Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Intra-arterial stem cell therapy modulates neuronal calcineurin and confers neuroprotection after ischemic stroke.

Aim: Calcineurin (CaN) is a threonine/phosphatase which play roles in neuronal homeostasis. Ischemic stroke induces hyperactivation of CaN which further triggers apoptotic signaling. CaN inhibition has limited therapeutic output and neurotoxicity due to its intricate roles in the neuronal network and requires a strategic modulation. Intra-arterial (IA) mesenchymal stem cells (MSCs) have shown to interact with the milieu in a paracrine manner as compared to CaN inhibitors to ameliorate the neuronal damage triggered by ischemia/reperfusion injury. The present study investigates the role of IA MSCs in modulating neuronal CaN after stroke onset. Materials and methods: To validate, middle-aged ovariectomized female rats exposed to MCAo (90 min) were treated with IA MSCs (1 × 105 MSCs) or phosphate-buffered saline (PBS) at 6 hours to check CaN expression in different groups.Tests for assessing functional and motor coordination were performed along with biochemical estimations. Furthermore, an inhibition study by non-selective inhibitor of neuronal calcium channel, flunarizine, was performed to explore the possible underlying mechanism by which IA MSCs may interact with CaN. Results: The study suggests that IA MSCs seemingly reduce the expression of CaN after ischemic stroke. IA MSCs have shown to improve the functional outcome and normalize oxidative parameters. Conclusion: Our study provides a preliminary evidence of role of IA MSCs in modulating CaN expression.

Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K+ Channel Dysfunction in DRG Neurons.

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI.SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K+ channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1).

The effect of RCAN1 on the biological behaviors of small cell lung cancer.

Bone is the third most common site of cancer metastasis. In total, 30%-40% of lung cancer cases can develop skeletal metastasis for which no effective therapy in clinic is available. RCAN1 (regulator of calcineurin 1) is an important regulator in angiogenesis which is vital to tumor growth. In this study, we investigated the changes of biological behaviors in SBC-5 and SBC-3 cells after the RCAN1 expression level was changed. Briefly, overexpression of RCAN1 significantly attenuated their malignancy, including decreased ability of proliferation, colony formation, migration, invasion, and bone adherence. Furthermore, the cell cycle progression was impeded. Although the opposite changes were observed in SBC-3 cells after the RCAN1 expression was suppressed by RNA interference, the apoptosis rate was not affected by the expression level of RCAN1 in these cells. So, our research revealed that RCAN1 was involved in the development of small cell lung cancer, and it might be a cancer-inhibiting gene for the formation of bone metastases in small cell lung cancer.

Effect of the knockdown of Cabin1 on p53 in glomerular podocyte.

Calcineurin binding protein 1 (Cabin1) is a natural inhibitor of calcineurin (CN). Moreover, Cabin1 retards tumor cell apoptosis by regulating p53. This study was designed to observe the expression of Cabin1 during podocyte injury, as well as its relationship with p53. Sprague-Dawley rats were used for the establishment of 5/6 nephrectomized rat model. Sham-operated rats underwent ventral laparotomy without nephrectomy. Then, rats were sacrificed at 8 and 12 weeks after nephrectomy. WT-1, a podocyte nuclear protein, was used for indicating the localization of Cabin1 in glomeruli. As tacrolimus protects podocyte via inhibiting AngiotensinII (AngII) induced CN activation. Cultured podocytes were injured by AngII or restored by tacrolimus. The protein expression and localization was detected by western blot or immunofluorescence staining. Cabin1 was knocked down by siRNA in cultured podocytes. In 5/6 nephrectomized rats, the colocalization of Cabin1 and WT-1 became more obviously in podocyte nuclei. Cabin1 protein was markedly increased in rats at 8 and 12 weeks after nephrectomy, as well as in AngII injured podocytes at 48 h (0.99 ± 0.12 in AngII group versus 0.80 ± 0.16 in control group). Cabin1 and p53 colocalized in cultured podocyte nuclei, p53 expression was significantly decreased (0.21 ± 0.05 in siRNA group versus 0.31 ± 0.05 in negative control group) after Cabin1 was being knocked down. In conclusion, Cabin1 expression significantly increases during podocyte injury. Knockdown of Cabin1 induces p53 expression decrease in cultured podocyte. Cabin1 may provide a new target to investigate podocyte injury.

[Expression profile of calcineurin pathway genes in myocardium tissues in relation to ischemic heart remodeling in humans].

Calcineurin pathway plays the critical role in the cardiac remodeling of various origin, development of chambers dilatation and progression of heart failure. Components of calcineurin pathway are involved in myocardium hypertrophy regulation, angiogenesis and apoptosis. Results of quantitative expression profiling study of main calcineurin pathway genes PPP3CA, PPP3R1, PPP3CB, GATA4 and NFATC4 in myocardium of right atrium auricle of patients with a coronary heart disease, exposed to various types of surgical treatments depending on weight of a clinical finding (surgical reconstruction of the geometry of left ventricle (LV) (postinfarction aneurysm) or coronary artery bypass grafting in case of unaltered morphology of LV) are presented. In patients with sizable postinfarction LV dilatation (n = 21) expression level of calcineurin catalytic subunit genes PPP3CA and PPP3CB was 1.3 and 1.6 times lower (p = 0.018 and 0.023, accordingly) compared to patients with unaltered shape of the heart (n = 34). Expression level of PPP3R1 gene encoding calcineurin regulatory subunit B and GATA4 and NFATC4 genes for transcription factors did not differ in studied subgroups of patients. Thus, lower expression of PPP3CA and PPP3CB genes in atrium myocardium can be related to expressed postinfarction LV remodeling. Further studies of relation quantitative expression profiling of calcineurin pathway genes with the level of damage of myocardium is essential what may have important outcome for the prevention of adverse events of cardiosurgical treatments in patients with postinfarction remodeling.

Calcineurin and Akt expression in hypertrophied bladder in STZ-induced diabetic rat.

Diabetes causes significant increases in bladder weight but the natural history and underlying mechanisms are not known. In this study, we observed the temporal changes of detrusor muscle cells (DMC) and the calcineurin (Cn) and Akt expressions in detrusor muscle in the diabetic rat. Male Sprague-Dawley rats were divided into 3 groups: streptozotocin-induced diabetics, 5% sucrose-induced diuretics, and age-matched controls. The bladders were removed 1, 2, or 9weeks after disease induction and the extent of hypertrophy was examined by bladder weights and cross sectional area of DMC. Cn and Akt expression were evaluated by immunoblotting. Both diabetes and diuresis caused significant increases in bladder weight. The mean cross sectional areas of DMC were increased in both diabetic and diuretic animals 1, 2, or 9weeks after disease induction. The expression levels of both the catalytic A (CnA) and regulatory B (CnB) subunits of Cn were increased at 1 and 2weeks, but not at 9weeks. Expression of Akt was similar among control, diabetic, and diuretic rat bladder at all time points. In conclusion, diabetes and diuresis induce similar hypertrophy of detrusor muscle during the first 9weeks, indicating that bladder hypertrophy in the early stage of diabetes is in response to the presence of increased urine output in diabetes. Our results suggest that the Cn, but not the Akt signaling pathway may be involved in the development of bladder hypertrophy.

Mechanism of activation of Saccharomyces cerevisiae calcineurin by Mn2+.

Saccharomyces cerevisiae calcineurin (CN) consists of a catalytic subunit CNA1 or CNA2 and a regulatory subunit CNB1. The kinetics of activation of yeast CN holoenzymes and their catalytic domains by Mn2+ were investigated. We report that the in vitro phosphatase reaction activated by Mn2+ typically has a pronounced initial lag phase caused by slow conformational rearrangement of the holoenzyme-Mn2+. A similar lag phase was detected using just the catalytic domain of yeast CN, indicating that the slowness of Mn2+-induced conformational change of CN results from a rearrangement within the catalytic domain. The Mn2+-activation of CN was reversible. The dissociation constant of the CN heterodimer containing the CNA2 subunit in the presence of Mn2+ was 3-fold higher than that of CN containing the CNA1 subunit and that of the catalytic domains of CNA1 and CNA2, pointing to differences between the residues surrounding the Mn2+-binding sites of CNA1 and CNA2.

Effect of chronic stress or nicotine on hypothyroidism-induced enhancement of LTD: electrophysiological and molecular studies.

We have shown recently that either hypothyroidism or chronic psychosocial stress enhances the expression of LTD, which is reversed by chronic nicotine treatment. In this study, we investigated the effect of combining chronic psychosocial stress with hypothyroidism on LTD. We have also investigated the levels of signaling molecules important for LTD in hypothyroid, stressed-hypothyroid and nicotine-treated hypothyroid rats. Following paired pulse stimulation, LTD was evoked in the CA1 region of anesthetized rats. Combining chronic psychosocial stress with hypothyroidism does not further enhance LTD magnitude compared to either alone. Western blot analysis conducted 1 h after induction of LTD, showed that the levels of calcineurin and P-CaMKII were increased in hypothyroid and stressed-hypothyroid groups compared to that of the control group. However, the levels of calcineurin and P-CaMKII after paired pulsed stimulation were not further increased in stressed-hypothyroid group compared to the hypothyroid only group. In addition, these levels were normalized by chronic nicotine treatment. No change was detected in any of the groups in the levels of calmodulin, PKCgamma, and BDNF after paired pulse stimulation. Our results indicate that changes in the levels of calcineurin and P-CaMKII during expression of LTD in the CA1 region may explain the enhanced magnitude of LTD in hypothyroid rats, and its reversal by chronic nicotine treatment.

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion.

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca2+ concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca2+-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.

Nifedipine suppresses Th1/Th2 cytokine production and increased apoptosis of anti-CD3 + anti-CD28-activated mononuclear cells from patients with systemic lupus erythematosus via calcineurin pathway.

Increased Ca(2+) influx is found in mononuclear cells (MNC) of patients with systemic lupus erythematosus (SLE). The role of calcineurin and potential implication of calcium channel blocker to suppress the abnormal Ca(2+) influx in SLE remain to be determined. In the present study, we found that the expression and phosphatase activity of calcineurin, but not calcineurin inhibitor in SLE-MNC were greater than normal MNC. Functionally, 1 microM nifedipine could suppress SLE-MNC IFN-gamma secretion but 10 microM nifedipine was required for suppressing that of normal MNC. IL-10 secretion by both SLE-MNC and normal MNC was suppressed by 1 microM nifedipine. However, high dose of nifedipine (50 microM) suppressed NFATc1 activation in SLE-MNC and enhanced apoptosis of anti-CD3 + anti-CD28-activated SLE-MNC irrelevant to expression of Fas ligand. These data suggest that SLE-MNC overexpressed calcineurin and hyper-responded to L-type Ca(2+) channel blocker-mediated apoptosis and cytokine suppression. We proposed that L-type Ca(2+) channel blocker maybe a potential medication for controlling SLE.

Regulation of calcineurin through transcriptional induction of the calcineurin A beta promoter in vitro and in vivo.

The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin A(beta) (CnA(beta)) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnA(beta) upregulation is not clear. Here, we isolated the mouse CnA(beta) promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnA(beta) promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated beta-galactosidase (LacZ) containing transgenic mice under the control of the CnA(beta) 2.3-kb promoter. CnA(beta)-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnA(beta)-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnA(beta) 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnA(beta) expression as an additional means of regulating calcineurin activity in the heart.

Locally overexpressing hepatocyte growth factor prevents post-ischemic heart failure by inhibition of apoptosis via calcineurin-mediated pathway and angiogenesis.

BACKGROUND: Myocardial infarction is a significant cause of heart failure. Currently, therapies are limited and novel revascularization methods may play a role. We investigated the effects of hepatocyte growth factor (HGF) expressed by bone marrow-derived mesenchymal stem cells (MSCs) on post-ischemic heart failure. METHODS: Four weeks after myocardial infarction (MI), Sprague Dawley rats were randomly divided into saline control group, MSC-GFP group, MSC-HGF group, and MSC-HGF+CsA group. After another 4 weeks, hearts were analyzed for ventricular geometry, myocardial function, angiogenesis and endothelial cell density, apoptosis and the expression of calcineurin, Akt, and Bcl-2 protein. RESULTS: In MSC-HGF group, rats exhibited better LV systolic and diastolic function compared with other groups after 8 weeks of MI. Angiogenesis was significantly enhanced by HGF through inducing proliferation of endothelial cells. The effects of HGF on apoptosis were associated with the expression level of calcineurin protein. CONCLUSIONS: Our findings suggest that overexpression of HGF improved ischemic cardiac function through angiogenesis and reduction of apoptosis partly mediated by upregulation of calcineurin.

An insight into new strategies to combat antifungal drug resistance.

Invasive fungal infections especially in immunocompromised patients represent a dominating cause of mortality. The most commonly used antifungal agents can be divided into three broad categories, including triazoles, echinocandins and polyenes. Antifungal resistance is on the increase, posing a growing threat to the stewardship of immunocompromised patients with fungal infections. The paucity of currently available antifungals leads to the rapid emergence of drug resistance and thus aggravates the refractoriness of invasive fungal infections. Therefore, deep exploration into mechanisms of drug resistance and search for new antifungal targets are required. This review highlights the therapeutic strategies targeting Hsp90, calcineurin, trehalose biosynthesis and sphingolipids biosynthesis, in an attempt to provide clinical evidence for overcoming drug resistance and to form the rationale for combination therapy of conventional antifungals and agents with novel mechanisms of action. What's more, this review also gives a concise introduction of three new-fashioned antifungals, including carboxymethyl chitosan, silver nanoparticles and chromogranin A-N46.

Methamphetamine toxicity-induced calcineurin activation, nuclear translocation of nuclear factor of activated T-cells and elevation of cyclooxygenase 2 levels are averted by calpastatin overexpression in neuroblastoma SH-SY5Y cells.

Methamphetamine (METH) is an addictive stimulant drug that has many negative consequences, including toxic effects to the brain. Recently, the induction of inflammatory processes has been identified as a potential contributing factor to induce neuronal cell degeneration. It has been demonstrated that the expression of inflammatory agents, such as cyclooxygenase 2 (COX-2), depends on the activation of calcineurin (CaN) and nuclear factor of activated T-cells (NFAT). Moreover, the excessive elevation in cytosolic Ca2+ levels activates the cell death process, including calpain activation in neurons, which was diminished by the overexpression of the calpain inhibitor protein, calpastatin. However, it is unclear whether calpain mediates CaN-NFAT activation in the neurotoxic process. In the present study, we observed that the toxic high dose of METH-treated neuroblastoma SH-SY5Y cells significantly decreased cell viability but increased apoptotic cell death, the active cleaved form of calcineurin, the nuclear translocation of NFAT, and COX-2 levels. Nevertheless, these toxic effects were diminished in METH-treated calpastatin-overexpressing SH-SY5Y cells. These findings might emphasize the role of calpastatin against METH-induced toxicity by a mechanism related to calpain-dependent CaN-NFAT activation-induced COX-2 expression.

Death, cardiac dysfunction, and arrhythmias are increased by calmodulin kinase II in calcineurin cardiomyopathy.

BACKGROUND: Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. METHODS AND RESULTS: We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to "clamp" CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy. CONCLUSIONS: CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.

Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions.

Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here, we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first 2 weeks after birth are particularly dynamic for differential gene expression and alternative splicing transitions, and calcium-handling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of the three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin target Nfatc3, and/or affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of alternative splicing during muscle postnatal development.

Reduced Cardiac Calcineurin Expression Mimics Long-Term Hypoxia-Induced Heart Defects in Drosophila.

BACKGROUND: Hypoxia is often associated with cardiopulmonary diseases, which represent some of the leading causes of mortality worldwide. Long-term hypoxia exposures, whether from disease or environmental condition, can cause cardiomyopathy and lead to heart failure. Indeed, hypoxia-induced heart failure is a hallmark feature of chronic mountain sickness in maladapted populations living at high altitude. In a previously established Drosophila heart model for long-term hypoxia exposure, we found that hypoxia caused heart dysfunction. Calcineurin is known to be critical in cardiac hypertrophy under normoxia, but its role in the heart under hypoxia is poorly understood. METHODS AND RESULTS: In the present study, we explore the function of calcineurin, a gene candidate we found downregulated in the Drosophila heart after lifetime and multigenerational hypoxia exposure. We examined the roles of 2 homologs of Calcineurin A, CanA14F, and Pp2B in the Drosophila cardiac response to long-term hypoxia. We found that knockdown of these calcineurin catalytic subunits caused cardiac restriction under normoxia that are further aggravated under hypoxia. Conversely, cardiac overexpression of Pp2B under hypoxia was lethal, suggesting that a hypertrophic signal in the presence of insufficient oxygen supply is deleterious. CONCLUSIONS: Our results suggest a key role for calcineurin in cardiac remodeling during long-term hypoxia with implications for diseases of chronic hypoxia, and it likely contributes to mechanisms underlying these disease states.