Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation.

BACKGROUND: Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. METHODS: Here, we present a method to obtain high-quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. RESULTS: After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression, we could identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton-associated protein 4 as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Cytoskeleton-associated protein 4 inhibition in activated fibroblasts treated with transforming growth factor ß triggered a greater increase in the expression of genes related to activated fibroblasts compared with control, suggesting a role of cytoskeleton-associated protein 4 in modulating fibroblast activation in the injured heart. CONCLUSIONS: Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

Stress-Activated Kinase Mitogen-Activated Kinase Kinase-7 Governs Epigenetics of Cardiac Repolarization for Arrhythmia Prevention.

BACKGROUND: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death. METHODS: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell-derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K+ channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts. CONCLUSIONS: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported.

Targeted deletion of ERK2 in cardiomyocytes attenuates hypertrophic response but provokes pathological stress induced cardiac dysfunction.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of cardiac hypertrophy and myocyte survival. Extracellular signal regulated protein kinase 1 and 2 (ERK1/2) are key components in the MAPK signaling pathways. Dysfunction of ERK1/2 in congenital heart diseases (Noonan syndrome and LEOPARD syndrome) leads to cardiac hypertrophy. ERK2 contributes 70% of protein content to total ERK1/2 content in myocardium; however, the specific role of ERK2 in regulating cardiac hypertrophy is yet to be further defined. To investigate the specific role of ERK2 played in the cardiomyocytes, we generated and examined mice with cardiomyocyte-specific deletion of the erk2 gene (ERK2(cko) mice). Following short-term pathological hypertrophic stresses, the mutant mice showed attenuated hypertrophic remodeling characterized by a blunted increase in the cross-sectional area of individual myocytes, downregulation of hypertrophic foetal gene markers (ANP and BNP), and less interstitial fibrosis. However, increased cardiomyocyte apoptosis was observed. Upon prolonged stimulation, ERK2(cko) mice developed deterioration in cardiac function. However, absence of ERK2 did not affect physiological hypertrophy induced by 4weeks of swimming exercise. These results revealed an essential role for ERK2 in cardiomyocytes in the development of pathological hypertrophic remodeling and resistance to cell death.

Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy is a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death, with currently no effective treatment. Plakophilin 2 (PKP2) is the most frequently affected gene. Here we show that adeno-associated virus (AAV)-mediated delivery of PKP2 in PKP2c.2013delC/WT induced pluripotent stem cell-derived cardiomyocytes restored not only cardiac PKP2 levels but also the levels of other junctional proteins, found to be decreased in response to the mutation. PKP2 restoration improved sodium conduction, indicating rescue of the arrhythmic substrate in PKP2 mutant induced pluripotent stem cell-derived cardiomyocytes. Additionally, it enhanced contractile function and normalized contraction kinetics in PKP2 mutant engineered human myocardium. Recovery of desmosomal integrity and cardiac function was corroborated in vivo, by treating heterozygous Pkp2c.1755delA knock-in mice. Long-term treatment with AAV9-PKP2 prevented cardiac dysfunction in 12-month-old Pkp2c.1755delA/WT mice, without affecting wild-type mice. These findings encourage clinical exploration of PKP2 gene therapy for patients with PKP2 haploinsufficiency.

PITX2 induction leads to impaired cardiomyocyte function in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive disease characterized by electrophysiological and structural remodeling of the ventricles. However, the disease-causing molecular pathways, as a consequence of desmosomal mutations, are poorly understood. Here, we identified a novel missense mutation within desmoplakin in a patient clinically diagnosed with ACM. Using CRISPR-Cas9, we corrected this mutation in patient-derived human induced pluripotent stem cells (hiPSCs) and generated an independent knockin hiPSC line carrying the same mutation. Mutant cardiomyocytes displayed a decline in connexin 43, NaV1.5, and desmosomal proteins, which was accompanied by a prolonged action potential duration. Interestingly, paired-like homeodomain 2 (PITX2), a transcription factor that acts a repressor of connexin 43, NaV1.5, and desmoplakin, was induced in mutant cardiomyocytes. We validated these results in control cardiomyocytes in which PITX2 was either depleted or overexpressed. Importantly, knockdown of PITX2 in patient-derived cardiomyocytes is sufficient to restore the levels of desmoplakin, connexin 43, and NaV1.5.

Desmosomal protein degradation as an underlying cause of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 (PKP2). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

Pak1 as a novel therapeutic target for antihypertrophic treatment in the heart.

BACKGROUND: Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy. METHODS AND RESULTS: Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation. CONCLUSIONS: These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Oxidized low-density lipoproteins as a novel risk factor and therapeutic target for ACM.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease involving arrhythmia in young adults accompanied by structural changes at later stages. In this issue of EMBO Molecular Medicine, Sommariva et al (2021) identified a positive correlation between circulating levels of oxidized low-density lipoproteins (oxLDL) and ACM disease penetrance, which contributes to fibro-fatty cardiac remodeling via the oxLDL/CD36/PPARγ axis. These data identify oxidized low-density lipoproteins as a risk factor for ACM and uncover a novel therapeutic intervention option to block disease pathogenesis.

The p21-activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration.

p21-activated kinase 1 (Pak1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1. It has been previously demonstrated to be involved in cardiac protection. Based on recent studies, this review provides an overview of the role of Pak1 in cardiac diseases including disrupted Ca2+ homoeostasis-related cardiac arrhythmias, adrenergic stress- and pressure overload-induced hypertrophy, and ischaemia/reperfusion injury. These findings demonstrate the important role of Pak1 mediated through the phosphorylation and transcriptional modification of hypertrophy and/or arrhythmia-related genes. This review also discusses the anti-arrhythmic and anti-hypertrophic, protective function of Pak1 and the beneficial effects of fingolimod (an FDA-approved sphingolipid drug), a Pak1 activator, and its ability to prevent arrhythmias and cardiac hypertrophy. These findings also highlight the therapeutic potential of Pak1 signalling in the treatment and prevention of cardiac diseases. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Smad3 Couples Pak1 With the Antihypertrophic Pathway Through the E3 Ubiquitin Ligase, Fbxo32.

Pathological cardiac hypertrophy is regarded as a critical intermediate step toward the development of heart failure. Many signal transduction cascades are demonstrated to dictate the induction and progression of pathological hypertrophy; however, our understanding in regulatory mechanisms responsible for the suppression of hypertrophy remains limited. In this study, we showed that exacerbated hypertrophy induced by pressure overload in cardiac-deleted Pak1 mice was attributable to a failure to upregulate the antihypertrophic E3 ligase, Fbxo32, responsible for targeting proteins for the ubiquitin-degradation pathway. Under pressure overload, cardiac overexpression of constitutively active Pak1 mice manifested strong resilience against pathological hypertrophic remodeling. Mechanistic studies demonstrated that subsequent to Pak1 activation, the binding of Smad3 on a critical singular AGAC(-286)-binding site on the FBXO32 promoter was crucial for its transcriptional regulation. Pharmacological upregulation of Fbxo32 by Berberine ameliorated hypertrophic remodeling and improved cardiac performance in cardiac-deficient Pak1 mice under pressure overload. Our findings discover Smad3 and Fbxo32 as novel downstream components of the Pak1-dependent signaling pathway for the suppression of hypertrophy. This discovery opens a new venue for opportunities to identify novel targets for the management of cardiac hypertrophy.

Novel insights into mechanisms for Pak1-mediated regulation of cardiac Ca(2+) homeostasis.

A series of recent studies report novel roles for Pak1, a key member of the highly conserved family of serine-threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1, in cardiac physiology and cardioprotection. Previous studies had identified Pak1 in the regulation of hypertrophic remodeling that could potentially lead to heart failure. This article provides a review of more recent findings on the roles of Pak1 in cardiac Ca(2+) homeostasis. These findings identified crucial roles for Pak1 in cardiomyocyte Ca(2+) handling and demonstrated that it functions through unique mechanisms involving regulation of the post-transcriptional activity of key Ca(2+)-handling proteins, including the expression of Ca(2+)-ATPase SERCA2a, along with the speculative possibility of an involvement in the maintenance of transverse (T)-tubular structure. They highlight important regulatory functions of Pak1 in Ca(2+) homeostasis in cardiac cells, and identify novel potential therapeutic strategies directed at manipulation of Pak1 signaling for the management of cardiac disease, particularly heart failure.

Pak1 is required to maintain ventricular Ca²âº homeostasis and electrophysiological stability through SERCA2a regulation in mice.

BACKGROUND: Impaired sarcoplasmic reticular Ca(2+) uptake resulting from decreased sarcoplasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under both normal physiological and acute and chronic ß-adrenergic stress conditions. METHODS AND RESULTS: Mice with a cardiomyocyte-specific Pak1 deletion (Pak1(cko)), but not controls (Pak1(f/f)), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute ß-adrenergic or chronic ß-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1(cko) ventricular myocytes correspondingly showed aberrant cellular Ca(2+) homeostasis. Pak1(cko) hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1-infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a. CONCLUSIONS: These findings indicate that Pak1 is required to maintain ventricular Ca(2+) homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.

Mkk4 is a negative regulator of the transforming growth factor beta 1 signaling associated with atrial remodeling and arrhythmogenesis with age.

BACKGROUND: Atrial fibrillation (AF), often associated with structural, fibrotic change in cardiac tissues involving regulatory signaling mediators, becomes increasingly common with age. In the present study, we explored the role of mitogen-activated protein kinase kinase 4 (Mkk4), a critical component of the stress-activated mitogen-activated protein kinase family, in age-associated AF. METHODS AND RESULTS: We developed a novel mouse model with a selective inactivation of atrial cardiomyocyte Mkk4 (Mkk4(ACKO)). We characterized and compared electrophysiological, histological, and molecular features of young (3- to 4-month), adult (6-month), and old (1-year) Mkk4(ACKO) mice with age-matched control littermates (Mkk4(F/F)). Aging Mkk4(ACKO) mice were more susceptible to atrial tachyarrhythmias than the corresponding Mkk4(F/F) mice, showing characteristic slow and dispersed atrial conduction, for which modeling studies demonstrated potential arrhythmic effects. These differences paralleled increased interstitial fibrosis, upregulated transforming growth factor beta 1 (TGF-ß1) signaling and dysregulation of matrix metalloproteinases in Mkk4(ACKO), compared to Mkk4(F/F), atria. Mkk4 inactivation increased the sensitivity of cultured cardiomyocytes to angiotensin II-induced activation of TGF-ß1 signaling. This, in turn, enhanced expression of profibrotic molecules in cultured cardiac fibroblasts, suggesting cross-talk between these two cell types in profibrotic signaling. Finally, human atrial tissues in AF showed a Mkk4 downregulation associated with increased production of profibrotic molecules, compared to findings in tissue from control subjects in sinus rhythm. CONCLUSIONS: These findings together demonstrate, for the first time, that Mkk4 is a negative regulator of the TGF-ß1 signaling associated with atrial remodeling and arrhythmogenesis with age, establishing Mkk4 as a new potential therapeutic target for treating AF.

A novel immunomodulator, FTY-720 reverses existing cardiac hypertrophy and fibrosis from pressure overload by targeting NFAT (nuclear factor of activated T-cells) signaling and periostin.

BACKGROUND: Hypertension or aortic stenosis causes pressure overload, which evokes hypertrophic myocardial growth. Sustained cardiac hypertrophy eventually progresses to heart failure. Growing evidence indicates that restraining hypertrophy could be beneficial; here, we discovered that FTY-720, an immunomodulator for treating multiple sclerosis, can reverse existing cardiac hypertrophy/fibrosis. METHODS AND RESULTS: Male C57/Bl6 mice underwent transverse aortic constriction (TAC) for 1 week followed by FTY-720 treatment for 2 weeks under continuing TAC. Compared with vehicle-treated TAC hearts, FTY-720 significantly reduced ventricular mass, ameliorated fibrosis, and improved cardiac performance. Mechanistic studies led us to discover that FTY-720 appreciably inhibited nuclear factor of activated T-cells (NFAT) activity. Moreover, we found that in primary cardiomyocytes (rat and human) pertussis toxin (Gi-coupled receptor inhibitor) substantially blocked the antihypertrophic effect of FTY-720. This observation was confirmed in a mouse model of pressure overload. Interestingly, gene array analysis of TAC hearts revealed that FTY-720 profoundly decreased gene expression of a group of matricellular proteins, of which periostin was prominent. Analysis of periostin protein expression in TAC-myocardium, as well as in rat and human cardiac fibroblasts, confirmed the array data. Moreover, we found that FTY-720 treatment or knockdown of periostin protein was able to inhibit transforming growth factor-ß responsiveness and decrease collagen expression. CONCLUSIONS: FTY-720 alleviates existing cardiac hypertrophy/fibrosis through mechanisms involving negative regulation of NFAT activity in cardiomyocytes and reduction of periostin expression allowing for a more homeostatic extracellular compartment milieu. Together, FTY-720 or its analogues could be a promising new approach for treating hypertrophic/fibrotic heart disease.