Signaling In Cardiac Fibroblasts Research Articles

Transcriptional coactivation of NRF2 signaling in cardiac fibroblasts promotes resistance to oxidative stress

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.

Intracellular and extracellular Cyclophilin a promote cardiac fibrosis through TGF-β signaling in response to angiotensin Ⅱ

Cardiac fibrosis is characterized by abnormal proliferation of cardiac fibroblasts (CFs) and ventricular remodeling, which finally leads to heart failure. Inflammation and oxidative stress play a central role in the development of cardiac fibrosis. CyPA (Cyclophilin A) is a main proinflammatory cytokine secreted under the conditions of oxidative stress. The mechanisms by which intracellular and extracellular CyPA interact with CFs are unclear. Male C57BL/6 J mice received angiotensin Ⅱ (Ang Ⅱ) or vehicle for 4 weeks. Inhibition of CyPA significantly reversed Ang Ⅱ-induced cardiac hypertrophy and fibrosis. Mechanically, TGF-β (Transforming growth factor-β) signaling was found to be an indispensable downstream factor of CyPA-mediated myofibroblast differentiation and proliferation. Furthermore, intracellular CyPA and extracellular CyPA activate TGF-β signaling through NOD-like receptor protein 3 (NLRP3) inflammasome and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, respectively. Pharmacological inhibition of CyPA and its receptor CD147 implemented by Triptolide also attenuated the expression of TGF-β signaling and cardiac fibrosis in Ang Ⅱ-model. These studies elucidate a novel mechanism by which CyPA promotes TGF-β and its downstream signaling in CFs and identify CyPA (both intracellular and extracellular) as plausible therapeutic targets for preventing or treating cardiac fibrosis induced by chronic Ang Ⅱ stimulation.

Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit.

Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

Regulation of cardiac fibroblast cell death by unfolded protein response signaling.

The endoplasmic reticulum (ER) is a tightly regulated organelle that requires specific environmental properties to efficiently carry out its function as a major site of protein synthesis and folding. Embedded in the ER membrane, ER stress sensors inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) serve as a sensitive quality control system collectively known as the unfolded protein response (UPR). In response to an accumulation of misfolded proteins, the UPR signals for protective mechanisms to cope with the cellular stress. Under prolonged unstable conditions and an inability to regain homeostasis, the UPR can shift from its original adaptive response to mechanisms leading to UPR-induced apoptosis. These UPR signaling pathways have been implicated as an important feature in the development of cardiac fibrosis, but identifying effective treatments has been difficult. Therefore, the apoptotic mechanisms of UPR signaling in cardiac fibroblasts (CFs) are important to our understanding of chronic fibrosis in the heart. Here, we summarize the maladaptive side of the UPR, activated downstream pathways associated with cell death, and agents that have been used to modify UPR-induced apoptosis in CFs.

TAX1BP1 downregulation by STAT3 in cardiac fibroblasts contributes to diabetes-induced heart failure with preserved ejection fraction

Heart failure (HF) with preserved ejection fraction (HFpEF) is now the most common form of HF and has been reported to be closely related to diabetes. Accumulating evidence suggests that HFpEF patients exhibit cardiac fibrosis. This study investigates whether direct targeted inhibition of the activation of cardiac fibroblasts (CFs), the main effector cells in cardiac fibrosis, improves diabetes-induced HFpEF and elucidates the underlying mechanisms. Twenty-week-old db/db mice exhibited HFpEF, as confirmed by echocardiography and hemodynamic measurements. Proteomics was performed on CFs isolated from the hearts of 20-week-old C57BL/6 and db/db mice. Bioinformatic prediction was used to identify target proteins. Experimental validation was performed in both high glucose (HG)-treated neonatal mouse CFs (NMCFs) and diabetic hearts. TAX1 binding protein 1 (TAX1BP1) was identified as the most significantly differentially expressed protein between 20-week-old C57BL/6 and db/db mice. TAX1BP1 mRNA and protein were markedly downregulated in CFs from diabetic hearts and HG-cultured NMCFs. Overexpression of TAX1BP1 profoundly inhibited HG/diabetes-induced NF-κB nuclear translocation and collagen synthesis in CFs, improved cardiac fibrosis, hypertrophy, inflammation and HFpEF in diabetic mice. Mechanistically, signal transducer and activator of transcription 3 (STAT3), which is phosphorylated and translocated from the cytoplasm into the nucleus under hyperglycemic conditions, bound to TAX1BP1 promoter and blocked TAX1BP1 transcriptional activity, consequently promoting NF-κB nuclear translocation and collagen synthesis in CFs, aggravating cardiac fibrosis, hypertrophy and inflammation, leading to HFpEF in db/db mice. Taken together, our findings demonstrate that targeting regulation of STAT3-TAX1BP1-NF-κB signaling in CFs may be a promising therapeutic approach for diabetes-induced HFpEF.

Abstract 15535: SGK1 is a Key Mediator of Pathological Cardiac Fibrosis

Background: Cardiac fibroblasts initially respond to injury adaptively, proliferating and differentiating into myofibroblasts to maintain cardiac structure and function. However, prolonged cardiac stress leads to pathological cardiac fibrosis, ultimately resulting in heart failure (HF). Thus, there is a major interest in developing therapeutic strategies to constrain fibroblast differentiation and sustained activation. We recently identified the cytosolic protein kinase, serum/glucocorticoid regulated kinase 1 (SGK1), as being largely involved with pathological remodeling in the cardiac myocyte. While there have been reports that SGK1 may also play a role in fibrosis, the mechanistic role for SGK1 in cardiac fibroblasts in response to injury remains unknown. Hypothesis: SGK1 promotes pathological cardiac fibrosis and exacerbates progression to HF. Methods: Transforming growth factor beta, (TGFβ) was used in primary cardiac fibroblasts (CFBs), in vitro , and transverse aortic constriction (TAC) was used as a HF model in mice, in vivo . CFBs were transfected with siRNA to knockdown SGK1, in vitro , and SGK1 floxed mice were injected with a novel AAV9 utilizing a myofibroblast-specific promoter to knockout SGK1 specifically in myofibroblasts (SGK1 mfbKO), in vivo . Results: SGK1 signaling was elevated in cardiac explants obtained from ischemic human HF patients which led us to further evaluate SGK1 activity in CFBs. SGK1 knockdown in CFBs attenuated TGFβ-mediated differentiation and fibrotic signaling. SGK1 mfbKO mice subjected to TAC showed preserved systolic and diastolic cardiac function, measured by echocardiography. At the biochemical level, pathological remodeling and gene markers of fibrosis were attenuated in SGK1 mfbKO mice as determined by qRT-PCR of cardiac extracts. Finally, treatment of CFBs with a small molecule SGK1 inhibitor attenuated differentiation and fibrotic signaling in response to TGFβ treatment. Conclusion: SGK1 knockdown is protective against cardiac fibrosis, attenuating differentiation and fibrotic signaling in CFBs. Further, SGK1 mfbKO protected against pathological cardiac remodeling and functional decline in a mouse model of HF, highlighting the specific pathophysiological contributions of CFBs.

Computational screen for sex-specific drug effects in a cardiac fibroblast signaling network model

Heart disease is the leading cause of death in both men and women. Cardiac fibrosis is the uncontrolled accumulation of extracellular matrix proteins, which can exacerbate the progression of heart failure, and there are currently no drugs approved specifically to target matrix accumulation in the heart. Computational signaling network models (SNMs) can be used to facilitate discovery of novel drug targets. However, the vast majority of SNMs are not sex-specific and/or are developed and validated using data skewed towards male in vitro and in vivo samples. Biological sex is an important consideration in cardiovascular health and drug development. In this study, we integrate a cardiac fibroblast SNM with estrogen signaling pathways to create sex-specific SNMs. The sex-specific SNMs demonstrated high validation accuracy compared to in vitro experimental studies in the literature while also elucidating how estrogen signaling can modulate the effect of fibrotic cytokines via multi-pathway interactions. Further, perturbation analysis and drug screening uncovered several drug compounds predicted to generate divergent fibrotic responses in male vs. female conditions, which warrant further study in the pursuit of sex-specific treatment recommendations for cardiac fibrosis. Future model development and validation will require more generation of sex-specific data to further enhance modeling capabilities for clinically relevant sex-specific predictions of cardiac fibrosis and treatment.

Abstract P3094: Cell-autonomous Immune Signaling In Human Cardiac Fibroblasts Modulates Fibrosis

Cardiac fibrosis is the pathophysiological hallmark of injury in an adult heart. Immune response in the injury niche modulates fibrosis. However, how cell-autonomous immune signaling in cardiac fibroblasts modulates fibrosis is unknown. In this report, we demonstrate that cardiac fibroblasts express TLR4 receptors, and inhibition of TLR4 signaling inhibits fibrotic changes such as proliferation and myofibroblast conversion in human cardiac fibroblasts. Using FACS, and single-cell data of cardiac fibroblasts from Colagen1 GFP mouse, we demonstrate that TLR4 is the predominant TLR and approximately 90 % of human cardiac fibroblasts express TLR4 receptors. Using EdU labeling, cardiac fibroblasts taking up EdU have reduced from 91.65 ± 3.89% from control conditions to 71.15 % ± 2.05 % (mean ± SEM, n=3, p< 0.05) in TLR4 inhibited condition. Ki67 staining shows a similar reduction from 93.1 ± 2.34 % to 59.3% ± 3.54%. TLR4 inhibition in TGF-β treated human cardiac fibroblasts reduced the expression of Periostin expression by 2.6 ± 0.58 and Sma1 by 1.46-± 0.18(mean ± SEM, n=3)fold. Further, migration of cardiac fibroblasts demonstrated a 76.3± 10.23 % (mean ± SEM, n=3, p< 0.05) reduction in the presence of TLR4 inhibitor. Transcriptome analysis of TLR4-inhibited cardiac fibroblasts shows downregulation of proliferation-related genes and pathways. Ingenuity pathway analysis (IPA) of differentially expressed genes in TLR4-inhibited TGF-β -treated cells compared to TGF-β alone shows a reduced expression of a subset of target genes of TGF-β signaling. Further, compared to healthy controls, single-cell data of heart failure patients show significant elevation of Histone 3 and Histone 4 in the fibroblast cluster along with chromatin readers, Brd4 and Nox4. Inhibition of TLR4 in human cardiac fibroblasts reduced the expression of Histone 3 and Histone 4, with Nox4 modulating the chromatin architecture required for fibroblast activation. Thus our data demonstrate that cardiac fibroblast act as an immune cell having cell-autonomous immune signaling, regulating fibrotic changes. Ongoing experiments in 3D human cardiac organoid injury models will reveal how TLR4 signaling regulates fibrotic activation of human cardiac fibroblasts in a heterogeneous system.

Tissue kallikrein-related peptidase8 accentuates cardiac fibrosis after myocardial ischemia-reperfusion injury via regulation of cardiac fibroblasts

AimsTissue kallikrein-related peptidase8 (KLK8) has been found to mitigate acute myocardial ischemia-reperfusion (IR) injury. However, the effect of KLK8 on cardiac remodeling in response to IR injury has not been determined. Materials and methodsKLK8 overexpressing transgenic rat (KLK8-TG) was used as the animal model. IR injury was induced by ligating the left anterior descending coronary artery for 1 h and subsequent reperfusion. The functional and morphological changes of the heart were examined 14 days after the injury. Neonatal rat cardiac fibroblasts (CFs) were used to investigate the molecular mechanisms in vitro. Key findingsKLK8 overexpression enhanced cardiac diastolic dysfunction, fibrosis, and hypertrophy after IR injury, indicating that KLK8 accentuated cardiac remodeling in response to IR injury. Moreover, KLK8 overexpression increased epidermal growth factor (EGF) release and promoted the phosphorylation of EGF receptor (EGFR) and ERK1/2 in the heart after IR injury. It was interesting to find that both EGFR antagonist (AG 1478) and MEK inhibitor (PD98059) attenuated the KLK8-induced proliferation and activation of CFs in vitro, indicating that EGFR signaling might mediate the pro-fibrotic action of KLK8. SignificanceKLK8 plays a crucial role in cardiac remodeling after myocardial infarction. KLK8 accentuates cardiac fibrosis after IR injury, possibly mediated by EGFR signaling in CFs.

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-β. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.

Intercellular cardiomyocyte to cardiac fibroblast signaling regulates global proteome and phosphoproteome landscape to promote oxidative stress via CK2

Intercellular cardiomyocyte to cardiac fibroblast signaling regulates global proteome and phosphoproteome landscape to promote oxidative stress via CK2

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-β1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor β1 (TGF-β1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-β1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-β1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-β1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-β1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-β1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-β1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Abstract P1071: Concurrent Stat1 And Stat3 Signaling Modulates Tlr4/nfkb-mediated Immunomodulatory And Antifibrotic Effects Of Cardiomyocyte-damaged Released S100a1 Protein In Cardiac Fibroblasts

Background: We previously showed that S100A1 released from damaged cardiomyocytes can accelerate post-myocardial infarction in vivo healing. The action of S100A1 as a damage-associated molecular pattern seemed to occur via an immunomodulatory (e.g., IL10 increase) and antifibrotic impact (e.g., COL1A1 and ACTA2 downregulation) in cardiac fibroblasts (CFs) via a TRL4/NKFB pathway. The signaling network in CFs conveying this effect has not been characterized yet. Methods and Results: We performed a 48h time-resolved transcriptome analysis on cultured adult rat CF stimulated with rec. human S100A1 protein (10 -6 M). Bioinformatic GSEA yielded an instant and almost exclusive significant upregulation of the chemokine activity GO-term with a delayed suppression of ECM genes. A transcription factor enrichment analysis for the differentially regulated transcripts indicated an immediate activation of NFKB/RELA with a concurrent temporary STAT1 but lasting STAT3 signaling. A secretome LC-MS/MS-based analysis confirmed the shift of S100A1-treated CFs to mostly chemokine secreting cells. With CCL2 as most secreted chemokine and COL1A1 and ACTA2 as established antifibrotic marker genes for released S100A1, we used a RT-PCR readout for these genes in response to siRNA-based knock-down (KD) of STAT1 and STAT3 in S100A1-treated CFs, while TLR4 KD was used to abrogate NFKB/RELA signaling due to known mechanisms (n=10 each group). S100A1 treatment upregulated CCL2 (16-fold*) and downregulated COL1A1 (3.1-fold*) and ACTA2 (2.9-fold*) mRNA expression vs control (*P<0.01) that was blunted by TLR4 KD. STAT3 KD doubled the CCL2 mRNA increase (32.9-fold*), attenuated COL1A1 mRNA downregulation by S100A1 treatment to 2-fold* but left ACTA2 downregulation unaffected vs contr. (*P<0.05). Conversely, STAT1 KD completely blocked the S100A1 mediated downregulation of ACTA2 but neither impacted S100A1-mediated changes in CCL2 nor COL1A1. Conclusion: Our study identifies concurrent STAT1 and STAT3 activation as a modulator of S100A1-mediated TLR4-NFKB signaling in CFs. As S100A1-activated CFs express e.g., IL-10, we speculate that IL-gp130 trans signaling may convey this effect guiding further studies on S100A1’s extracellular effects in the diseased heart.

Fibrotic Signaling in Cardiac Fibroblasts and Vascular Smooth Muscle Cells: The Dual Roles of Fibrosis in HFpEF and CAD.

Patients with heart failure with preserved ejection fraction (HFpEF) and atherosclerosis-driven coronary artery disease (CAD) will have ongoing fibrotic remodeling both in the myocardium and in atherosclerotic plaques. However, the functional consequences of fibrosis differ for each location. Thus, cardiac fibrosis leads to myocardial stiffening, thereby compromising cardiac function, while fibrotic remodeling stabilizes the atherosclerotic plaque, thereby reducing the risk of plaque rupture. Although there are currently no drugs targeting cardiac fibrosis, it is a field under intense investigation, and future drugs must take these considerations into account. To explore similarities and differences of fibrotic remodeling at these two locations of the heart, we review the signaling pathways that are activated in the main extracellular matrix (ECM)-producing cells, namely human cardiac fibroblasts (CFs) and vascular smooth muscle cells (VSMCs). Although these signaling pathways are highly overlapping and context-dependent, effects on ECM remodeling mainly act through two core signaling cascades: TGF-β and Angiotensin II. We complete this by summarizing the knowledge gained from clinical trials targeting these two central fibrotic pathways.

Mitochondrial PKD Activates Mitochondrial Fission and Proliferative Signaling in Cardiac Fibroblasts

IntroductionCardiac fibrosis, characterized by wall stiffening, reduced contractility, and impaired overall heart performance, is a self‐reinforcing process in response to injury (e.g., myocardial infarction) and pressure overload (e.g., systemic hypertension and pulmonary arterial hypertension [PAH]). The severity of cardiac fibrosis is associated with cardiac dysfunction, increased risk of arrhythmia, and mortality. However, there is a lack of effective therapies designed to inhibit or reverse cardiac fibrosis. Hyperproliferation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts are the key contributors to cardiac fibrosis. We previously showed that mitochondrial reactive oxygen species (mROS) are one of the regulators for activating proliferative signaling in rat neonatal CFs. Moreover, we reported that phosphorylation of dynamin‐related protein 1 (DRP1) by a stress‐responsive protein kinase D (PKD) at the outer mitochondrial membrane (OMM) promotes mROS generation via increased mitochondrial fission in H9c2 cardiac myoblasts.HypothesisMitochondrial PKD‐DRP1 signaling increases mitochondrial fission leading to increased mROS levels, which activates proliferative signaling.MethodsPrimary adult CFs from human ventricles and the right ventricles (RVs) of a SU5415/hypoxia‐induced rat PAH model were used. Mitochondrial morphology and mROS levels were measured by confocal microscopy.ResultsOverexpression of PKD1 increased DRP1 phosphorylation at Ser637, and activates proliferative signaling pathways including ERK1/2 and p38 in human CFs, but did not promote their differentiation into myofibroblasts, assessed by the amount of a‐smooth muscle actin. To specifically identify the involvement of OMM‐localized PKD activity in these processes, we generated an OMM‐targeted dominant‐negative PKD1 (termed mt‐PKD‐DN) by adding an OMM‐target sequence (amino acid 1‐33 from human TOM20) at the N‐terminus of PKD1‐K612W. Importantly, mt‐PKD‐DN was able to inhibit mitochondrial fission, decrease mROS, hyperpolarize mitochondrial membrane potential, and ultimately decrease proliferation in human CFs. To further understand the role of PKD‐DRP1 signaling in vivo, we employed a preclinical PAH animal model. Immunofluorescence staining of RVs from PAH rats revealed that PKD activity and DRP1 phosphorylation increased only in CFs, but not in ventricular myocytes. RV‐CFs isolated from PAH rats exhibited increased mitochondrial fission, mROS levels, and proliferative signaling compared to RV‐CFs from control rats, and these changes were abolished by adenoviral expression of mt‐PKD‐DN.ConclusionMitochondrial mROS elevation via PKD‐DRP1‐dependent mitochondrial fission promotes CF proliferation. Targeting mitochondrial PKD may be a potential therapeutic strategy to reduce cardiac fibrosis under pathological conditions such as PAH.

Wnt is coming: A role for serum Wnt2 and 4 in fibrotic remodeling following acute myocardial infarction.

Wnt is coming: A role for serum Wnt2 and 4 in fibrotic remodeling following acute myocardial infarction.

Curcumin Ameliorates Cardiac Fibrosis by Regulating Macrophage-Fibroblast Crosstalk via IL18-P-SMAD2/3 Signaling Pathway Inhibition.

Ethnopharmacological relevance: Curcumin is a bright yellow chemical produced by plants of the Curcuma longa species. Chemically, curcumin is a diarylheptanoid, belonging to the group of curcuminoids. The therapeutic potential of curcumin has been widely investigated, including its utilization in various of cardiovascular diseases. However, its effect in cardiac remodeling post myocardial infarction and underlying mechanism remains to be uncover. Aim: To evaluate the therapeutic effect and underlying mechanism of curcumin on cardiac fibrosis after myocardial infarction via macrophage-fibroblast crosstalk. Methods: Male C57BL/6 (C57) mice were subjected to left anterior descending coronary artery ligation to establish myocardial infarction and intragastrically fed vehicle or curcumin (50 mg/kg or 100 mg/kg) for 4 weeks. In parallel, neonatal rat cardiac fibroblasts were isolated and co-cultured with liposaccharide (LPS− or LPS+) curcumin-treated macrophages, followed by TGF-β stimulation for 24 h. Cardiac function was determined by 2-dimensional echocardiography, and cardiac fibrosis was measured by picrosirius red staining. Apoptosis of macrophages was investigated by flow cytometry; all pro-fibrotic protein expression (EDA-Fibronectin, Periostin, Vimentin, and α-SMA) as well as TGF-βR1 downstream signaling activation reflected by phosphorylated SMAD2/3 (p-SMAD2 and p-SMAD3) were demonstrated by western blotting. Results: Curcumin significantly ameliorated the inflammation process subsequent to myocardial infarction, reflected by decreased expression of CD68+ and CD3+ cells, accompanied by dramatically improved cardiac function compared with the placebo group. In addition, cardiac fibrosis is inhibited by curcumin administration. Interestingly, no significant reduction in fibrotic gene expression was observed when isolated cardiac fibroblasts were directly treated with curcumin in vitro; however, pro-fibrotic protein expression was significantly attenuated in CF, which was co-cultured with LPS-stimulated macrophages under curcumin treatment compared with the placebo group. Mechanistically, we discovered that curcumin significantly downregulated pro-inflammatory cytokines in macrophages, which in turn inhibited IL18 expression in co-cultured cardiac fibroblasts using bulk RNA sequencing, and the TGF-β1-p-SMAD2/3 signaling network was also discovered as the eventual target downstream of IL18 in curcumin-mediated anti-fibrosis signaling. Conclusion: Curcumin improves cardiac function and reduces cardiac fibrosis after myocardial infarction. This effect is mediated by the inhibition of macrophage-fibroblast crosstalk in the acute phase post-MI and retrained activation of IL18-TGFβ1-p-SMAD2/3 signaling in cardiac fibroblasts.

Ultrasonically Enhanced Cardioprotective Effects of Royal Jelly Targeting Molecular and Cellular Signaling in Cardiac Fibroblasts

Ultrasonically Enhanced Cardioprotective Effects of Royal Jelly Targeting Molecular and Cellular Signaling in Cardiac Fibroblasts

Hippo signaling in cardiac fibroblasts during development, tissue repair, and fibrosis.

The evolutionarily conserved Hippo signaling pathway plays key roles in regulating the balance between cell proliferation and apoptosis, cell differentiation, organ size control, tissue repair, and regeneration. Recently, the Hippo pathway has been shown to regulate heart fibrosis, defined as excess extracellular matrix (ECM) deposition and increased tissue stiffness. Cardiac fibroblasts (CFs) are the primary cell type that produces, degrades, and remodels the ECM during homeostasis, aging, inflammation, and tissue repair and regeneration. Here, we review the available evidence from the current literature regarding how the Hippo pathway regulates the formation and function of CFs during heart development and tissue repair.