Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

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.

Interleukin-33 Mediates Cardiomyopathy After Acute Kidney Injury by Signaling to Cardiomyocytes.

BACKGROUND: Acute kidney injury (AKI) is a short-term life-threatening condition that, if survived, can lead to renal insufficiency and development of chronic kidney disease. The pathogenesis of AKI and chronic kidney disease involves direct effects on the heart and the development of hypertrophy and cardiomyopathy. METHODS: We used mouse models of ischemia/reperfusion AKI and unilateral ureteral obstruction to investigate the role of IL-33 (interleukin-33) and its receptor-encoding gene Il1rl1 (also called ST2L [suppression of tumorigenicity 2]) in cardiac remodeling after AKI. Mice with cell type-specific genetic disruption of the IL-33/ST2L axis were used, and IL-33 monoclonal antibody, adeno-associated virus encoding IL-33 or ST2L, and recombinant IL-33, as well. RESULTS: Mice deficient in Il33 were refractory to cardiomyopathy associated with 2 models of kidney injury. Treatment of mice with monoclonal IL-33 antibody also protected the heart after AKI. Moreover, overexpression of IL-33 or injection of recombinant IL-33 induced cardiac hypertrophy or cardiomyopathy, but not in mice lacking Il1rl1. AKI-induced cardiomyopathy was also reduced in mice with cardiac myocyte-specific deletion of Il1rl1 but not in endothelial cell- or fibroblast-specific deletion of Il1rl1. Last, overexpression of the ST2L receptor in cardiac myocytes recapitulated induction of cardiac hypertrophy. CONCLUSIONS: These results indicate that IL-33 released from the kidney during AKI underlies cardiorenal syndrome by directly signaling to cardiac myocytes, suggesting that antagonism of IL-33/ST2 axis would be cardioprotective in patients with kidney disease.

Skeletal muscle fibers count on nuclear numbers for growth.

Skeletal muscle cells are noteworthy for their syncytial nature, with each myofiber accumulating hundreds or thousands of nuclei derived from resident muscle stem cells (MuSCs). These nuclei are accrued through cell fusion, which is controlled by the two essential fusogens Myomaker and Myomerger that are transiently expressed within the myogenic lineage. While the absolute requirement of fusion for muscle development has been known for decades, the underlying need for the magnitude of multinucleation in muscle remains mysterious. Possible advantages of multinucleation include the potential it affords for transcriptional diversity within these massive cells, and as a means of increasing DNA content to support optimal cell size and function. In this article, we review recent advances that elucidate the relationship between myonuclear numbers and establishment of myofiber size, and discuss how this new information refines our understanding of the concept of myonuclear domains (MND), the cytoplasmic volumes that each resident myonucleus can support. Finally, we explore the potential consequences and costs of multinucleation and its impacts on myonuclear transcriptional reserve capacity, growth potential, myofiber size regulation, and muscle adaptability. We anticipate this report will not only serve to highlight the latest advances in the basic biology of syncytial muscle cells but also provide information to help design the next generation of therapeutic strategies to maintain muscle mass and function.

Nuclear numbers in syncytial muscle fibers promote size but limit the development of larger myonuclear domains.

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

Heparin-induced thrombocytopenia in patients readmitted after open cardiac surgical procedures: A case series.

Objectives: Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication that occurs in a small percentage of patients exposed to heparin. Concerns of HIT are particularly high in patients undergoing cardiac procedures requiring cardiopulmonary bypass, as they are exposed to high doses of heparin intraoperatively. Our aim was to identify and assess the hospital courses of patients who were diagnosed with HIT during readmission following cardiac surgery. Methods: A retrospective review of patients who underwent open cardiac surgical procedures from June 2017 through October 2019 was performed. Of these, we identified patients who were newly diagnosed with HIT upon readmission. HIT positivity was defined as a positive anti-PF4 antibody screening test, plus a positive serotonin release assay. Results: Of the 2496 patients identified, 13 patients were HIT positive on index admission and were excluded. Of the remaining 2483 patients, 351 were readmitted within 30 days. Six were newly diagnosed with HIT during readmission, 5 of whom presented with thrombotic complications. One patient was readmitted with thrombocytopenia and was started on argatroban; the remaining 5 did not have a significantly lower platelet count on readmission. Of the 12 patients readmitted for venous thromboembolism, 4 tested positive for HIT. Conclusions: HIT can have a delayed appearance following open heart surgery. Venous thromboembolism appears to be a significant indicator for HIT during readmission, even in the absence of thrombocytopenia. This may support the use of non-heparin anticoagulation for cardiac surgery patients readmitted with thromboembolism until HIT status is determined.

Myomaker and Myomerger Work Independently to Control Distinct Steps of Membrane Remodeling during Myoblast Fusion.

Classic mechanisms for membrane fusion involve transmembrane proteins that assemble into complexes and dynamically alter their conformation to bend membranes, leading to mixing of membrane lipids (hemifusion) and fusion pore formation. Myomaker and Myomerger govern myoblast fusion and muscle formation but are structurally divergent from traditional fusogenic proteins. Here, we show that Myomaker and Myomerger independently mediate distinct steps in the fusion pathway, where Myomaker is involved in membrane hemifusion and Myomerger is necessary for fusion pore formation. Mechanistically, we demonstrate that Myomerger is required on the cell surface where its ectodomains stress membranes. Moreover, we show that Myomerger drives fusion completion in a heterologous system independent of Myomaker and that a Myomaker-Myomerger physical interaction is not required for function. Collectively, our data identify a stepwise cell fusion mechanism in myoblasts where different proteins are delegated to perform unique membrane functions essential for membrane coalescence.

Developmental vascular regression is regulated by a Wnt/ß-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death.

Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/ß-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/ß-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/ß-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.

Gata4-Dependent Differentiation of c-Kit+-Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

BACKGROUND: Although c-Kit+ adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation, we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells. METHODS: Kit allele-dependent lineage tracing and fusion analysis were performed in mice following simultaneous Gata4 and Gata6 cell type-specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit+ cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. RESULTS: Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart, and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion because of defective endothelial cell differentiation in the absence of Gata4. CONCLUSIONS: Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit+ cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit+ endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development.

Despite the importance of cell fusion for mammalian development and physiology, the factors critical for this process remain to be fully defined, which has severely limited our ability to reconstitute cell fusion. Myomaker (Tmem8c) is a muscle-specific protein required for myoblast fusion. Expression of myomaker in fibroblasts drives their fusion with myoblasts, but not with other myomaker-expressing fibroblasts, highlighting the requirement of additional myoblast-derived factors for fusion. Here we show that Gm7325, which we name myomerger, induces the fusion of myomaker-expressing fibroblasts. Thus, myomaker and myomerger together confer fusogenic activity to otherwise non-fusogenic cells. Myomerger is skeletal muscle-specific and genetic deletion in mice results in a paucity of muscle fibres demonstrating its requirement for normal muscle formation. Myomerger deficient myocytes differentiate and harbour organized sarcomeres but are fusion-incompetent. Our findings identify myomerger as a fundamental myoblast fusion protein and establish a system that begins to reconstitute mammalian cell fusion.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and ß-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

Cutaneous adnexal carcinoma with apocrine differentiation.

Primary cutaneous apocrine carcinomas are uncommon malignant neoplasms that can be difficult, if not impossible, to distinguish histologically from metastatic breast carcinomas. We present the case of a 71-year-old man with a 5-year history of extensive ulcerated plaques on the posterior neck and posterior scalp. Biopsy revealed a poorly differentiated infiltrating adenocarcinoma consistent with either primary cutaneous apocrine carcinoma or occult metastatic breast carcinoma. Immunohistochemical analysis demonstrated positive staining for cytokeratin (CK) 7, estrogen receptor, and progesterone receptor, and negative staining for p63, podoplanin, CK20, and thyroid transcription factor 1. Extensive radiologic imaging studies showed no evidence of occult breast or other internal malignancies. Based on the indolent clinical course, lack of evidence for an internal primary site, and immunohistochemical staining, the lesion was determined to be consistent with a cutaneous neoplasm with features of apocrine differentiation. This case highlights the distinction between apocrine carcinoma and other primary adnexal carcinomas for which p63 and D2-40 have been reported to be sensitive and specific markers but are negative in apocrine carcinomas.

SERCA2 Haploinsufficiency in a Mouse Model of Darier Disease Causes a Selective Predisposition to Heart Failure.

Null mutations in one copy of ATP2A2, the gene encoding sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease in humans, a skin condition involving keratinocytes. Cardiac function appears to be unimpaired in Darier disease patients, with no evidence that SERCA2 haploinsufficiency itself causes heart disease. However, SERCA2 deficiency is widely considered a contributing factor in heart failure. We therefore analyzed Atp2a2 heterozygous mice to determine whether SERCA2 haploinsufficiency can exacerbate specific heart disease conditions. Despite reduced SERCA2a levels in heart, Atp2a2 heterozygous mice resembled humans in exhibiting normal cardiac physiology. When subjected to hypothyroidism or crossed with a transgenic model of reduced myofibrillar Ca(2+)-sensitivity, SERCA2 deficiency caused no enhancement of the disease state. However, when combined with a transgenic model of increased myofibrillar Ca(2+)-sensitivity, SERCA2 haploinsufficiency caused rapid onset of hypertrophy, decompensation, and death. These effects were associated with reduced expression of the antiapoptotic Hax1, increased levels of the proapoptotic genes Chop and Casp12, and evidence of perturbations in energy metabolism. These data reveal myofibrillar Ca(2+)-sensitivity to be an important determinant of the cardiac effects of SERCA2 haploinsufficiency and raise the possibility that Darier disease patients are more susceptible to heart failure under certain conditions.

Ablation of plasma membrane Ca(2+)-ATPase isoform 4 prevents development of hypertrophy in a model of hypertrophic cardiomyopathy.

The mechanisms linking the expression of sarcomeric mutant proteins to the development of pathological hypertrophy in hypertrophic cardiomyopathy (HCM) remain poorly understood. We investigated the role of the plasma membrane Ca(2+)-ATPase PMCA4 in the HCM phenotype using a transgenic model that expresses mutant (Glu180Gly) α-tropomyosin (Tm180) in heart. Immunoblot analysis revealed that cardiac PMCA4 expression was upregulated early in Tm180 disease pathogenesis. This was accompanied by an increase in levels of the L-type Ca(2+)-channel, which is implicated in pathological hypertrophy. When Tm180 mice were crossed with a PMCA4-null line, loss of PMCA4 caused the abrogation of hypertrophy in Tm180/PMCA4-null double mutant mice. RT-PCR analysis of Tm180/PMCA4-null hearts revealed blunting of the fetal program and reversion of pro-fibrotic Col1a1 and Col3a1 gene expression to wild-type levels. This was accompanied by evidence of reduced L-type Ca(2+)-channel expression, and diminished calcineurin activity. Expression of the metabolic substrate transporters glucose transporter 4 and carnitine palmitoyltransferase 1b was preserved and Tm180-related changes in mRNA levels of various contractile stress-related proteins including the cardiac ankyrin protein CARP and the N2B isoform of titin were reversed in Tm180/PMCA4-null hearts. cGMP levels were increased and phosphorylation of vasodilator-stimulated phosphoprotein was elevated in Tm180/PMCA4-null hearts. These changes were associated with a sharp reduction in left ventricular end-diastolic pressure in Tm180/PMCA4-null hearts, which occurred despite persistence of Tm180-related impairment of relaxation dynamics. These results reveal a novel and specific role for PMCA4 in the Tm180 hypertrophic phenotype, with the "protective" effects of PMCA4 deficiency encompassing multiple determinants of HCM-related hypertrophy.

Incidence of contrast material-induced nephropathy after neuroendovascular procedures.

PURPOSE: To report the incidence and risk factors for contrast material ( CM contrast material )-induced nephropathy ( CIN CM-induced nephropathy ) in patients with no history of chronic kidney disease and estimated glomerular filtration rate that exceeded 30 mL/min/1.73 m(2) after a relatively high dose of CM contrast material (≥250 mL) during neuroendovascular procedures. MATERIALS AND METHODS: An institutional review board-approved retrospective chart review was performed for all patients who received a dose of CM contrast material 250 mL or greater while they underwent a neuroendovascular procedure between January 2011 and February 2013. The control group consisted of comparable patients who received a CM contrast material dose of 75-249 mL during the same period. Patients with pre-existing estimated glomerular filtration rate of 30 mL/min/1.73 m(2) or less or documented history of chronic kidney disease were excluded. CIN CM-induced nephropathy was defined as an increase in serum creatinine 50% above the baseline or an absolute increase of 0.3 mg/dL at either 24 or 48 hours after the procedure. Statistical analysis was performed with the Student t test, χ(2) analysis, and mixed-model analysis of variance. RESULTS: Clinical characteristics between the control and high-dose group were similar for age (95% confidence interval [CI]: -3.69, 5.48; P = .70), sex (95% CI: 0.28, 0.43; P = .62), and ethnicity (95% CI: 0.42, 0.58; P = .47). The average volume of CM contrast material administered was 172 mL in the control group and 326 mL in the high-dose cohort (95% CI: 131.78, 175.05; P < .001). Of the 79 cases in the high-dose cohort, 36 (46%) received a CM contrast material dose between 250 and 299 mL, 29 (37%) received 300-399 mL, nine (11%) received 400-499 mL, and five (6%) received greater than 500 mL. By 48 hours, a statistically significant decrease in serum creatinine was seen in two of the four high-dose CM contrast material dose categories: 250-299 mL (decrease of 24%; [95% CI: 0.04, 0.36]; P = .003) and greater than 500 mL (decrease of 14% [95% CI: -0.33, 0.57]; P = .007). There were four cases (5%) of CIN CM-induced nephropathy : three (4%) at 24 hours and one (1%) at 48 hours. The comorbid rate of diabetes (25% vs 15% [95% CI: -0.01, 0.04]; P < .001) was found to be higher among those who developed CIN CM-induced nephropathy compared with those who did not within the high-dose cohort. No cases of CIN CM-induced nephropathy occurred in the control group. CONCLUSION: Risk of developing CIN CM-induced nephropathy is relatively low in patients who undergo neuroendovascular procedures with CM contrast material doses of 250 mL or greater.

Novel technique for preoperative pedicle localization in spinal surgery with challenging anatomy.

Accurately localizing a spine level in the thoracic spine is often not easily achieved with the existing imaging modalities available in the operating room. The coordination of the preoperative imaging pathology with intraoperative imaging is even more difficult in patients with challenging anatomy. Using standard percutaneous techniques, the authors placed a radiopaque embolization coil into the pedicle of interest under biplanar fluoroscopy in 1 patient. Thoracic spine MRI along with scout MRI was then performed to confirm coil marker placement in relation to the actual spine pathology prior to surgical intervention. No complications were observed during placement of the radiopaque marker. Intraoperatively, the marker was immediately and easily visualized, leading to a confident identification of the correct thoracic spinal level. The preoperative placement of a radiopaque marker into the vertebral pedicle of the identified pathological level combined with postplacement MRI verification provides an advantage over previously proposed techniques in the literature.

Pipeline endovascular reconstruction of traumatic dissecting aneurysms of the intracranial internal carotid artery.

A 22-year-old woman was involved in a motor vehicle collision resulting in multiple facial fractures and extensive internal carotid artery (ICA) injury including a right carotid-cavernous fistula, complex dissection flap and dissecting aneurysms. Endovascular coil embolization was initially performed to treat the cavernous carotid fistula and then again on two separate occasions to treat expanding dissecting aneurysms. Parent vessel reconstruction of the right ICA was subsequently performed with the Pipeline embolization device, resulting in complete anatomical restoration of this vessel.

In vivo epithelial wound repair requires mobilization of endogenous intracellular and extracellular calcium.

We report that a localized intracellular and extracellular Ca(2+) mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca(2+)-sensitive protein (yellow cameleon 3.0) report that intracellular Ca(2+) selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca(2+) increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca(2+) increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca(2+) mobilization. Indomethacin and verapamil also inhibit the luminal Ca(2+) increase. Intracellular Ca(2+) chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca(2+) increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N'-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca(2+) and unevenly inhibits late-phase intracellular Ca(2+) mobilization. Both modes of Ca(2+) chelation slow gastric repair. In plasma membrane Ca-ATPase 1(+/-) mice, but not plasma membrane Ca-ATPase 4(-/-) mice, there is slowed epithelial repair and a diminished gastric surface Ca(2+) increase. We conclude that endogenous Ca(2+), mobilized by signaling pathways and transmembrane Ca(2+) transport, causes increased Ca(2+) levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.

Tmem64 modulates calcium signaling during RANKL-mediated osteoclast differentiation.

Osteoclast maturation and function primarily depend on receptor activator of NF-κB ligand (RANKL)-mediated induction of nuclear factor of activated T cells c1 (NFATc1), which is further activated via increased intracellular calcium ([Ca(2+)](i)) oscillation. However, the coordination mechanism that mediates Ca(2+) oscillation during osteoclastogenesis remains ill defined. Here, we identified transmembrane protein 64 (Tmem64) as a regulator of Ca(2+) oscillation during osteoclastogenesis. We found that Tmem64-deficient mice exhibit increased bone mass due in part to impaired osteoclast formation. Using in vitro osteoclast culture systems, we show here that Tmem64 interacts with sarcoplasmic endoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and modulates its activity. Consequently, Tmem64 deficiency significantly diminishes RANKL-induced [Ca(2+)](i) oscillation, which results in reduced Ca(2+)/calmodulin-dependent protein kinases (CaMK) IV and mitochondrial ROS, both of which contribute to achieving the CREB activity necessary for osteoclast formation. These data demonstrate that Tmem64 is a positive modulator of osteoclast differentiation via SERCA2-dependent Ca(2+) signaling.