Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match ...energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu−/− mice display no overt baseline phenotype and are protected against mCa2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu−/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metabolism during acute stress.
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•The MCU is dispensable for baseline homeostatic cardiac function•Deletion of Mcu protects against myocardial IR injury by reducing MPTP activation•The MCU is required to match energetics with contractile demand during stress•A slow MCU-independent uptake mechanism may maintain basal matrix mCa2+ content
Luongo et al. show, using a conditional knockout mouse model, that the mitochondrial Ca2+ uniporter (MCU), although dispensable for homeostatic function, is necessary for the cardiac “fight-or-flight” contractile response and a significant contributor to mitochondrial permeability transition during ischemia-reperfusion injury.
TRPM channels are a subgroup of the transient receptor potential (TRP) channel superfamily whose members have important roles in cell proliferation and survival. TRPM2, the second subfamily member to ...be cloned, is expressed in many tissues including brain, heart, vasculature and haematopoietic cells. TRPM2 is activated by oxidative stress and several other extracellular signals including tumour necrosis factor α (TNF‐α) and amyloid β‐peptide, which increase production of ADP‐ribose (ADPR). ADPR binds to the TRPM2 C‐terminal NUDT9‐H domain, activating the channel. Early studies support the paradigm that TRPM2 activation induces cell death by sustained Ca2+ influx or by enhancing cytokine production, aggravating inflammation and tissue injury. However, more recent data show that for a number of physiological processes, TRPM2 is protective. TRPM2 protects lungs from endotoxin‐induced injury by reducing reactive oxygen species (ROS) production by phagocytes. It protects hearts from oxidative damage after ischaemia–reperfusion or hypoxia–reoxygenation by maintaining better mitochondrial bioenergetics and by decreasing ROS. Sustained Ca2+ entry through TRPM2 is required to maintain cellular bioenergetics and protect against hypoxia–reoxygenation injury. TRPM2 also protects neuroblastoma from moderate oxidative stress by decreasing ROS through increased levels of forkhead box transcription factor 3a (FOXO3a) and a downstream effector, superoxide dismutase 2. TRPM2 is important for tumour growth and cell survival through modulation of hypoxia‐inducible transcription factor expression, mitochondrial function and mitophagy. These findings in cardiac ischaemia and in neuroblastoma suggest that TRPM2 has a basic role in sustaining mitochondrial function and in cell survival that applies to a number of physiological systems and pathophysiological processes including ischaemia–reperfusion injury.
Although activation of the ion channel TRPM2 can induce cell death in some circumstances, TRPM2 can also preserve cell viability and protect against tissue damage following oxidative stress and ischaemia–reperfusion. TRPM2 dependent Ca2+ entry can modulate HIF‐1/2α expression. One mechanism through which this may occur is through enhancement of calcineurin activity through TRPM2‐dependent Ca2+ entry, which may increase HIF‐1/2α stability. HIF‐1/2α enhances expression of a number of target genes including those involved in energy metabolism, antioxidant expression and mitophagy. Ca2+ entry through TRPM2 may also directly influence mitochondrial Ca2+ uptake. Together, the impact on mitochondrial function results in reduced ROS production and reduced cell death. In contrast, in the TRPM2 KO, Ca2+ influx is reduced after oxidative stress and HIF‐1/2α expression is decreased, as are proteins downstream of HIF‐1/2α including BNIP3, SOD1/2, and NDUFA4L2. In addition, mitochondrial Ca2+ uptake is reduced, which may contribute to dysfunctional mitochondria along with decreased NDUFA4L2, and reduced mitochondrial bioenergetics. Decreased BNIP3, which results in reduced mitophagy, contributes to an accumulation of dysfunctional mitochondria and along with decreased SOD1/2 antioxidant activity, increased ROS. The cell has reduced tolerance to a further rise in ROS, for example following ischaemia or doxorubicin, leading to reduced cell survival and increased cell death in the absence of TRPM2.
Transient receptor potential channel TRPM2 is highly expressed in many cancers and involved in regulation of key physiological processes including mitochondrial function, bioenergetics, and oxidative ...stress. In Stage 4 non-MYCN amplified neuroblastoma patients, high TRPM2 expression is associated with worse outcome. Here, neuroblastoma cells with high TRPM2 expression demonstrated increased migration and invasion capability. RNA sequencing, RT-qPCR, and Western blotting demonstrated that the mechanism involved significantly greater expression of integrins α1, αv, β1, and β5 in cells with high TRPM2 expression. Transcription factors HIF-1α, E2F1, and FOXM1, which bind promoter/enhancer regions of these integrins, were increased in cells with high TRPM2 expression. Subcellular fractionation confirmed high levels of α1, αv, and β1 membrane localization and co-immunoprecipitation confirmed the presence of α1β1, αvβ1, and αvβ5 complexes. Inhibitors of α1β1, αvβ1, and αvβ5 complexes significantly reduced migration and invasion in cells highly expressing TRPM2, confirming their functional role. Increased pAkt
and pERK
, which promote migration through mechanisms including integrin activation, were found in cells highly expressing TRPM2. TRPM2 promotes migration and invasion in neuroblastoma cells with high TRPM2 expression through modulation of integrins together with enhancing cell survival, negatively affecting patient outcome and providing rationale for TRPM2 inhibition in anti-neoplastic therapy.
Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by ...depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca
-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.
Resting mitochondrial matrix Ca2+ 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 Ca2+ threshold for mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.
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•Mitochondrial Ca2+ is maintained by MICU1 inhibition of mitochondrial uniporter•MICU1 localizes to the mitochondrial matrix and forms homo-oligomers•MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain•MICU1 EF hands determine MCU activity, but not binding
Resting mitochondrial matrix Ca2+ is maintained through a MICU1-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. Madesh and colleagues show that MICU1 localizes to the mitochondrial matrix and that MICU1/MCU interaction is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to endothelial cell migration. MICU1 dysregulation may thus enhance vascular dysfunction.
Current Landscape of Heart Failure Gene Therapy Kieserman, Jake M; Myers, Valerie D; Dubey, Praveen ...
Journal of the American Heart Association,
05/2019, Letnik:
8, Številka:
10
Journal Article
G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine ...as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.
Cardiovascular disease is a leading cause of co-morbidity in HIV-1 positive patients, even those in whom plasma virus levels are well-controlled. The pathogenic mechanism of HIV-1-associated ...cardiomyopathy is unknown, but has been presumed to be mediated indirectly, owing to the absence of productive HIV-1 replication in cardiomyocytes. We sought to investigate the effect of the HIV-1 auxiliary protein, Nef, which is suspected of extracellular release by infected CD4+ T cells on protein quality control and autophagy in cardiomyocytes. After detection of Nef in the serum of HIV-1 positive patients and the accumulation of this protein in human and primate heart tissue from HIV-1/SIV-infected cells we employed cell and molecular biology approaches to investigate the effect of Nef on cardiomyocyte-homeostasis by concentrating on protein quality control (PQC) pathway and autophagy. We found that HIV-1 Nef-mediated inhibition of autophagy flux leads to cytotoxicity and death of cardiomyocytes. Nef compromises autophagy at the maturation stage of autophagosomes by interacting with Beclin 1/Rab7 and dysregulating TFEB localization and cellular lysosome content. These effects were reversed by rapamycin treatment. Our results indicate that HIV-1 Nef-mediated inhibition of cellular PQC is one possible mechanism involved in the development of HIV-associated cardiomyopathy.
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