Calcium Regulation of Mitochondrial Metabolism.

The following is an abstract of the article discussed in the subsequent letter: Drew, Barry and Christiaan Leeuwenburgh. Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol

Is the Failing Heart Starved of Mitochondrial Calcium?

Although the recent identification of the mitochondrial Ca2+ uniporter (MCU) has resolved a long-standing mystery as to how Ca2+ freely enters the mitochondria, it has also evoked additional questions such as its mode of regulation and the identity of other associated factors. In an article recently published in Nature , Joiner et al1 provide data demonstrating that in the heart, matrix-localized Ca2+/calmodulin-dependent protein kinase II (CaMKII) can upregulate MCU activity in a manner requiring phosphorylation of the channel N terminus. They showed that inhibition of CaMKII-dependent MCU activity protected the heart from ischemic injury by presumably reducing Ca2+ influx and desensitizing the mitochondrial permeability transition pore (MPTP) to opening. Although these results demonstrate convincingly that CaMKII plays an important role in MCU regulation and subsequent response to cardiac injury, several questions remain unanswered. The ability of mitochondria to take up and sequester Ca2+ plays an important role in the buffering of cytosolic Ca2+, regulation of ATP production via the citric acid cycle, and regulation of apoptotic and necrotic cell death pathways.2 Although mitochondrial Ca2+ uptake was first described in the 1960s3 and the electrophysiological properties of the MCU were reported in 2004,4 it was not until 2011 that 2 articles were published revealing the genetic identity of the MCU.5,6 This pioneering work has initiated a search for additional members of the MCU complex (such as MICU17 and the recently discovered MCUR1),8 as well as an attempt to understand how MCU-mediated Ca2+ influx participates in the regulation of whole-cell Ca2+ signaling and whether well-described pathways that regulate other Ca2+ handling processes can similarly modulate MCU-dependent Ca2+ uptake. In the featured article, Joiner et al1 show that CaMKII serves …

Mitochondrial bioenergetics and neurodegeneration: a paso doble

Diabetes mellitus is a growing health care problem, resulting in significant cardiovascular morbidity and mortality. Diabetes also increases the risk for heart failure (HF) and decreased cardiac myocyte function, which are linked to changes in cardiac mitochondrial energy metabolism. The free mitochondrial calcium level ([Ca2+] m ) is fundamental in activating the mitochondrial respiratory chain complexes and ATP production and is also known to regulate pyruvate dehydrogenase complex (PDC) activity. The mitochondrial calcium uniporter (MCU) complex (MCUC) plays a major role in mediating mitochondrial Ca2+ import, and its expression and function therefore have a marked impact on cardiac myocyte metabolism and function. Here, we investigated MCU's role in mitochondrial Ca2+ handling, mitochondrial function, glucose oxidation, and cardiac function in the heart of diabetic mice. We found that diabetic mouse hearts exhibit altered expression of MCU and MCUC members and a resulting decrease in [Ca2+] m , mitochondrial Ca2+ uptake, mitochondrial energetic function, and cardiac function. Adeno-associated virus-based normalization of MCU levels in these hearts restored mitochondrial Ca2+ handling, reduced PDC phosphorylation levels, and increased PDC activity. These changes were associated with cardiac metabolic reprogramming toward normal physiological glucose oxidation. This reprogramming likely contributed to the restoration of both cardiac myocyte and heart function to nondiabetic levels without any observed detrimental effects. These findings support the hypothesis that abnormal mitochondrial Ca2+ handling and its negative consequences can be ameliorated in diabetes by restoring MCU levels via adeno-associated virus-based MCU transgene expression.

Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

BackgroundExcessive proliferation of vascular smooth muscle cells (VSMCs) is a consequence of type 2 diabetes (T2D) that increases the risk for atherosclerosis and restenosis after angioplasty. Here, we sought to determine whether and how mitochondrial dysfunction in T2D drives VSMC proliferation with a focus on increased reactive oxygen species and intracellular [Ca2+] that both drive cell proliferation, occur in T2D, and are regulated by the mitochondrial Ca2+ uniporter (MCU).MethodsUsing a mouse model of T2D, we performed in vivo phenotyping after mechanical injury and established the mechanisms of excessive proliferation in cultured VSMCs. The T2D model was induced by high‐fat diet and low‐dose streptozotocin in both wild type mice and mice with the VSMC‐specific inhibition of the mtCaMKII (mitochondrial Ca2+/calmodulin‐dependent kinase IImtCaMKII), a regulator of Ca2+ entry via the MCU.ResultsIn VSMCs from T2D model mice, MCU inhibition reduced both in vivo neointima formation after mechanical injury, as well as in vitro proliferation of cultured VSMCs. Further, in VSMCs from T2D mice, the composition of the MCU complex and MCU activity were altered with loss of MICU1 (mitochondrial calcium uptake 1). In addition, VSMC mitochondrial reactive oxygen species was elevated and mitochondrial respiration blunted. The increase in cytosolic reactive oxygen species induced activation of G6PD (glucose‐6‐phosphate dehydrogenase), a key enzyme of the pentose phosphate pathway. However, inhibiting MCU or MICU1 overexpression on VSMCs from T2D mice decreased intracellular reactive oxygen species, preserved mitochondrial respiration and ATP production, decreased activity of G6PD, and normalized cell proliferation. These data suggest the MCU complex controls a T2D‐induced metabolic switch that promotes cell proliferation.ConclusionsCollectively, these data indicate that MCU complex remodeling in T2D drives neointimal restenosis, suggesting MCU as a therapeutic target.

Mitochondrial Ca2+ uniporter (MCU) provides a Ca2+ influx pathway from the cytosol into the mitochondrial matrix and a moderate mitochondrial Ca2+ rise stimulates ATP production and cell growth. MCU is highly expressed in various cancer cells including breast cancer cells, thereby increasing the capacity of mitochondrial Ca2+ uptake, ATP production, and cancer cell proliferation. The objective of this study was to examine MCU inhibition as an anti-cancer mechanism. The effects of MCU-i4, a newly developed MCU inhibitor, on cell viability, apoptosis, cytosolic Ca2+, mitochondrial Ca2+ and potential, glycolytic rate, generation of ATP, and reactive oxygen species, were examined in breast cancer BT474 cells. MCU-i4 caused apoptotic cell death, and it decreased and increased, respectively, mitochondrial and cytosolic Ca2+ concentration. Inhibition of MCU by MCU-i4 revealed that cytosolic Ca2+ elevation resulted from endoplasmic reticulum (ER) Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RYR). Unexpectedly, MCU-i4 enhanced glycolysis and ATP production; it also triggered a large production of reactive oxygen species (ROS) and mitochondrial membrane potential collapse. Cytotoxic mechanisms of MCU-i4 in cancer cells involved enhanced glycolysis and heightened formation of ATP and ROS. It is conventionally believed that cancer cell death could be caused by inhibition of glycolysis. Our observations suggest cancer cell death could also be induced by increased glycolytic metabolism.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Overexpression of RyR1 Enhances Ca2+-Induced Mitochondrial ATP Production in Cardiac H9C2 Cells

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

Piezo1 channels are mechanosensitive cationic channels that are activated by mechanical stretch or shear stress. Endothelial Piezo1 activation by shear stress caused by blood flow induces ATP release from endothelial cells; however, the link between shear stress and endothelial ATP production is unclear. The mitochondrial respiratory function of cells was measured by using high-resolution respirometry system Oxygraph-2k. The intracellular Ca2+ concentration was evaluated by using Fluo-4/AM and mitochondrial Ca2+ concentration by Rhod-2/AM. The specific Piezo1 channel activator Yoda1 or its analogue Dooku1 increased [Ca2+ ]i in human umbilical vein endothelial cells (HUVECs), and both Yoda1 and Dooku1 increased mitochondrial oxygen consumption rates (OCRs) and mitochondrial ATP production in HUVECs and primary cultured rat aortic endothelial cells (RAECs). Knockdown of Piezo1 inhibited Yoda1- and Dooku1-induced increases of mitochondrial OCRs and mitochondrial ATP production in HUVECs. The shear stress mimetics, Yoda1 and Dooku1, and the Piezo1 knock-down technique also demonstrated that Piezo1 activation increased glycolysis in HUVECs. Chelating extracellular Ca2+ with EGTA or chelating cytosolic Ca2+ with BAPTA-AM did not affect Yoda1- and Dooku1-induced increases of mitochondrial OCRs and ATP production, but chelating cytosolic Ca2+ inhibited Yoda1- and Dooku1-induced increase of glycolysis. Confocal microscopy showed that Piezo1 channels are present in mitochondria of endothelial cells, and Yoda1 and Dooku1 increased mitochondrial Ca2+ in endothelial cells. Piezo1 channel activation stimulates ATP production through enhancing mitochondrial respiration and glycolysis in vascular endothelial cells, suggesting a novel role of Piezo1 channel in endothelial ATP production.

Thyroid hormones act as master regulators of cellular metabolism. Thereby, the biologically active triiodothyronine (T3) induces the expression of genes to enhance mitochondrial metabolic function. Notably, Ca2+ ions are necessary for the activity of dehydrogenases of the tricarboxylic acid cycle and, thus, mitochondrial respiration.We investigated whether treating HeLa cells with T3 causes alterations in mitochondrial Ca2+ ([Ca2+]mito) levels. Real-time measurements by fluorescence microscopy revealed that treatment with T3 for 3 h induces a significant increase in basal [Ca2+]mito levels and [Ca2+]mito uptake upon the depletion of the endoplasmic reticulum (ER) Ca2+ store, while cytosolic Ca2+ levels remained unchanged. T3 incubation was found to upregulate mRNA expression levels of uncoupling proteins 2 and 3 (UCP2, UCP3) and of protein arginine methyltransferase 1 (PRMT1). Live-cell imaging revealed that T3-induced enhancement of mitochondrial Ca2+ uptake depends on the mitochondrial Ca2+ uniporter (MCU), UCP2, and PRMT1 that are essential for increased mitochondrial ATP ([ATP]mito) production after T3 treatment. Besides, increased [Ca2+]mito and [ATP]mito levels correlated with enhanced production of reactive oxygen species (ROS) in mitochondria. Notably, ROS scavenging causes mitochondrial Ca2+ elevation and outplays the impact of T3 on [Ca2+]mito homeostasis.Based on these results, we assume that thyroid hormones adjust [Ca2+]mito homeostasis by modulating the UCP2- and PRMT1-balanced [Ca2+]mito uptake via MCU in case of physiological ROS levels to convey their impact on mitochondrial ATP and ROS production.

Multifidus muscles maintain the stability of the lumbar spine and play a crucial role in the pathogenesis of nonspecific lower back pain. Previous studies have shown that electroacupuncture (EA) can relieve the symptoms of low back pain and reduce injury to the lumbar multifidus muscles. In this study, a rat model of lumbar multifidus muscle injury was established by 0.05% bupivacaine injection and subsequently treated with EA at bilateral "Weizhong" (BL40) acupoints. Disruption of the function and structure of multifidus muscles, increased cytosolic Ca2+ in multifidus myocytes, and reduced mitochondrial fission and ATP production were observed in the model group. Additionally, increased expression of the mitochondrial calcium uniporter (MCU) promoted mitochondrial reuptake of Ca2+ , reversing the excessive increase in cytoplasmic Ca2+ . However, the excessive increase in MCU not only aggravated the increased cytoplasmic Ca2+ but also decreased the expression of the mitochondrial division proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (MFF). EA inhibited the overexpression of MCU, promoted mitochondrial reuptake of Ca2+ , and reversed cytosolic Ca2+ overload. Furthermore, EA regulated the expression of the mitochondrial fission proteins Drp1 and MFF and promoted the production of ATP, helping the recovery of mitochondrial function after multifidus injury. Therefore, EA can protect against bupivacaine-induced mitochondrial dysfunction, possibly by attenuating MCU overexpression in the inner mitochondrial membrane and reducing Ca2+ overloading in muscle cells, thereby protecting mitochondrial function and maintaining the normal energy demand of muscle cells.

How mitochondrial ATP production dynamically responds to cellular metabolic needs is poorly understood. Here we report on quantitative investigations in heart muscle that identify the key molecular and cellular controls of ATP production. We show that Ca2+ influx into the mitochondrial matrix through the mitochondrial calcium uniporter (MCU) is small with no cytosolic Ca2+ threshold and tissue‐specific Ca2+ sensitive gating. MCU controls ATP production by its influence on matrix Ca2+ concentration ([Ca2+]m) which regulates pyruvate and oxoglutarate dehydrogenases and hence Krebs cycle production of NADH. The NADH level affects the voltage‐gradient across the inner mitochondrial membrane (Δψm) which in turn regulates ATP synthase. Our first report of the voltage‐dependence of ATP synthase in mammalian system is determined by measuring its ATP production. By this means, we show that the Δψm dependence of ATP synthase is different from the previous “gold standard”, the voltage‐dependence of bacterial ATP synthase. Additionally, we show how ATP production in cardiac mitochondria depends on both [Ca2+]m and on cytosolic [ADP]. Higher heart rates and contraction under load increase both Ca2+ sensitive and Ca2+ insensitive ATP production. These findings provide a new and different understanding of how mitochondria generate ATP in response to metabolic need, how ATP production is powered by Δψm, and how Δψm is regulated by mitochondrial Ca2+ signaling.Support or Funding InformationThis research was supported by American Heart Association grants SDG 15SDG22100002 (to L.B.) and 16PRE31030023 (to A.P.W.); by NIH R01 HL106056, R01 HL105239, U01 HL116321, 1R01HL142290, 1 R01 HL140934 and 1R01 AR071618 (to W.J.L.); by the Medical Scientist Training Program and Training Program in Integrative Membrane Biology, NIH 2T32GM092237‐06 and 5T32GM008181‐28 (to A.P.W.).