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Cardiac fibrosis in hypertrophic cardiomyopathy: elucidating the
role of cardiac fibroblasts

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Hypertrophic cardiomyopathy (HCM) is one of the most common genetic heart diseases and the leading cause of sudden cardiac death in young people. Cardiac fibrosis, the excess deposition of extracellular matrix protein (ECM), is an important histologic feature of HCM that may interfere with the electrical conductance of the heart and accelerate the progression of heart failure. In the centre of this fibrotic process are the cardiac fibroblast (cFB) that - in response to pathological stimuli - proliferate, migrate and differentiate into myofibroblasts (MyoFB), the predominant ECM-synthesizing cells. The molecular mechanisms involved in cardiac fibrosis in HCM are incompletely understood and to date, there is no effective treatment to attenuate the fibrotic process. TGF-β-signalling and dysregulated intracellular Ca2+ handling both play a key role for the differentiation of cFB into MyoFB and may therefore represent a relevant therapeutic target for cardiac fibrosis in HCM. The overall aim of this project is to elucidate the molecular and cellular mechanisms that lead to the development of cardiac fibrosis in HCM in primary human cFB. The specific objectives are to study cFB dysfunction with respect to their proliferation, migration and differentiation; to establish the molecular basis of pathways involved in TGF-β-induced fibrosis; and to determine the role of altered intracellular Ca2+ signalling for fibrogenesis in HCM. The project will be completed over a total duration of 24 months.

Background and AimHypertrophic cardiomyopathy (HCM) is a genetic heart disease characterized by hypertrophy of the left ventricular walls that can lead to blood flow obstruction and heart failure. HCM affects approximately 1 in 500 individuals and represents the leading cause of sudden cardiac death in young people. The underlying causes of the disease are incompletely understood but several mutations in genes encoding for structural proteins of the cardiomyocytes (CM) have been identified in association with HCM. At the cellular level, HCM induces hypertrophy and sarcomeric disarray of the CM. A much less studied but equally important histologic feature is the development of cardiac fibrosis, which manifests as morphological changes of the collagen network and enhanced deposition of extracellular matrix (ECM) proteins. A histological study with hearts from young HCM patients who died of sudden cardiac death revealed an 8-fold increase of cardiac collagen compared to controls and another study confirmed enhanced cardiac collagen turnover in HCM patients in vivo. Importantly, fibrotic tissue may stiffen the ventricular walls, impair the cardiac conductance and accelerate the progression of heart failure. Cardiac fibroblasts (cFB) are key players in the fibrotic process. Under physiological conditions, cFB provide structural support to the heart by regulating the synthesis and degradation of ECM proteins. Upon pathological stimuli, cFB proliferate, migrate and differentiate into myofibroblasts (MyoFB). Sustained activation of MyoFB leads to an excess deposition of ECM proteins and cardiac fibrosis. Activated MyoFB moreover secrete matrix metalloproteinases (MMPs) that are involved in cardiac remodelling and cytokines, such as TGF-β, that may further perpetuate the fibrogenesis cascade. In a recent animal study, Meng et al. have demonstrated that MyoFB-specific TGF-β signalling plays a central role for the maintenance and exacerbation of cardiac fibrosis even though the initial pathological stimuli originate from the CM. Blocking TGF-β signalling specifically in MyoFB moreover attenuated fibrosis and improved cardiac function even in an advanced disease stage, which is of particular relevance for patients with HCM as they are typically diagnosed late when fibrosis is already advanced. cFB- and MyoFB specific signalling pathways therefore represent an attractive and highly relevant therapeutic target to stop the vicious cycle of fibrosis in HCM. To date, these fibrotic pathways have not been investigated in human cFB in the context of HCM and it remains to be established whether the mechanisms discovered in animal studies can be translated to humans. The differentiation of quiescent cFB into MyoFB is a pivotal step in the fibrogenesis cascade and an increase in intracellular Ca2+ has emerged as a key aspect in this process. Several ion channels and transporters participate in Ca2+ signalling in cFB but in the context of fibrosis, the transient receptor potential (TRP) channels have gained particular attention. TRP channels promote non-selective influx of Ca2+ and have been shown to mediate cardiac fibrosis in atrial fibrillation and other fibrosis-associated heart diseases. The role of TRP channels and of altered Ca2+ signalling for cardiac fibrosis in HCM remains, however, to be establishedThe overall aim of this project is to elucidate, at the cell and molecular level, the role of primary human cFB for cardiac fibrosis development in HCM. In particular, we aim to establish the role of cFB-specific signalling pathways and the interaction with intracellular Ca2+ handling for the fibrotic process.To achieve this, the specific objectives of this project are to:i.) Study the dysfunction of primary human cFB isolated from patients with HCM with specific emphasis on their proliferation, migration, spontaneous differentiation and expression of profibrotic markersii.) Establish the molecular basis of the fibrotic process in response to TGF-β-stimulation in primary human cFB from patients with HCMiii.) To determine the involvement of altered intracellular Ca2+ signalling for cardiac fibrosis in primary human cFB from patients with HCM and to study the role of TRP channels as mediators of Ca2+- induced fibrogenesisTo achieve the overall aim and the specific outcomes, the project will be split into work packages (WPs) with specifically defined tasks:WP1: Generation of primary human cFB cell lines from patients with HCM and characterization of proliferation, migration, differentiation and fibrotic markers Aim: To create primary human cFB cell lines from patients with HCM and from healthy controls and to characterize their proliferation potential, migration, differentiation and expression of pro-fibrotic markers in culture.WP2: Assessment of the fibrotic response to TGF-β stimulation in HCM- and CTRL-cFB Aim: TGF-β induces the differentiation of cFB and the accumulation of ECM proteins. The aim of WP2 is to assess whether HCM-cFB have altered signalling mechanisms and/or an increased fibrotic response than CTRLHCM. Cell lines will be stimulated by TGF-β and fibrotic markers will be assessed.WP3: To determine the contribution of altered intracellular Ca2+ signalling for cardiac fibrosis in HCM and to study the role of TRP channels as mediators of Ca2+-induced fibrogenesis Aim: Increased intracellular Ca2+ has been associated with the differentiation of cFB into MyoFB in cardiac fibrosis and several members of the TRP channel family have emerged as mediators of cFB-specific Ca2+ signalling in this context. We first aim to study dysfunctions in intracellular Ca2+ signalling in HCM. Then, we aim to study the role of Ca2+ signalling on the development of fibrosis and last, we aim to identify TRP channels involved in the process.WP4: Training of the researcher through research, exchange with collaborators and courses. WP5: Dissemination and communication of the results of the project.  Results and Future workOverall summary:The research project has for the most part followed the project description. Small methodological changes have been implemented when necessary. Collaborations with partners in Firenze, Vienna and Milano have been initiated and visits including training and exchange were done in September 2021 (Firenze), December 2021 (Milano) and in April 2022 (Firenze) (WP4). Moreover, from April to June 2023 a PhD student from University of Firenze came in Eurac Research to learn how to work with primary cardiac fibroblasts. Another PhD student from University of Vienna came in June 2023 to learn how to produce spheroids from primary cardiac fibroblasts.As planned, primary human cFB were successfully dissociated from biopsies from six patients with HCM and seven healthy controls (Task 1.1. and 1.2.). All isolated cells were spindle-shaped and plastic adherent. Two cell lines tested positive for mycoplasma and were therefore treated with 50 µg/ml Mycoplasma Removal Agent (ECMC210A, EuroClone) for 7 days. Before amplifying the cells, a second test was performed to confirm that the treatment was successful.The immunophenotype of the isolated cells was determined by flow cytometry (Task 1.3.). All cell lines were positive for the mesenchymal markers CD13, CD29, CD44 and CD73 and negative for the endothelial and hematopoietic markers CD31, CD34, CD45, CD14 and HLA-DR whereas the expression of CD140a, CD105, CD90 and CD146 was heterogeneous. No statistically significant differences were observed between the CTRL and HCM cell lines for any of the markers.To determine the proliferation rate of the cell lines (Task 1.4.), cells were plated in 8 wells of a 12-welll plate at a density of 10.000 c/cm2. For four days, cells of two wells (duplicate measurements) were detached and counted using acridine orange dye and a LUNA-FLTM Automated Fluorescence Cell Counter. The mean of the duplicates were calculated. Two separate growth curve experiments were performed for each cell line. Cells proliferated visibly over the four days but no difference was detected between HCM and CTRL cells (p > 0.05 for RM-ANOVA interaction effect).Different protocols were tested to evaluate cell migration ability of the cell lines. Initial experiments were performed with a manual scratch wound assay followed by live cell imaging. In a second set of experiments, cells were seeded in specific silicone inserts which created a more reproducible wound. Due to technical difficulties with live cell imaging, pilot experiments using a high-throughput live cell imaging system (Incucyte) were performed at the Centro Cardiologico Monzino in Milano in December 2021. Final experiments were planned for the fall 2022, however, unfortunately due to technical problems with the device in Milano these experiments had to be cancelled.  To quantify the percentage of cFB that spontaneously differentiate into myofibroblasts (Task 1.6.) cells were plated in 35-mm microscopy dishes at a density of 7000 c/cm2. After three to four days, cells were fixed in 4% PFA and stained using antibodies for vimentin (ab16700, abcam), α smooth muscle actin (α-SMA) (M0851, Dako) and DAPI. The fixed cells were visualized under a fluorescent microscope and the percentage of spontaneously activated myofibroblasts was quantified with ImageJ. Under these basal conditions, less than 1% of the HCM and the CTRL cells were positive for α-SMA and there is  no significant difference between HCM and CTRL cells with regards to spontaneous activation. In order to quantify the expression of early fibrotic markers pellets were collected and frozen at -80°C to perform real time qPCR and western blotting (Task 1.7).To assess the TGF-β induced fibrotic response (WP 2), cells were plated in uncoated 60 mm petri dishes and in chambered polymer coverslips (ibidi, 80426) at densities of 15.000 and 10.000 cells/cm2, respectively. Cells were incubated for 48h in complete medium, washed with PBS and then starved for 24h in serum- and growth factor free starvation medium. The medium was then changed to 10 ng/ml recombinant human TGF-β1 (Miltenyi Biotec, 130-095-067) or with regular starvation medium as a control (Task 2.1.). Coverslips were fixed in 4% PFA for immunostaining after 24 and 48 h and pellets for mRNA and protein quantification were collected from the petri dishes after 24, 48 and 72 h. Fixed cells were stained and imaged (Task 2.2.) as described in the previous section. An increase in the number of activated cells was clearly visible after 48h both for CTR and HCM cells.For the Ca2+ signalling part of the project (WP 3), training and set up of the method was performed with collaborators in Firenze in September 2021 (Task 3.1.). Pilot experiments to measure intracellular Ca2+ concentration, Ca2+ release from intracellular stores and Ca2+ influx via plasma membrane in HCM-cFB and CTRL-cFB (Task 3.2.) were performed in Firenze in April 2022. Further experiments, also on the involvement of TRP channels (Tasks 3.4. and 3.5.), were performed in the meantime.In detail, to study the calcium entry in HCM-cFB and CTRL-cFB we used calcium imaging technique; we labelled calcium ions with a fluorescent probe (Cal520; ex/em 493/515) and thanks to an inverted microscope, a laser light source and a fast acquisition camera, we were able to capture the calcium entry in the fibroblast as a rising fluorescent signal emitted by the single cells in culture. The system is implemented with a perfusion system, to change the solution of the experimental bath during the recording. This allow to give the cells different pharmacological stimuli. In this study we switched from Tyrode solution with 0mM calcium (TYR) to one with 1.8mM calcium (TYN) to induce calcium entry in the fibroblast; during the recordings cells are exposed to Thapsigargin (Thapsi), a SERCA inhibitor, to avoid the calcium re-uptake from endoplasmic reticulum; we also used different pharmacological inhibitors, SKF (5µM), a TRP inhibitor, or Lacidipine (10µM) (Laci), a calcium channel type T inhibitor. To load the fibroblast with the calcium probe we used 1ml of culture medium added of 2µl of Cal520 (2.5µg/µl) and 5µl of pluronic solution, and we exposed cells for 30min at 37°C. Then we changed the medium with TYR, we set up the experimental chamber with the perfusion system and we started recording. As first step, we washed by perfusion with TYR solution added with 1µM Thapsi (since this time, thapsi is added to every solution we used), after signal stabilization we switch to TYN and we observed the first fluorescent peak in response to the increment of extracellular calcium concentration. We further switched to TYR, waiting until the stabilization of the signal and we finally switch to TYR added with SKF or LACI: here, no increment of the signal should be visible because there is no calcium in the extracellular solution. Consequently, we moved to TYN added with SKF or LACI, waiting for signal stabilization and then we switched again to TYR with SKF or LACI. After complete stabilization, we used ionomycin to induce the total release of intracellular calcium. The contribution of the sarcoplasmic reticulum to the intracellular calcium concentration should have been cancelled by the presence of Thapsi; so, the intracellular calcium concentration increment should be dependent only by the calcium permeable channels on the membrane. The obtained results indicate that the peak induced by the switch from TYR to TYN (Ca2+-induced peak), so from no-calcium to normal-calcium concentration of the extracellular solution, is larger in amplitude in HCM fibroblast then in control fibroblast derived from non-HCM cardiac samples. In parallel, the exposure to SKF induced a larger reduction of this peak in control (-60%) than in HCM fibroblasts (-33%). Our first hypothesis was that the larger amplitude of the Ca2+-induced peak observed in HCM fibroblasts is mediated by channels NON sensitive to SKF. We then analyzed the effect of LACI on cFB to verify the contribution of L-Type calcium channel to the intracellular calcium concentration. This time we observed a larger reduction of the Ca2+ -induced peak in HCM-cFB (-35%) than in CTRL-cFB (-22%), this confirmed that the larger amplitude of Ca2+-induced peak in HCM-cFB is actually mediated by L-type calcium channels and the contribution of TRP channels to intracellular calcium concentration is smaller in HCM-cFB than in CTRL-cFB. In the end we also tested the effect of 24h treatment with TGFβ on HCM- cFB and the response to SKF acute exposure. We observed that the exposure to TGFβ determine an increment of the amplitude of the Ca2+-induced peak compared to HCM-cFB not exposed to TGFβ and this increment seems not to be linked to TRP channels since their inhibition, mediated by SKF, doesn’t led to a larger reduction of the Ca2+-induced peak. For further analysis, we need to compare the HCM fibroblasts expose to TGFβ with control fibroblasts expose to TGFβ and to test, on both cultures, the LACI effect on the amplitude of Ca2+-induced peak.Taken together, these results indicate that, despite the fact that the HCM cFB did not differ from controls with respect to their immunophenotype, proliferation rate and α-SMA expression, they exhibit significant differences related to Ca2+ signalling. Whether HCM cFB differ from controls with regard to gene and protein expression remains to be established.An additional experimental part on the mechanical properties of collagen fibers of HCM and CTRL cFB was included in the project. A collaboration with experts on biomechanics from the Technical University of Vienna was initiated and samples were shipped to Vienna for pilot testing. For this part, a protocol for the cultivation of cFB in 3-dimensional spheroids using AggreWellTM Microwell Culture Plates was specifically established. These spheroids were frozen and shipped at the TU Vienna where biomechanics colleagues have developed a method to measure mechanical properties of biological tissue by parallel-plate compression testing. The results on force and displacement of the spheroid during parallel-plate compression and their validation will be presented at three different conferences (see outputs). Further experiments to compare the mechanical properties between HCM and control cells are currently ongoing. For WP5 (dissemination and communication of the results of the project), an article was published online in the Eurac Magazine and through social media channels (Facebook, Linkedin).  In June 2023 a review entitled “Myocardial fibrosis in hypertrophic cardiomyopathy: a perspective from fibroblasts.” was submitted at the European Heart Journal Open. Moreover 3 abstracts in collaboration with the Technical University of Vienna were submitted and accepted.The experimental part of the project has not been fully completed due to unpredictable experimental obstacles. However, we plan to move in two different directions. One is to collect the main results, in particular the differences in the Ca2+ signalling between HCM and CTRL cFB (which is a novelty in this field), with the aim to write a short report on this new findings. The second one is to write a proposal with the Technical University of Vienna in order to conclude the last planned work, add a new experimental part on hybrid spheroids made up with cFB but also with hIPSC derived cardiomyocytes and include all the results in a future original manuscript.

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