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Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7β1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability.

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.

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic- pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D., Introduction Obesity-induced insulin resistance is associated with an increase in circulating levels of cytokines and free fatty acids (1) that activate proinflammatory signaling pathways. Mitogen-activated protein kinase (MAPK) signaling plays [...]

ObjectiveAcute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established.DesignWe investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis.ResultsMPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished.ConclusionsThis work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

RationaleMitochondria 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.ObjectiveTo directly characterize tPTP occurrence during sarcoplasmic reticulum Ca(2+) release in adult cardiac myocytes.Methods and resultsHere, 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

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.

Latent transforming growth factor-β (TGFβ) binding proteins (LTBPs) bind to inactive TGFβ in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGFβ release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGFβ signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGFβ release and activity and decrease inflammation and muscle damage in muscular dystrophy.

RationaleThe Na+ / K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+ / Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance.ObjectiveTo determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart.Methods and resultsCardiac-specific transgenic mice overexpressing either NKA-α1 or NKA-α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-α1 had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-α2 overexpression but not NKA-α1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-α2 showed greater affinity for Na+ compared with NKA-α1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-α2 but not NKA-α1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal.ConclusionsOur results suggest that the protective effect produced by increased expression of NKA-α2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-α1.

RationaleThe Na+ / K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+ / Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance.ObjectiveTo determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart.Methods and resultsCardiac-specific transgenic mice overexpressing either NKA-α1 or NKA-α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-α1 had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-α2 overexpression but not NKA-α1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-α2 showed greater affinity for Na+ compared with NKA-α1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-α2 but not NKA-α1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal.ConclusionsOur results suggest that the protective effect produced by increased expression of NKA-α2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-α1.

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. [ABSTRACT FROM AUTHOR]

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic b- and c-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the nonsarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width. [ABSTRACT FROM AUTHOR]