Reduced O-GlcNAcylation diminishes cardiomyocyte Ca2+ dependent facilitation and frequency dependent acceleration of relaxation.

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIß isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-ß-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-ß (TGF-ß)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-ß receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-ß signaling. Pharmacological inhibition of the downstream TGF-ß effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-ß1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-ß receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-ß-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-ß-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-ß signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.