Cardiac fibrosis is the excess deposition of myocardial extracellular matrix components, which increases tissue stiffness and heterogeneity, causing impaired diastolic/systolic function and arrhythmias, and eventually leading to heart failure and death. There are no available treatments for cardiac fibrosis. Myofibroblasts mediate fibrosis, and are characterized by hypersynthesis of collagens, decreased migration, and increased α-smooth muscle actin, which is incorporated into stress fibers, imparting contractility. Scleraxis is a transcriptional regulator of collagen-rich tissues, increased in response to the same stimuli that drive the myofibroblast phenotype, such as cyclic stretch. We show that Scleraxis mediates the conversion of cardiac fibroblasts to myofibroblasts, by increasing myofibroblast marker expression and contraction, and decreasing migration. Additionally, a proximal 1500 bp human SCLERAXIS promoter is activated by stretch and is responsive to transforming growth factor-β1. Thus, Scleraxis is a specific mechanoresponsive regulator of the myofibroblast, representing a novel target for the treatment of cardiac fibrosis.

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Der spezifische Knockdown von RhoA in neonatalen kardialen Rattenfibroblasten führte auf molekularem Level zu einer Reduktion des Myofibroblastenmarkers α-Glattmuskelaktin und zu einem Anstieg im modifizierten acetylierten Tubulin. Auf subzellulärer Ebene konnte ein Verlust von Stressfasern, Aktinstrukturen höherer Ordnung und eine erhöhte Dichte des Golgi-Apparats beobachtet werden. Außerdem waren die Fokaladhäsionen kürzer und zufällig verteilt, was auf einen Verlust der Zellpolarität hinweist. Auf dem zellulären Level erhöhte der Knockdown von RhoA die Zellfläche aber nicht das Volumen. Diese Veränderungen führten zu einer schnelleren Adhäsion unabhängig vom Substrat, eine Reduktion der Migration in 2D und im Gegensatz dazu eine verbesserte Migration durch eine poröse Membran. Außerdem war die mitogene Antwort der Zellen auf einen Serumstimulus stark reduziert. Eine Veränderung in Zellviabilität konnte zudem nicht beobachtet werden. Die Expression und Sekretion des Fibrose-assoziierten Faktors CTGF war in gehungerten Zellen mit einer Reduktion in RhoA Expression signifikant vermindert, was jedoch in der Anwesenheit eines Serumstimulus aufgehoben werden konnte. Auf einer heterogenen multizellulären Ebene verringerte der Knockdown von RhoA die kontraktile Funktion von generierten künstlichen Herzgeweben unter Kalziumstimulation. Dies ging einher mit einer Reduktion der Expression von α-Glattmuskelaktin und Calsequestrin. Durch die Verwendung spezifischer Inhibitoren der Rho-assoziierten Kinase (ROCK) und HDAC6 konnten einige dieser zellulären Veränderungen imitiert und demensprechend einem Effektorprotein zugeordnet werden. Der ROCK Inhibitor Fasudil konnte die morphologischen Veränderungen und die reduzierte Migrationskapazität in Wildtyp-Fibroblasten abbilden, wobei eine Reduktion der Proliferation nach der Verwendung des HDAC6 Inhibitors Tubastatin A beobachtet wurde.

No description available.

Cardiac fibroblast to myofibroblast phenoconversion is a critical step during the development of cardiac fibrosis. Myofibroblasts chronically remodel extracellular matrix that results in myocardial stiffening, cardiac dysfunction and eventually heart failure. Previously we showed that Meox2, a homeobox transcription factor, can inhibit myofibroblast phenoconversion. Here we show that Zeb2, a repressor of Meox2, plays a crucial role during this phenoconversion process. Zeb2 overexpression significantly upregulates the expression of three key myofibroblast markers: α-SMA, SMemb and ED-A fibronectin in primary rat cardiac myofibroblast. We show that Zeb2 is highly expressed in myofibroblast nuclei whereas it is minimally expressed in fibroblast nuclei. Zeb2 overexpression in myofibroblasts results in a less migratory and more contractile mature myofibroblast phenotype. Moreover, Zeb2 overexpression represses Meox2 expression in endothelial cells. Thus, the current study enhances our understanding of the mechanism behind myofibroblast phenoconversion and provides a basis for developing Zeb2-based novel anti-fibrotic drug in the future. / February 2016

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.

Activation of cardiac fibroblasts is a key event in the progression of cardiac fibrosis that leads to heart failure. However, the molecular mechanisms underlying mechanical stress-induced cardiac fibroblast activation are complex and poorly understood. This study demonstrates that Pellino1, an E3 ubiquitin ligase, was activated in vivo in pressure overloaded rat hearts and in cultured neonatal rat cardiac fibroblasts (NRCFs) exposed to mechanical stretch in vitro. Suppression of the expression and activity of Pellino1 by adenovirus-mediated delivery of shPellino1 (adv-shpeli1) attenuated pressure overload-induced cardiac dysfunction and cardiac hypertrophy and decreased cardiac fibrosis in rat hearts. Transfection of adv-shpeli1 also significantly attenuated mechanical stress-induced proliferation, differentiation and collagen synthesis in NRCFs. Pellino1 silencing also abrogated mechanical stretch-induced polyubiquitination of tumor necrosis factor-alpha receptor association factor-6 (TRAF6) and receptor-interacting protein 1 (RIP1) and consequently decreased the DNA binding activity of nuclear factor-kappa B (NF-κB) in NRCFs. In addition, Pellino1 silencing prevented stretch-induced activation of p38 and activator protein 1 (AP-1) binding activity in NRCFs. Chromatin Immunoprecipitation (ChIP) and luciferase reporter assays showed that Pellino1 silencing prevented the binding of NF-κB and AP-1 to the promoter region of transforming growth factor-β1 (TGF-β1) thus dampening TGF-β1 transactivation. Our data reveal a previously unrecognized role of Pellino1 in extracellular matrix deposition and cardiac fibroblast activation in response to mechanical stress and provides a novel target for treatment of cardiac fibrosis and heart failure.

Introduction: Hypertrophic cardiomyopathy (HCM) is a heterogeneous disorder of the cardiac sarcomere, resulting in myocyte hypertrophy and disarray, interstitial fibrosis, and cardiac dysfunction. Our aim was to determine whether the amount of fibrosis in HCM correlates with echocardiographic measures of diastolic dysfunction, presence of HCM-susceptibility mutations, or polymorphisms in the renin-angiotensin-aldosterone system (RAAS). Methods: Surgical specimens from patients with obstructive HCM undergoing septal myectomy at the Mayo Clinic (2001-2004) were examined and compared with autopsy-derived tissues from age- and sex-matched normal controls. Digital image analysis was used to quantitate the fibrosis in representative microscopic sections. Genotyping was performed for myofilament-HCM using polymerase chain reaction, high-performance liquid chromatography, and direct DNA sequencing. RAAS polymorphism status was similarly established. Results: The study included 59 HCM cases and 44 controls. Patients with HCM exhibited more fibrosis (mean 17%, range 3-45%) than controls (mean 8%, range 3-17%) (P<.0001). A significant relationship existed between amount of fibrosis and maximum wall thickness (P=.02), left ventricular ejection fraction (P=.02), and peak early/late diastolic mitral annulus velocity (E/A ratio) (P=.002). Although there was no association between amount of fibrosis and myofilament-HCM genotype status or polymorphisms in the RAAS cascade, there was a trend toward more fibrosis in patients with ≥1 C-encoding allele in CYP11B2-encoded aldosterone synthase. Conclusions: Patients with HCM undergoing septal myectomy had significantly more myocardial interstitial fibrosis than controls. The amount of fibrosis in HCM patients correlated with degree of septal hypertrophy and left ventricular systolic and diastolic function. Notably, neither mutations in cardiac myofilament proteins or polymorphisms in RAAS exhibited strong associations with severity of myocardial fibrosis.

Fibroblast to myofibroblast differentiation is essential for the initial healing response, but excessive myofibroblast activation leads to pathological fibrosis. Upon injury, quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it contributes to organ dysfunction and failure. Cytosolic calcium (cCa2+) signaling is necessary for myofibroblast differentiation yet the role of mitochondrial calcium (mCa2+) has not been explored. cCa2+ signaling is rapidly integrated into the mitochondrial matrix via the mitochondrial calcium uniporter channel (mtCU), a mechanism theorized to integrate cellular energetic demand with metabolism and respiration. This is intriguing, as it is now appreciated that metabolic reprogramming is required for numerous cellular differentiation programs. The Mcu gene encodes the channel-forming subunit of the mtCU and is required for acute mCa2+ uptake. To examine the contribution of mCa2+ signaling to myofibroblast differentiation, we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu with adenovirus-expressing Cre recombinase. Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced cCa2+ transient amplitude when treated with ATP (purinergic, IP3-mediated Ca2+ release). Loss of Mcu promoted myofibroblast differentiation: increased alpha-smooth muscle actin (α-SMA) expression and contractile function (gel retraction), increased myofibroblast gene expression, and decreased proliferation. Further, we found that treatment of wild-type fibroblasts with fibrotic agonists – transforming growth factor beta (TGFβ) and Angiotensin II (AngII) – increased expression of the mtCU gatekeeper, MICU1, to modulate mtCU activity and down-regulate mCa2+ uptake. This suggests that fibrotic agonists signal to acutely inhibit mCa2+ uptake to initiate myofibroblast differentiation. Next, we evaluated the relationship between mCa2+ uptake, metabolism, and myofibroblast differentiation. Fibrotic stimuli increased glycolysis and loss of MCU augmented this phenotype. In addition, genetic activation of glycolysis promoted myofibroblast differentiation, while genetic inhibition of glycolysis ablated the increased differentiation observed in Mcu-/- MEFs. We hypothesize that loss of mCa2+ uptake promoted aerobic glycolysis by reducing the activity of key Ca2+-dependent enzymes such as pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogase (αKGDH). Metabolomic analysis revealed a multitude of changes induced by both TGFβ and the loss of MCU, including increased levels of pyruvate, consistent with inactive PDH. In addition, metabolite quantification showed TGFβ increased alpha-ketoglutarate (αKG) levels ~2-fold and this increase was augmented by loss of Mcu. This is interesting because αKG promotes histone and DNA demethylation by modulating αKG-dependent dioxygenases. Indeed we observed that TGFβ and loss of MCU induced demethylation of histone lysine residues. Further, using ChIP-qPCR we found that TGFβ decreased H3K27me2 marks at the periostin and platelet-derived growth factor receptor alpha loci, which are early and robust indicators fibroblast activation. Finally, to examine the contribution of mCa2+ in cardiac fibrosis, we generated conditional, fibroblast-specific knockout mice by crossbreeding Mcufl/fl mice with Col1a2-CreERT mice (Mcufl/fl x Col1a2-Cre), permitting tamoxifen-inducible gene deletion in adult mice. Loss of Mcu (Mcufl/fl x Col1a2-Cre) increased myofibroblast differentiation and exacerbated fibrosis following myocardial infarction or chronic AngII infusion. In summary, our data linked changes in mCa2+ uptake with metabolic alterations necessary for chromatin modifications and activation of the myofibroblast gene program. While mCa2+ signaling is most well known and studied for its role in cell death, we have demonstrated a previously unrecognized role for modulation of mCa2+ uptake as a key regulator of myofibroblast differentiation. / Biomedical Sciences