Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Dependence of Mitochondrial Calcium Signalling and Dynamics on the Disaggregase, CLPB

Cells utilize protein disaggregases to avoid abnormal protein aggregation that causes many diseases. Among these, caseinolytic peptidase B protein homolog (CLPB) is localized in the mitochondrial intermembrane space and linked to human disease. Upon CLPB loss, MICU1 and MICU2, regulators of the mitochondrial calcium uniporter complex (mtCU), and OPA1, a main mediator of mitochondrial fusion, become insoluble but the functional outcome remains unclear. In this work we demonstrate that CLPB is required to maintain mitochondrial calcium signalling and fusion dynamics. CLPB loss results in altered mtCU composition, interfering with mitochondrial calcium uptake independently of cytosolic calcium and mitochondrial membrane potential. Additionally, OPA1 decreases, and aggregation occurs, accompanied by mitochondrial fragmentation. Disease-associated mutations in the CLPB gene present in skin fibroblasts from patients also display mitochondrial calcium and structural changes. Thus, mtCU and fusion activity are dependent on CLPB, and their impairments might contribute to the disease caused by CLPB variants

Parvalbumin affects skeletal muscle trophism through modulation of mitochondrial calcium uptake

none9Parvalbumin (PV) is a cytosolic Ca2+-binding protein highly expressed in fast skeletal muscle, contributing to an increased relaxation rate. Moreover, PV is an “atrogene” downregulated in most muscle atrophy conditions. Here, we exploit mice lacking PV to explore the link between the two PV functions. Surprisingly, PV ablation partially counteracts muscle loss after denervation. Furthermore, acute PV downregulation is accompanied by hypertrophy and upregulation by atrophy. PV ablation has a minor impact on sarcoplasmic reticulum but is associated with increased mitochondrial Ca2+ uptake, mitochondrial size and number, and contacts with Ca2+ release sites. Mitochondrial calcium uniporter (MCU) silencing abolishes the hypertrophic effect of PV ablation, suggesting that mitochondrial Ca2+ uptake is required for hypertrophy. In turn, an increase of mitochondrial Ca2+ is required to enhance expression of the pro-hypertrophy gene PGC-1α4, whose silencing blocks hypertrophy due to PV ablation. These results reveal how PV links cytosolic Ca2+ control to mitochondrial adaptations, leading to muscle mass regulation.noneButera G.; Vecellio Reane D.; Canato M.; Pietrangelo L.; Boncompagni S.; Protasi F.; Rizzuto R.; Reggiani C.; Raffaello A.Butera, G.; Vecellio Reane, D.; Canato, M.; Pietrangelo, L.; Boncompagni, S.; Protasi, F.; Rizzuto, R.; Reggiani, C.; Raffaello, A

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions

Loss of mitochondrial calcium uniporter rewires skeletal muscle metabolism and substrate preference

Skeletal muscle mitochondria readily accumulate Ca2+ in response to SR store-releasing stimuli thanks to the activity of the mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ uptake. MCU positively regulates myofiber size in physiological conditions and counteracts pathological loss of muscle mass. Here we show that skeletal muscle-specific MCU deletion inhibits myofiber mitochondrial Ca2+ uptake, impairs muscle force and exercise performance, and determines a slow to fast switch in MHC expression. Mitochondrial Ca2+ uptake is required for effective glucose oxidation, as demonstrated by the fact that in muscle-specific MCU-/ (-)myofibers oxidative metabolism is impaired and glycolysis rate is increased. Although defective, mitochondrial activity is partially sustained by increased fatty acid (FA) oxidation. In MCU-/- myofibers, PDP2 overexpression drastically reduces FA dependency, demonstrating that decreased PDH activity is the main trigger of the metabolic rewiring of MCU-/- muscles. Accordingly, PDK4 overexpression in MCUfl/fl myofibers is sufficient to increase FA-dependent respiration. Finally, as a result of the muscle-specific MCU deletion, a systemic catabolic response impinging on both liver and adipose tissue metabolism occurs