Ryanodine Receptor Stabilization Therapy Suppresses Ca 2+ -Based Arrhythmias in a Novel Model of Metabolic HFpEF.

Heart Failure with preserved ejection fraction (HFpEF) is the most prevalent form of heart failure worldwide and its significant mortality is associated with a high rate of sudden cardiac death (SCD; 30% - 40%). Chronic metabolic stress is an important driver of HFpEF, and clinical data show metabolic stress as a significant risk factor for ventricular arrhythmias in HFpEF patients. The mechanisms of SCD and ventricular arrhythmia in HFpEF remain critically understudied and empirical treatment is ineffective. To address this important knowledge gap, we developed a novel preclinical model of metabolic-stress induced HFpEF using Western diet (High fructose and fat) and hypertension induced by nitric oxide synthase inhibition (with L-NAME) in wildtype C57BL6/J mice. After 5 months, mice display all clinical characteristics of HFpEF and present with stress-induced sustained ventricular tachycardia (VT). Mechanistically, we found a novel pattern of arrhythmogenic intracellular Ca 2+ handling that is distinct from the well-characterized changes pathognomonic for heart failure with reduced ejection fraction. In addition, we show that the transverse tubular system remains intact in HFpEF and that arrhythmogenic, intracellular Ca 2+ mobilization becomes hyper-sensitive to ß- adrenergic activation. Finally, in proof-of-concept experiments we show in vivo that the clinically used intracellular calcium stabilizer dantrolene, which acts on the Ca 2+ release channels of the sarcoplasmic reticulum (SR), the ryanodine receptors, acutely prevents stress-induced VT in HFpEF mice. Therapeutic control of SR Ca 2+ leak may present a novel mechanistic treatment approach in metabolic HFpEF.

Na + /K + ATPase-Ca v 1.2 nanodomain differentially regulates intracellular [Na + ], [Ca 2+ ] and local adrenergic signaling in cardiac myocytes.

Background: The intracellular Na + concentration ([Na + ] i ) is a crucial but understudied regulator of cardiac myocyte function. The Na + /K + ATPase (NKA) controls the steady-state [Na + ] i and thereby determines the set-point for intracellular Ca 2+ . Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na + and Ca 2+ homeostases in atrial and ventricular myocytes. Methods: Multicolor STORM super-resolution microscopy, Western Blot analyses, and in vivo examination of adrenergic regulation are employed to examine the organization and function of Na + nanodomains in cardiac myocytes. Quantitative fluorescence microscopy at high spatiotemporal resolution is used in conjunction with cellular electrophysiology to investigate intracellular Na + homeostasis in atrial and ventricular myocytes. Results: The NKAα1 (NKAα1) and the L-type Ca 2+ -channel (Ca v 1.2) form a nanodomain with a center-to center distance of ∼65 nm in both ventricular and atrial myocytes. NKAα1 protein expression levels are ∼3 fold higher in atria compared to ventricle. 100% higher atrial I NKA , produced by large NKA "superclusters", underlies the substantially lower Na + concentration in atrial myocytes compared to the benchmark values set in ventricular myocytes. The NKA's regulatory protein phospholemman (PLM) has similar expression levels across atria and ventricle resulting in a much lower PLM/NKAα1 ratio for atrial compared to ventricular tissue. In addition, a huge PLM phosphorylation reserve in atrial tissue produces a high ß-adrenergic sensitivity of I NKA in atrial myocytes. ß-adrenergic regulation of I NKA is locally mediated in the NKAα1-Ca v 1.2 nanodomain via A-kinase anchoring proteins. Conclusions: NKAα1, Ca v 1.2 and their accessory proteins form a structural and regulatory nanodomain at the cardiac dyad. The tissue-specific composition and local adrenergic regulation of this "signaling cloud" is a main regulator of the distinct global intracellular Na + and Ca 2+ concentrations in atrial and ventricular myocytes.

Outer mitochondrial membrane E3 Ub ligase MARCH5 controls mitochondrial steps in peroxisome biogenesis.

Peroxisome de novo biogenesis requires yet unidentified mitochondrial proteins. We report that the outer mitochondrial membrane (OMM)-associated E3 Ub ligase MARCH5 is vital for generating mitochondria-derived pre-peroxisomes. MARCH5 knockout results in accumulation of immature peroxisomes and lower expression of various peroxisomal proteins. Upon fatty acid-induced peroxisomal biogenesis, MARCH5 redistributes to newly formed peroxisomes; the peroxisomal biogenesis under these conditions is inhibited in MARCH5 knockout cells. MARCH5 activity-deficient mutants are stalled on peroxisomes and induce accumulation of peroxisomes containing high levels of the OMM protein Tom20 (mitochondria-derived pre-peroxisomes). Furthermore, depletion of peroxisome biogenesis factor Pex14 leads to the formation of MARCH5- and Tom20-positive peroxisomes, while no peroxisomes are detected in Pex14/MARCH5 dko cells. Reexpression of WT, but not MARCH5 mutants, restores Tom20-positive pre-peroxisomes in Pex14/MARCH5 dko cells. Thus, MARCH5 acts upstream of Pex14 in mitochondrial steps of peroxisome biogenesis. Our data validate the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process. Summary: The authors found that mitochondrial E3 Ub ligase MARCH5 controls the formation of mitochondria-derived pre-peroxisomes. The data support the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process.

Calcium and bicarbonate signaling pathways have pivotal, resonating roles in matching ATP production to demand.

Mitochondrial ATP production in ventricular cardiomyocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local cardiomyocyte metabolic signaling. However, these two regulatory systems do not fully account for the physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product of the oxidation of nutrients. It is a lipid soluble gas that rapidly permeates the inner mitochondrial membrane and produces bicarbonate in a reaction accelerated by carbonic anhydrase. The bicarbonate level is tracked physiologically by a bicarbonate-activated soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular cardiomyocytes where it generates cAMP when activated by bicarbonate. Our data strongly suggest that ATP production in these mitochondria is regulated by this cAMP signaling cascade operating within the inter-membrane space by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein-1). Thus, mitochondrial ATP production is increased by bicarbonate-triggered sAC-signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. We propose that the bicarbonate and calcium signaling arms function in a resonant or complementary manner to match mitochondrial ATP production to the full range of energy consumption in ventricular cardiomyocytes.

Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling.

The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. STATEMENT OF SIGNIFICANCE: This work shows non-tumorigenic breast epithelial cells increase intracellular Ca 2+ after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte's sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Understanding the Dynamics of the Transient and Permanent Opening Events of the Mitochondrial Permeability Transition Pore with a Novel Stochastic Model.

The mitochondrial permeability transition pore (mPTP) is a non-selective pore in the inner mitochondrial membrane (IMM) which causes depolarization when it opens under conditions of oxidative stress and high concentrations of Ca2+. In this study, a stochastic computational model was developed to better understand the dynamics of mPTP opening and closing associated with elevated reactive oxygen species (ROS) in cardiomyocytes. The data modeled are from "photon stress" experiments in which the fluorescent dye TMRM (tetramethylrhodamine methyl ester) is both the source of ROS (induced by laser light) and sensor of the electrical potential difference across the IMM. Monte Carlo methods were applied to describe opening and closing of the pore along with the Hill Equation to account for the effect of ROS levels on the transition probabilities. The amplitude distribution of transient mPTP opening events, the number of transient mPTP opening events per minute in a cell, the time it takes for recovery after transient depolarizations in the mitochondria, and the change in TMRM fluorescence during the transition from transient to permanent mPTP opening events were analyzed. The model suggests that mPTP transient open times have an exponential distribution that are reflected in TMRM fluorescence. A second multiple pore model in which individual channels have no permanent open state suggests that 5-10 mPTP per mitochondria would be needed for sustained mitochondrial depolarization at elevated ROS with at least 1 mPTP in the transient open state.

Pyruvate-Driven Oxidative Phosphorylation is Downregulated in Sepsis-Induced Cardiomyopathy: A Study of Mitochondrial Proteome.

BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is a major contributing factor for morbidity and mortality in sepsis. Accumulative evidence has suggested that cardiac mitochondrial oxidative phosphorylation is attenuated in sepsis, but the underlying molecular mechanisms remain incompletely understood. METHODS: Adult male mice of 9 to 12 weeks old were subjected to sham or cecal ligation and puncture procedure. Echocardiography in vivo and Langendorff-perfused hearts were used to assess cardiac function 24 h after the procedures. Unbiased proteomics analysis was performed to profile mitochondrial proteins in the hearts of both sham and SIC mice. Seahorse respirator technology was used to evaluate oxygen consumption in purified mitochondria. RESULTS: Of the 665 mitochondrial proteins identified in the proteomics assay, 35 were altered in septic mice. The mitochondrial remodeling involved various energy metabolism pathways including subunits of the electron transport chain, fatty acid catabolism, and carbohydrate oxidative metabolism. We also identified a significant increase of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) and inhibition of PDH activity in septic hearts. Furthermore, compared to sham mice, mitochondrial oxygen consumption of septic mice was significantly reduced when pyruvate was provided as a substrate. However, it was unchanged when PDH was bypassed by directly supplying the Complex I substrate NADH, or by using the Complex II substrate succinate, or using Complex IV substrate, or by providing the beta-oxidation substrate palmitoylcarnitine, neither of which require PDH for mitochondrial oxygen consumption. CONCLUSIONS: These data demonstrate a broad mitochondrial protein remodeling, PDH inactivation and impaired pyruvate-fueled oxidative phosphorylation during SIC, and provide a molecular framework for further exploration.

Calcium Signaling Silencing in Atrial Fibrillation: Implications for Atrial Sodium Homeostasis.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, affecting more than 33 million people worldwide. Despite important advances in therapy, AF's incidence remains high, and treatment often results in recurrence of the arrhythmia. A better understanding of the cellular and molecular changes that (1) trigger AF and (2) occur after the onset of AF will help to identify novel therapeutic targets. Over the past 20 years, a large body of research has shown that intracellular Ca2+ handling is dramatically altered in AF. While some of these changes are arrhythmogenic, other changes counteract cellular arrhythmogenic mechanisms (Calcium Signaling Silencing). The intracellular Na+ concentration ([Na+])i is a key regulator of intracellular Ca2+ handling in cardiac myocytes. Despite its importance in the regulation of intracellular Ca2+ handling, little is known about [Na+]i, its regulation, and how it might be changed in AF. Previous work suggests that there might be increases in the late component of the atrial Na+ current (INa,L) in AF, suggesting that [Na+]i levels might be high in AF. Indeed, a pharmacological blockade of INa,L has been suggested as a treatment for AF. Here, we review calcium signaling silencing and changes in intracellular Na+ homeostasis during AF. We summarize the proposed arrhythmogenic mechanisms associated with increases in INa,L during AF and discuss the evidence from clinical trials that have tested the pharmacological INa,L blocker ranolazine in the treatment of AF.

The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter.

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination.

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.

Calcium influx through the mitochondrial calcium uniporter holocomplex, MCUcx.

Ca2+ flux into the mitochondrial matrix through the MCU holocomplex (MCUcx) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca2+ in the mitochondrial matrix ([Ca2+]m) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca2+ into the mitochondrial matrix through MCUcx and efflux through the mitochondrial Na+ / Ca2+ exchanger (NCLX). Under these conditions [Ca2+]m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca2+]m continue to be reported in the literature. Here we review the new findings attributed to MCUcx function and provide a framework for understanding and investigating mitochondrial Ca2+ influx features, many of which remain controversial. The properties and functions of the MCUcx subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCUpore). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCUcx Ca2+ flux from heart and skeletal muscle. These MCUcx Ca2+ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca2+ conductances and with distinct extramitochondrial Ca2+ ([Ca2+]i) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca2+]i levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCUcx dependent matrix [Ca2+]m signaling provide a quantitative basis for on-going and new investigations of the roles of MCUcx in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca2+ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨm.

Regulation of Mitochondrial ATP Production: Ca2+ Signaling and Quality Control.

Cardiac ATP production primarily depends on oxidative phosphorylation in mitochondria and is dynamically regulated by Ca2+ levels in the mitochondrial matrix as well as by cytosolic ADP. We discuss mitochondrial Ca2+ signaling and its dysfunction which has recently been linked to cardiac pathologies including arrhythmia and heart failure. Similar dysfunction in other excitable and long-lived cells including neurons is associated with neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Central to this new understanding is crucial Ca2+ regulation of both mitochondrial quality control and ATP production. Mitochondria-associated membrane (MAM) signaling from the sarcoplasmic reticulum (SR) and the endoplasmic reticulum (ER) to mitochondria is discussed. We propose future research directions that emphasize a need to define quantitatively the physiological roles of MAMs, as well as mitochondrial quality control and ATP production.

Dynamics of the mitochondrial permeability transition pore: Transient and permanent opening events.

A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨM) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 ″super-resolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 s (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 min. The time to first depolarization events was 8.4 min (±1.1 SEM n = 21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 min) and virtually no permanent depolarization events occurred for over 1 h of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 µM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80% and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ±â€¯6 s (n = 478) at 37 °C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca2+. In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨM depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6,7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨM depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool ("Optical Stress-Probe"), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks.

Voltage-energized Calcium-sensitive ATP Production by Mitochondria.

Regulation of ATP production by mitochondria, critical to multicellular life, is poorly understood. Here we investigate the molecular controls of this process in heart and provide a framework for its Ca2+-dependent regulation. We find that the entry of Ca2+ into the matrix through the mitochondrial calcium uniporter (MCU) in heart has neither an apparent cytosolic Ca2+ threshold nor gating function and guides ATP production by its influence on the inner mitochondrial membrane (IMM) potential, ΔΨm. This regulation occurs by matrix Ca2+-dependent modulation of pyruvate and glutamate dehydrogenase activity and not through any effect of Ca2+ on ATP Synthase or on Electron Transport Chain Complexes II, III or IV. Examining the ΔΨm dependence of ATP production over the range of -60 mV to -170 mV in detail reveals that cardiac ATP synthase has a voltage dependence that distinguishes it fundamentally from the previous standard, the bacterial ATP synthase. Cardiac ATP synthase operates with a different ΔΨm threshold for ATP production than bacterial ATP synthase and reveals a concave-upwards shape without saturation. Skeletal muscle MCU Ca2+ flux, while also having no apparent cytosolic Ca2+ threshold, is substantially different from the cardiac MCU, yet the ATP synthase voltage dependence in skeletal muscle is identical to that in the heart. These results suggest that while the conduction of cytosolic Ca2+ signals through the MCU appears to be tissue-dependent, as shown by earlier work1, the control of ATP synthase by ΔΨm appears to be broadly consistent among tissues but is clearly different from bacteria.

Author Correction: Voltage-energized calcium-sensitive ATP production by mitochondria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Real-time local oxygen measurements for high resolution cellular imaging.

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O2 (pO2) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O2-micro-sensor created by adsorbing O2-sensitive and O2-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O2, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 µm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO2 and micro-gradients of pO2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO2 and intracellular events can be imaged simultaneously. In addition, the response-time (t0.5 = 0.7 s, 0-160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨm); and (2) OxySplots measuring pO2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca2+]i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein.

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5(-/-) cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5(-/-) cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.