MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Mechanisms of reduced myocardial energetics of the dystrophic heart.

Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD), characterized by the progressive replacement of contractile tissue with scar tissue. Effective therapies for dystrophic cardiomyopathy will require addressing the disease before the onset of fibrosis, however, the mechanisms of the early disease are poorly understood. To understand the pathophysiology of DMD, we perform a detailed functional assessment of cardiac function of the mdx mouse, a model of DMD. These studies use a combination of functional, metabolomic, and spectroscopic approaches to fully characterize the contractile, energetic, and mitochondrial function of beating hearts. Through these innovative approaches, we demonstrate that the dystrophic heart has reduced cardiac reserve and is energetically limited. We show that this limitation does not result from poor delivery of oxygen. Using spectroscopic approaches, we provide evidence that mitochondria in the dystrophic heart have attenuated mitochondrial membrane potential and deficits in the flow of electrons in complex IV of the electron transport chain. These studies provide evidence that poor myocardial energetics precede the onset of significant cardiac fibrosis and likely results from mitochondrial dysfunction centered around complex IV and reduced membrane potential. The multimodal approach used here implicates specific molecular components in the etiology of reduced energetics. Future studies focused on these targets may provide therapies that improve the energetics of the dystrophic heart leading to improved resiliency against damage and preservation of myocardial contractile tissue.NEW & NOTEWORTHY Dystrophic hearts have poor contractile reserve that is associated with a reduction in myocardial energetics. We demonstrate that oxygen delivery does not contribute to the limited energy production of the dystrophic heart even with increased workloads. Cytochrome optical spectroscopy of the contracting heart reveals alterations in complex IV and evidence of depolarized mitochondrial membranes. We show specific alterations in the electron transport chain of the dystrophic heart that may contribute to poor myocardial energetics.

Distinct effects of cardiac mitochondrial calcium uniporter inactivation via EMRE deletion in the short and long term.

Transport of Ca2+ into mitochondria is thought to stimulate the production of ATP, a critical process in the heart's fight or flight response, but excess Ca2+ can trigger cell death. The mitochondrial Ca2+ uniporter complex is the primary route of Ca2+ transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differed from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca2+ uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca2+, had lower basal mitochondrial Ca2+ levels, and displayed attenuated Ca2+-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca2+ handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.

Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes.

BACKGROUND: Silica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2. RESULTS: The cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 µg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 µg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP. CONCLUSION: The aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure.

Mitochondrial Hyperacetylation in the Failing Hearts of Obese Patients Mediated Partly by a Reduction in SIRT3: The Involvement of the Mitochondrial Permeability Transition Pore.

BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.

Therapeutic silencing of HPV 16 E7 by systemic administration of siRNA-neutral DOPC nanoliposome in a murine cervical cancer model with obesity.

PURPOSE: To evaluate the effectiveness of a neutral DOPC nanoliposome system for the delivery of siRNA to tumor cells in an obese murine cervical cancer model. METHODS: In vitro silencing of E6-E7 mRNA and E7 protein using siRNAE6 or siRNAE7 was analyzed in TC-1 cells by RT-PCR and Western blot. Silencing and antitumor capacities of siRNAE7-DOPC-nanoparticles (NP) were tested in vivo in both normal and obese mice using qPCR. These NPs were administered twice a week for 15 days and tumor volume and weight were recorded. RESULTS: Levels of in vitro E6-E7 silencing were 90% for mRNA and 60% for protein when siRNAE7 was used. On the other hand when siRNAE6 was used, the levels of silencing were 50% for E6-E7 mRNA and only 20% for protein. In vivo E7 mRNA silencing by siRNAE7-DOPC-NP was similar (60%) in both non-obese and obese mouse models. The therapeutic study showed a 65% decrease in tumor volume and a 57% reduction in tumor weight as compared to the control groups. CONCLUSION: There was no negative impact of obesity on the antitumor activity of siRNA-DOPC-NP in obese mice.

Resveratrol Prevents Right Ventricle Dysfunction, Calcium Mishandling, and Energetic Failure via SIRT3 Stimulation in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential (ΔΨm); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca2+ transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH.

A single session of physical activity restores the mitochondrial organization disrupted by obesity in skeletal muscle fibers.

BACKGROUND: Several studies have proved that physical activity (PA) regulates energetic metabolism associated with mitochondrial dynamics through AMPK activation in healthy subjects. Obesity, a condition that induces oxidative stress, mitochondrial dysfunction, and low AMPK activity leads to mitochondrial fragmentation. However, few studies describe the effect of PA on mitochondrial dynamics regulation in obesity. AIM: The present study aimed to evaluate the effect of a single session of PA on mitochondrial dynamics regulation as well as its effect on mitochondrial function and organization in skeletal muscles of obese rats (Zucker fa/fa). MAIN METHODS: Male Zucker lean and Zucker fa/fa rats aged 12 to 13 weeks were divided into sedentary and subjected-to-PA (single session swimming) groups. Gastrocnemius muscle was dissected into isolated fibers, mitochondria, mRNA, and total proteins for their evaluation. KEY FINDINGS: The results showed that PA increased the Mfn-2 protein level in the lean and obese groups, whereas Drp1 levels decreased in the obese group. OMA1 protease levels increased in the lean group and decreased in the obese group. Additionally, AMPK analysis parameters (expression, protein level, and activity) did not increase in the obese group. These findings correlated with the partial restoration of mitochondrial function in the obese group, increasing the capacity to maintain the membrane potential after adding calcium as a stressor, and increasing the transversal organization level of the mitochondria analyzed in isolated fibers. SIGNIFICANCE: These results support the notion that obese rats subjected to PA maintain mitochondrial function through mitochondrial fusion activation by an AMPK-independent mechanism.

Resveratrol Prevents Right Ventricle Remodeling and Dysfunction in Monocrotaline-Induced Pulmonary Arterial Hypertension with a Limited Improvement in the Lung Vasculature.

Pulmonary arterial hypertension (PAH) is a life-threatening disease that is characterized by an increase in pulmonary vascular pressure, leading to ventricular failure and high morbidity and mortality. Resveratrol, a phenolic compound and a sirtuin 1 pathway activator, has known dietary benefits and is used as a treatment for anti-inflammatory and cardiovascular diseases. Its therapeutic effects have been published in the scientific literature; however, its benefits in PAH are yet to be precisely elucidated. Using a murine model of PAH induced by monocrotaline, the macroscopic and microscopic effects of a daily oral dose of resveratrol in rats with PAH were evaluated by determining its impact on the lungs and the right and left ventricular function. While most literature has focused on smooth muscle cell mechanisms and lung pathology, our results highlight the relevance of therapy-mediated improvement of right ventricle and isolated cardiomyocyte physiology in both ventricles. Although significant differences in the pulmonary architecture were not identified either micro- or macroscopically, the effects of resveratrol on right ventricular function and remodeling were observed to be beneficial. The values for the volume, diameter, and contractility of the right ventricular cardiomyocytes returned to those of the control group, suggesting that resveratrol has a protective effect against ventricular dysfunction and pathological remodeling changes in PAH. The effect of resveratrol in the right ventricle delayed the progression of findings associated with right heart failure and had a limited positive effect on the architecture of the lungs. The use of resveratrol could be considered a future potential adjunct therapy, especially when the challenges to making a diagnosis and the current therapy limitations for PAH are taken into consideration.

Changes in the Stoichiometry of Uniplex Decrease Mitochondrial Calcium Overload and Contribute to Tolerance of Cardiac Ischemia/Reperfusion Injury in Hypothyroidism.

Background: Thyroid hormone status in hypothyroidism (HT) downregulates key elements in Ca2+ handling within the heart, reducing contractility, impairing the basal energetic balance, and increasing the risk of cardiovascular disease. Mitochondrial Ca2+ transport is reduced in HT, and tolerance to reperfusion damage has been documented, but the precise mechanism is not well understood. Therefore, we aimed to determine the stoichiometry and activity of the mitochondrial Ca2+ uniporter or uniplex in an HT model and the relevance to the opening of the mitochondrial permeability transition pores (mPTP) during ischemia/reperfusion (I/R) injury. Methods: An HT model was established in Wistar rats by treatment with 6-propylthiouracil for 28 days. Uniplex composition and activity were determined in cardiac mitochondria. Hearts were perfused ex vivo to induce I/R injury, and functional parameters related to contractility and tissue viability were evaluated. Results: The cardiac stoichiometry between two subunits of the uniplex (MICU1/MCU) increased by 25% in animals with HT. The intramitochondrial Ca2+ content was reduced by 40% and was less prone to the mPTP opening. After I/R injury, ischemic contracture and the onset of ventricular fibrillation were delayed in animals with HT, concomitant with a reduction in oxidative damage and mitochondrial dysfunction. Conclusions: Our results suggest that HT is associated with an increase in the cardiac MICU1/MCU ratio, thereby changing the stoichiometry between these subunits to increase the threshold to cytosolic Ca2+ and reduce mitochondrial Ca2+ overload. Our results also demonstrate that this HT model can be used to explore the role of mitochondrial Ca2+ transport in cardiac diseases due to its induced tolerance to cardiac damage.

Microbial Competition of Rhodotorula mucilaginosa UANL-001L and E. coli increase biosynthesis of Non-Toxic Exopolysaccharide with Applications as a Wide-Spectrum Antimicrobial.

Bacterial species are able to colonize and establish communities in biotic and abiotic surfaces. Moreover, within the past five decades, incidence of bacterial strains resistant to currently used antibiotics has increased dramatically. This has led to diverse health issues and economical losses for different industries. Therefore, there is a latent need to develop new and more efficient antimicrobials. This work reports an increased production of an exopolysaccharide in a native yeast strain isolated from the Mexican Northeast, Rhodotorula mucilaginosa UANL-001L, when co-cultured with E. coli. The exopolysaccharide produced is chemically and physically characterized and its applications as an antimicrobial and antibiofilm are explored. The exopolysaccharide is capable of inhibiting planktonic growth and biofilm formation in Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, the exopolysaccharide studied here does not exhibit cytotoxic effects when assessed both, in vitro against an H9c2 mammalian cell line, and in vivo in a murine toxicity model. Taken together, the properties of this exopolysaccharide indicate that it has potential applications to inhibit bacterial colonization in medical and industrial settlings.

Ex Vivo Cardiotoxicity of Antineoplastic Casiopeinas Is Mediated through Energetic Dysfunction and Triggered Mitochondrial-Dependent Apoptosis.

Casiopeinas are a group of copper-based antineoplastic molecules designed as a less toxic and more therapeutic alternative to cisplatin or Doxorubicin; however, there is scarce evidence about their toxic effects on the whole heart and cardiomyocytes. Given this, rat hearts were perfused with Casiopeinas or Doxorubicin and the effects on mechanical performance, energetics, and mitochondrial function were measured. As well, the effects of Casiopeinas-triggered cell death were explored in isolated cardiomyocytes. Casiopeinas III-Ea, II-gly, and III-ia induced a progressive and sustained inhibition of heart contractile function that was dose- and time-dependent with an IC50 of 1.3 ± 0.2, 5.5 ± 0.5, and 10 ± 0.7 µM, correspondingly. Myocardial oxygen consumption was not modified at their respective IC50, although ATP levels were significantly reduced, indicating energy impairment. Isolated mitochondria from Casiopeinas-treated hearts showed a significant loss of membrane potential and reduction of mitochondrial Ca2+ retention capacity. Interestingly, Cyclosporine A inhibited Casiopeinas-induced mitochondrial Ca2+ release, which suggests the involvement of the mitochondrial permeability transition pore opening. In addition, Casiopeinas reduced the viability of cardiomyocytes and stimulated the activation of caspases 3, 7, and 9, demonstrating a cell death mitochondrial-dependent mechanism. Finally, the early perfusion of Cyclosporine A in isolated hearts decreased Casiopeinas-induced dysfunction with reduction of their toxic effect. Our results suggest that heart cardiotoxicity of Casiopeinas is similar to that of Doxorubicin, involving heart mitochondrial dysfunction, loss of membrane potential, changes in energetic metabolites, and apoptosis triggered by mitochondrial permeability.

Small Interfering RNA Targeting Mitochondrial Calcium Uniporter Improves Cardiomyocyte Cell Viability in Hypoxia/Reoxygenation Injury by Reducing Calcium Overload.

Intracellular Ca2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca2+ (mCa2+) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy.