Summary After injury or cytokine stimulation, fibroblasts transdifferentiate into myofibroblasts, contractile cells that secrete extracellular matrix for wound healing and tissue remodeling. Here, a genome-wide screen identified TRPC6, a Ca2+ channel necessary and sufficient for myofibroblast transformation. TRPC6 overexpression fully activated myofibroblast transformation, while fibroblasts lacking Trpc6 were refractory to transforming growth factor-β (TGFβ) and angiotensin II-induced transdifferentiation. Trpc6 gene-deleted mice showed impaired dermal and cardiac wound healing after injury. The pro-fibrotic ligands TGFβ and angiotensin II induced TRPC6 expression through p38 mitogen-activated protein kinase (MAPK) - serum response factor (SRF) signaling via the TRPC6 promoter. Once induced, TRPC6 activates the Ca2+-responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPC6-dependent transdifferentiation and dermal wound healing. These results demonstrate an obligate function for TRPC6 and calcineurin in promoting myofibroblast differentiation, suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGFβ, p38 MAPK and SRF.

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.DOI: http://dx.doi.org/10.7554/eLife.00772.001

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally1, somatic deletion in RMCs of the Wnt ligand transporter Wntless2,3 results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF)4-6. These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor that partners with Orai1 to elicit Ca2+ entry in response to endoplasmic reticulum (ER) Ca2+ store depletion. While store-operated Ca2+ entry (SOCE) is important for maintaining ER Ca2+ homeostasis in non-excitable cells, it is unclear what role it plays in the heart, although STIM1 is expressed in the heart and upregulated during disease. Here we analyzed transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to disease. As expected, STIM1 transgenic myocytes showed enhanced Ca2+ entry following store depletion and partial co-localization with the type 2 ryanodine receptor (RyR2) within the sarcoplasmic reticulum (SR), as well as enrichment around the sarcolemma. STIM1 transgenic mice exhibited sudden cardiac death as early as 6 weeks of age, while mice surviving past 12 weeks of age developed heart failure with hypertrophy, induction of the fetal gene program, histopathology and mitochondrial structural alterations, loss of ventricular functional performance and pulmonary edema. Younger, pre-symptomatic STIM1 transgenic mice exhibited enhanced pathology following pressure overload stimulation or neurohumoral agonist infusion, compared to controls. Mechanistically, cardiac myocytes isolated from STIM1 transgenic mice displayed spontaneous Ca2+ transients that were prevented by the SOCE blocker SKF-96365, increased L-type Ca2+ channel (LTCC) current, and enhanced Ca2+ spark frequency. Moreover, adult cardiac myocytes from STIM1 transgenic mice showed both increased diastolic Ca2+ and maximal transient amplitude but no increase in total SR Ca2+ load. Associated with this enhanced Ca2+ profile was an increase in cardiac nuclear factor of activated T-cells (NFAT) and Ca2+/calmodulin-dependent kinase II (CaMKII) activity. We conclude that STIM1 has an unexpected function in the heart where it alters communication between the sarcolemma and SR resulting in greater Ca2+ flux and a leaky SR compartment.

Rationale The 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. Objective To determine if 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 Results Cardiac-specific transgenic mice overexpressing either the NKA-α1 or α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that while 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 two transgenic models, as 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. Moreover, 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. Conclusions Our 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.

Muscular dystrophy (MD) refers to a clinically and genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. Although the primary defect underlying most forms of MD typically results from a loss of sarcolemmal integrity, the secondary molecular mechanisms leading to muscle degeneration and myofiber necrosis is debated. One hypothesis suggests that elevated or dysregulated cytosolic calcium is the common transducing event, resulting in myofiber necrosis in MD. Previous measurements of resting calcium levels in myofibers from dystrophic animal models or humans produced equivocal results. However, recent studies in genetically altered mouse models have largely solidified the calcium hypothesis of MD, such that models with artificially elevated calcium in skeletal muscle manifest fulminant dystrophic-like disease, whereas models with enhanced calcium clearance or inhibited calcium influx are resistant to myofiber death and MD. Here, we will review the field and the recent cadre of data from genetically altered mouse models, which we propose have collectively mostly proven the hypothesis that calcium is the primary effector of myofiber necrosis in MD. This new consensus on calcium should guide future selection of drugs to be evaluated in clinical trials as well as gene therapy-based approaches.

We present a structurally dynamic model for nucleotide-and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solventfilled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.double electron-electron resonance ͉ dipolar interaction ͉ actomyosin M yosin motors use the energy derived from ATP hydrolysis to generate force for a diverse repertoire of biological tasks (1). Class II myosins produce muscle contraction and are involved in cytokinesis and cell motility. The present study focuses on Dictyostelium discoideum (Dicty) myosin II, which offers optimal facility for expression and purification of mutants, and is functionally similar to muscle myosin II (2, 3).

f Unregulated Ca 2؉ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na ؉ -Ca 2؉ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in ␦-sarcoglycan (Sgcd ؊/؊ ), Dysf ؊/؊ , and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd ؊/؊ mice. Measured increases in baseline Na ؉ and Ca 2؉ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca 2؉ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca 2؉ levels. Indeed, Atp1a2 ؉/؊ (encoding Na ؉ -K ؉ ATPase ␣2) mice, which have reduced Na ؉ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na ؉ -K ؉ ATPase inhibitor digoxin. Treatment of Sgcd ؊/؊ mice with ranolazine, a broadly acting Na ؉ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology. M uscular dystrophy (MD) is characterized by myofiber degeneration that results in muscle loss, functional impairment, and eventually death. MD is generally caused by genetic mutations in genes encoding proteins that are either part of the membrane-stabilizing dystrophin-glycoprotein complex (DGC) or otherwise impact some aspect of sarcolemmal integrity and membrane channel activity (1). Such alterations cause enhanced Ca 2ϩ entry through microtears or Ca 2ϩ channels/exchangers (2). Downstream consequences of increased Ca 2ϩ entry include altered signaling, calpain activation leading to unregulated intracellular protein degradation, and induction of necrosis through opening of the mitochondrial permeability transition pore with mitochondrial rupture (3, 4). However, the hypothesis that Ca 2ϩ elevations directly induce myofiber necrosis and lead to MD is controversial (2). While some groups have indeed reported global or even subsarcolemmal increases in Ca 2ϩ in dystrophic myofibers (5-10), such measurements are often technically difficult, which may be the reason why other studies have not observed a significant increase (11-13). Recent studies in transgenic (TG) mice have supported the Ca 2ϩ hypothesis of disease. For example, overexpression of dominant-negative transient receptor potential canonical 6 (dnTRPC6) or dnTRPV2 was sufficient to abrogate the dystrophic phenotype in mice by inhibiting a type of storeoperated Ca 2ϩ entry that characterizes these channels (14, 15). TRPC3 overexpression in skeletal muscle, which dramatically enhanced Ca 2ϩ entry, was sufficient to induce MD in mice (14). Finally, overexpre...