Mitochondrial citrate carrier SLC25A1 is a dosage-dependent regulator of metabolic reprogramming and morphogenesis in the developing heart.

The developing mammalian heart undergoes an important metabolic shift from glycolysis toward mitochondrial oxidation, such that oxidative phosphorylation defects may present with cardiac abnormalities. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mice with systemic loss of the mitochondrial citrate carrier SLC25A1. Slc25a1 null embryos displayed impaired growth, cardiac malformations, and aberrant mitochondrial function. Importantly, Slc25a1 haploinsufficient embryos, which are overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 dose-dependent effects. Supporting clinical relevance, we found a near-significant association between ultrarare human pathogenic SLC25A1 variants and pediatric congenital heart disease. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of PPARγ to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.

Sex differences in the involvement of skeletal and cardiac muscles in myopathic Ano5-/- mice.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E). Although ANO5 myopathy is not X-chromosome linked, we performed a meta-analysis of the research literature and found that three-quarters of patients with LGMD-R12 are males. Females are less likely to present with moderate to severe skeletal muscle and/or cardiac pathology. Because these sex differences could be explained in several ways, we compared males and females in a mouse model of LGMD-R12. This model recapitulates the sex differences in human LGMD-R12. Only male Ano5-/- mice had elevated serum creatine kinase after exercise and exhibited defective membrane repair after laser injury. In contrast, by these measures, female Ano5-/- mice were indistinguishable from wild type. Despite these differences, both male and female Ano5-/- mice exhibited exercise intolerance. Although exercise intolerance of male mice can be explained by skeletal muscle dysfunction, echocardiography revealed that Ano5-/- female mice had features of cardiomyopathy that may be responsible for their exercise intolerance. These findings heighten concerns that mutations of ANO5 in humans may be linked to cardiac disease.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome.

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Heterogeneous Expression of Nuclear Encoded Mitochondrial Genes Distinguishes Inhibitory and Excitatory Neurons.

Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single-cell mRNA sequencing (scRNAseq) could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained from microdissected brain tissue segregated the hippocampus, striatum, and cortex from each other. Nuclear-encoded mitochondrial transcripts could only segregate cell types and brain regions when the analysis was performed at the single-cell level. In fact, single-cell mitochondrial transcriptomes were able to distinguish glutamatergic and distinct types of GABAergic neurons from one another. Within these cell categories, unique SLC25A transporters were able to identify distinct cell subpopulations. Our results demonstrate heterogeneous mitochondrial composition across brain regions and cell types. We postulate that mitochondrial heterogeneity influences regional and cell type-specific mechanisms in health and disease.

Mitochondrial functional resilience after TFAM ablation in the adult heart.

The nuclear genome-encoded mitochondrial DNA (mtDNA) transcription factor A (TFAM) is indispensable for mitochondrial energy production in the developing and postnatal heart; a similar role for TFAM is inferred in adult heart. Here, we provide evidence that challenges this long-standing paradigm. Unexpectedly, conditional Tfam ablation in vivo in adult mouse cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function, despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting maintenance of respiratory chain assembly/function. Long-term Tfam inactivation, however, downregulated the core mtDNA transcription and replication machinery, leading to mitochondrial dysfunction and cardiomyopathy. Collectively, in contrast to the developing heart, these data reveal a striking resilience of the differentiated adult heart to acute insults to mtDNA regulation.

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.

Mitochondrial dysfunction and oxidative stress in heart disease.

Beyond their role as a cellular powerhouse, mitochondria are emerging as integral players in molecular signaling and cell fate determination through reactive oxygen species (ROS). While ROS production has historically been portrayed as an unregulated process driving oxidative stress and disease pathology, contemporary studies reveal that ROS also facilitate normal physiology. Mitochondria are especially abundant in cardiac tissue; hence, mitochondrial dysregulation and ROS production are thought to contribute significantly to cardiac pathology. Moreover, there is growing appreciation that medical therapies designed to mediate mitochondrial ROS production can be important strategies to ameliorate cardiac disease. In this review, we highlight evidence from animal models that illustrates the strong connections between mitochondrial ROS and cardiac disease, discuss advancements in the development of mitochondria-targeted antioxidant therapies, and identify challenges faced in bringing such therapies into the clinic.

Systems Analysis of the 22q11.2 Microdeletion Syndrome Converges on a Mitochondrial Interactome Necessary for Synapse Function and Behavior.

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts from both sexes and mouse brains carrying a 22q11.2-like defect, Df(16)A+/- Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A1 or SLC25A4 orthologues, dSLC25A1-sea and dSLC25A4-sesB, affected synapse morphology, neurotransmission, plasticity, and sleep patterns. Our findings indicate that synapses are sensitive to partial loss of function of mitochondrial solute transporters. We propose that mitoproteomes regulate synapse development and function in normal and pathological conditions in a cell-specific manner.SIGNIFICANCE STATEMENT We address the central question of how to comprehensively define molecular mechanisms of the most prevalent and penetrant microdeletion associated with neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. This complex mutation reduces gene dosage of ∼63 genes in humans. We describe a disruption of the mitoproteome in 22q11.2 patients and brains of a 22q11.2 mouse model. In particular, we identify a network of inner mitochondrial membrane transporters as a hub required for synapse function. Our findings suggest that mitochondrial composition and function modulate the risk of neurodevelopmental disorders, such as schizophrenia.

Cardiac-specific deficiency of the mitochondrial calcium uniporter augments fatty acid oxidation and functional reserve.

The mitochondrial calcium uniporter (MCU) relays cytosolic Ca2+ transients to the mitochondria. We examined whether energy metabolism was compromised in hearts from mice with a cardiac-specific deficiency of MCU subjected to an isoproterenol (ISO) challenge. Surprisingly, isolated working hearts from cardiac MCU-deficient mice showed higher cardiac work, both in the presence or absence of ISO. These hearts were not energy-starved, with ISO inducing a similar increase in glucose oxidation rates compared to control hearts, but a greater increase in fatty acid oxidation rates. This correlated with lower levels of the fatty acid oxidation inhibitor malonyl CoA, and to an increased stimulatory acetylation of its degrading enzyme malonyl CoA decarboxylase and of the fatty acid ß-oxidation enzyme ß-hydroxyacyl CoA dehydrogenase. We conclude that impaired mitochondrial Ca2+ uptake does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation that provides a higher energy reserve during acute adrenergic stress.

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.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

The mitochondrial calcium uniporter in the heart: energetics and beyond.

Ca2+ and mitochondria are inextricably linked to cardiac function and dysfunction. Ca2+ is central to cardiac excitation-contraction coupling and stimulates mitochondrial energy production to fuel contraction. Under pathological conditions of dysregulated Ca2+ cycling, mitochondrial Ca2+ overload activates cellular death pathways. Thus, in the cardiomyocyte, the mitochondrial Ca2+ microdomain is where contraction, energy and death collide. A key component of mitochondrial Ca2+ signalling is the mitochondrial Ca2+ uniporter complex (uniplex), an inner membrane Ca2+ transporter and major pathway of mitochondrial Ca2+ entry. Once known only as the unidentified target for ruthenium red and related compounds, in recent years, the uniplex has evolved into a complex multiprotein assembly. The identification of the molecular constituents of the uniplex has made possible the generation of targeted genetic models to interrogate uniplex function in vivo. This review will summarize our current understanding of the molecular structure of the uniplex, its impact on mitochondrial energetics and cardiac physiology, its contribution to cardiomyocyte death, and its expanding roles in cardiac biology.

Thrombospondin expression in myofibers stabilizes muscle membranes.

Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of ßPS integrin. This functional conservation emphasizes the fundamental importance of Thbs' as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.

Inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum-mitochondrial crosstalk influences adenosine triphosphate production via mitochondrial Ca2+ uptake through the mitochondrial ryanodine receptor in cardiac myocytes.

AIMS: Elevated levels of inositol 1,4,5-trisphosphate (IP3) in adult cardiac myocytes are typically associated with the development of cardiac hypertrophy, arrhythmias, and heart failure. IP3 enhances intracellular Ca(2+ )release via IP3 receptors (IP3Rs) located at the sarcoplasmic reticulum (SR). We aimed to determine whether IP3-induced Ca(2+ )release affects mitochondrial function and determine the underlying mechanisms. METHODS AND RESULTS: We compared the effects of IP3Rs- and ryanodine receptors (RyRs)-mediated cytosolic Ca(2+ )elevation achieved by endothelin-1 (ET-1) and isoproterenol (ISO) stimulation, respectively, on mitochondrial Ca(2+ )uptake and adenosine triphosphate (ATP) generation. Both ET-1 and isoproterenol induced an increase in mitochondrial Ca(2+ )(Ca(2 +) m) but only ET-1 led to an increase in ATP concentration. ET-1-induced effects were prevented by cell treatment with the IP3 antagonist 2-aminoethoxydiphenyl borate and absent in myocytes from transgenic mice expressing an IP3 chelating protein (IP3 sponge). Furthermore, ET-1-induced mitochondrial Ca(2+) uptake was insensitive to the mitochondrial Ca(2+ )uniporter inhibitor Ru360, however was attenuated by RyRs type 1 inhibitor dantrolene. Using real-time polymerase chain reaction, we detected the presence of all three isoforms of IP3Rs and RyRs in murine ventricular myocytes with a dominant presence of type 2 isoform for both receptors. CONCLUSIONS: Stimulation of IP3Rs with ET-1 induces Ca(2+ )release from the SR which is tunnelled to mitochondria via mitochondrial RyR leading to stimulation of mitochondrial ATP production.

Individual Cardiac Mitochondria Undergo Rare Transient Permeability Transition Pore Openings.

RATIONALE: Mitochondria produce ATP, especially critical for survival of highly aerobic cells, such as cardiac myocytes. Conversely, opening of mitochondrial high-conductance and long-lasting permeability transition pores (mPTP) causes respiratory uncoupling, mitochondrial injury, and cell death. However, low conductance and transient mPTP openings (tPTP) might limit mitochondrial Ca(2+) load and be cardioprotective, but direct evidence for tPTP in cells is limited. OBJECTIVE: To directly characterize tPTP occurrence during sarcoplasmic reticulum Ca(2+) release in adult cardiac myocytes. METHODS AND RESULTS: Here, we measured tPTP directly as transient drops in mitochondrial [Ca(2+)] ([Ca(2+)]mito) and membrane potential (ΔΨm) in adult cardiac myocytes during cyclic sarcoplasmic reticulum Ca release, by simultaneous live imaging of 500 to 1000 individual mitochondria. The frequency of tPTPs rose at higher [Ca(2+)]mito, [Ca(2+)]i, with 1 µmol/L peroxide exposure and in myocyte from failing hearts. The tPTPs were suppressed by preventing mitochondrial Ca(2+) influx, by mPTP inhibitor cyclosporine A, sanglifehrin, and in cyclophilin D knockout mice. These tPTP events were 57±5 s in duration, but were rare (occurring in <0.1% of myocyte mitochondria at any moment) such that the overall energetic cost to the cell is minimal. The tPTP pore size is much smaller than for permanent mPTP, as neither Rhod-2 nor calcein (600 Da) were lost. Thus, proteins and even molecules the size of NADH (663 Da) will be retained during these tPTP. CONCLUSIONS: We conclude that tPTP openings (MitoWinks) may be molecularly related to pathological mPTP, but are likely to be normal physiological manifestation that benefits mitochondrial (and cell) survival by allowing individual mitochondria to reset themselves with little overall energetic cost.

MBNL1-mediated regulation of differentiation RNAs promotes myofibroblast transformation and the fibrotic response.

The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of Mbnl1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aß. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3'UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1.

The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart.

In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.

Physiological and pathological roles of the mitochondrial permeability transition pore in the heart.

Prolonged mitochondrial permeability transition pore (MPTP) opening results in mitochondrial energetic dysfunction, organelle swelling, rupture, and typically a type of necrotic cell death. However, acute opening of the MPTP has a critical physiologic role in regulating mitochondrial Ca(2+) handling and metabolism. Despite the physiological and pathological roles that the MPTP orchestrates, the proteins that comprise the pore itself remain an area of ongoing investigation. Here, we will discuss the molecular composition of the MPTP and its role in regulating cardiac physiology and disease. A better understanding of MPTP structure and function will likely suggest novel cardioprotective therapeutic approaches.

P38α MAPK underlies muscular dystrophy and myofiber death through a Bax-dependent mechanism.

Muscular dystrophies are a group of genetic diseases that lead to muscle wasting and, in most cases, premature death. Cytokines and inflammatory factors are released during the disease process where they promote deleterious signaling events that directly participate in myofiber death. Here, we show that p38α, a kinase in the greater mitogen-activated protein kinase (MAPK)-signaling network, serves as a nodal regulator of disease signaling in dystrophic muscle. Deletion of Mapk14 (p38α-encoding gene) in the skeletal muscle of mdx- (lacking dystrophin) or sgcd- (δ-sarcoglycan-encoding gene) null mice resulted in a significant reduction in pathology up to 6 months of age. We also generated MAPK kinase 6 (MKK6) muscle-specific transgenic mice to model heightened p38α disease signaling that occurs in dystrophic muscle, which resulted in severe myofiber necrosis and many hallmarks of muscular dystrophy. Mechanistically, we show that p38α directly induces myofiber death through a mitochondrial-dependent pathway involving direct phosphorylation and activation of the pro-death Bcl-2 family member Bax. Indeed, muscle-specific deletion of Bax, but not the apoptosis regulatory gene Tp53 (encoding p53), significantly reduced dystrophic pathology in the muscles of MKK6 transgenic mice. Moreover, use of a p38 MAPK pharmacologic inhibitor reduced dystrophic disease in Sgcd(-/-) mice suggesting a future therapeutic approach to delay disease.