Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

AIMS: Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. METHODS AND RESULTS: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. CONCLUSIONS: S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion.

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during ß-adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during ß-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-AR stimulation. Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (SNP; 200 µM) and/or ß-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent ß-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain ß-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

RhoA signaling increases mitophagy and protects cardiomyocytes against ischemia by stabilizing PINK1 protein and recruiting Parkin to mitochondria.

Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.

Co-occurrence of BAP1 and SF3B1 mutations in uveal melanoma induces cellular senescence.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Recurrent mutations in BRCA1-associated protein 1 (BAP1) and splicing factor 3B subunit 1 (SF3B1) display a mutually exclusive pattern in UM, but the underlying mechanism is unknown. We show that combined BAP1 deficiency and SF3B1 hotspot mutation lead to senescence and growth arrest in human UM cells. Although p53 protein expression is induced, deletion of TP53 (encoding p53) only modestly rescues the observed senescent phenotype. UM cells with BAP1 loss or SF3B1 mutation are more sensitive to chemotherapeutic drugs compared with their isogenic parental cells. Transcriptome analysis shows that DNA-repair genes are downregulated upon co-occurrence of BAP1 deletion and SF3B1 mutation, thus leading to impaired DNA damage response and the induction of senescence. The co-occurrence of these two mutations reduces invasion of UM cells in zebrafish xenograft models and suppresses growth of melanoma xenografts in nude mice. Our findings provide a mechanistic explanation for the mutual exclusivity of BAP1 and SF3B1 mutations in human UM.

The contribution of the cardiomyocyte to tissue inflammation in cardiomyopathies.

Cardiac injury triggers an acute immune response that drives tissue healing and remodeling via the activation of compensatory mechanisms. Over time, remodeling and inflammation become chronic and have adverse effects that lead to a depression of cardiac function and eventual heart failure. Cardiac inflammation is characterized by dynamic spatial and temporal crosstalk between the resident cells of the heart and recruitment of circulating leukocytes. Until recently, the cardiomyocyte has not been accepted as a direct contributor to cardiac inflammation. It has now emerged as a key initiator of the acute immune response via its ability to produce cytokines and may also synchronize leukocyte recruitment post-injury. This review will focus on the role of the cardiomyocyte in the acute immune response to ischemic and non-ischemic injury and the mechanisms by which it may influence the course of cardiac remodeling and failure.

CaMKIIδC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy.

RATIONALE: CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. OBJECTIVE: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. METHODS AND RESULTS: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. CONCLUSIONS: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Inflammation in nonischemic heart disease: initiation by cardiomyocyte CaMKII and NLRP3 inflammasome signaling.

There is substantial evidence that chronic heart failure in humans and in animal models is associated with inflammation. Ischemic interventions such as myocardial infarction lead to necrotic cell death and release of damage associated molecular patterns, factors that signal cell damage and induce expression of proinflammatory chemokines and cytokines. It has recently become evident that nonischemic interventions are also associated with increases in inflammatory genes and immune cell accumulation in the heart and that these contribute to fibrosis and ventricular dysfunction. How proinflammatory responses are elicited in nonischemic heart disease which is not, at least initially, associated with cell death is a critical unanswered question. In this review we provide evidence supporting the hypothesis that cardiomyocytes are an initiating site of inflammatory gene expression in response to nonischemic stress. Furthermore we discuss the role of the multifunctional Ca2+/calmodulin-regulated kinase, CaMKIIδ, as a transducer of stress signals to nuclear factor-κB activation, expression of proinflammatory cytokines and chemokines, and priming and activation of the NOD-like pyrin domain-containing protein 3 (NLRP3) inflammasome in cardiomyocytes. We summarize recent evidence that subsequent macrophage recruitment, fibrosis and contractile dysfunction induced by angiotensin II infusion or transverse aortic constriction are ameliorated by blockade of CaMKII, of monocyte chemoattractant protein-1/C-C chemokine receptor type 2 signaling, or of NLRP3 inflammasome activation.

Inflammation and NLRP3 Inflammasome Activation Initiated in Response to Pressure Overload by Ca2+/Calmodulin-Dependent Protein Kinase II δ Signaling in Cardiomyocytes Are Essential for Adverse Cardiac Remodeling.

BACKGROUND: Inflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to nonischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) in cardiomyocytes (CMs) in response to pressure overload elicits inflammatory responses leading to adverse remodeling. METHODS: Mice in which CaMKIIδ was selectively deleted from CMs (cardiac-specific knockout [CKO]) and floxed control mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKIIδ deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation, and fibrosis were assessed by quantitative polymerase chain reaction, histochemistry, and ventricular remodeling by echocardiography. RESULTS: TAC induced increases in cardiac mRNA levels for proinflammatory chemokines and cytokines in ≤3 days, and these responses were significantly blunted when CM CaMKIIδ was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression, which were markedly attenuated in CKO. Priming and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome, assessed by measuring interleukin-1ß and NOD-like receptor pyrin domain-containing protein 3 mRNA levels, caspase-1 activity, and interleukin-18 cleavage, were increased at day 3 after TAC in control hearts and in CMs isolated from these hearts. These responses were dependent on CaMKIIδ and associated with activation of Nuclear Factor-kappa B and reactive oxygen species. Accumulation of macrophages observed at days 7 to 14 after TAC was diminished in CKO and, by blocking Monocyte Chemotactic Protein-1 signaling, deletion of CM Monocyte Chemotactic Protein-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 after TAC were diminished in the CKO. Inhibition of CaMKII, Nuclear Factor-kappa B, inflammasome, or Monocyte Chemotactic Protein-1 signaling in the first 1 or 2 weeks after TAC decreased remodeling, but inhibition of CaMKII after 2 weeks did not. CONCLUSIONS: Activation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis, and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by CM CaMKIIδ signaling to prevent progression to heart failure.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

RhoA regulates Drp1 mediated mitochondrial fission through ROCK to protect cardiomyocytes.

Cardiac ischemia/reperfusion, loss of blood flow and its subsequent restoration, causes damage to the heart. Oxidative stress from ischemia/reperfusion leads to dysfunction and death of cardiomyocytes, increasing the risk of progression to heart failure. Alterations in mitochondrial dynamics, in particular mitochondrial fission, have been suggested to play a role in cardioprotection from oxidative stress. We tested the hypothesis that activation of RhoA regulates mitochondrial fission in cardiomyocytes. Our studies show that expression of constitutively active RhoA in cardiomyocytes increases phosphorylation of Dynamin-related protein 1 (Drp1) at serine-616, and leads to localization of Drp1 at mitochondria. Both responses are blocked by inhibition of Rho-associated Protein Kinase (ROCK). Endogenous RhoA activation by the GPCR agonist sphingosine-1-phosphate (S1P) also increases Drp1 phosphorylation and its mitochondrial translocation in a RhoA and ROCK dependent manner. Consistent with the role of mitochondrial Drp1 in fission, RhoA activation in cardiomyocytes leads to formation of smaller mitochondria and this is attenuated by inhibition of ROCK, by siRNA knockdown of Drp1 or by expression of a phosphorylation-deficient Drp1 S616A mutant. In addition, activation of RhoA prevents cell death in cardiomyocytes challenged by oxidative stress and this protection is blocked by siRNA knockdown of Drp1 or by Drp1 S616A expression. Taken together our findings demonstrate that RhoA activation can regulate Drp1 to induce mitochondrial fission and subsequent cellular protection, implicating regulation of fission as a novel mechanism contributing to RhoA-mediated cardioprotection.

Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice.

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.

Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes.

BACKGROUND: Sphingosine 1-phosphate (S1P) signals through G protein-coupled receptors to elicit a wide range of cellular responses. In CNS injury and disease, the blood-brain barrier is compromised, causing leakage of S1P from blood into the brain. S1P can also be locally generated through the enzyme sphingosine kinase-1 (Sphk1). Our previous studies demonstrated that S1P activates inflammation in murine astrocytes. The S1P1 receptor subtype has been most associated with CNS disease, particularly multiple sclerosis. S1P3 is most highly expressed and upregulated on astrocytes, however, thus we explored the involvement of this receptor in inflammatory astrocytic responses. METHODS: Astrocytes isolated from wild-type (WT) or S1P3 knockout (KO) mice were treated with S1P3 selective drugs or transfected with short interfering RNA to determine which receptor subtypes mediate S1P-stimulated inflammatory responses. Interleukin-6 (IL-6), and vascular endothelial growth factor A (VEGFa) messenger RNA (mRNA) and cyclooxygenase-2 (COX-2) mRNA and protein were assessed by q-PCR and Western blotting. Activation of RhoA was measured using SRE.L luciferase and RhoA implicated in S1P signaling by knockdown of Gα12/13 proteins or by inhibiting RhoA activation with C3 exoenzyme. Inflammation was simulated by in vitro scratch injury of cultured astrocytes. RESULTS: S1P3 was highly expressed in astrocytes and further upregulated in response to simulated inflammation. Studies using S1P3 knockdown and S1P3 KO astrocytes demonstrated that S1P3 mediates activation of RhoA and induction of COX-2, IL-6, and VEGFa mRNA, with some contribution from S1P2. S1P induces expression of all of these genes through coupling to the Gα12/13 proteins which activate RhoA. Studies using S1P3 selective agonists/antagonists as well as Fingolimod (FTY720) confirmed that stimulation of S1P3 induces COX-2 expression in astrocytes. Simulated inflammation increased expression of Sphk1 and consequently activated S1P3, demonstrating an autocrine pathway through which S1P is formed and released from astrocytes to regulate COX-2 expression. CONCLUSIONS: S1P3, through its ability to activate RhoA and its upregulation in astrocytes, plays a unique role in inducing inflammatory responses and should be considered as a potentially important therapeutic target for CNS disease progression.

CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α.

Deletion of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been shown to protect against in vivo ischemia/reperfusion (I/R) injury. It remains unclear which CaMKIIδ isoforms and downstream mechanisms are responsible for the salutary effects of CaMKIIδ gene deletion. In this study we sought to compare the roles of the CaMKIIδB and CaMKIIδC subtypes and the mechanisms by which they contribute to ex vivo I/R damage. WT, CaMKIIδKO, and mice expressing only CaMKIIδB or δC were subjected to ex vivo global ischemia for 25min followed by reperfusion. Infarct formation was assessed at 60min reperfusion by triphenyl tetrazolium chloride (TTC) staining. Deletion of CaMKIIδ conferred significant protection from ex vivo I/R. Re-expression of CaMKIIδC in the CaMKIIδKO background reversed this effect and exacerbated myocardial damage and dysfunction following I/R, while re-expression of CaMKIIδB was protective. Selective activation of CaMKIIδC in response to I/R was evident in a subcellular fraction enriched for cytosolic/membrane proteins. Further studies demonstrated differential regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and tumor necrosis factor alpha (TNF-α) expression by CaMKIIδB and CaMKIIδC. Selective activation of CaMKIIδC was also observed and associated with NF-κB activation in neonatal rat ventricular myocytes (NRVMs) subjected to oxidative stress. Pharmacological inhibition of NF-κB or TNF-α significantly ameliorated infarct formation in WT mice and those that re-express CaMKIIδC, demonstrating distinct roles for CaMKIIδ subtypes in I/R and implicating acute activation of CaMKIIδC and NF-κB in the pathogenesis of reperfusion injury.

Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection.

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1, S1P2, and S1P3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12, Gαq, or Gαi. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.

Exercise training reverses myocardial dysfunction induced by CaMKIIδC overexpression by restoring Ca2+ homeostasis.

Several conditions of heart disease, including heart failure and diabetic cardiomyopathy, are associated with upregulation of cytosolic Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIδC) activity. In the heart, CaMKIIδC isoform targets several proteins involved in intracellular Ca(2+) homeostasis. We hypothesized that high-intensity endurance training activates mechanisms that enable a rescue of dysfunctional cardiomyocyte Ca(2+) handling and thereby ameliorate cardiac dysfunction despite continuous and chronic elevated levels of CaMKIIδC CaMKIIδC transgenic (TG) and wild-type (WT) mice performed aerobic interval exercise training over 6 wk. Cardiac function was measured by echocardiography in vivo, and cardiomyocyte shortening and intracellular Ca(2+) handling were measured in vitro. TG mice had reduced global cardiac function, cardiomyocyte shortening (47% reduced compared with WT, P < 0.01), and impaired Ca(2+) homeostasis. Despite no change in the chronic elevated levels of CaMKIIδC, exercise improved global cardiac function, restored cardiomyocyte shortening, and reestablished Ca(2+) homeostasis to values not different from WT. The key features to explain restored Ca(2+) homeostasis after exercise training were increased L-type Ca(2+) current density and flux by 79 and 85%, respectively (P < 0.01), increased sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) function by 50% (P < 0.01), and reduced diastolic SR Ca(2+) leak by 73% (P < 0.01), compared with sedentary TG mice. In conclusion, exercise training improves global cardiac function as well as cardiomyocyte function in the presence of a maintained high CaMKII activity. The main mechanisms of exercise-induced improvements in TG CaMKIIδC mice are mediated via increased L-type Ca(2+) channel currents and improved SR Ca(2+) handling by restoration of SERCA2a function in addition to reduced diastolic SR Ca(2+) leak.

Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs.

Although c-kit(+) cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. G-protein coupled receptors (GPCRs) play an important role in regulating these processes in mammalian cell types thus we assessed GPCR mRNA expression in c-kit(+) cells isolated from adult mouse hearts. Our data provide the first comprehensive overview of the distribution of this fundamental class of cardiac receptors in CPCs and reveal notable distinctions from that of adult cardiomyocytes. We focused on GPCRs that couple to RhoA activation in particular those for sphingosine-1-phosphate (S1P). The S1P2 and S1P3 receptors are the most abundant S1P receptor subtypes in mouse and human CPCs while cardiomyocytes express predominantly S1P1 receptors. Treatment of CPCs with S1P, as with thrombin and serum, increased proliferation through a pathway requiring RhoA signaling, as evidenced by significant attenuation when Rho was inhibited by treatment with C3 toxin. Further analysis demonstrated that both S1P- and serum-induced proliferation are regulated through the S1P2 and S1P3 receptor subtypes which couple to Gα12/13 to elicit RhoA activation. The transcriptional co-activator MRTF-A was activated by S1P as assessed by its nuclear accumulation and induction of a RhoA/MRTF-A luciferase reporter. In addition S1P treatment increased expression of cardiac lineage markers Mef2C and GATA4 and the smooth muscle marker GATA6 through activation of MRTF-A. In conclusion, we delineate an S1P-regulated signaling pathway in CPCs that introduces the possibility of targeting S1P2/3 receptors, Gα12/13 or RhoA to influence the proliferation and commitment of c-kit(+) CPCs and improve the response of the myocardium following injury.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic ß-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB.

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory ß-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of ß-adrenergic receptors (ß-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic ß-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic ß-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by ß-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as ß-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic ß-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.