An increase in cytoplasmic Ca2+ concentration activates multiple cellular activities, including cell division, metabolism, growth, contraction and death. In smooth muscle Ca2+ entry via voltage-dependent Ca2+ channels leads to a relatively uniform increase in cytoplasmic Ca2+ levels that facilitates co-ordinated contraction throughout the cell. However certain functions triggered by voltage-dependent Ca2+ channels require periodic, pulsatile Ca2+ changes. The mechanism by which Ca2+ entry through voltage-dependent channels supports both co-ordinated contraction and distinct cellular responses driven by pulsatile Ca2+ changes is unclear. Here in intact resistance arteries we show that Ca2+ entry via voltage-dependent Ca2+ channels evokes Ca2+ release via inositol triphosphate receptors (IP3Rs), generating repetitive Ca2+ oscillations and waves. We also show that mitochondria play a vital role in regulating Ca2+ signals evoked by voltage-dependent Ca2+ entry by selectively modulating Ca2+ release via IP3Rs. Depolarizing the mitochondrial membrane inhibits Ca2+ release from internal stores, reducing the overall signal-generated Ca2+ influx without altering the signal resulting from voltage-dependent Ca2+ entry. Notably neither Ca2+ entry via voltage-dependent Ca2+ channels nor Ca2+ release via IP3Rs alters mitochondrial location or mitochondrial membrane potential in intact smooth muscle cells. Collectively these results demonstrate that activation of voltage-dependent Ca2+ channels drives Ca2+ entry, which subsequently triggers Ca2+ release from the internal store in smooth muscle cells. Mitochondria selectively regulate this process by modulating IP3R-mediated amplification of Ca2+ signals, ensuring that different cellular responses are precisely controlled. KEY POINTS: In smooth muscle Ca2⁺ entry via voltage-dependent channels produces a uniform Ca2⁺ increase, enabling co-ordinated contraction in each cell. Certain functions, however, require large, pulsatile Ca2⁺ changes rather than a uniform increase. Using advanced imaging in intact arteries, we discovered that voltage-dependent Ca2⁺ entry triggers internal store Ca2⁺ release via IP₃ receptors, generating repetitive Ca2⁺ oscillations and waves. Mitochondria selectively modulate these signals by regulating only IP₃ receptor-mediated release; neither mitochondrial location nor membrane potential is altered by either type of Ca2+ signal. These findings demonstrate how voltage-dependent Ca2⁺ entry supports both co-ordinated contraction and pulsatile Ca2⁺-driven biological responses.

Introduction Breast cancer remains a prevalent disease in women worldwide. Though significant advancements in the standard of care for breast cancer have contributed to improved patient survival and quality of life, a breast cancer diagnosis and subsequent treatment interventions have a long-lasting impact on patients’ lived experiences. A high-quality healthcare system uses a patient-centred approach to healthcare, with patient engagement being a central pillar in the delivery of patient-centred care. However, the disconnect between patients and researchers can translate into research lacking real-world relevance to patient health needs. Here, we report a patient and stakeholder engagement workshop series that was conceptualized with the goal of promoting dialogue between patients with breast cancer, breast cancer researchers and the clinician involved in their care. We present the collaborative learning process and emerging opportunities from this patient engagement workshop series as a community-academic partnership. Method We report on a three-part storytelling workshop, with the scope of the workshops including topics related to raising awareness of the patient lived experience following a breast cancer diagnosis, breast cancer research activities undertaken by researchers, and the approach used by multidisciplinary healthcare teams in the management of breast cancer using storytelling as a tool. We used an iterative approach to cohort trust and relationship building, narrative development, and the use of multiple media formats to capture patient stories. This included the use of object memories, storytelling prompt cards and open-mic audio format to capture patient stories from diagnosis to treatment, and remission. Results 20 patients shared their stories with key themes emerging from the qualitative analysis of audio recordings. For many, this was the first time they had spoken about their breast cancer experience beyond family and friends. Emerging themes included common public misconceptions about a breast cancer diagnosis, the importance of self-advocacy in patient decision making about treatment, and the complex emotional journey experienced by patients diagnosed with breast cancer. The group-based storytelling approach provided collective empowerment to share personal experiences and connect meaningfully across the peer community. Conclusion While a breast cancer diagnosis can be overwhelming from a physical, social, emotional and cognitive perspective, storytelling as a patient engagement approach can build patient trust in researchers, ensuring that as key stakeholders they are involved in the process of research. Understanding the patient perspective of a breast cancer diagnosis and subsequent experiences can support healthcare professionals in developing an empathetic approach to sharing information, and involving patients in shared decision making about their healthcare.

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca 2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca 2+ /calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects. Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red. Results: Treatment of CFs with sunitinib (0.1–10 μM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca 2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93. Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca 2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.

Background Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. Methods We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. Results We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. Conclusion We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Introduction Ca2+/calmodulin dependent protein kinase II (CaMKII) is a central mediator of Ca2+-induced signalling in the heart and regulates both normal cardiac physiology and pathology. Sunitinib malate is an oral Type I tyrosine kinase inhibitor (TKI) known to inhibit more than 50 kinases, with anti-angiogenic and anti-proliferative effects affiliated with off-target cardiotoxicity. Previous work has shown that chronic sunitinib treatment significantly increases CaMKII expression and activity and this correlates with significant cardiac dysfunction in vivo.1 Mitochondrial dysfunction, mediated by increased mitochondrial Ca2+ and resultant mitochondrial ROS production, has been proposed as an underlying mechanism for TKI-induced cardiotoxicity in cardiomyocytes.2 However, little is known of how TKIs may affect the non-contractile cells of the heart. Here, we have investigated whether sunitinib treatment increases mitochondrial ROS production in cardiac fibroblasts (CF) and whether CaMKII may play a role in this potential cardiotoxic mechanism. Methods CF were isolated from adult Sprague-Dawley rats (male, 250–350 g, n=8) via bulk collagenase digestion and were maintained in culture before treatment with sunitinib (Cayman Chemical Company, Michigan, USA) (1–10µM) for 16 hours in the presence of serum (DMEM supplemented with 20% FBS). Mitochondrial superoxide production was assessed using MitoSOX Red (ThermoFisher Scientific, UK). CF were pre-treated with 5µM KN-93 (2 hours) Sigma-Aldrich, UK) to inhibit CaMKII before the addition of 10µM sunitinib for 16 hours. MitoSOX Red analysis was then repeated as before. Data are presented as mean values ± S.E.M of n observations, where n represents the number of samples. Comparisons were assessed by one-way ANOVA with post hoc Dunnett’s test. Results MitoSOX fluorescence imaging revealed a significant increase in mitochondrial superoxide production in sunitinib treated CF (144.6±1.1 vs 526.2±24.0 vs 178.8±4.6 relative fluorescent units; control vs 10µM sunitinib vs 10µM antimycin A, n=3, p<0.05). However, KN-93 pre-treatment significantly reduced mitochondrial ROS production in sunitinib treated CF (440.2±0.6 vs 680.5±5.6 vs 576.1±11.4 relative fluorescent units; control vs 10µM sunitinib vs 10µM sunitinib + 5µM KN-93, n=3, p<0.05). Conclusion Sunitinib treatment increases mitochondrial superoxide production in CF, supporting a previously proposed TKI-induced cardiotoxic mechanism mediated by mitochondrial dysfunction. This cardiotoxic mechanism may be mediated by CaMKII (activated via oxidation), although further work is required to corroborate this. Future work will determine the effect this mechanism has on mitochondrial function and establish whether targeted CaMKII inhibition can reduce/reverse TKI-induced cardiotoxic effects. References Mooney, L., et al. British Journal of Pharmacology. 2015;172:4342–4354. Kerkela, R., et al. Nature Medicine. 2006;12:908–916.

Raw RNA sequencing dataset spilt into files comparing either UN with LPS or Toxoplasma gondii and also LPS with Toxoplasma gondii. Each comparison folder should have gene expression tables of all genes, gene expression table of just significant genes, the FPKM values of all genes and then the FPKM of just top genes. Type II Toxoplasma gondii was used and DCs were co-cultured for 6 hours before RNA isolation was performed. Primary analysis was performed by GATC biotech

Toxoplasma gondii is capable of actively invading almost any mammalian cell type including phagocytes. Early events in phagocytic cells such as dendritic cells are not only key to establishing parasite infection, but conversely play a pivotal role in initiating host immunity. It is now recognized that in addition to changes in canonical immune markers and mediators, alteration in metabolism occurs upon activation of phagocytic cells. These metabolic changes are important for supporting the developing immune response, but can affect the availability of nutrients for intracellular pathogens including T. gondii. However, the interaction of T. gondii with these cells and particularly how infection changes their metabolism has not been extensively investigated. Herein, we use a multi-omics approach comprising transcriptomics and metabolomics validated with functional assays to better understand early events in these cells following infection. Analysis of the transcriptome of T. gondii infected bone marrow derived dendritic cells (BMDCs) revealed significant alterations in transcripts associated with cellular metabolism, activation of T cells, inflammation mediated chemokine and cytokine signaling pathways. Multivariant analysis of metabolomic data sets acquired through non-targeted liquid chromatography mass spectroscopy (LCMS) identified metabolites associated with glycolysis, the TCA cycle, oxidative phosphorylation and arginine metabolism as major discriminants between control uninfected and T. gondii infected cells. Consistent with these observations, glucose uptake and lactate dehydrogenase activity were upregulated in T. gondii infected BMDC cultures compared with control BMDCs. Conversely, BMDC mitochondrial membrane potential was reduced in T. gondii-infected cells relative to mitochondria of control BMDCs. These changes to energy metabolism, similar to what has been described following LPS stimulation of BMDCs and macrophages are often termed the Warburg effect. This metabolic reprogramming of cells has been suggested to be an important adaption that provides energy and precursors to facilitate phagocytosis, antigen processing and cytokine production. Other changes to BMDC metabolism are evident following T. gondii infection and include upregulation of arginine degradation concomitant with increased arginase-1 activity and ornithine and proline production. As T. gondii is an arginine auxotroph the resultant reduced cellular arginine levels are likely to curtail parasite multiplication. These results highlight the complex interplay of BMDCs and parasite metabolism within the developing immune response and the consequences for adaptive immunity and pathogen clearance.

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+- ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype.

This chapter describes how the structure and positioning of mitochondria contribute to the control of Ca2+ signaling, including a previously unrecognized ability of the position of the organelles to increase local Ca2+ entry via voltage-dependent Ca2+ channels. In native smooth muscle cells, mitochondria contribute to the localization of Ca2+ signals and to the modulation of the amplitude of Ca2+ signals. In smooth muscle, mitochondria control contractility, proliferation, and growth through regulation of cytoplasmic Ca2+ concentrations. The precise structure and position of mitochondria have been studied most extensively in cultured smooth muscle cells because of the relative ease that the organelles can be visualized in these cells. Live cell imaging is required to appreciate the precise relationship between position and structure of mitochondria and the control of Ca2+ signaling in fully-differentiated cells. Features of Ca2+ signals including the amplitude, duration, frequency and location are encoded by various physiological stimuli.

Abstract MYC is one of the most commonly mutated and highly amplified oncogenes in human breast cancer. MYC amplifications occur most frequently in triple-negative breast cancers (TNBCs). TNBCs can be divided into two molecular subtypes: basal-like and claudin-low breast cancers. These cancers tend to be extremely aggressive and are strongly associated with disease recurrence, poor prognosis and high mortality. In particular, claudin-low tumors are classified by a loss of tight junctions and cell-to-cell contacts and an enrichment for genes associated with an epithelial-to-mesenchymal transition (EMT) and mammary stem cells (also known as tumor-initiating cells). Despite the high level of disease severity, there are no targeted therapies for claudin-low TNBCs. To address this unmet need, we utilized human mammary epithelial cells (HuMECs) that express oncogenic levels of MYC and a mutant MYC (T58A) to characterize the behavioral and metabolic changes that occur during the formation of MYC-driven breast cancers. We found that MYC regulates the expression of genes associated with cell stemness, EMT, lipid metabolism, and calcium (Ca2+) signaling and that the expression of this gene signature promotes cell growth, survival, migration, and metabolic plasticity. The gene signature of MYC-expressing HuMECs highly correlates with the gene signature of claudin-low breast cancers, therefore highlighting the relevance of our HuMEC model to human claudin-low breast cancer. We found the major drivers underlying the MYC-dependent changes in cell behavior to be stimulation of Ca2+ signaling and strong activation of lipid metabolism. Ca2+ signaling is stimulated through the MYC-dependent repression of Ca2+ efflux mechanisms; elevated cytosolic Ca2+ then consequently stimulates a Ca2+/calmodulin kinase kinase 2 (CAMKK2)/AMPK signaling axis that activates fatty acid scavenging and transport, as well as β-oxidation. Enhanced lipid metabolism thereby provides the necessary biomass (fatty acids) for phospholipid biosynthesis and energy (ATP) to support the metabolically demanding processes of cell growth, proliferation, and migration. In all, our findings provide a strong rationale for targeting lipid metabolism and the Ca2+/CAMKK2/AMPK signaling axis in MYC-driven, and potentially claudin-low, breast cancers. Citation Format: Jessica C. Casciano, Adam Cohen-Nowak, Johan Vande Voorde, Qifeng Zhang, Susan Chalmers, Mairi Sandison, Ann Hedley, Tony McBryan, Thomas Beer, Hsin-Yao Tang, David W. Speicher, Peter Adams, Xiufeng Liu, Richard Schlegel, John McCarron, Michael J. Wakelam, Eyal Gottlieb, Zachary T. Schug. MYC expression promotes lipid metabolism and metabolic plasticity in human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1441.

The endothelium is the innermost layer of vascular cells, with a central role in maintaining vascular health. Within endothelial cells, mitochondria play important roles in calcium homeostasis, reactive oxygen species production and, to a lesser extent, ATP generation. The balance of mitochondrial fission, fusion and motility is likely to provide fine control of subcellular location and interactions of the organelle; however, the outcome of perturbation of mitochondrial dynamics on endothelial function remains unclear. We sought to address this gap by investigating the effects of mitochondrial fission inhibitor, Mdivi-1, on endothelial cells. Treatment of cultured endothelial cells with Mdivi-1 (1 or 10 µM, 48 hour) increased mitochondrial length and branching extent compared to control, consistent with inhibition of fission. Mdivi-1 increased branched, twisted and looped endothelial mitochondrial morphologies, whilst also reducing net mitochondrial speed. No acute toxicity was observed after Mdivi-1 treatment (10 µM, 48 hour), however Mdivi-1 did decrease the intracellular content of the glycoprotein von Willebrand Factor (produced, stored and released by endothelial cells to aid thrombosis). Endothelial gap junction communication was also assessed as a function of confluent cells’ ability to inter-cellularly transfer the dye, Lucifer yellow; Mdivi-1 decreased dye transfer rates, suggesting reduced intercellular gap junction communication. In conclusion, Mdivi-1 treatment altered endothelial mitochondrial morphology and dynamics, and also decreased von Willebrand Factor content and gap junction communication, however the mechanistic links remain unclear. Clarification is important, as modulation of mitochondrial dynamics has been proposed as a novel target against the cell proliferation associated with vascular disease.

In smooth muscle, Ca release from the internal store into the cytoplasm occurs via inositol trisphosphate (IP3R) and ryanodine receptors (RyR). The internal Ca stores containing IP3R and RyR may be arranged as multiple separate compartments with various IP3R and RyR arrangements, or there may be a single structure containing both receptors. The existence of multiple stores is proposed to explain several physiological responses which include the progression of Ca waves, graded Ca release from the store and various local responses and sensitivities. We suggest that, rather than multiple stores, a single luminally-continuous store exists in which Ca is in free diffusional equilibrium throughout. Regulation of Ca release via IP3R and RyR by the local Ca 2+ concentration within the stores explains the apparent existence of multiple stores and physiological processes such as graded Ca release and Ca waves. Close positioning of IP3R on the store with mitochondria or with receptors on the plasma membrane creates ‘IP3 junctions’ to generate local responses on the luminally-continuous store.

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