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RationaleMitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types1–3. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection1, 4. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure1. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models1, 4, 5. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue6, 7. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling.ObjectiveThis study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication.Methods and ResultsUsing high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound β-ACTIN only exists in MAM and not in the purified mitochondria.Additionally, co-immunoprecipitation pulls down both DRP1 and β-ACTIN together. Inhibition of β-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and β-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts.ConclusionsThe results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with β-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through β-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.

SUMMARY Mitochondrial Ca2+ elevations enhance ATP production, but uptake must be balanced by efflux to avoid overload. Uptake is mediated by the mitochondrial Ca2+ uniporter channel complex (MCUC), and extrusion is controlled largely by the Na+/Ca2+ exchanger (NCLX), both driven electrogenically by the inner membrane potential (Δ𝚿m). MCUC forms hotspots at the cardiac mitochondria-junctional SR (jSR) association to locally receive Ca2+ signals; however, the distribution of NCLX is unknown. Our fractionation-based assays reveal that extensively jSRassociated mitochondrial segments contain a minor portion of NCLX and lack Na+-dependent Ca2+ extrusion. This pattern is retained upon in vivo NCLX overexpression, suggesting extensive targeting to non-jSR-associated submitochondrial domains and functional relevance. In cells with non-polarized MCUC distribution, upon NCLX overexpression the same given increase in matrix Ca2+ expends more Δ𝚿m. Thus, cardiac mitochondrial Ca2+ uptake and extrusion are reciprocally polarized, likely to optimize the energy efficiency of local calcium signaling in the beating heart., In Brief Calcium signals control mitochondrial fuel generation. De La Fuente et al. report that in heart mitochondria, calcium uptake and extrusion are spatially separated; the most calcium-exposed area is an uptake hotspot, but it lacks extrusion to optimize signaling efficiency and minimize the energy expense of controlling mitochondrial function by calcium., Graphical Abstract

Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.

Aims Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. Methods and results In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption. Conclusions Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

Background: Endothelial progenitor cells (EPCs) were indicated in vascular repair, angiogenesis of ischemic organs, and inhibition of formation of initial hyperplasia. Differentiation of endothelial cells (ECs) from human induced pluripotent stem cells (hiPSC)-derived endothelial cells (hiPSC-ECs) provides an unlimited supply for clinical application. Furthermore, magnetic cell labelling offers an effective way of targeting and visualization of hiPSC-ECs and is the next step towards in vivo studies. Methods: ECs were differentiated from hiPSCs and labelled with uncoated superparamagnetic iron-oxide nanoparticles (uSPIONs). uSPION uptake was compared between hiPSC-ECs and mature ECs isolated from patients by software analysis of microscopy pictures after Prussian blue cell staining. The acute and long-term cytotoxic effects of uSPIONs were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and Annexin assay. Results: We showed, for the first time, uptake of uncoated SPIONs (uSPIONs) by hiPSC-ECs. In comparison with mature ECs of identical genetic background hiPSC-ECs showed lower uSPION uptake. However, all the studied endothelial cells were effectively labelled and showed magnetic properties even with low labelling concentration of uSPIONs. uSPIONs prepared by microwave plasma synthesis did not show any cytotoxicity nor impair endothelial properties. Conclusion: We show that hiPSC-ECs labelling with low concentration of uSPIONs is feasible and does not show any toxic effects in vitro, which is an important step towards animal studies.

Aims Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. Methods and results In the present study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Electrocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the sarco-endoplasmic reticulum membranes. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough endoplasmic reticulum were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads and in the vicinity of mitochondria. At a functional level, ER stress resulted in a substantial decrease in mitochondrial biogenesis as demonstrated by the decrease of the expression of PGC-1α and of its targets NRF1, Tfam, CS and COXIV. ER stress also led to an impairment of mitochondrial oxidative phosphorylation and to a metabolic remodeling characterized by a shift from fatty acid to glycolytic substrate consumption. Conclusion Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

Photosensitizers (PSs) in photodynamic therapy (PDT) are administered systemically with preferential accumulation in malignant cells; however, exposure of non-malignant cells to PS may be clinically relevant if PS molecules affect the pro-apoptotic cascade without illumination. The important PS characteristic is low dark cytotoxicity. Originally, Hypericin (Hyp) as PS displayed minimal dark cytotoxicity and preferential accumulation in tumor cells, however recently, evidence to contrary appeared. The molecular mechanisms underlying Hyp dark toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and they are not understood in detail. Our previous study demonstrated that in human glioma U87 MG cells, Hyp affected localization of pro-apoptotic Bax and Bak, from Bcl2 family of proteins that are key regulators of apoptosis and mitogenesis. To understand the mechanisms underlying Hyp dark toxicity better, we investigated the Hyp effect on mitochondrial function and cell metabolism in U87 MG and human coronary artery endothelial (HCAEC) cells. We have found that Hyp in HCAEC displayed significant dark cytotoxicity in contrast to U87 MG cells. Further, Hyp significantly affected mitochondria function in U87 MG by turning them towards glycolysis, but it did not have this effect in HCAEC. In addition, in both cell types Hyp in the dark triggered morphological changes in mitochondria and the ER and GA ultrastructure indicative of altered protein synthesis. Lastly, in U87 MG and HCAEC Hyp affected the distribution of Bcl2 and Bax proteins via hydrophobic interaction at their BH1-BH3 domain. Overall, our results indicate that Hyp in the dark had effects at several sub-cellular levels similar to small mitochondria targeting molecules (mitocans), and thus Hyp should be explored as mitocan. Support: EU 7FP PIRG06-GA-2009-256580, CELIM 316310; EU SF ITMS 26240120040; VEGA -1-0111-12, APVV-0134-11 and APVV-0242-11