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Rationale: Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinase (GRK) 5 is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and upregulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium- calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of NFAT. Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts (CFs), however its pathophysiological role in these crucial cardiac cells is unknown. Objective: The aim of this study is to elucidate the role of GRK5 in the activation of cardiac fibroblasts in vitro and cardiac fibrosis after injury in vivo. Methods and Results: We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits Angiotensin II (AngII) mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice decreased fibrosis and cardiac hypertrophy after chronic AngII infusion compared to non-transgenic littermate controls (NLCs). Fibroblast-specific deletion of GRK5 was also protective in mice after ischemic injury as they presented with preserved systolic function, decreased fibrosis, and decreased hypertrophy compared to NLCs. Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. Conclusions: We present novel data demonstrating that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrates the potential beneficial effects of GRK5 inhibition in the context of cardiac disease. / Biomedical Sciences

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

Device anchoring of artificial cornea implants, through tissue integration of stromal tissue, is necessary to ensure long-term success. In this work, the delivery of basic fibroblast growth factor (FGF-2), a key modulator in corneal wound healing, via heparin modified materials was investigated as a means of sustained, soluble growth factor delivery for stimulation of device anchorage. Two materials types, commonly used for ophthalmic applications and currently under investigation for use in artificial cornea applications, were utilized. Poly (dimethyl siloxane) (PDMS) is currently under investigation as the base material for keratoprosthetic devices; dendrimer crosslinked collagen has been examined as the basis for use as a tissue engineered corneal equivalent. PDMS surfaces were modified directly or indirectly, through a poly (ethylene oxide) (PEO) spacer, to contain functionalized reactive NSC groups capable of binding heparin and FGF-2 Surface modifications were characterized with attenuated total reflection Fourier transform infrared spectrophotometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angles. Heparin coverage was assessed with metachromatic and bioactivity assays. Heparinized collagen gels were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and polypropyleneimine octaamine G2 dendrimers. Gel integrity was assessed with water uptake, differential sr::anning calorimetry, and heparin and dendrimer stability. Both materials were exposed to radiolabelled FGF-2 and growth factor immobilization and delivery were quantified. Heparinized PDMS surfaces were capable of binding on average 100 ng/cm2 ofFGF-2, while heparinized collagen gels had higher FGF-2 immobilization, 300 ng, likely attributed to their higher heparin densities and the fact that the bulk gel rather than the surface only was modified. Delivery of FGF-2 from the heparinized materials revealed a first order release profile, with an initial burst of FGF-2, followed by gradual growth factor release. Release rates, over a 2 week period, reached 6.5% and 50%, for 1 day and 3 day FGF-2 exposed heparinized PDMS modified surfaces, while hepruinized dendrimer crosslinked collagen gels released 40%. / Thesis / Master of Applied Science (MASc)

Eukaryotic cell chemotaxis, or directed cell migration in response to a chemoeffector gradient, plays a central role in many important biological process such as wound healing, cancer metastasis, and embryogenesis. In vivo, cells migrate on fibrous ECM, but chemotaxis studies are typically conducted on flat substrates which fail to recapitulate ECM or 3D gel environments with heterogeneous and poorly defined biophysical properties. To address these challenges, this thesis focused on developing a microfluidic assay device which utilizes a reductionist approach to study single cell chemotaxis on aligned, suspended ECM-mimicking nanofibers. The device is comprised of a network of microfluidic mixing channels which produce a temporally invariant, linear chemical gradient over nanofiber scaffolds in an observation channel. The microfluidic device design was guided by a numerical model and validated with experimental testing. This device was used to study mouse embryonic fibroblast NIH/3T3 response to platelet derived growth factor (PDGF) on flat polystyrene and suspended, polystyrene nanofibers with small (15 μm), and large (25 μm) spacing. Cell aspect ratio is lowest for flat polystyrene (spread morphology) and highest for large-spaced fibers (spindle morphology). Cells migrating on fibers begin to show a chemotaxis response to a PDGF gradient 10 times shallower than that required for chemotaxis response on a flat substrate. Furthermore, cells with spindle morphology maintain a robust and strong response over a broad range of chemoattractant concentration. These cells also had a 45% increase in speed and 26% increase in persistence over cells on flat polystyrene. The findings of this thesis suggest that 2D substrates may not be sufficient for studying physiologically relevant chemotaxis. / Master of Science