Muhammad Rizwan Alam

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic β-Cells

Background: The molecular contributors of the mitochondrial Ca2+ uptake, which is essential for metabolism-secretion coupling in β-cells, are unknown. Results: Knockdown of MICU1 and MCU reduced agonist- and depolarization-induced mitochondrial Ca2+ sequestration, ATP production, and d-glucose-stimulated insulin secretion. Conclusion: MICU1 and MCU are integral to metabolism-secretion coupling in β-cells. Significance: The presented data identify MICU1 and MCU as important contributors to pancreatic β-cell function. In pancreatic β-cells, uptake of Ca2+ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca2+ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca2+ uptake 1 (MICU1), mitochondrial Ca2+ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca2+ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca2+ uptake upon both intracellular Ca2+ release and Ca2+ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca2+ uptake in response to either intracellular Ca2+ release or Ca2+ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca2+ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca2+ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca2+ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.

Inhibition of Autophagy Rescues Palmitic Acid-induced Necroptosis of Endothelial Cells

Background: Accumulation of palmitic acid in endothelial cells induces cellular dysfunction and death. Results: Palmitic acid triggers Ca2+-dependent autophagy, which results in programmed necrotic death (necroptosis) of endothelial cells. Conclusion: Autophagy promotes lipotoxic signaling of palmitic acid in endothelial cells leading to necroptosis. Significance: Showing a new molecular mechanism of palmitic acid-induced cytotoxicity may reveal novel strategies in the treatment of diseases related to lipid overload. Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca2+ triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Studying mitochondrial Ca2+ uptake – A revisit

Highlights ► Mitochondrial Ca2+ sequestration was tested by various techniques. ► Kinetics and Ca2+ sensitivity of mitochondrial Ca2+ uptake depends on the techniques chosen. ► By electrophysiology, 2 and 3 distinct Ca2+ inward currents were measured in HeLa and endothelial cell mitoplasts. ► Mitoplast Ca2+ inward currents differ in frequency of appearance and conductance ranging from 7.6 to 74.3 pS. ► Mitochondrial Ca2+ uptake routes/modes exists and might be cell specific.

Inhibition of myocardial reperfusion injury by ischemic postconditioning requires sirtuin 3-mediated deacetylation of cyclophilin D

RATIONALE How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP. OBJECTIVE The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD. METHODS AND RESULTS Using hypoxia-reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871 ± 266 vs 1193 ± 263 nmoles Ca(2+)/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compared to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice. CONCLUSIONS Our study suggests that the increased acetylation of CyPD following myocardial ischemia-reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance.

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca2+ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca2+ ([Ca2+]cyto) can be excellently quantified with the ratiometric Ca2+ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca2+ sensors, the cameleons, are efficiently used to specifically measure Ca2+ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca2+]cyto and mitochondrial Ca2+ ([Ca2+]mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca2+ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca2+]cyto and [Ca2+]mito within same individual cells. Our data indicate that depending on the kinetics of [Ca2+]cyto rises there is a significant lag between onset of [Ca2+]cyto and [Ca2+]mito signals, pointing to a certain threshold of [Ca2+]cyto necessary to activate mitochondrial Ca2+ uptake. The temporal correlation between [Ca2+]mito and [Ca2+]cyto as well as the efficiency of the transfer of Ca2+ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca2+ extrusion and a desensitization of mitochondrial Ca2+ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca2+ oscillations of pancreatic beta-cells in response to D-glucose.

Mitochondrial Ca 2+ uniporter (MCU)-dependent and MCU-independent Ca 2+ channels coexist in the inner mitochondrial membrane

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca2+ uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca2+ channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca2+ and Na+ currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca2+ current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca2+ current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca2+ channels.

Molecularly distinct routes of mitochondrial Ca2+ uptake are activated depending on the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).

Background: Mitochondria may utilize different proteins to decode high and low cytosolic Ca2+. Results: Inhibition of SERCA shifts mitochondrial Ca2+ uptake from being UCP3-dependent to Letm1-dependent. Conclusion: Depending on the mode of intracellular Ca2+ release, two different mitochondrial Ca2+ uptake pathways are engaged. Significance: The dissection of two molecularly distinct mitochondrial Ca2+ uptake routes depending on SERCA activity points to the complexity of the mitochondrial Ca2+ uptake machinery. The transfer of Ca2+ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca2+ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca2+ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca2+ uptake pathway or establish distinct routes for mitochondrial Ca2+ sequestration. In this study, we show that a modulation of Ca2+ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca2+ signals and consequently switches mitochondrial Ca2+ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca2+ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca2+ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca2+ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca2+ rises.

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia-reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca(2+) transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP3Rs) Ca(2+) channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP3R with the Ca(2+) channeling complex, impaired SR/ER Ca(2+) release and reduced the histamine-stimulated Ca(2+) exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP3R phosphorylation, which leads to enhanced transfer of Ca(2+) from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP3R phosphorylation and SR/ER Ca(2+) release, which consequently diminished both cytosolic and mitochondrial Ca(2+) concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca(2+) leak from IP3R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca(2+) overload and subsequent cell death.

Delayed low pressure at reperfusion: A new approach for cardioprotection

OBJECTIVES The aims of this study were to evaluate whether the delayed application of low-pressure reperfusion could reduce lethal reperfusion injury and whether the inhibition of the opening of the mitochondrial permeability transition pore is involved in this protection. METHODS Isolated rat hearts (n = 120) underwent 40 minutes of global ischemia followed by 60 minutes of reperfusion. Hearts were randomly assigned to the following groups: control, postconditioning (comprising 2 episodes of 30 seconds of ischemia and 30 seconds of reperfusion), and low-pressure reperfusion (using a reduction of perfusion pressure at 70 cm H2O for 10 minutes). In additional groups, postconditioning and low-pressure reperfusion were applied after a delay of 3, 10, and 20 minutes after the initial 40-minute ischemic insult. RESULTS As expected, infarct size (triphenyltetrazolium chloride staining) and lactate dehydrogenase release were significantly reduced in low-pressure reperfusion and postconditioning versus controls (P < .01), whereas functional parameters (coronary flow, rate pressure product) were improved (P < .01). Although delaying postconditioning by more than 3 minutes resulted in a loss of protection, low-pressure reperfusion still significantly reduced infarct size when applied as late as 20 minutes after reperfusion. This delayed low-pressure reperfusion protection was associated with an improved mitochondrial respiration, lower reactive oxygen species production, and enhanced calcium retention capacity, related to inhibition of permeability transition pore opening. CONCLUSIONS We demonstrated for the first time that low-pressure reperfusion can reduce lethal myocardial reperfusion injury even when performed 10 to 20 minutes after the initiation of reperfusion.