Inhibition of the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca(2+) stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca(2+) content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca(2+) content by CaMKII. PLN(-/-) mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN(-/-) mice, indicating the involvement of a Ca(2+)-dependent pathway. AC3-I and AC3-C mice were bred into a PLN(-/-) background. Loss of PLN increased and equalized SR Ca(2+) content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca(2+) content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.

Glaucoma is a common cause of vision loss or blindness and reduction of intraocular pressure (IOP) has been proven beneficial in a large fraction of glaucoma patients. The IOP is maintained by the trabecular meshwork (TM) and the elevation of IOP in open-angle glaucoma is associated with dysfunction and loss of the postmitotic cells residing within this tissue. To determine if IOP control can be maintained by replacing lost TM cells, we transplanted TM-like cells derived from induced pluripotent stem cells into the anterior chamber of a transgenic mouse model of glaucoma. Transplantation led to significantly reduced IOP and improved aqueous humor outflow facility, which was sustained for at least 9 wk. The ability to maintain normal IOP engendered survival of retinal ganglion cells, whose loss is ultimately the cause for reduced vision in glaucoma. In vivo and in vitro analyses demonstrated higher TM cellularity in treated mice compared with littermate controls and indicated that this increase is primarily because of a proliferative response of endogenous TM cells. Thus, our study provides in vivo demonstration that regeneration of the glaucomatous TM is possible and points toward novel approaches in the treatment of this disease.G laucoma is one of the most common causes of irreversible vision loss and blindness worldwide; ∼60 million suffer from this disease, and of these, 7 million are blind (1). By definition all glaucoma involves some degree of vision loss, which is because of the death of retinal ganglion cells (RGC), as well as degeneration of the optic nerve head, the optic nerve, and the lateral geniculate nucleus (2, 3). Advanced age and elevated intraocular pressure (IOP) are the two most significant risk factors for the development of glaucoma. Elevated IOP is typically a result of disturbances in the balance of aqueous humor production and drainage. Aqueous humor is continuously synthesized within the eye and drained primarily through the trabecular meshwork (TM), a specialized structure located anterior to the root of the iris. Although occlusion of the aqueous humor outflow pathways can occur through several mechanisms, in the United States and other Western populations the most common form of glaucoma is primary open angle glaucoma (POAG), which manifests no gross abnormalities to the anterior portion of the eye.Randomized clinical trials have shown that reduction of IOP slows the onset and progression of glaucoma, even in patients without suspicious elevation of IOP (4, 5). Although there has been increasing awareness that factors other than elevated IOP contribute to glaucomatous damage, to date all treatments for glaucoma remain aimed at reducing IOP either through surgical or medical means, which has resulted in significant preservation of vision and increased quality of life for millions of glaucoma patients (6, 7).Although the TM in eyes with POAG appears relatively normal at a gross morphological level, a number of more subtle changes influencing the mechanical properties of the TM collage...

Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. Not only do these cells represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM, resulting in re-establishment of IOP.

PurposePrimary open-angle glaucoma (POAG) is particularly common in older individuals and associated with pathologic degeneration of the trabecular meshwork (TM). We have shown previously that transplantation of induced pluripotent stem cell (iPSC) derived TM cells restores aqueous humor dynamics in young transgenic mice expressing a pathogenic form of human myocilin (Tg-MYOCY437H). This study was designed to determine if this approach is feasible in older mice with more pronounced TM dysfunction.MethodsMouse iPSC were differentiated toward a TM cell phenotype (iPSC-TM) and injected into the anterior chamber of 6-month-old Tg-MYOCY437H or control mice. IOP and aqueous humor outflow facility were recorded for up to 3 months. Transmission electron microscopy, Western blot, and immunohistochemistry were performed to analyze TM morphology, quantify endoplasmic reticulum (ER) stress, and assess TM cellularity.ResultsA 12 weeks after transplantation, IOP in iPSC-TM recipients was statistically lower and outflow facility was significantly improved compared to untreated controls. The number of endogenous TM cells increased significantly in iPSC-TM recipients along with the appearance of TM cells immmunopositive for a marker of cellular division. Morphologically, transplantation of iPSC-TM preserves ER structure 12 weeks after transplantation. However, myocilin and calnexin expression levels remain elevated in transplanted eyes of these 9-month-old Tg-MYOCY437H mice, indicating that ER stress persists within the TM.ConclusionsTransplantation of iPSC-TM can restore IOP and outflow facility in aged Tg-MYOCY437H mice. This type of stem cell–based therapy is a promising possibility for restoration of IOP control in some glaucoma patients.

Abstract-The multifunctional Ca2ϩ /calmodulin-dependent protein kinase II ␦ C (CaMKII␦ C ) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca 2ϩ release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKII␦ C via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKII␦ C expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing ␤-galactosidase (␤-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca 2ϩ transients was slowed, accelerated, or unchanged, whereas spontaneous Ca 2ϩ spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKII␦ C groups, respectively. When challenged by high extracellular 4 which is thought to be arrhythmogenic for disturbance of cardiac electrical activity. Given its central role in cardiac EC coupling and Ca 2ϩ signaling, RyR2 activity is under the exquisite control of an array of molecular partners found in the RyR2 macromolecular signaling complex, including protein kinase A (PKA), Ca 2ϩ /calmodulindependent protein kinase II (CaMKII), protein kinase C, and protein phosphatases 1, 2A, and 2B (calcineurin). 5,6 Among these, CaMKII-dependent phosphorylation of RyR2 is of particular interest, because the waxing and waning of high microdomain Ca 2ϩ accompanying the channel gating might activate and deactivate this kinase, affording a local feedback regulation to SR Ca 2ϩ release. 7 Early studies have suggested that RyR2 possesses a unique CaMKII phosphorylation site at Ser2809, which is also phosphorylated by PKA to a lesser extent. 8 In contrast, more recent studies proposed that Ser2809 is a PKA-specific phosphorylation site in RyR2, 6,9 whereas our own data The functional consequences of RyR2 phosphorylation in the healthy and diseased heart are highly controversial, and the conflicting literature falls into 4 categories: enhancement, inhibition, no function, or complicated regulation, with the enhancement view being prevalent. 12 Using canine cardiac junctional SR vesicles or partially purified RyR2 fused into planar bilayers, it has been shown that CaMKII phosphorylation activates the channel by increasing open channel probability (P o ). 8,13 However, Hain et al 14 found that activation of endogenous CaMKII led to RyR2 channel closure, whereas exogenous CaMKII activated the channel. Using photolytic Ca 2ϩ steps, Valdivia et al 15 showed that RyR2 response to CaMKII-or PKA-dependent phosphorylation is rather complex, characterized by a higher peak P o , a markedly accelerated "adaptation," and a reduced steady-state P o . The...

IntroductionSeveral studies have indicated that autoimmune and neuroinflammatory processes contribute to the neurodegeneration of retinal ganglion cells in human glaucoma patients and in animal models. To test the involvement of cellular immune processes in the pathophysiology of retinal ganglion cell degeneration in vivo, we carried out adoptive transfer experiments from two independent genetic mouse models of glaucoma into normal recipient mice.ResultsOur findings indicate that transfer results in a progressive loss of retinal ganglion cells and their axons despite normal intraocular pressure in recipient mice. Signs of pan-retinal inflammation were not detected. Similar findings were obtained following transfer of isolated T-lymphocytes, but not after transfer of splenocytes from immune deficient glaucomatous mice. Transferred lymphocytes were detected integrated in the spleen and in the retinal ganglion cell layer of recipient animals, albeit at very low frequencies. Furthermore, we observed cell-cell interaction between transferred T-cells and recipient microglia along with focal microglial activation in recipient eyes.ConclusionThis study demonstrates that the pathophysiology of glaucomatous degeneration in the tested animal models includes T-cell mediated events that are capable of causing loss of healthy retinal ganglion cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0234-y) contains supplementary material, which is available to authorized users.

the trabecular meshwork's (tM) physiological role is to maintain normal intraocular pressure by regulating aqueous humor outflow. With age, and particularly in eyes with primary open angle glaucoma, the number of cells residing within the tM is markedly decreased and the function of the tissue is compromised. Here we evaluate if transplantation of induced pluripotent stem cell derived tM like cells (ipSc-tM) restores tM cellularity and function in human eyes obtained from older human donors. Human iPSC were differentiated into iPSC-TM and compared to primary TM cells by RNAseq. ipSc-tM were then injected into the anterior segments of human eyes maintained in perfusion culture. Seven and 14 days eyes after injection eyes that received iPSC-TM contained significantly more cells in the TM. Fewer than 1% of all cells appeared to be iPSC-TM, but significantly more cells in these eyes were immunopositive for Ki 67 and incorporated BrdU. Our study demonstrates that transplantation ipSc-tM stimulates proliferation of endogenous tM cells in perfusion cultured human eyes from aged donors. These data, in concert with our previous findings in animal models, suggest that functional regeneration of the tM may be possible in human eyes with primary open angle glaucoma.

The present studies were conducted to better define the mechanism of action of polyethylene hexamethylene biguanide (PEHMB) (designated herein as NB325), which was shown in previous studies to inhibit infection by the human immunodeficiency virus type 1 (HIV-1). Fluorescence-activated flow cytometric analyses of activated human CD4؉ T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 epitope recognition in the absence of altered recognition of selected CD4 or CD3 epitopes. NB325 also inhibited chemotaxis of CD4 ؉ T lymphocytes induced by the CXCR4 ligand CXCL12. However, NB325 did not cause CXCR4 internalization (unlike CXCL12) and did not interfere with CXCL12 binding. Additional flow cytometric analyses using antibodies with distinct specificities for extracellular domains of CXCR4 demonstrated that NB325 specifically interfered with antibody binding to extracellular loop 2 (ECL2). This interaction was confirmed using competitive binding analyses, in which a peptide derived from CXCR4 ECL2 competitively inhibited NB325-mediated reductions in CXCR4 epitope recognition. Collectively, these results demonstrate that the biguanide-based compound NB325 inhibits HIV-1 infection by specifically interacting with the HIV-1 coreceptor CXCR4.