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Objective Skeletal muscle insulin signaling is a major determinant of muscle growth and glucose homeostasis. Protein kinase B/Akt plays a prominent role in mediating many of the metabolic effects of insulin. Mice and humans harboring systemic loss-of-function mutations in Akt2, the most abundant Akt isoform in metabolic tissues, are glucose intolerant and insulin resistant. Since the skeletal muscle accounts for a significant amount of postprandial glucose disposal, a popular hypothesis in the diabetes field suggests that a reduction in Akt, specifically in skeletal muscle, leads to systemic glucose intolerance and insulin resistance. Despite this common belief, the specific role of skeletal muscle Akt in muscle growth and insulin sensitivity remains undefined. Methods We generated multiple mouse models of skeletal muscle Akt deficiency to evaluate the role of muscle Akt signaling in vivo. The effects of these genetic perturbations on muscle mass, glucose homeostasis and insulin sensitivity were assessed using both in vivo and ex vivo assays. Results Surprisingly, mice lacking Akt2 alone in skeletal muscle displayed normal skeletal muscle insulin signaling, glucose tolerance, and insulin sensitivity despite a dramatic reduction in phosphorylated Akt. In contrast, deletion of both Akt isoforms (M-AktDKO) prevented downstream signaling and resulted in muscle atrophy. Despite the absence of Akt signaling, in vivo and ex vivo insulin-stimulated glucose uptake were normal in M-AktDKO mice. Similar effects on insulin sensitivity were observed in mice with prolonged deletion (4 weeks) of both skeletal muscle Akt isoforms selectively in adulthood. Conversely, short term deletion (2 weeks) of skeletal muscle specific Akt in adult muscles impaired insulin tolerance paralleling the effect observed by acute pharmacological inhibition of Akt in vitro. Mechanistically, chronic ablation of Akt induced mitochondrial dysfunction and activation of AMPK, which was required for insulin-stimulated glucose uptake in the absence of Akt. Conclusions Together, these data indicate that chronic reduction in Akt activity alone in skeletal muscle is not sufficient to induce insulin resistance or prevent glucose uptake in all conditions. Therefore, since insulin-stimulated glucose disposal in skeletal muscle is markedly impaired in insulin-resistant states, we hypothesize that alterations in signaling molecules in addition to skeletal muscle Akt are necessary to perturb glucose tolerance and insulin sensitivity in vivo., Highlights • Deletion of skeletal muscle Akt2 alone does not reduce downstream insulin signaling or alter glucose homeostasis. • Inhibition of both skeletal muscle Akt isoforms prevents downstream signaling and results in muscle atrophy. • Chronic ablation of Akt in skeletal muscle does not block insulin-stimulated glucose uptake in vivo. • Prolonged Akt deficiency activates AMPK, which is required for insulin-stimulated glucose uptake in muscle lacking Akt.

Calcium (Ca 2+ ) uptake into the mitochondrial matrix occurs via the mitochondrial Ca 2+ uniporter channel (mtCU) and tunes mitochondrial metabolism to meet acute changes in cellular ATP demand. However, the role of mtCU-dependent mitochondrial Ca 2+ ( m Ca 2+ ) uptake in regulating homeostatic heart function and adaptation to chronic increases in workload remain controversial. We subjected mice with tamoxifen-inducible, cardiomyocyte-specific gain (flox-stop-MCU x αMHC-MCM, MCU-Tg) or loss ( Mcu fl/fl x αMHC-MCM , Mcu- cKO) of MCU function to 2wk isoproterenol (iso) infusion to test the hypothesis that m Ca 2+ uptake through MCU contributes to functional adaptation to sustained catecholamine signaling. Neither gain nor loss of MCU function altered baseline cardiac structure or function. Fractional shortening was increased after 2d of iso infusion in MCM control mice, but loss of MCU blocked this effect. In contrast, fractional shortening declined significantly after 7-14d of iso in MCU-Tg mice. MCU-Tg mice also exhibited increased LV dilation, heart mass, and lung edema compared to controls after 14d of iso. Acute treatment of MCU-Tg cardiomyocytes in vitro with the Ca 2+ ionophore ionomycin revealed increased ROS production and a trend towards increased sensitivity to Ca 2+ -induced cell death. In agreement, Evans blue dye exclusion assays revealed increased cardiomyocyte necrosis in MCU-Tg hearts following iso. Therefore, we deleted the mPTP regulator cyclophilin D (CypD) in MCU-Tg mice to test whether cardiomyocyte dropout due to m Ca 2+ overload-induced mPTP contributed to iso-induced contractile dysfunction. Unexpectedly, CypD deletion failed to attenuate contractile dysfunction and hypertrophic remodeling, and increased rather than attenuated iso-induced cardiomyocyte death in MCU-Tg mice. We conclude that while MCU-dependent m Ca 2+ uptake is required for the heart’s initial contractile response to catecholaminergic stress, under prolonged stimulation augmented m Ca 2+ uptake becomes deleterious and predisposes to cardiomyocyte death and heart failure. Our findings support the notion that the detrimental effects of sustained MCU-dependent m Ca 2+ uptake are mediated by mechanisms distinct from mitochondrial permeability transition.

The mitochondrial calcium uniporter (MCU) forms the pore of the mitochondrial calcium uniporter channel (mtCU) and is required for rapid mitochondrial Ca 2+ ( m Ca 2+ ) uptake. MCU is necessary to increase cardiac energetics to fuel an increase in cardiac contractility during acute sympathetic stimulation. However, little is known about how MCU-dependent Ca 2+ flux may contribute to the heart’s adaptations to chronic stress. We therefore compared mice with adult cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu-cKO) or gain (CAG-CAT-MCU; MCU-Tg) of MCU function to examine the role of MCU-dependent m Ca 2+ uptake in a model of chronic catecholamine overload. In vitro characterization of ACMs confirmed that MCU overexpression enhanced and Mcu deletion inhibited acute m Ca 2+ uptake. Neither loss nor gain of MCU function altered baseline contractile function in vivo . In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion. This initial increase in contractility was attenuated in MCU-cKO mice. In contrast, MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on contractile function was associated with increased LV dilation, HW/BW ratio, and lung edema. MCU-Tg cardiomyocytes in vitro exhibited increased ROS production and a trend towards increased cell death upon elevation of cytosolic Ca 2+ with ionomycin. These data prompted us to test the hypothesis that isoproterenol-induced contractile dysfunction in MCU-Tg hearts is caused by cardiomyocyte dropout due to m Ca 2+ overload and mitochondrial permeability transition. However, genetic deletion of the mPTP component cyclophilin D did not prevent the decline in contractile function, diminish cardiomyocyte death, or attenuate LV remodeling in MCU-Tg animals during chronic isoproterenol infusion. We conclude that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure. Our data suggest that this maladaptive response occurs via mechanisms independent of cyclophilin D-mediated permeability transition.

Since the initial identification of the mitochondrial calcium uniporter (MCU) in 2011, several studies employing genetic models have attempted to decipher the role of mitochondrial calcium uptake in cardiac physiology. Confounding results in various mutant mouse models have led to an ongoing debate regarding the function of MCU in the heart. In this review, we evaluate and discuss the totality of evidence for mitochondrial calcium uptake in the cardiac stress response. We highlight recent reports that implicate MCU in the control of homeostatic cardiac metabolism and function. This review concludes with a discussion of current gaps in knowledge and remaining experiments to define how MCU contributes to contractile function, cell death, metabolic regulation, and heart failure progression.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3–5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)—an essential6,7 mitochondrial protein of previously unknown function—as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial—but not whole-cell—NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria. SLC25A51 is identified as a transporter of intact NAD+ into mammalian mitochondria and is required to maintain the mitochondrial NAD+ pool and respiratory function.

Objective Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo. Methods We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes. Results Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test. Conclusions Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis., Highlights • Inhibition of adipose mTORC1 causes hypertriglyceridemia prior to lipodystrophy. • Genetically inhibiting lipolysis reverses the increase in plasma TG. • Acute pharmacological inhibition of lipolysis reverses the increase in plasma TG caused by rapamycin treatment. • Unrestrained lipolysis impairs LPL activity and decreases TG clearance.

The mitochondrial calcium uniporter (mtCU) is a ~700 kD multi-subunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. Mitochondrial Calcium Uniporter B ( MCUB ) is reported to negatively regulate m Ca 2+ uptake, but its precise functional role and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of ventricular mitochondria revealed MCUB was absent from high-molecular weight (MW) mtCU complexes in sham animals, but present 24 hours following myocardial ischemia-reperfusion injury (IR). To investigate MCUB ’s contribution to mtCU regulation we created a MCUB -/- cell line by CRISPR-Cas9n. MCUB deletion increased histamine-mediated [ m Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO1). MCUB deletion increased mtCU capacitance (mitoplast patch-clamp) and rate of [ m Ca 2+ ] uptake. Size-exclusion chromatography revealed loss of MCUB increased MCU incorporation into high-MW mtCU, suggesting stoichiometric replacement and overall more functional mtCU’s. To examine MCUB’s role in cardiac physiology we generated a cardiac-specific, tamoxifen-inducible MCUB mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg). FPLC revealed MCUB was undetected in high-MW mtCU complexes of Cre controls, but enriched in MCUB-Tg hearts. MCUB incorporation decreased the presence of channel gatekeepers, MICU1/2, and decreased the MW of the mtCU complex. Immunoprecipitations suggest MCUB interacts with MCU but not MICU1/2. MCUB-Tg adult cardiomyocytes (ACMs) expressing AAV9-mitycam ( m Ca 2+ reporter) were paced and displayed a ~30% decrease in m Ca 2+ transient peak amplitude with significantly reduced m Ca 2+ uptake rates vs controls. A reduction in OxPhos reserve capacity correlated with a severe impairment in cardiac contractile reserve (LV invasive hemodynamics during isoproterenol infusion). MCUB-Tg cardiac mitochondria were resistant to Ca 2+ -induced permeability transition and MCUB-Tg mice displayed ~50% decrease in infarct size per area-at-risk after in vivo IR-injury. These data suggest MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive to cardiac energetic responsiveness.

Mitochondrial calcium ( m Ca 2+ ) uptake couples acute changes in cardiomyocyte bioenergetic demand to ATP production, but in excess triggers mitochondrial permeability transition and cardiomyocyte necrosis, as occurs during cardiac injury. Despite established roles for m Ca 2+ flux in response to acute stress, the role of m Ca 2+ signaling in chronic stress is poorly defined. As m Ca 2+ regulates the TCA cycle with the potential to affect mitochondrial signaling and/or metabolite pools required for biosynthesis, we reasoned that altered m Ca 2+ homeostasis may be an essential mechanism underlying hypertrophic growth and remodeling of the myocardium. Here, we used mice with cardiac-specific overexpression (OE) of the mitochondrial Na + /Ca 2+ exchanger (NCLX), the primary mediator of m Ca 2+ efflux in the heart, to test the hypothesis that m Ca 2+ signaling contributes to cardiac remodeling during sustained hemodynamic stress. We subjected NCLX OE (TRE-NCLX x α-MHC-tTA) and control (αMHC-tTA) mice to 12-wk transverse aortic constriction (TAC) or 4-wk infusion with angiotensin II + phenylephrine (AngII/PE) as experimental models of cardiac hypertrophy and non-ischemic heart failure. Cardiac function of NCLX OE and control mice was monitored throughout these studies via echocardiography and remodeling was assessed via tissue gravimetrics, histology, and qPCR gene expression analysis. Cardiac NCLX OE preserved ejection fraction, prevented afterload-induced hypertrophy and fibrosis, and attenuated the induction of both hypertrophic ( Nppa , Nppb , Acta1 ) and fibrotic ( Postn1 , Spp1 ) gene programs in mice subjected to 12-wk TAC. Examination at 2-wk post-TAC revealed attenuated hypertrophy and blunted hypertrophic and fibrotic gene expression in NCLX OE mice. These data indicate that increased capacity for m Ca 2+ efflux mitigates TAC-induced remodeling, prior to the development of contractile dysfunction. NCLX OE similarly attenuated atrial hypertrophy and the induction of hypertrophic and fibrotic gene programs in mice infused with AngII/PE. Together, these findings support a critical role for m Ca 2+ in driving pathological remodeling in non-ischemic heart disease and point to NCLX as a potent therapeutic target for cardiovascular disease.

OBJECTIVES/GOALS: Nicotinamide adenine dinucleotide (NAD) plays essential roles in energy metabolism and cell signaling pathways. NAD functions as a coenzyme by accepting electrons during glycolysis and the TCA cycle and subsequently donates them to complex I of the electron transport chain providing the driving force for ATP production. NAD also acts as a co-substrate for several classes of enzymes, including sirtuin deacetylases. Both NAD and the enzyme that is rate limiting for synthesis, Nicotinamide phosphoribosyltransferase (Nampt), are depleted in the failing heart, concurrent with hyperacetylation and mitochondrial dysfunction. Moreover, treatment with NAD precursors reduced cardiac injury in several heart failure models. However, NAD precursors may have systemic effects, and it remains unproven whether depletion of myocardial NAD is causative or merely correlative for the onset and progression of heart failure. METHODS/STUDY POPULATION: To test this, we generated a cardiac-specific tamoxifen-inducible (αMHC-MerCreMer) model for deletion of Nampt (Nampt cKO) in cardiomyocytes. Adult mice were administered tamoxifen for 5 days leading to deletion of Nampt, resulting in a 72% reduction in myocardial NAD after two-weeks. RESULTS/ANTICIPATED RESULTS: Echocardiography revealed that Nampt cKO mice displayed a significant reduction in left ventricular (LV) contractility as well as cardiac hypertrophy. Despite the further loss of NAD, the majority of animals survived to 8 weeks of age before experiencing sudden deaths resulting in significant mortality over the next several weeks. Remarkably, we observed only a slight increase in acetylation of mitochondrial proteins, and cardiac mitochondria isolated from Nampt-null mice even at 8 weeks displayed a normal or higher oxygen consumption rate. We found that mitochondrial NAD levels were preferentially maintained and depleted at a slower rate compared to those in bulk tissue. DISCUSSION/SIGNIFICANCE OF IMPACT: While mild depletion of cardiac NAD has been reported in heart failure, our data indicate that the heart can adapt to much more severe loss of NAD prior to the loss of viability.

The mitochondrial calcium uniporter (MCU) constitutes the pore-forming unit of the mitochondrial calcium uniporter channel (mtCU) and is necessary for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. We previously reported that MCU is required to increase cardiac energetics and contractility in response to acute β-adrenergic stimulation. However, deletion of MCU has no detrimental effect on basal cardiac function or metabolism. While these findings support a role for MCU-dependent Ca 2+ flux in functional adaptations to acute stress, little is known about the involvement of MCU in cardiac responses to sustained stress. We compared mice with adult, cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu -cKO) or gain (CAG-CAG-MCU; MCU-Tg) of MCU function to explore the role of m Ca 2+ uptake in a model of chronic catecholamine overload. Biophysical characterization confirmed that MCU overexpression enhanced and MCU deletion inhibited ACM m Ca 2+ uptake. Neither loss nor gain of MCU had a significant impact on baseline contractile function. In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion (Fig 1). This early increase in contractility was attenuated in Mcu -cKO mice. MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on LV function was associated with increased LV dilation, HW/BW ratio, and lung edema as compared to αMHC-MCM controls. These data suggest that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure.

The mitochondrial calcium uniporter channel (mtCU) resides in the inner mitochondrial membrane and is a multi-subunit complex required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. RNA-seq and PCR analysis of mtCU component expression revealed that MCUB expression increases after myocardial infarction. To investigate how MCUB , a paralog of the MCU gene, contributes to mtCU regulation we created a MCUB -/- cell line (HeLa) using CRISPR-Cas9n. MCUB deletion increased histamine-mediated m [Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO). Biophysical characterization found MCUB deletion increased mtCU capacitance and rate of m [Ca 2+ ] uptake. FPLC fractionation of mtCU revealed loss of MCUB increased MCU incorporation into the supercomplex, suggesting MCUB replacement of MCU and overall increase in functional mtCU supercomplexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (mito genetic calcium reporter) and adult cardiomyocytes were isolated to record [ m Ca 2+ ] transients during pacing. Increasing the MCUB/MCU ratio decreased [ m Ca 2+ ] peak amplitude by ~30% and significantly reduced the [ m Ca 2+ ] uptake rate. FPLC assessment of the mtCU revealed MCUB was undetectable in the supercomplex fraction (~700kD) of Cre controls, but enriched in MCUB -Tg hearts. Examining the oxygen consumption rate of cardiomyocytes isolated from MCUB- Tg hearts found decreased maximal respiration and spare reserve capacity, which correlated with severe impairment in isoproterenol-induced contractile reserve. Mitochondria from MCUB -Tg mouse hearts were resistant to Ca 2+ induced mitochondrial swelling suggesting MCUB protects against permeability transition. MCUB -Tg mice subjected to myocardial ischemia reperfusion injury had a ~50% decrease in infarct size suggesting increased MCUB expression prevents m Ca 2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive in mitochondrial energetic responsiveness.

Fibroblast to myofibroblast differentiation is crucial for the initial healing response but excessive myofibroblast activation leads to pathological fibrosis. Therefore, it is imperative to understand the mechanisms underlying myofibroblast formation. Here we report that mitochondrial calcium (mCa2+) signaling is a regulatory mechanism in myofibroblast differentiation and fibrosis. We demonstrate that fibrotic signaling alters gating of the mitochondrial calcium uniporter (mtCU) in a MICU1-dependent fashion to reduce mCa2+ uptake and induce coordinated changes in metabolism, i.e., increased glycolysis feeding anabolic pathways and glutaminolysis yielding increased α-ketoglutarate (αKG) bioavailability. mCa2+-dependent metabolic reprogramming leads to the activation of αKG-dependent histone demethylases, enhancing chromatin accessibility in loci specific to the myofibroblast gene program, resulting in differentiation. Our results uncover an important role for the mtCU beyond metabolic regulation and cell death and demonstrate that mCa2+ signaling regulates the epigenome to influence cellular differentiation., Myofibroblast differentiation contributes to extracellular matrix remodeling and fibrosis. Here, the authors report that alterations in mitochondrial calcium uptake is essential for metabolic reprogramming and epigenetic signaling for activation of the myofibroblast gene program.

The mitochondrial calcium uniporter (MCU) is a multicomponent channel that is the primary mechanism for mitochondrial Ca 2+ uptake ( m Ca 2+ ). We previously reported that the MCU is required for energetic signaling to meet contractile demand during the ‘fight or flight’ response. In addition, we showed that deletion of the pore-forming component ( Mcu gene) protected against mitochondria permeability transition pore (MPTP) opening and ischemia-reperfusion injury. However, results from our study and others questioned the physiological relevance of MCU-mediated Ca 2+ uptake during chronic stress states featuring sustained intracellular Ca 2+ load ( i Ca 2+ ). To address this, we deleted Mcu from cardiomyocytes in adult mice ( Mcu cKO) and implanted osmotic pumps to deliver the β adrenergic agonist isoproterenol (iso, 70 mg/kg/day for 14d). In contrast to controls, Mcu cKO mice lacked contractile responsiveness to chronic βAR stimulation with evidence of LV dysfunction and failure by d14 ( Fig 1 ). Next, we crossed the Mcu cKO with mice overexpressing the β2a subunit (β2a-Tg) of the L-type Ca 2+ channel (LTCC). This model displays enhanced LTCC activity and cardiac contractility, but with added stress such as iso infusion, Ca 2+ overload eventually leads to MPTP-dependent cell death and heart failure. Surprisingly, loss of Mcu in this model was lethal with all mice dying by d13 ( Fig 2 ). Baseline echocardiography revealed that loss of Mcu ablated all β2a-mediated enhancements in LV contractility and accelerated dysfunction post-iso. These findings demonstrate that MCU-mediated m Ca 2+ uptake is critical to meet energetic demand during chronic stress states featuring sustained i Ca 2+ load.

Tyrosine sulfate-mediated interactions play an important role in HIV-1 entry. After engaging the CD4 receptor at the cell surface, the HIV-1 gp120 glycoprotein binds to the CCR5 co-receptor via an interaction that requires two tyrosine sulfates, at positions 10 and 14 in the CCR5-N terminus. Building on previous structure determinations of this interaction, here we report the targeting of these tyrosine sulfate binding sites for drug design through in silico screening of small molecule libraries, identification of lead compounds, and characterization of biological activity. A class of tyrosine sulfate-mimicking small molecules containing a "phenyl sulfonate-linker-aromatic" motif was identified that specifically inhibited binding of gp120 to the CCR5-N terminus as well as to sulfated antibodies that recognize the co-receptor binding region on gp120. The most potent of these compounds bound gp120 with low micromolar affinity and its CD4-induced conformation with K(D)'s as tight as ∼50 nM. Neutralization experiments suggested the targeted site to be conformationally inaccessible prior to CD4 engagement. Primary HIV-1 isolates were weakly neutralized, preincubation with soluble CD4 enhanced neutralization, and engineered isolates with increased dependence on the N terminus of CCR5 or with reduced conformational barriers were neutralized with IC(50) values as low as ∼1 μM. These results reveal the potential of targeting the tyrosine sulfate interactions of HIV-1 and provide insight into how mechanistic barriers, evolved by HIV-1 to evade antibody recognition, also restrict small-molecule-mediated neutralization.

Mitochondrial calcium ( m Ca 2+ ) signaling is critical for both energy production and the activation of cell death pathways in the heart. Further, m Ca 2+ overload is hypothesized to be a significant contributor to the development and progression of heart failure (HF). The mitochondrial sodium/calcium exchanger (mNCX) is hypothesized to be the primary mechanism of m Ca 2+ efflux, but to date no study has genetically confirmed its identity or function in an in vivo system. To investigate the role of mNCX in HF, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene, Slc8b1 (also known as NCLX) , and crossed them with a tamoxifen (tamox)-inducible cardiac-specific Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Cardiomyocytes isolated from mNCX-cKO mice displayed a significant reduction in m Ca 2+ efflux rate and Ca 2+ uptake capacity. Tamoxifen-induced ablation of mNCX resulted in sudden death with only 54% of mice surviving 8d post-tamoxifen treatment (Fig 1). Assessment of mNCX-cKO hearts 2d post-tamox revealed significant remodeling characterized by dilation and a decrease in %EF (Fig 2). Next, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to evaluate if increased m Ca 2+ efflux would alter the progression of HF. mNCX-Tg and controls were subjected to both myocardial infarction (LCA ligation) and pressure-overload induced HF (transverse aortic constriction). mNCX-Tg mice displayed preserved LV function, structure and a reduction in HF indices in both models. For the first time we show that mNCX is essential for m Ca 2+ efflux in cardiomyocytes and that mNCX represents a novel therapeutic target in HF.

Summary Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca 2+ ), 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 m Ca 2+ 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 Ca 2+ -dependent metabolism during acute stress.

SummaryThe site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

Background. Alzheimer's disease (AD) is characterized by neurodegeneration, specifically the progressive loss of neuronal populations in the frontal cortex and hippocampus. Numerous studies have shown that neuronal cell death and metabolic dysregulation are fundamental cellular mechanisms driving the progression of AD and other dementia-related diseases. Previous studies have suggested numerous mechanisms whereby intracellular Ca2+ load is increased in AD and thereby likely significantly impacts mCa2+ signaling. Methods. To discern if mCa2+ signaling is causative in the progression of AD, human AD brain samples, an AD mutant mouse model (3xTg) and an AD-mutant cell line (N2a/APPswe) were examined for mitochondrial alterations in: Ca2+ handling, ROS generation, membrane potential (ΔΨ), permeability transition pore activation, OxPhos, APP metabolism and cell death. Results. 3xTg-AD mice and human AD brain samples displayed significant alterations in the expression of key mCa2+ exchange genes, most notably a reduction in the expression of the mitochondrial Na+/Ca2+ exchanger (mNCX, SLC8B1), the major efflux pathway in excitable cells. We discovered that mCa2+ efflux and mCa2+ retention capacity was severely impaired in N2a/APPswe cells. Rescue of mCa2+ extrusion, via adenoviral expression of mNCX, enhanced the clearance of pathogenic mCa2+, recovered (ΔΨ), enhanced OxPhos, reduced extracellular Aβ1-40 levels and protected from ionomycin-, glutamate- and ROS-induced cell death. Conclusions. Our data suggest that impaired mCa2+ exchange is a central contributor to neuronal cell death in AD and that mNCX represents a new therapeutic target to inhibit or reverse AD progression.

When the heart is injured, fibroblasts transition from a structural role into contractile, synthetic myofibroblasts. This is crucial for scar formation after myocardial infarction to prevent ventricular rupture, but excessive fibrosis is maladaptive and leads to heart failure. While intracellular calcium (iCa2+) elevation has been shown to be necessary for myofibroblast transdifferentiation, the mitochondrial calcium (mCa2+) domain has not been explored. Mcu encodes the channel-forming portion of the mitochondrial calcium uniporter complex and is required for acute mCa2+ uptake. We generated a conditional, fibroblast-restricted Mcu knockout mouse by crossing Mcufl/fl mice with a mouse expressing a tamoxifen-inducible Cre recombinase under control of the collagen1a2 promoter (fibroblast Mcu-cKO). Fibroblast Mcu-cKO mice and controls were subjected to ligation of the left coronary artery and cardiac function was examined weekly by echocardiography. Deletion of fibroblast Mcu worsened left ventricular dysfunction and increased fibrosis. To examine cellular mechanisms responsible we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu using Cre-adenovirus. When challenged with pro-fibrotic ligands, Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced iCa2+ transient amplitude. Mcu-/- MEFs displayed enhanced myofibroblast transdifferentiation as evidenced by decreased migration and proliferation as well as increased α-SMA expression and gel contraction. Metabolically, Mcu-/- MEFs display a shift towards more glycolysis with less oxidative phosphorylation, correlating with increased phosphorylation (inactivation) of the pyruvate dehydrogenase subunit E3. These results suggest that alterations in mitochondrial buffering of pro-fibrotic iCa2+ signaling and changes in energetic pathways may be mechanisms driving myofibroblast transdifferentiation and fibrosis.

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

Summary The interface between the HIV-1 gp120 envelope glycoprotein and the CD4 receptor contains an unusual interfacial cavity, the "Phe43 cavity", which CD4-mimetic miniproteins with nonnatural extensions can potentially utilize to enhance their neutralization of HIV-1. Here, we report cocrystal structures of HIV-1 gp120 with miniproteins M48U1 and M48U7, which insert cyclohexylmethoxy and 5-hydroxypentylmethoxy extensions, respectively, into the Phe43 cavity. Both inserts displayed flexibility and hydrophobic interactions, but the M48U1 insert showed better shape complementarity with the Phe43 cavity than the M48U7 insert. Subtle alteration in the gp120 conformation played a substantial role in optimizing fit. With M48U1, these translated into a YU2-gp120 affinity of 0.015 nM and neutralization of all 180 circulating HIV-1 strains tested, except clade-A/E isolates with noncanonical Phe43 cavities. Ligand chemistry, shape complementarity, surface burial, and gp120 conformation act in concert to modulate binding of ligands to the gp120-Phe43 cavity and, when optimized, can effect near-pan-neutralization of HIV-1.

Background HIV-1 enters cells by sequentially binding the CD4 receptor and a coreceptor, either CCR5 or CXCR4. Functional constraints result in a high degree of conservation of the receptor-binding sites making them potential targets for intervention. The coreceptor-binding site on HIV-1 envelope gp120 glycoprotein is protected from the humoral immune system by conformational masking and steric occlusion. The site becomes available after a conformational change in gp120 following CD4 engagement, but at that point in the entry process, the proximity of the viral and cellular membranes makes the site inaccessible to bulky antibody molecules. Thus, in spite of being highly conserved, this region has not been considered a viable vaccine target.

Background The design of gp120 monomeric probes with modified antigenic profiles that are specific for a target epitope has been successfully used for the isolation of broadly neutralizing HIV-1 antibodies. Existing probes, however, do not possess sufficient specificity and can bind antibodies with undesired properties (e.g., weakly neutralizing antibodies targeting an overlapping epitope). To achieve improved epitope specificity, positive and negative design stages can be incorporated into the probe design process.