Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in ...response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap+/flox;Creα-MHC (YAP-CHKO) and Yap+/flox mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.
A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes physiological hypertrophy in vitro. The purpose of this study is to determine if AKIP1 promotes physiological ...cardiomyocyte hypertrophy in vivo. Therefore, adult male mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and wild type (WT) littermates were caged individually for four weeks in the presence or absence of a running wheel. Exercise performance, heart weight to tibia length (HW/TL), MRI, histology, and left ventricular (LV) molecular markers were evaluated. While exercise parameters were comparable between genotypes, exercise-induced cardiac hypertrophy was augmented in AKIP1-TG vs. WT mice as evidenced by an increase in HW/TL by weighing scale and in LV mass on MRI. AKIP1-induced hypertrophy was predominantly determined by an increase in cardiomyocyte length, which was associated with reductions in p90 ribosomal S6 kinase 3 (RSK3), increments of phosphatase 2A catalytic subunit (PP2Ac) and dephosphorylation of serum response factor (SRF). With electron microscopy, we detected clusters of AKIP1 protein in the cardiomyocyte nucleus, which can potentially influence signalosome formation and predispose a switch in transcription upon exercise. Mechanistically, AKIP1 promoted exercise-induced activation of protein kinase B (Akt), downregulation of CCAAT Enhancer Binding Protein Beta (C/EBPβ) and de-repression of Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4 (CITED4). Concludingly, we identified AKIP1 as a novel regulator of cardiomyocyte elongation and physiological cardiac remodelling with activation of the RSK3-PP2Ac-SRF and Akt-C/EBPβ-CITED4 pathway. These findings suggest that AKIP1 may serve as a nodal point for physiological reprogramming of cardiac remodelling.
Glioblastoma multiforme (GBM), the most common malignant brain tumor, is associated with upregulation of a number of G‐protein coupled receptors. TCGA analysis of glioblastoma reveals that Gα12 mRNA ...levels are elevated in 28% of GBM patient tumors, the highest rate for all tumor types surveyed. There are concomitant alterations in expression of a subset of GPCRs that couple to Gα12. Gα12 signals through activation of RhoA and its downstream effectors. Our earlier studies revealed that gene programs induced through the RhoA‐regulated transcriptional co‐activators YAP and MRTF‐A contribute to glioma stem cell properties and their in vivo growth as orthotopic (brain) tumors (Yu et al., Oncogene 2018). To determine if Gα12 signaling pathways transduce the effects of known and orphan GPCRs activated in the tumor environment we knocked down Gα12 using shRNA in two human GBM cancer stem cell lines, GSC‐23 and HK‐281. Gα12 mRNA levels decreased by 60‐80% without compensatory upregulation of mRNA for the homologous Gα13. Loss of Gα12 significantly attenuated the stem cell properties of GSC‐23 cells as determined by limiting dilution assays and also reduced mRNA levels for multiple canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. Tumor growth of GSC‐23 control and Gα12 knockdown (KD) cells was examined following orthotopic injection into mouse brain. Surprisingly, Gα12 deletion had no evident effect on tumor size nor did it prolong mouse survival. On the other hand, histopathological analyses revealed striking differences at the tumor border, indicative of diminished invasive properties and this was confirmed by immunohistochemical staining for the human nucleoli antigen. RNA sequencing and DESeq2 analysis in WT and KD tumors identified a number of genes encoding extra and intracellular proteins related to cell adhesion and migration dynamics which were differentially expressed in Gα12 KDs. Thrombospondin‐1 (Thbs‐1) was one of the most repressed genes in the Gα12 knockdown tumors and it was previously identified as a YAP‐dependent gene in these cells. Expression of Thbs‐1 and Gα12 is highly correlated in the more aggressive mesenchymal‐types of GBM by Gliovis and IvyGAP tools, and clinical reports show elevated Thbs‐1 as a marker of poor prognosis in GBM patients. To assess the effect of Gα12 and Thbs‐1 on glioblastoma stem cell migration we performed in vitro Transwell‐based assays. These studies demonstrated that both Gα12 and its transcriptional target Thbs‐1 are required for agonist‐stimulated GSC‐23 cell migration and invasion. We also demonstrated through chemogenetic activation of Gα12 by DREADD expression that Gα12 signaling in GSC‐23 cells is sufficient to drive Thbs‐1 expression and migration. Our findings indicate that Gα12 is a focal point for GPCR control of transcriptional programs for stemness and invasiveness in glioma stem cells derived tumors and suggest targeting this site as a therapeutic intervention to disrupt tumor cell invasion.
As rib fractures are a common injury in the geriatric trauma population and can result in increased morbidity and mortality, we sought to understand predicting outcomes in this population. We ...hypothesized that frail geriatric rib fracture patients would have worse outcomes than their non-frail counterparts. This single-center retrospective study includes patients from July 2019 to June 2022 who were ≥65 years-old, had ≥ 2 rib fractures, and a documented Clinical Frailty Scale score. Univariate analysis was conducted comparing frail vs non-frail, and ≤3 rib fractures vs >3 rib fractures. Multivariate logistic regressions for risk of mortality and of frailty were performed. We found higher mortality in patients with >3 rib fractures on univariate analysis; however, this did not hold true on multivariate analysis. Frail patients were less likely discharged home and had a lower functional status at discharge. Further investigation is needed to effectively improve outcomes for geriatric trauma patients with rib fractures.
RATIONALE:Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca needed to maintain cardiac function when systolic wall stress is increased. ...Hypertrophic Ca binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known.
OBJECTIVE:This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn.
METHODS AND RESULTS:Green fluorescent protein–tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKIIδc were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9±1%, P<0.01 versus control) NFAT nuclear translocation (Control12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca increased Cn-dependent NFAT translocation (to 71.7±7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts.
CONCLUSION:These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.
1 Biomedical Sciences Graduate Program and Departments of 2 Pharmacology and 3 Medicine, University of California, San Diego, La Jolla, California; and 4 Department of Molecular Biology, The Scripps ...Research Institute, La Jolla, California
Submitted 5 December 2006
; accepted in final form 6 February 2007
Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P 2 or S1P 3 receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P 2,3 receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P 2,3 receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P 2,3 receptor knockout mouse hearts. Neither S1P 2 nor S1P 3 receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P 2 and S1P 3 receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P 2 or S1P 3 receptor knockout mice and completely abolished in the S1P 2,3 receptor double-knockout myocytes. Our data demonstrate that activation of S1P 2 and S1P 3 receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.
cardioprotection; mitogen-activated kinase; G protein-coupled receptors; infarct
Address for reprint requests and other correspondence: J. H. Brown, Dept. of Pharmacology, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA, 92093-0636 (e-mail: jhbrown{at}ucsd.edu )
Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are ...emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.
A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt ...kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7 ± 2.7% versus 22.9 ± 3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia.
Considerable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized ...by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant cross talk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study, we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF.
Immunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with β-AR blockers revealed no difference in CaMKII activity when compared with non-β-AR blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδ
transgenic mice were treated with the β
-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29;
<0.001), attenuated the development of cardiac hypertrophy (-14±6% heart weight/tibia length;
<0.05), and strongly reduced ventricular arrhythmias (-70±22% premature ventricular contractions;
<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A-dependent phospholamban and ryanodine receptor 2 phosphorylation (-42±9% for P-phospholamban-S16 and -22±7% for P-ryanodine receptor 2-S2808;
<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by β-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol.
Chronic β-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of β-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to β-AR blockade.
Contrary to previous assumptions, G proteins do not permanently reside on the plasma membrane, but are constantly monitoring the cytoplasmic surfaces of the plasma membrane and endomembranes. Here, ...we report that the Gαq and Gα11 proteins locate at the mitochondria and play a role in a complex signaling pathway that regulates mitochondrial dynamics. Our results provide evidence for the presence of the heteromeric G protein (Gαq/11βγ) at the outer mitochondrial membrane and for Gαq at the inner membrane. Both localizations are necessary to maintain the proper equilibrium between fusion and fission; which is achieved by altering the activity of mitofusin proteins, Drp1, OPA1 and the membrane potential at both the outer and inner mitochondrial membranes. As a result of the absence of Gαq/11, there is a decrease in mitochondrial fusion rates and a decrease in overall respiratory capacity, ATP production and OXPHOS-dependent growth. These findings demonstrate that the presence of Gαq proteins at the mitochondria serves as a physiological function: stabilizing elongated mitochondria and regulating energy production in Drp1 and Opa1 dependent mechanisms. This thereby links organelle dynamics and physiology.
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