Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms

Mitochondrial [Ca²⁺] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca²⁺ overload. This regulation depends on Ca²⁺ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca²⁺ uniporter (MCU). Here, we have studied the mitochondrial Ca²⁺ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca²⁺ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca²⁺] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca²⁺ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca²⁺ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca²⁺ pathway.     

Effects of MICU1-Mediated Mitochondrial Calcium Uptake on Energy Metabolism and Quality of Vitrified-Thawed Mouse Metaphase II Oocytes

Background: Oocyte vitrification has been widely used in the treatment of infertility and fertility preservation. However, vitrification-induced mitochondrial damage adversely affects oocyte development. Several studies have reported that mitochondrial calcium uptake protein 1 (MICU1) regulates the uptake of mitochondrial calcium by the mitochondrial calcium uniporter (MCU) and subsequently controls aerobic metabolism and oxidative stress in mitochondria, but research considering oocytes remains unreported. We evaluated whether the addition of MICU1 modulators enhances mitochondrial activity, pyruvate metabolism, and developmental competence after warming of MII oocytes. Methods: Retrieved MII oocytes of mice were classified as vitrified or control groups. After thawing, oocytes of vitrified group were cultured with or without DS16570511 (MICU1 inhibitor) and MCU-i4 (MICU1 activator) for 2 h. Results: Mitochondrial Ca²⁺ concentration, pyruvate dephosphorylation level, and MICU1 expression of MII oocytes were significantly increased after vitrification. These phenomena were further exacerbated by the addition of MCU-i4 and reversed by the addition of DS16570511 after warming. However, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) in vitrified-warmed MII oocytes drop significantly after vitrification, which was improved after MCU-i4 treatment and decreased significantly after DS16570511 treatment. The vitrification process was able to elicit a development competence reduction. After parthenogenetic activation, incubation of the thawed oocytes with MCU-i4 did not alter the cleavage and blastocyst rates. Moreover, incubation of the thawed oocytes with DS16570511 reduced the cleavage and blastocyst rates. Conclusions: MICU1-mediated increasing mitochondrial calcium uptake after vitrification of the MII oocytes promoted the pyruvate oxidation, and this process may maintain oocyte development competence by compensating for the consumption of ATP under stress state.     

The Regulatory Roles of Mitochondrial Calcium and the Mitochondrial Calcium Uniporter in Tumor Cells

Mitochondria, as the main site of cellular energy metabolism and the generation of oxygen free radicals, are the key switch for mitochondria-mediated endogenous apoptosis. Ca²⁺ is not only an important messenger for cell proliferation, but it is also an indispensable signal for cell death. Ca²⁺ participates in and plays a crucial role in the energy metabolism, physiology, and pathology of mitochondria. Mitochondria control the uptake and release of Ca²⁺ through channels/transporters, such as the mitochondrial calcium uniporter (MCU), and influence the concentration of Ca²⁺ in both mitochondria and cytoplasm, thereby regulating cellular Ca²⁺ homeostasis. Mitochondrial Ca²⁺ transport-related processes are involved in important biological processes of tumor cells including proliferation, metabolism, and apoptosis. In particular, MCU and its regulatory proteins represent a new era in the study of MCU-mediated mitochondrial Ca²⁺ homeostasis in tumors. Through an in-depth analysis of the close correlation between mitochondrial Ca²⁺ and energy metabolism, autophagy, and apoptosis of tumor cells, we can provide a valuable reference for further understanding of how mitochondrial Ca²⁺ regulation helps diagnosis and therapy.     

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca²⁺) mishandling. Ca²⁺ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca²⁺ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca²⁺ concentration was increased, but RYR1-mediated Ca²⁺ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca²⁺ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca²⁺ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca²⁺ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.     

A Calcium Guard in the Outer Membrane: Is VDAC a Regulated Gatekeeper of Mitochondrial Calcium Uptake?

Already in the early 1960s, researchers noted the potential of mitochondria to take up large amounts of Ca²⁺. However, the physiological role and the molecular identity of the mitochondrial Ca²⁺ uptake mechanisms remained elusive for a long time. The identification of the individual components of the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane in 2011 started a new era of research on mitochondrial Ca²⁺ uptake. Today, many studies investigate mitochondrial Ca²⁺ uptake with a strong focus on function, regulation, and localization of the MCUC. However, on its way into mitochondria Ca²⁺ has to pass two membranes, and the first barrier before even reaching the MCUC is the outer mitochondrial membrane (OMM). The common opinion is that the OMM is freely permeable to Ca²⁺. This idea is supported by the presence of a high density of voltage-dependent anion channels (VDACs) in the OMM, forming large Ca²⁺ permeable pores. However, several reports challenge this idea and describe VDAC as a regulated Ca²⁺ channel. In line with this idea is the notion that its Ca²⁺ selectivity depends on the open state of the channel, and its gating behavior can be modified by interaction with partner proteins, metabolites, or small synthetic molecules. Furthermore, mitochondrial Ca²⁺ uptake is controlled by the localization of VDAC through scaffolding proteins, which anchor VDAC to ER/SR calcium release channels. This review will discuss the possibility that VDAC serves as a physiological regulator of mitochondrial Ca²⁺ uptake in the OMM.     

Characterizing SERCA Function in Murine Skeletal Muscles after 35–37 Days of Spaceflight

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca²⁺ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) pump actively brings cytosolic Ca²⁺ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca²⁺ ([Ca²⁺]_i). SERCA dysfunction contributes to elevations in [Ca²⁺]_i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35–37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca²⁺ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca²⁺ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.     

Mitochondrial Dynamics and Mitophagy in Skeletal Muscle Health and Aging

The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca²⁺ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy—the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.     

Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca²⁺ microdomain (HCMD) generated by Ca²⁺ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca²⁺ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na⁺/Ca²⁺ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K⁺-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca²⁺ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365"}}CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) K⁺-challenged BCCs, and (v) non-inactivated [Ca²⁺]_c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca²⁺ handling during K⁺ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.     

Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca²⁺-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca²⁺, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca²⁺]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca²⁺]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca²⁺-dependent regulation of actin–myosin interactions.     

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca²⁺ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca²⁺ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca²⁺ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca²⁺ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.     

Thyroid Hormone Induces Ca2+-Mediated Mitochondrial Activation in Brown Adipocytes

Thyroid hormones, including 3,5,3′-triiodothyronine (T₃), cause a wide spectrum of genomic effects on cellular metabolism and bioenergetic regulation in various tissues. The non-genomic actions of T₃ have been reported but are not yet completely understood. Acute T₃ treatment significantly enhanced basal, maximal, ATP-linked, and proton-leak oxygen consumption rates (OCRs) of primary differentiated mouse brown adipocytes accompanied with increased protein abundances of uncoupling protein 1 (UCP1) and mitochondrial Ca²⁺ uniporter (MCU). T₃ treatment depolarized the resting mitochondrial membrane potential (Ψ_m) but augmented oligomycin-induced hyperpolarization in brown adipocytes. Protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were activated by T₃, leading to the inhibition of autophagic degradation. Rapamycin, as an mTOR inhibitor, blocked T₃-induced autophagic suppression and UCP1 upregulation. T₃ increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) in brown adipocytes. Most of the T₃ effects, including mTOR activation, UCP1 upregulation, and OCR increase, were abrogated by intracellular Ca²⁺ chelation with BAPTA-AM. Calmodulin inhibition with W7 or knockdown of MCU dampened T₃-induced mitochondrial activation. Furthermore, edelfosine, a phospholipase C (PLC) inhibitor, prevented T₃ from acting on [Ca²⁺]_i, UCP1 abundance, Ψ_m, and OCR. We suggest that short-term exposure of T₃ induces UCP1 upregulation and mitochondrial activation due to PLC-mediated [Ca²⁺]_i elevation in brown adipocytes.     

Dynamin-Related Protein 1 Inhibitors Protect against Ischemic Toxicity through Attenuating Mitochondrial Ca2+ Uptake from Endoplasmic Reticulum Store in PC12 Cells

Intracellular calcium homeostasis disorder and mitochondrial dysfunction are involved in many acute and chronic brain diseases, including ischemic brain injury. An imbalance in mitochondrial fission and fusion is one of the most important structural abnormalities found in a large number of mitochondrial dysfunction related diseases. Here, we investigated the effects of mitochondrial division inhibitor A (mdivi A) and mdivi B, two small molecule inhibitors of mitochondrial fission protein dunamin-related protein 1 (Drp-1), in neuronal injury induced by oxygen-glucose deprivation (OGD) in PC12 cells. We found that mdivi A and mdivi B inhibited OGD-induced neuronal injury through attenuating apoptotic cell death. These two inhibitors also preserved mitochondrial function, as evidenced by reduced reactive oxygen species (ROS) generation and cytochrome c release, as well as prevented loss of mitochondrial membrane potential (MMP). Moreover, mdivi A and mdivi B significantly suppressed mitochondrial Ca²⁺ uptake, but had no effect on cytoplasmic Ca²⁺ after OGD injury. The results of calcium imaging and immunofluorescence staining showed that Drp-1 inhibitors attenuated endoplasmic reticulum (ER) Ca²⁺ release and prevented ER morphological changes induced by OGD. These results demonstrate that Drp-1 inhibitors protect against ischemic neuronal injury through inhibiting mitochondrial Ca²⁺ uptake from the ER store and attenuating mitochondrial dysfunction.     

Study of the Expression and Function of Calcium-Sensing Receptor in Human Skeletal Muscle

Skeletal muscle has an outstanding capacity for regeneration in response to injuries, but there are disorders in which this process is seriously impaired, such as sarcopenia. Pharmacological treatments to restore muscle trophism are not available, therefore, the identification of suitable therapeutic targets that could be useful for the treatment of skeletal reduced myogenesis is highly desirable. In this in vitro study, we explored the expression and function of the calcium-sensing receptor (CaSR) in human skeletal muscle tissues and their derived satellite cells. The results obtained from analyses with various techniques of gene and protein CaSR expression and of its secondary messengers in response to calcium (Ca²⁺) and CaSR drugs have demonstrated that this receptor is not present in human skeletal muscle tissues, neither in the established satellite cells, nor during in vitro myogenic differentiation. Taken together, our data suggest that, although CaSR is a very important drug target in physiology and pathology, this receptor probably does not have any physiological role in skeletal muscle in normal conditions.     

Influence of dietary n-3 fatty acids on the membrane properties of skeletal muscle in pigs

The aim of the experiment was the in vivo modification of long-chain fatty acids in phospholipids and to investigate the impact on Ca²⁺ transport of sarcoplasmic reticulum (SR). Ten pigs were fed daily a diet containing 1.3 g n-3 fatty acids/kg diet (control), and ten pigs were fed a diet containing 14 g n-3 fatty acids/kg diet (n-3 diet) during the growing-finishing period. The intake of dietary n-3 fatty acids increased the percentages of these fatty acids in the phospholipids of skeletal muscle sarcoplasmic reticulum (21.6% n-3 fatty acids) and in the polar fraction of total muscle homogenates (30.7% n-3 fatty acids) significantly, compared with control (3.0% and 3.9% n-3 fatty acids, respectively), while the n-6 fatty acid concentration was reduced. In phosphatidylethanolamine of skeletal muscle polar lipids eico-sapentaenoic (EPA) and docosahexaenoic acids (DPA) were increased seven times compared with control. There were no differences in the maximal rate of Ca²⁺ uptake in skeletal muscle SR between the groups. However, the activity of Ca²⁺-ATPase of skeletal muscle SR was elevated in the n-3 diet group. It is suggested, that n-3 fatty acid enriched diet can change the complex membrane composition dependent on experimental conditions and animal species leading to different effects on membrane protein activities.     

Intracellular Ca2+ Signaling in Protozoan Parasites: An Overview with a Focus on Mitochondria

Ca²⁺ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca²⁺ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca²⁺ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca²⁺ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca²⁺-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca²⁺ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca²⁺ uptake by pathogenic protists.     

Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors

The mitochondrial Ca²⁺ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca²⁺ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca²⁺ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca²⁺ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.     

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.     

Exploiting Peptidomimetics to Synthesize Compounds That Activate Ryanodine Receptor Calcium Release Channels

Ryanodine receptor (RyR) Ca²⁺‐release channels are essential for contraction in skeletal and cardiac muscle and are prime targets for modification of contraction in disorders that affect either the skeletal or heart musculature. We designed and synthesized a number of compounds with structures based on a naturally occurring peptide ( A peptides) that modifies the activity of RyRs. In total, 34 compounds belonging to eight different classes were prepared. The compounds were screened for their ability to enhance Ca²⁺ release from isolated cardiac sarcoplasmic reticulum (SR) vesicles, with 25 displaying enhanced Ca²⁺ release. Competition studies with the parent peptides indicated that the synthetic compounds act at a competing site. The activity of the most effective of the compounds, BIT 180, was further explored using Ca²⁺ release from skeletal SR vesicles and contraction in intact skeletal muscle fibers. The compounds did not alter tension in intact fibers, indicating that (as expected) they are not membrane permeable, but importantly, that they are not toxic to the intact cells. Proof in principal that the compounds would be effective in intact muscle fibers if rendered membrane permeable was obtained with a structurally related membrane‐permeable scorpion toxin (imperatoxin A), which was found to enhance contraction.     

MiR-25 Protects Cardiomyocytes against Oxidative Damage by Targeting the Mitochondrial Calcium Uniporter

MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H₂O₂, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3'' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H₂O₂-induced elevation of mitochondrial Ca²⁺ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.