruitment of CC motif chemokine receptor 2 (CCR2)-positive monocytes/macrophages⁴⁹, was shown to be a Yap target gene in cultured cardiac fibroblasts and was upregulated following Yap expression in vivo. A similar phenotype was observed in mice genetically engineered to selectively express active mutant Yap in cardiac fibroblasts²⁵. After MI, Yap transgenic mice showed augmented myocardial fibrosis, elevated myofibroblast burden, and enhanced inflammatory gene expression compared to controls. Taken together, these studies indicate activation of cardiac fibroblast Yap can induce and enhance injury-related fibrotic and inflammatory remodeling in the heart.

Function of Yap/Taz in cardiac macrophages and epicardium to influence inflammation

Macrophages are critical mediators of cardiac inflammation and fibrosis in response to injury. Mice with myeloid selective Yap/Taz deletion (LysM-Cre) showed improved cardiac function following MI injury⁵⁰. Reduced fibrotic area, along with reduced cardiomyocyte hypertrophy, and enhanced expression of angiogenic markers including vascular endothelial growth factor (Vegf) and cluster of differentiation 31 (CD31) were also observed in Yap/Taz double knockout mice. Conversely, using LysM-Cre to drive ectopic expression of activated mutant Yap in the myeloid compartment resulted in increased collagen deposition post-infarct. To investigate a potential mechanism, transcriptome profiling of Yap/Taz double knockout bone marrow-derived macrophages (BMDMs) was performed and revealed altered gene expression patterns that indicated impaired pro-inflammatory polarization and an enhanced reparative phenotype. The ability of Yap to directly promote IL-6 expression while transcriptionally repressing arginase 1 (Arg1) via engagement of an HDAC3-nuclear receptor corepressor (NCor1) complex was demonstrated and likely contributed to the shift in polarization and the aberrant inflammatory phenotype. These findings are consistent with reports that Yap exerts pro-inflammatory functions in macrophages in other systems^51–54, and supports the hypothesis that interventions to target Yap/Taz inhibition may antagonize pathological remodeling through cell autonomous and non-autonomous mechanisms.

The function of Yap/Taz in the adult epicardium has also been investigated⁵⁵. Conditional deletion of both genes resulted in a hyperinflammatory response and diffuse fibrosis throughout the LV posterior wall, and was associated with worsened cardiac function and increased mortality after MI. Yap/Taz deletion caused dysregulated cytokine expression, and further analysis identified interferon gamma (IFNγ) as a direct transcriptional target of Yap-TEAD. Blunted IFNγ expression in Yap/Taz deficient mice prevented Treg recruitment and accentuated inflammation in response to infarct. These results demonstrate the importance of Yap/Taz function in the epicardium during ischemic injury to restrain inflammation and pathological remodeling, and serve to highlight once again the cell type-specificity of Hippo-Yap function in the regulation of wound healing processes in the heart.

Implications for human heart disease

The potential relevance of Hippo-Yap in fibrotic remodeling in human heart failure is beginning to emerge. Recent work has reported that Yap expression was downregulated in myocardium from heart failure patients³⁹, however this analysis did not discriminate by cell type and likely reflects Yap status in cardiomyocytes. In contrast, Yap expression and activation were selectively increased in cardiac fibroblasts isolated from heart tissue of dilated cardiomyopathy (DCM) patients⁵⁶, indicating that differences in cell type-specific regulation of Hippo-Yap observed in mice may also occur in humans. Additional evidence to suggest Yap involvement in cardiac fibrosis in patients was revealed using RNA sequencing. Enrichment of genes associated with ECM organization and ECM-membrane interactions were found to overlap between DCM cardiac fibroblasts and healthy cardiac fibroblasts expressing active Yap. Moreover, forced Yap expression in healthy human cardiac fibroblasts upregulated myofibroblast-associated gene expression²⁶ and stimulated the ability to contract collagen lattice, while cardiac fibroblasts from DCM patients showed comparable enhanced contractile capability that was abolished by Yap pharmacological inhibition using verteporfin⁵⁶. These findings implicate Yap activity in diseased cardiac fibroblasts as a potential pathological driver of ECM remodeling in heart failure patients, although more studies are needed to further refine this hypothesis.

Conclusion

Cardiac injury initiates adverse remodeling of the myocardium characterized by excessive deposition of the ECM, increased stiffness, and impaired contractility that contributes to the progression of heart failure. The fibrotic response is incredibly complex, is mediated by multiple integrated signaling pathways, and involves interactions between distinct cell types, yet our understanding of the molecular circuitry that governs it remains incomplete. The Hippo-Yap pathway has emerged as an important mechanism regulating stress responses in cardiomyocytes, cardiac fibroblasts, macrophages, and others, and has evident implications for fibrosis, inflammation, and wound healing. The question remains, how do we leverage this knowledge to bridge the translational gap to patients? Future studies should continue to elucidate cell type specific Hippo-Yap functions, which thus far have been shown to mediate selective and sometimes opposing outcomes on cardiac physiology and disease progression. A focus on potential differences in transcription factor accessibility and partnering with Yap/Taz may prove important. Moreover, mechanisms governing how these signals influence cell-to-cell interactions to modulate pathological remodeling of the heart are only beginning to manifest and require further investigation. For example, Hippo-Yap mediates bidirectional communication between immune and cardiac resident cells^{50, 55}, yet how these paracrine actions can be leveraged to improve outcomes remains unclear. It will also be imperative that disease model be carefully considered (e.g. ischemia versus PO) when interpreting the efficacy of Hippo pathway modulation, as the type of injury can elicit distinct responses and may necessitate selective interventions. Ultimately, therapeutics aimed at manipulating this signaling pathway still hold great promise, yet will likely require targeted approaches to alleviate fibrotic responses while maintaining myocardial integrity and function.