Restoring Lost Anti-HER-2 Th1 Immunity in Breast Cancer: A Crucial Role for Th1 Cytokines in Therapy and Prevention

The ErbB/B2 (HER-2/neu) oncogene family plays a critical role in the development and metastatic spread of several tumor types including breast, ovarian and gastric cancer. In breast cancer, HER-2/neu is expressed in early disease development in a large percentage of DCIS lesions and its expression is associated with an increased risk of invasion and recurrence. Targeting HER-2 with antibodies such as trastuzumab or pertuzumab has improved survival, but patients with more extensive disease may develop resistance to therapy. Interestingly, response to HER-2 targeted therapies correlates with presence of immune response genes in the breast. Th1 cell production of the cytokines interferon gamma (IFNγ) and TNFα can enhance MHC class I expression, PD-L1 expression, augment apoptosis and tumor senescence, and enhances growth inhibition of many anti-breast cancer agents, including anti-estrogens and HER-2 targeted therapies. Recently, we have identified that a loss of anti-HER-2 CD4 Th1 in peripheral blood occurs during breast tumorigenesis and is dramatically diminished, even in Stage I breast cancers. The loss of anti-HER-2 Th1 response is specific and not readily reversed by standard therapies. In fact, this loss of anti-HER-2 Th1 response in peripheral blood correlates with lack of complete response to neoadjuvant therapy and diminished disease-free survival. This defect can be restored with HER-2 vaccinations in both DCIS and IBC. Correcting the anti-HER-2 Th1 response may have significant impact in improving response to HER-2 targeted therapies. Development of immune monitoring systems for anti-HER-2 Th1 to identify patients at risk for recurrence could be critical to improving outcomes, since the anti-HER-2 Th1 response can be restored by vaccination. Correction of the cellular immune response against HER-2 may prevent recurrence in high-risk patients with DCIS and IBC at risk of developing new or recurrent breast cancer.Fil: Nocera, Nadia F.. University of Pennsylvania; Estados UnidosFil: Lee, M. Catherine. H. Lee Moffitt Cancer Center; Estados UnidosFil: De La Cruz, Lucy M.. University of Pennsylvania; Estados UnidosFil: Rosemblit, Cinthia. University of Pennsylvania; Estados UnidosFil: Czerniecki, Brian J.. H. Lee Moffitt Cancer Center; Estados Unido

Progestins induce transcriptional activation of signal transducer and activator of transcription 3 (Stat3) via a Jak- and Src-dependent mechanism in breast cancer cells

Interactions between steroid hormone receptors and signal transducer and activator of transcription (Stat)-mediated signaling pathways have already been described. In the present study, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in BALB/c mice and in the human breast cancer cell line T47D. We found that C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 and that MPA treatment of C4HD cells up-regulated Stat3 protein expression. In addition, MPA induced rapid, nongenomic Stat3, Jak1, and Jak2 tyrosine phosphorylation in C4HD and T47D cells. MPA treatment of C4HD cells also resulted in rapid c-Src tyrosine phosphorylation. These effects were completely abolished by the progestin antagonist RU486. Abrogation of Jak1 and Jak2 activity by transient transfection of C4HD cells with dominant negative (DN) Jak1 or DN Jak2 vectors, or inhibition of Src activity by preincubation of cells with the Src family kinase inhibitor PP2, blocked the capacity of MPA to induce Stat3 phosphorylation. Treatment of C4HD cells with MPA induced Stat3 binding to DNA. In addition, MPA promoted strong Stat3 transcriptional activation in C4HD and T47D cells that was inhibited by RU486 and by blockage of Jak1, Jak2, and Src activities. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F mutant had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Finally, we addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells' ability to form tumors in syngeneic mice. Our results have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation, which is in turn an obligatory requirement for progestin stimulation of both in vitro and in vivo breast cancer growth.Fil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Salatino, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rosemblit, Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Carnevale, Romina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Molinolo, Alfredo. National Institutes of Health; Estados UnidosFil: Frahm, Isabel. Sanatorio Mater Dei Hermanas de María de Schoenstatt; ArgentinaFil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Oncodriver inhibition and CD4+ Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and antioncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.Fil: Rosemblit, Cinthia. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Datta, Jashodeep. University of Pennsylvania; Estados UnidosFil: Lowenfeld, Lea. University of Pennsylvania; Estados UnidosFil: Xu, Shuwen. University of Pennsylvania; Estados UnidosFil: Basu, Amrita. H. Lee Moffitt Cancer Center; Estados UnidosFil: Kodumudi, Krithika. H. Lee Moffitt Cancer Center; Estados UnidosFil: Wiener, Doris. H. Lee Moffitt Cancer Center; Estados UnidosFil: Czerniecki, Brian J.. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unido

Transcriptional regulation of oncogenic protein kinase Cε (PKCε) by STAT1 and Sp1 proteins

Overexpression of PKCε, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCε expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCε gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCε mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCε and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCε and its effectors in cancer therapeutics.Centro de Investigaciones Inmunológicas Básicas y AplicadasFacultad de Ciencias Médica

p42/p44 MAPK-mediated Stat3 Ser727 phosphorylation is required for progestin-induced full activation of Stat3 and breast cancer growth

Stat3 is a signaling node for multiple oncogenic pathways and is therefore frequently active in breast cancer. As experimental and clinical evidence reveals that progestins are key players in controlling mammary gland tumorigenesis, we studied Stat3 participation in this event. We have previously shown that progestins induce Stat3Tyr705 phosphorylation and its transcriptional activation in breast cancer cells. In this study, we demonstrate that progestins also induce Stat3 phosphorylation at Ser727 residue, which occurs via activation of c-Src/p42/p44 MAPK pathways in murine progestin-dependent C4HD cells and in T-47D cells. Expression of a Stat3S727A vector, which carries a serine-to-alanine substitution at codon 727, shows that Stat3Ser727 phosphorylation is required for full transcriptional activation of cyclin D1 gene expression by progestins and for in vivo Stat3 recruitment on cyclin D1 promoter. Transfection of Stat3S727A in murine and human breast cancer cells abolished progestin-induced in vitro and in vivo growth. Moreover, we found a positive correlation between progesterone receptor expression and nuclear localization of Stat3Ser727 phosphorylation in breast cancer biopsies. These data highlight Stat3 phosphorylation in Ser727 residue as a nongenomic action by progestins, necessary to promote breast cancer growth.Fil: Tkach, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rosemblit, Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rivas, Martin Alfredo. Vall d; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Díaz Flaqué, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Beguelin, Wendy. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Maronna, Esteban. Sanatorio Mater Dei; ArgentinaFil: Guzman, Pablo. Universidad de la Frontera. Facultad de Medicina; ChileFil: Gercovich, Felipe G.. Instituto Oncológico Henry Moore; ArgentinaFil: Gil Deza, Ernesto. Instituto Oncológico Henry Moore; ArgentinaFil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT

Inhibition diminishes cell viability via PKC alpha (PKCa) in thyroid cancer cells

Thyroid carcinoma (TC) is the most common endocrine neoplasia.Its incidence has increased in the last 40 years worldwide. It comprises a group of tumors of different lineage and biological behavior.About half of TC are driven by an acquired activating mutation inthe BRAF oncogene. While targeted therapies have improved outcomes in melanoma patients, most TC patients become resistant orrecur suggesting that new or additive non-cross-reactive therapiesare needed. We have previously shown that PKCa mediates TSHand thyroid hormones proliferative effects in TC. Recent evidenceindicates that together PKCa overexpression and BRAF mutationshould contribute to tumorigenesis and resistance to anticancertherapies. We found that by inhibiting BRAF expression with RNAi inanaplastic TC cells with BRAF mutation, PKCa expression decreases as well, suggesting that the latter is found downstream of BRAF.Furthermore, a decrease in the expression of the cell proliferationmarker PCNA was observed in BRAF-depleted cells by westernblot analysis. Also, TC cells were sensitive to increasing doses ofthe BRAF inhibitor widely used in the clinic vemurafenib/PLX4032in a dose-dependent manner (p<0.0001) by Cell Titer Blue (CTB)assay. To begin to study the combined inhibition of PKC and BRAF,CTB assays were performed with increasing doses of vemurafenibin presence or absence of the PKC inhibitor GF109203X at selective concentrations in follicular TC cells carrying BRAF mutation. Weconfirmed the dose-dependency of vemurafenib and found that thecombination leads to a significant decrease in cell viability (p<0.5).Our results establish that the effective dual PKCa and BRAF blockade can significantly drive tumor proliferation inhibition. The results obtained could provide new therapeutic targets and alternatives tothe treatments currently used for this disease. Despite its increasingincidence and mortality in many cases, TC constitutes a very poorlystudied area in our country.Fil: Campos Haedo, Mateo Nicolas. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Díaz Flaqué, María Celeste. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Díaz Albuja, Johanna Abigail. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Perona, Marina. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Debernardi, Maria Mercedes. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Cayrol, Maria Florencia. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Barreiro Arcos, María Laura. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Sterle, Helena Andrea. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Juvenal, Guillermo Juan. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cremaschi, Graciela Alicia. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rosemblit, Cinthia. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaLXV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LXVIII Reunión Anual de la Sociedad Argentina de Inmunología y Reunión Anual de la Sociedad Argentina de FisiologíaArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de FisiologíaSociedad Argentina de Inmunologí

Detection of Maedi-Visna Virus from Sheep Bronchoalveolar Lavage by Nested PCR Evaluation of Different Primers Pairs

Background: Small ruminant lentiviruses (SRLV) are characterized by a high degree of genetic variability related to your replication process, resulting in several strains in different geographic regions. The Polymerase Chain Reaction (PCR) is very successful in the detection of proviral DNA of SRLV, however, due to the high variability of the lentivirus genome, the efficiency and sensibility of PCR depends mainly on the specificity of the primers designed and the choice of the amplified target viral region. The aim of this study was to compare detection of Maedi Visna Virus (MVV) from bronco alveolar lavage samples of sheep by Nested PCR using primers for the gag and LTR genes.Materials, Methods & Results: Samples of sheep bronchoalveolar lavage (n = 58) from slaughterhouse in the Metropolitan Region of Fortaleza were previously tested by nested PCR using primers for region gag. Thereafter, these samples were tested by nested PCR with primers designed for the LTR region. Both tests were conducted using thermocycler (Biocycler®) under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing of primers at 56°C for 1 min and extension of DNA at 72°C for 45 s with a final extension at 72 for 7 min. The first and second round were performed under the same conditions. Every amplification was performed using a positive control MVV-K1514 and water RNA/DNA free with a negative control. After the amplification, the PCR products were separated by agarose gel electrophoresis at 1% stained with ethidium bromide in TBE buffer. The tests revelead only 1 sample (P1) was detected exclusively for the primer of gag gene, while 8 samples were positive only for the test performed with primers of the LTR region, 5 samples were positive for both sets of primers tested and 30 samples were negative for all tests. The test with the LTR gene demonstrated 37.93% (22/58) positives of Maedi Visna in the samples studied.Discussion: In recent years, with advances in molecular biology techniques, some PCR protocols have been developed for the diagnosis of SRLV. However, these viruses exhibit a high instability and mutation rate becomes very difficult to use the same primers in different geographic regions. In this study, comparing the MVV detection capability by nested PCR with differents primer sets was possible to demonstrate that primers LTR gene were more efficient in detecting positive animals when compared with the primers designed for the gag region. In all tests, only the animal (P1) was positive for the nested PCR performed with the primers for the gag gene, not being detected by the LTR gene. Some studies suggest success in the detection of MVV using primers for the gag gene. However, for more efficient detection of MVV in sheep samples, many studies have shown that the choice of primers for the LTR region is more accurate, since these primers have better MVV detection capability even when it has a large range of circulating virus strains. it is known that the genetic diversity of SRLV generate difficulties in carrying out molecular tests, since the molecular diagnostic tests depend on factors such as the percentage of identity of nucleotides of the viral populations circulating in the herds and the sequences used for testing. In this study it is possible to conclude that the effective control of lentiviruses diagnostic methods should be chosen properly in order to be applied in disease control programs

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF