Cell-to-Cell Variation in p53 Dynamics Leads to Fractional Killing.

Many chemotherapeutic drugs kill only a fraction of cancer cells, limiting their efficacy. We used live-cell imaging to investigate the role of p53 dynamics in fractional killing of colon cancer cells in response to chemotherapy. We found that both surviving and dying cells reach similar levels of p53, indicating that cell death is not determined by a fixed p53 threshold. Instead, a cell's probability of death depends on the time and levels of p53. Cells must reach a threshold level of p53 to execute apoptosis, and this threshold increases with time. The increase in p53 apoptotic threshold is due to drug-dependent induction of anti-apoptotic genes, predominantly in the inhibitors of apoptosis (IAP) family. Our study underlines the importance of measuring the dynamics of key players in response to chemotherapy to determine mechanisms of resistance and optimize the timing of combination therapy.

MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Global Inhibition with Specific Activation: How p53 and MYC Redistribute the Transcriptome in the DNA Double-Strand Break Response.

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.

Mechanisms of reduced myocardial energetics of the dystrophic heart.

Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD), characterized by the progressive replacement of contractile tissue with scar tissue. Effective therapies for dystrophic cardiomyopathy will require addressing the disease before the onset of fibrosis, however, the mechanisms of the early disease are poorly understood. To understand the pathophysiology of DMD, we perform a detailed functional assessment of cardiac function of the mdx mouse, a model of DMD. These studies use a combination of functional, metabolomic, and spectroscopic approaches to fully characterize the contractile, energetic, and mitochondrial function of beating hearts. Through these innovative approaches, we demonstrate that the dystrophic heart has reduced cardiac reserve and is energetically limited. We show that this limitation does not result from poor delivery of oxygen. Using spectroscopic approaches, we provide evidence that mitochondria in the dystrophic heart have attenuated mitochondrial membrane potential and deficits in the flow of electrons in complex IV of the electron transport chain. These studies provide evidence that poor myocardial energetics precede the onset of significant cardiac fibrosis and likely results from mitochondrial dysfunction centered around complex IV and reduced membrane potential. The multimodal approach used here implicates specific molecular components in the etiology of reduced energetics. Future studies focused on these targets may provide therapies that improve the energetics of the dystrophic heart leading to improved resiliency against damage and preservation of myocardial contractile tissue.NEW & NOTEWORTHY Dystrophic hearts have poor contractile reserve that is associated with a reduction in myocardial energetics. We demonstrate that oxygen delivery does not contribute to the limited energy production of the dystrophic heart even with increased workloads. Cytochrome optical spectroscopy of the contracting heart reveals alterations in complex IV and evidence of depolarized mitochondrial membranes. We show specific alterations in the electron transport chain of the dystrophic heart that may contribute to poor myocardial energetics.

Distinct effects of cardiac mitochondrial calcium uniporter inactivation via EMRE deletion in the short and long term.

Transport of Ca2+ into mitochondria is thought to stimulate the production of ATP, a critical process in the heart's fight or flight response, but excess Ca2+ can trigger cell death. The mitochondrial Ca2+ uniporter complex is the primary route of Ca2+ transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differed from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca2+ uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca2+, had lower basal mitochondrial Ca2+ levels, and displayed attenuated Ca2+-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca2+ handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.

Is MCU dispensable for normal heart function?

The uptake of Ca2+ into mitochondria is thought to be an important signal communicating the need for increased energy production. However, dysregulated uptake leading to mitochondrial Ca2+ overload can trigger opening of the mitochondrial permeability transition pore and potentially cell death. Thus mitochondrial Ca2+ entry is regulated via the activity of a Ca2+-selective channel known as the mitochondrial calcium uniporter. The last decade has seen enormous momentum in the discovery of the molecular identities of the multiple proteins comprising the uniporter. Increasing numbers of studies in cultured cells and animal models have provided insight into how disruption of uniporter proteins affects mitochondrial Ca2+ regulation and impacts tissue function and physiology. This review aims to summarize some of these recent findings, particularly in the context of the heart.

The In Vivo Biology of the Mitochondrial Calcium Uniporter.

The identification of the molecular composition of the mitochondrial calcium uniporter has allowed for the genetic manipulation of its components and the creation of various in vivo genetic models. Here, we review the initial attempts to modulate the expression of components of the calcium uniporter in a range of organisms from plants to mammals. This analysis has confirmed the strict requirement for the uniporter for in vivo mitochondrial calcium uptake and for maintaining mitochondrial calcium homeostasis. We further discuss the physiological effects following genetic manipulation of the uniporter on tissue bioenergetics and the threshold for cell death. Finally, we analyze the limited information regarding the role of various uniporter components in human disease.

Bacterial adaptation through loss of function.

The metabolic capabilities and regulatory networks of bacteria have been optimized by evolution in response to selective pressures present in each species' native ecological niche. In a new environment, however, the same bacteria may grow poorly due to regulatory constraints or biochemical deficiencies. Adaptation to such conditions can proceed through the acquisition of new cellular functionality due to gain of function mutations or via modulation of cellular networks. Using selection experiments on transposon-mutagenized libraries of bacteria, we illustrate that even under conditions of extreme nutrient limitation, substantial adaptation can be achieved solely through loss of function mutations, which rewire the metabolism of the cell without gain of enzymatic or sensory function. A systematic analysis of similar experiments under more than 100 conditions reveals that adaptive loss of function mutations exist for many environmental challenges. Drawing on a wealth of examples from published articles, we detail the range of mechanisms through which loss-of-function mutations can generate such beneficial regulatory changes, without the need for rare, specific mutations to fine-tune enzymatic activities or network connections. The high rate at which loss-of-function mutations occur suggests that null mutations play an underappreciated role in the early stages of adaption of bacterial populations to new environments.

Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter.

Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient of MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU(-/-) mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20months of age. Moreover, the physiological response of MCU(-/-) mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU(-/-) mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

Mitochondrial calcium and reactive oxygen species in cardiovascular disease.

Cardiomyocytes are one of the most mitochondria-rich cell types in the body, with ∼30-40% of the cell volume being composed of mitochondria. Mitochondria are well established as the primary site of adenosine triphosphate (ATP) generation in a beating cardiomyocyte, generating up to 90% of its ATP. Mitochondria have many functions in the cell, which could contribute to susceptibility to and development of cardiovascular disease (CVD). Mitochondria are key players in cell metabolism, ATP production, reactive oxygen species (ROS) production, and cell death. Mitochondrial calcium (Ca2+) plays a critical role in many of these pathways, and thus the dynamics of mitochondrial Ca2+ are important in regulating mitochondrial processes. Alterations in these varied and in many cases interrelated functions play an important role in CVD. This review will focus on the interrelationship of mitochondrial energetics, Ca2+, and ROS and their roles in CVD. Recent insights into the regulation and dysregulation of these pathways have led to some novel therapeutic approaches.

EMRE is essential for mitochondrial calcium uniporter activity in a mouse model.

The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE's impact on organismal physiology is less understood. Here we characterize a mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE-/- mice are born less frequently; however, the mice that are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.

Mitochondrial Permeability Transition Pore and Calcium Handling.

The opening of a large conductance channel in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (PTP), has been shown to be a primary mediator of cell death in the heart subjected to ischemia-reperfusion injury. Inhibitors of the PTP have been shown to reduce cardiac ischemia-reperfusion injury in many animal models. Furthermore, most cardioprotective strategies appear to reduce ischemic cell death either by reducing the triggers for the opening of the PTP, such as reducing calcium overload or reactive oxygen species, or by inhibiting PTP modulators. This chapter will focus on key issues in the study of the PTP and provide some methods for measuring PTP opening in isolated mitochondria.

MICU1 Serves as a Molecular Gatekeeper to Prevent In Vivo Mitochondrial Calcium Overload.

MICU1 is a component of the mitochondrial calcium uniporter, a multiprotein complex that also includes MICU2, MCU, and EMRE. Here, we describe a mouse model of MICU1 deficiency. MICU1(-/-) mitochondria demonstrate altered calcium uptake, and deletion of MICU1 results in significant, but not complete, perinatal mortality. Similar to afflicted patients, viable MICU1(-/-) mice manifest marked ataxia and muscle weakness. Early in life, these animals display a range of biochemical abnormalities, including increased resting mitochondrial calcium levels, altered mitochondrial morphology, and reduced ATP. Older MICU1(-/-) mice show marked, spontaneous improvement coincident with improved mitochondrial calcium handling and an age-dependent reduction in EMRE expression. Remarkably, deleting one allele of EMRE helps normalize calcium uptake while simultaneously rescuing the high perinatal mortality observed in young MICU1(-/-) mice. Together, these results demonstrate that MICU1 serves as a molecular gatekeeper preventing calcium overload and suggests that modulating the calcium uniporter could have widespread therapeutic benefits.

Stem cells: balancing resistance and sensitivity to DNA damage.

Embryonic stem cells (ESCs) are known to be very sensitive to DNA damage and undergo rapid apoptosis even after low-damage doses. By contrast, adult stem cells show variable sensitivity to damage. Here we describe the multiple pathways that have been proposed to affect the sensitivity of stem cells to damage, including proximity to the apoptotic threshold (mitochondrial priming) and the p53 signaling pathway, through activation of transcription or direct interaction with proapoptotic proteins in the cytoplasm. We also discuss which cellular factors might connect mitochondrial priming with pluripotency and the potential therapeutic advances that can be achieved by better understanding of the molecular mechanisms leading to sensitivity or resistance of embryonic or adult stem cells from different tissues.

High mitochondrial priming sensitizes hESCs to DNA-damage-induced apoptosis.

Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.