Mitochondrial calcium uniporter affects neutrophil bactericidal activity during Staphylococcus aureus infection

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus ; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1 -/- ) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1 -/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus . Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1 -/- neutrophils alone are not more antibacterial towards S. aureus , but rather enhanced suicidal NETosis by MICU1 -/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1 -/- neutrophils in the heart produced higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

MICU3 regulates mitochondrial Ca2+-dependent antioxidant response in skeletal muscle aging

Age-related loss of skeletal muscle mass and function, termed sarcopenia, could impair the quality of life in the elderly. The mechanisms involved in skeletal muscle aging are intricate and largely unknown. However, more and more evidence demonstrated that mitochondrial dysfunction and apoptosis also play an important role in skeletal muscle aging. Recent studies have shown that mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium affects skeletal muscle mass and function by affecting mitochondrial function. During aging, we observed downregulated expression of mitochondrial calcium uptake family member3 (MICU3) in skeletal muscle, a regulator of MCU, which resulted in a significant reduction in mitochondrial calcium uptake. However, the role of MICU3 in skeletal muscle aging remains poorly understood. Therefore, we investigated the effect of MICU3 on the skeletal muscle of aged mice and senescent C2C12 cells induced by d-gal. Downregulation of MICU3 was associated with decreased myogenesis but increased oxidative stress and apoptosis. Reconstitution of MICU3 enhanced antioxidants, prevented the accumulation of mitochondrial ROS, decreased apoptosis, and increased myogenesis. These findings indicate that MICU3 might promote mitochondrial Ca2+ homeostasis and function, attenuate oxidative stress and apoptosis, and restore skeletal muscle mass and function. Therefore, MICU3 may be a potential therapeutic target in skeletal muscle aging.

Mitochondrial calcium uptake regulates tumour progression in embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is characterized by a failure of cells to complete skeletal muscle differentiation. Although ERMS cells are vulnerable to oxidative stress, the relevance of mitochondrial calcium homeostasis in oncogenesis is unclear. Here, we show that ERMS cell lines as well as primary tumours exhibit elevated expression of the Mitochondrial Calcium Uniporter (MCU). MCU knockdown resulted in impaired mitochondrial calcium uptake and a reduction in mitochondrial reactive oxygen species (mROS) levels. Phenotypically, MCU knockdown cells exhibited reduced cellular proliferation and motility, with an increased propensity to differentiate in vitro and in vivo. RNA-sequencing of MCU knockdown cells revealed a significant reduction in genes involved in TGF? signalling that play prominent roles in oncogenesis and inhibition of myogenic differentiation. Interestingly, modulation of mROS production impacted TGF? signalling. Our study elucidates mechanisms by which mitochondrial calcium dysregulation promotes tumour progression and suggests that targeting the MCU complex to restore mitochondrial calcium homeostasis could be a therapeutic avenue in ERMS.

The mitochondrial calcium uniporter promotes arrhythmias caused by high-fat diet

Obesity and diabetes increase the risk of arrhythmia and sudden cardiac death. However, the molecular mechanisms of arrhythmia caused by metabolic abnormalities are not well understood. We hypothesized that mitochondrial dysfunction caused by high fat diet (HFD) promotes ventricular arrhythmia. Based on our previous work showing that saturated fat causes calcium handling abnormalities in cardiomyocytes, we hypothesized that mitochondrial calcium uptake contributes to HFD-induced mitochondrial dysfunction and arrhythmic events. For experiments, we used mice with conditional cardiac-specific deletion of the mitochondrial calcium uniporter (Mcu), which is required for mitochondrial calcium uptake, and littermate controls. Mice were used for in vivo heart rhythm monitoring, perfused heart experiments, and isolated cardiomyocyte experiments. MCU KO mice are protected from HFD-induced long QT, inducible ventricular tachycardia, and abnormal ventricular repolarization. Abnormal repolarization may be due, at least in part, to a reduction in protein levels of voltage gated potassium channels. Furthermore, isolated cardiomyocytes from MCU KO mice exposed to saturated fat are protected from increased reactive oxygen species (ROS), mitochondrial dysfunction, and abnormal calcium handling. Activation of calmodulin-dependent protein kinase (CaMKII) corresponds with the increase in arrhythmias in vivo. Additional experiments showed that CaMKII inhibition protects cardiomyocytes from the mitochondrial dysfunction caused by saturated fat. Hearts from transgenic CaMKII inhibitor mice were protected from inducible ventricular tachycardia after HFD. These studies identify mitochondrial dysfunction caused by calcium overload as a key mechanism of arrhythmia during HFD. This work indicates that MCU and CaMKII could be therapeutic targets for arrhythmia caused by metabolic abnormalities.

MCU-complex-mediated mitochondrial calcium signaling is impaired in Barth syndrome

Calcium signaling via mitochondrial calcium uniporter (MCU) complex coordinates mitochondrial bioenergetics with cellular energy demands. Emerging studies show that the stability and activity of the pore-forming subunit of the complex, MCU, is dependent on the mitochondrial phospholipid, cardiolipin (CL), but how this impacts calcium-dependent mitochondrial bioenergetics in CL-deficiency disorder like Barth syndrome (BTHS) is not known. Here we utilized multiple models of BTHS including yeast, mouse muscle cell line, as well as BTHS patient cells and cardiac tissue to show that CL is required for the abundance and stability of the MCU-complex regulatory subunit MICU1. Interestingly, the reduction in MICU1 abundance in BTHS mitochondria is independent of MCU. Unlike MCU and MICU1/MICU2, other subunit and associated factor of the uniporter complex, EMRE and MCUR1, respectively, are not affected in BTHS models. Consistent with the decrease in MICU1 levels, we show that the kinetics of MICU1-dependent mitochondrial calcium uptake is perturbed and acute stimulation of mitochondrial calcium signaling in BTHS myoblasts fails to activate pyruvate dehydrogenase, which in turn impairs the generation of reducing equivalents and blunts mitochondrial bioenergetics. Taken together, our findings suggest that defects in mitochondrial calcium signaling could contribute to cardiac and skeletal muscle pathologies observed in BTHS patients.

Abstract P467: Cardiac-specific Deletion Of Voltage Dependent Anion Channel 2 Leads To Dilated Cardiomyopathy By Altering Calcium Homeostasis

Voltage dependent anion channel 2 (VDAC2) is a mitochondrial outer membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in cellular calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. Previous literature suggests that improving mitochondrial calcium uptake via VDAC2 rescues arrhythmia phenotypes in genetic models of impaired cellular calcium signaling. However, the direct role of VDAC2 in intracellular calcium signaling and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac-specific deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium during development causes severe impairment in excitation-contraction coupling by reducing mitochondrial calcium uptake (n=3, p<0.05) and thereby impairing intracellular calcium signaling. VDAC2 knock-out mice showed a significant reduction in RYR-mediated calcium release (F/F 0 ) and rate of calcium uptake by SERCA2a [tau(msec)] compared to control mice (N=3, WT=54, KO=38, p<0.0001 (F/F 0 ) and p<0.05 (tau)). We also observed adverse cardiac remodeling which progressed to severe dilated cardiomyopathy and death (N=6, p<0.0001). Reintroducing VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype evident from improvement in ejection fraction and fractional shortening (n=3, p<0.05). Improving mitochondrial calcium uptake via VDAC2 using a VDAC2 agonist efsevin, increased cardiac contractile force in a mouse model of pressure-overload induced heart failure (N=8, n=22, p<0.05). In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing mitochondrial and cellular calcium signaling. Through this role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

Abstract P401: The Anti-arrhythmic Effects Of VDAC Isoforms Are Mediated By Glutamate 73 Through Regulation Of Mitochondrial Calcium Transport

Mitochondria critically regulate cellular processes such as bioenergetics, metabolism, calcium homeostasis and apoptosis. VDAC proteins are abundant proteins that control the passage of ions and metabolites across the outer mitochondrial membrane. We have previously shown that activation of VDAC2, is able to buffer excess calcium and thereby suppress calcium overload induced arrhythmogenic events in vitro and in vivo. However, the mechanism by which VDAC2 regulates calcium transport and cardiac contractions remained unclear. It is also unclear whether all three VDAC isoforms (VDAC1,2 and 3) possess similar cardioprotective activity. The zebrafish tremblor/ncx1 mutant lacks functional NCX1 in cardiomyocytes leading to calcium overload, and the manifestation of fibrillation-like phenotypes. Using the tremblor/ncx1 mutant as a model, we observed isoform-specific differences between the VDAC family members. VDAC1 and VDAC2 enhanced mitochondrial calcium trafficking and restore rhythmic contraction in tremblor mutants, whereas, VDAC3 did not. We found that the differing rescue capabilities of VDAC proteins were dependent upon residues in their N-terminal halves. Phylogenetic analysis further revealed the presence of an evolutionarily conserved glutamate at position 73 (E73) within VDAC1 and VDAC2, but a glutamine (Q73) in VDAC3. Excitingly, we showed that replacing VDAC2 E73 with Q73 ablated its calcium transporting activity. Conversely, substituting the Q73 with E73 allows VDAC3 to gain calcium trafficking and cardioprotective abilities. Overall, our study demonstrates an essential role for the evolutionarily conserved glutamate-73 in determining the anti-arrhythmic effect of VDAC isoforms through their regulation of mitochondrial calcium uptake.

Abstract P339: Examining Cardiac Phenotypes Of EF Hand Containing Domain 1 Protein (EFHD1) Knockout Mice

Mitochondrial dysfunction due to calcium overload is common in heart disease and results in heart failure. In a mouse model of mitochondrial cardiomyopathies we noted a substantial downregulation of the gene encoding for the EF hand containing domain 1 protein (EFHD1). EFHD1 is a mitochondrial protein which binds calcium and has been shown to stimulate both apoptosis and mitoflashes. These processes involve mitochondrial calcium overload. We therefore hypothesized that EFHD1 down regulation could represent a natural response by failing hearts to prevent mitochondrial calcium overload. We therefore studied the calcium handling properties of mice where Efhd1 had been knocked out (Efhd1-KO). These mice are viable and have no obvious adverse cardiac or metabolic phenotypes. We find that EFHD1 is expressed in the cytoplasm and mitochondrial outer membrane and intermembrane space. It associates strongly with endoplasmic reticulum (ER)-associated mitochondrial membranes. Moreover, mitochondria isolated from Efhd1-KO mice also exhibited 250±10 % improvement in calcium retention in liver but not heart, while neonatal mouse hearts were 3 times more resistant to hypoxia. These findings suggest that EFHD1 may modulate mitochondrial calcium uptake.

Role of Melatonin in Angiotensin and Aging

The cellular utilization of oxygen leads to the generation of free radicals in organisms. The accumulation of these free radicals contributes significantly to aging and several age-related diseases. Angiotensin II can contribute to DNA damage through oxidative stress by activating the NAD(P)H oxidase pathway, which in turn results in the production of reactive oxygen species. This radical oxygen-containing molecule has been linked to aging and several age-related disorders, including renal damage. Considering the role of angiotensin in aging, melatonin might relieve angiotensin-II-induced stress by enhancing the mitochondrial calcium uptake 1 pathway, which is crucial in preventing the mitochondrial calcium overload that may trigger increased production of reactive oxygen species and oxidative stress. This review highlights the role and importance of melatonin together with angiotensin in aging and age-related diseases.