MCU genetically altered mice suggest how mitochondrial Ca2+ regulates metabolism.

Skeletal muscle has a major impact on total body metabolism and obesity, and is characterized by dynamic regulation of substrate utilization. While it is accepted that acute increases in mitochondrial matrix Ca2+ increase carbohydrate usage to augment ATP production, recent studies in mice with deleted genes for components of the mitochondrial Ca2+ uniporter (MCU) complex have suggested a more complicated regulatory scenario. Indeed, mice with a deleted Mcu gene in muscle, which lack acute mitochondrial Ca2+ uptake, have greater fatty acid oxidation (FAO) and less adiposity. By contrast, mice deleted for the inhibitory Mcub gene in skeletal muscle, which have greater acute mitochondrial Ca2+ uptake, antithetically display reduced FAO and progressive obesity. In this review we discuss the emerging concept that dynamic fluxing of mitochondrial matrix Ca2+ regulates metabolism.

Thbs1 regulates skeletal muscle mass in a TGFß-Smad2/3-ATF4-dependent manner.

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy.

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle.

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.

Col1a2-Deleted Mice Have Defective Type I Collagen and Secondary Reactive Cardiac Fibrosis with Altered Hypertrophic Dynamics.

RATIONALE: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. OBJECTIVE: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. METHODS AND RESULTS: We generated and characterized Col1a2-/- mice using standard gene targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFß activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2-/- mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. CONCLUSIONS: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

Rpl3l gene deletion in mice reduces heart weight over time.

Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight.

Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy.

Skeletal muscle can repair and regenerate due to resident stem cells known as satellite cells. The muscular dystrophies are progressive muscle wasting diseases underscored by chronic muscle damage that is continually repaired by satellite cell-driven regeneration. Here we generate a genetic strategy to mediate satellite cell ablation in dystrophic mouse models to investigate how satellite cells impact disease trajectory. Unexpectedly, we observe that depletion of satellite cells reduces dystrophic disease features, with improved histopathology, enhanced sarcolemmal stability and augmented muscle performance. Mechanistically, we demonstrate that satellite cells initiate expression of the myogenic transcription factor MyoD, which then induces re-expression of fetal genes in the myofibers that destabilize the sarcolemma. Indeed, MyoD re-expression in wildtype adult skeletal muscle reduces membrane stability and promotes histopathology, while MyoD inhibition in a mouse model of muscular dystrophy improved membrane stability. Taken together these observations suggest that satellite cell activation and the fetal gene program is maladaptive in chronic dystrophic skeletal muscle.

MCUb Induction Protects the Heart From Postischemic Remodeling.

RATIONALE: Mitochondrial Ca2+ loading augments oxidative metabolism to match functional demands during times of increased work or injury. However, mitochondrial Ca2+ overload also directly causes mitochondrial rupture and cardiomyocyte death during ischemia-reperfusion injury by inducing mitochondrial permeability transition pore opening. The MCU (mitochondrial Ca2+ uniporter) mediates mitochondrial Ca2+ influx, and its activity is modulated by partner proteins in its molecular complex, including the MCUb subunit. OBJECTIVE: Here, we sought to examine the function of the MCUb subunit of the MCU-complex in regulating mitochondria Ca2+ influx dynamics, acute cardiac injury, and long-term adaptation after ischemic injury. METHODS AND RESULTS: Cardiomyocyte-specific MCUb overexpressing transgenic mice and Mcub gene-deleted (Mcub-/-) mice were generated to dissect the molecular function of this protein in the heart. We observed that MCUb protein is undetectable in the adult mouse heart at baseline, but mRNA and protein are induced after ischemia-reperfusion injury. MCUb overexpressing mice demonstrated inhibited mitochondrial Ca2+ uptake in cardiomyocytes and partial protection from ischemia-reperfusion injury by reducing mitochondrial permeability transition pore opening. Antithetically, deletion of the Mcub gene exacerbated pathological cardiac remodeling and infarct expansion after ischemic injury in association with greater mitochondrial Ca2+ uptake. Furthermore, hindlimb remote ischemic preconditioning induced MCUb expression in the heart, which was associated with decreased mitochondrial Ca2+ uptake, collectively suggesting that induction of MCUb protein in the heart is protective. Similarly, mouse embryonic fibroblasts from Mcub-/- mice were more sensitive to Ca2+ overload. CONCLUSIONS: Our studies suggest that Mcub is a protective cardiac inducible gene that reduces mitochondrial Ca2+ influx and permeability transition pore opening after ischemic injury to reduce ongoing pathological remodeling.

The mitochondrial calcium uniporter underlies metabolic fuel preference in skeletal muscle.

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.

Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure.

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

MiRNA-Mediated Macrophage Polarization and its Potential Role in the Regulation of Inflammatory Response.

Monocytes and macrophages are important components of the immune system, specialized in either removing pathogens as part of innate immunity or contributing to adaptive immunity through antigen presentation. Essential to such functions is classical activation (M1) and alternative activation (M2) of macrophages. M1 polarization of macrophages is characterized by production of pro-inflammatory cytokines, antimicrobial and tumoricidal activity, whereas M2 polarization of macrophages is linked to immunosuppression, tumorigenesis, wound repair, and elimination of parasites. MiRNAs are small non-coding RNAs with the ability to regulate gene expression and network of cellular processes. A number of studies have determined miRNA expression profiles in M1 and M2 polarized human and murine macrophages using microarray and RT-qPCR arrays techniques. More specifically, miR-9, miR-127, miR-155, and miR-125b have been shown to promote M1 polarization while miR-124, miR-223, miR-34a, let-7c, miR-132, miR-146a, and miR-125a-5p induce M2 polarization in macrophages by targeting various transcription factors and adaptor proteins. Further, M1 and M2 phenotypes play distinctive roles in cell growth and progression of inflammation-related diseases such as sepsis, obesity, cancer, and multiple sclerosis. Hence, miRNAs that modulate macrophage polarization may have therapeutic potential in the treatment of inflammation-related diseases. This review highlights recent findings in miRNA expression profiles in polarized macrophages from murine and human sources, and summarizes how these miRNAs regulate macrophage polarization. Last, therapeutic potential of miRNAs in inflammation-related diseases through modulation of macrophage polarization is also discussed.