Nicholas E. Hoffman

MICU1 is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake That Regulates Cell Survival

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

Bone-Derived Stem Cells Repair the Heart after Myocardial Infarction Through Transdifferentiation and Paracrine Signaling Mechanisms

Rationale: Autologous bone marrow–derived or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials, but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is the key toward improving clinical outcomes. Objective: To determine the mechanism by which novel bone-derived stem cells support the injured heart. Methods and Results: Cortical bone–derived stem cells (CBSCs) and cardiac-derived stem cells were isolated from enhanced green fluorescent protein (EGFP+) transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis, and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction with injection of CBSCs (n=67), cardiac-derived stem cells (n=36), or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor), and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to cardiac-derived stem cells– or saline-treated myocardial infarction controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle, and endothelial cells could be identified in CBSC-treated, but not in cardiac-derived stem cells–treated, animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP− myocytes. Conclusions: CBSCs improve survival, cardiac function, and attenuate remodeling through the following 2 mechanisms: (1) secretion of proangiogenic factors that stimulate endogenous neovascularization, and (2) differentiation into functional adult myocytes and vascular cells.

SLC25A23 augments mitochondrial Ca2+ uptake, interacts with MCU, and induces oxidative stress–mediated cell death

Knockdown of SLC25A23 decreases mitochondrial Ca2+ uptake, and SLC25A23 interacts with MCU and MICU1, components of mitochondrial Ca2+ uniporter. Expression of SLC25A23 EF-hand-domain mutants has a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. It also attenuates basal ROS and oxidant-induced ATP decline and cell death.

MICU1 motifs define mitochondrial calcium uniporter binding and activity.

Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

Rationale: The cellular and molecular basis for post–myocardial infarction (MI) structural and functional remodeling is not well understood. Objective: Our aim was to determine if Ca2+ influx through transient receptor potential canonical (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results: TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte–specific expression of a dominant-negative (loss-of-function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival in mice. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 overexpression in adult feline myocytes induced calcineurin (Cn)-nuclear factor of activated T-cells (NFAT)–mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in adult feline myocytes increased rested state contractions and increased spontaneous sarcoplasmic reticulum Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady-state pacing, likely because of enhanced sarcoplasmic reticulum Ca2+ leak. Conclusions: Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function.

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury ROLE OF MITOCHONDRIA

Background: TRPM2 channels are present in the heart, but their function is unknown. Results: Genetic ablation of TRPM2 results in cardiac mitochondrial dysfunction, enhanced ROS production, and exacerbated cardiac ischemic injury. Conclusion: TRPM2 channels preserve cardiac mitochondrial bioenergetics and protect cardiac myocytes from ischemic injury. Significance: TRPM2 is a rational target for treatment of ischemic heart disease. Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU

Calcium signaling stimulates the accumulation of the mitochondrial calcium uniporter to regulate mitochondrial metabolism. Maintaining mitochondrial calcium uptake The calcium uniporter complex, which includes the protein MCU, mediates mitochondrial calcium uptake, a process that buffers excess cytosolic calcium and regulates mitochondrial metabolism. Shanmughapriya et al. examined mitochondrial calcium uptake and function in a B lymphocyte cell line deficient in one or more proteins necessary for mediating two types of calcium signals—IICR, calcium released from the endoplasmic reticulum through the calcium-permeable IP3 receptors, and SOCE, calcium influx through store-operated calcium channels. Without IICR or SOCE, the activity of the transcription factor CREB, which bound to the MCU promoter, and the expression and abundance of MCU were reduced, mitochondrial calcium uptake was compromised, and mitochondrial metabolism was altered. Cells deficient in IICR or SOCE lacked an oscillating basal calcium signal. Thus, IICR and SOCE control the capacity of mitochondria to uptake calcium and therefore regulate mitochondrial metabolism. Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB (cyclic adenosine monophosphate response element–binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation

Dysregulation of mitochondrial Ca2+‐dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca2+ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1‐mediated Ca2+ transport remains unresolved. This study examines LETM1‐mediated mitochondrial Ca2+ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf‐Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca2+ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, D676A D688KLETM1 mutant‐overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS‐derived fibroblasts, the mitochondrial Ca2+ uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch‐clamp current (IMCU) was largely unaffected in LETM1‐knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca2+ transport and bioenergetics. These findings reveal the role of LETM1‐dependent mitochondrial Ca2+ flux in shaping cellular bioenergetics.—Doonan, P J., Chandramoorthy, H. C., Hoffman, N. E., Zhang, X., Cárdenas, C., Shanmughapriya, S., Rajan, S., Vallem, S., Chen, X., Foskett, J. K., Cheung, J. Y., Houser, S. R., Madesh, M., LETM1‐dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation. FASEB J. 28, 4936–4949 (2014). www.fasebj.org

Convergence of G protein-coupled receptor and S-nitrosylation signaling determines the outcome to cardiac ischemic injury.

S-nitrosothiols protect against ischemic injury in the heart by limiting signaling downstream of G protein–coupled receptors. NO More Heart Damage Damage caused by the lack of oxygen and nutrients that occurs during myocardial ischemia can result in heart failure. A therapeutic strategy that helps to limit the effects of heart failure is to increase signaling through G protein–coupled receptors (GPCRs) by inhibiting GRK2 (GPCR kinase 2), a kinase that desensitizes GPCRs. Another therapeutic strategy provides S-nitrosothiols, such as nitric oxide, which can be added to proteins in a posttranslational modification called S-nitrosylation. Huang et al. found that the ability of S-nitrosothiols to enhance cardiomyocyte survival after ischemic injury required the S-nitrosylation of GRK2, a modification that inhibits this kinase. Mice bearing a form of GRK2 that could not be S-nitrosylated were more susceptible to cardiac damage after ischemia. These results suggest that therapeutic strategies that promote the S-nitrosylation of GRK2 could be used to treat heart failure after myocardial ischemia. Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein–coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, required GRK2 activation, and we found that cardioprotection mediated by inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS) and was associated with S-nitrosylation of GRK2. Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.