Salivary microbial shifting in hypertensive patients with chronic periodontitis after scaling and root surface debridement.

OBJECTIVES: This study aimed to evaluate the impact of scaling and root surface debridement (SRP) on salivary bacterial counts and systolic and diastolic blood pressure in hypertensive patients with chronic periodontitis, with a focus on clinical significance. METHODS: An observational trial included 24 chronic periodontitis patients, eleven of them were hypertensive patients. Non-surgical periodontal treatment was administered to all patients, with clinical parameters including gingival index (GI), plaque index (PI), and probing pocket depth (PPD) recorded. Saliva samples were collected before and after SRP to quantify total bacterial counts and specific bacterial counts. RESULTS: Two months following SRP, PI and PPD in every subject under study demonstrated good responses. In hypertension patients, the salivary bacterial count was significantly higher following SRP (P = 0.0221). The incidence of Porphyromonas gingivalis in hypertension patients significantly decreased after treatment (P = 0.0386). Despite this, there was no discernible decrease in blood pressure following treatment. CONCLUSIONS: SRP alone was ineffective in reducing overall bacterial counts, but P. gingivalis levels responded favorably. Regular periodontal assessment is crucial for hypertensive individuals to mitigate cardiovascular risk. CLINICAL SIGNIFICANCE: Periodontal therapy in hypertensive patients may improve oral health but might not significantly impact blood pressure. Regular periodontal evaluation is essential for managing cardiovascular risk in hypertension.

Type I interferon receptor signalling deficiency results in dysregulated innate immune responses to SARS-CoV-2 in mice.

SARS-CoV-2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID-19). The type I interferon (IFN) pathway is of particular importance for anti-viral defense and recent studies identified that type I IFNs drive early inflammatory responses to SARS-CoV-2. Here, we use a mouse model of SARS-CoV-2 infection, facilitating viral entry by intranasal recombinant Adeno-Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN receptor-1 deficient (Ifnar1-/- ) mice, to study the role of type I IFN signalling and innate immune responses during SARS-CoV-2 infection. Our data show that type I IFN signalling is essential for inducing anti-viral effector responses to SARS-CoV-2, control of virus replication, and to prevent enhanced disease. Furthermore, hACE2-Ifnar1-/- mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as reduced and delayed production of monocyte-recruiting chemokine CCL2. hACE2-Ifnar1-/- mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS-CoV-2 infection, with a shift from Ly6C+ to Ly6C- expressing cells. Together, our findings suggest that type I IFN signalling deficiency results in a dysregulated innate immune response to SARS-CoV-2 infection.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Changes in the level of cytokines in the saliva of hypertensive patients with chronic periodontitis after scaling and root planning.

INTRODUCTION: Cytokines have a key role in the pathogenesis of both hypertension and periodontitis. Salivary diagnosis is a promising field with numerous clinical applications. Since limited studies have been carried out on how salivary inflammatory cytokines can be determined and how well periodontal disease and hypertension might react to scaling and root planning (SRP). The goal of this study was to identify the pattern of changes in salivary inflammatory cytokines in chronic periodontitis subjects with hypertension after nonsurgical periodontal therapy. METHODS: It included observational trial recruited 94 chronic periodontitis patients, 44 of whom had hypertension. All subjects have undergone non- surgical periodontal treatment. The clinical periodontal parameters included gingival index (GI), plaque index (PI), and probing of pocket depth (PPD). Unstimulated saliva was collected to determine the inflammatory biomarkers (using a commercial Elisa kit) both before and after SRP RESULTS: In comparison to non-hypertensive participants, the periodontal PPD was significantly higher in hypertensive subjects. All clinical parameters in the first examination, except for PI, were significantly higher in hypertensive than in non-hypertensive subjects. Plaque Index, GI, and PPD parameters at first visit and after finishing treatment positively correlated with salivary IL-1ß, excluding pretreatment GI. The current results demonstrate the presence of a positive correlation between diastolic blood pressure and TNF (r = 0.330 and P = 0.029). All patients enrolled in this study showed a significant increase in the salivary levels of IL-4 after SRP. CONCLUSIONS: The current study offer important and valuable information concerning the practical application of pro-inflammatory and anti-inflammatory cytokines as useful biomarkers and indicators for determining the outcome of SRP and progression of chronic periodontitis in patients with hypertension.

Exploring nitric oxide as a crucial prognostic biomarker of coronary artery disease.

PURPOSE: The study aimed to examine if the polymorphism of the endothelial nitric oxide synthase (eNOS) gene variable number of tandem repeats (VNTR) and the serum NO levels are associated with CAD. MATERIALS/METHODS: Case-control study, 70 CAD and 30 control subjects were enrolled. The eNOS gene polymorphism was measured by polymerase chain reaction-agarose gel electrophoresis and the serum NO was assessed by using an ELISA plate and reader covering 540 nm. RESULTS: Uncovering the area under curve (AUC) for serum NO, which was (0.6821), indicating that NO seemed to be a critical prognostic biomarker of CAD; also, glucose, serum creatinine and total bilirubin proved to be significant predictors of CAD with AUC (0.6793, 0.6717 and 0.6662) respectively. Furthermore, higher serum NO levels were associated with the eNOS (ab) genotype. Revealing the intron (a) allele was protective against CAD. Moreover, diminished levels of serum NO in CAD groups compared to controls (P < 0.05). Additionally, Multiple logistic regression analysis shows a significantly high Odds ratio associated with CAD in the Duhok population. CONCLUSIONS: The eNOS (ab) variant seems to be a protective CAD factor for patients. Low serum NO levels are another risk factor for the advancement of CAD, suggesting their involvement in atherosclerosis. The (a) allele's protective effect is mediated through changes in eNOS promoter activity and higher NO levels.

Exploring the role of Sirtuin 3 gene polymorphisms and oxidative stress markers in the susceptibility to coronary artery disease.

OBJECTIVE: Coronary artery disease (CAD) is a complex disorder influenced by genetic and environmental factors. This case-control study investigated the association between Sirtuin SIRT3 gene polymorphisms, serum malondialdehyde (MDA) levels, and CAD susceptibility. METHODS: Blood samples were collected from 70 CAD cases and 30 controls at the Cardiac Center, Azadi Teaching Hospital, Duhok, Iraq. Genomic DNA was extracted, and PCR-based allele genotyping determined SIRT3 rs11246029 T/C polymorphisms. Serum MDA levels were measured using ELISA. Statistical analysis included t-tests, Mann-Whitney tests, and Spearman correlations. Odds ratios (OR) with 95% confidence intervals (CI) assessed genotypes/alleles and CAD associations. The accuracy of serum MDA in predicting the severity of CAD was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: There were no significant variations in serum MDA levels between controls and CAD patients in the study. The diagnostic accuracy of serum MDA for CAD severity prediction was modest (Area Under Curve (AUC) = 0.56). Correlations revealed associations between MDA and total bilirubin (negative) and Troponin (positive). CRP correlated positively with LDH, glucose, cholesterol, LDL, CKmB, and Troponin. CKmB and Troponin are positively associated with clinical characteristics. Genotype analysis identified a significantly higher CAD risk with the CC genotype compared to controls. CONCLUSION: These findings shed light on the potential role of SIRT3 gene polymorphisms and serum MDA levels in CAD susceptibility. Further research is needed to understand underlying mechanisms and therapeutic implications based on these markers. TRIAL REGISTRATION: 15092021-9-12. Registered 15 September 2021.

Lung directed antibody gene transfer confers protection against SARS-CoV-2 infection.

BACKGROUND: The COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. AIMS: We hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. METHODS: We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. CONCLUSIONS: A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.

Components of Volatile Fractions from Eucalyptus camaldulensis Leaves from Iraqi-Kurdistan and Their Potent Spasmolytic Effects.

Inhalation of vapors from a hot tea of Eucalyptus camaldulensis Dehnh. leaves is considered by Iraqi-Kurdistan people an effective spasmolytic and antipyretic remedy for the treatment of respiratory diseases. The constituents of volatile fractions isolated by hydrodistillation from dried leaves of the plant collected in Kurdistan were determined by GC-FID and GC-MS analyses. More than 90% components were identified. The most abundant constituents were 1,8-cineole, p-cymene, α-pinene, terpinen-4-ol, aromadendrene, and α-terpineol. The different volatile fractions induced relaxation on rat isolated aortic and tracheal rings in concentration-dependent manner. These effects appeared to be due to a complex interaction between various terpenoid components rather than being only due to the main oil constituent, 1,8-cineole. The KCa channel and the NO pathway were not significantly involved in the relaxation mechanism, while Ca2+ channels played a major role in the spasmolytic effects.

Exploring the therapeutic potential of pomegranate juice for uterine relaxation.

BACKGROUND: The effects of pomegranate juice (PJ) and its components on uterine smooth muscle are unknown. Hence, this study unequivocally demonstrates that pomegranate juice (PJ) significantly impacts myometrial function, providing crucial insights into its relaxant properties and their potential therapeutic applications for uterine-related disorders. RESEARCH DESIGN AND METHODS: Rat uterine smooth muscle horn strips were suspended in Krebs solution organ baths. Contractions were measured isometrically using a transducer (AD instrument Australia). The effects of PJ were evaluated on contractile activity elicited by potassium chloride (KCl 60 Mm) depolarization. Inhibitors of nitric oxide (L-NAME 3 X 10-4), guanylate cyclase (methylene blue 1 X 10-5), and Prostaglandin I2 (indomethacin 3 X 10-5), as well as Potassium Channels blockers, were determined. RESULTS: The juice at concentrations from 1.5-5 mg/ml significantly decreased the rat uterine horn contraction induced by KCl. The NO, cGMP, and PGI2 inhibitors did not block the relaxation response. Furthermore, the PGI2 inhibitor significantly enhanced the relaxation effects; K+ channel blockers had no inhibitory effects on the relaxation responses. Contrarily, GLIB improved considerably relaxation. CONCLUSION: Research suggests pomegranate juice's active ingredient may reduce uterine contractions and treat uterotonic disorders, potentially preventing preterm birth and dysmenorrhea. Further research is needed to determine its mechanism of action. TRIAL REGISTRATION: Code: AEC-013.

Nitric oxide-cyclic GMP role in Ang II induced hyperpolarization in bovine aortic endothelium cell line (BAE-1).

Angiotensin II (Ang II), a mitogen-activated peptide, exerts numerous effects on the cardiovascular system including the regulation of blood pressure. The current study focused on the potential mechanisms that seem to be involved in Ang II vasodilation using bovine aortic endothelial cells (BAE-1) cell lines. Expression of the Ang II receptor (AT2) in BAE-1 was checked by western blots in the presence of valsartan (AT1 inhibitor). To check if Ang II's vasodilator impact was mediated by the nitric oxide (NO) pathway, the Griess reagent was used. Furthermore, cell-attached patch-clamp and fire-polished borosilicate electrodes with a resistance of 3-5 MΩ in the working solutions was used to record membrane currents from treated BAE-1. BEA-1 revealed 50 kDa immunoreactive bands that matched AT2. The concentration of AT2 was elevated in valsartan-treated cells in comparison to control cells. The biochemical experimental data indicated that the NO level increased in a concentration-dependent manner. Meanwhile, Ang II at a concentration of 1 µM, the level of NO increased more than at 100 µM. In patch-clamp experiments, K current and chord conductance were enhanced after incubation of Ang II with valsartan. When 100 µM Ang II was added, the current peaked rapidly and after 15 min of incubation, the maximum value was obtained, as opposed to 10 min and control (110.9 ± 13.3 pA control, 141.4 ± 30.4 pA after 10 min and 174.4 ± 49.3 pA after 15 min). Ang II type two receptor inhibitor (PD1231777) reduced the current and conductance induced by Ang II. The presented data revealed that Ang II released NO via the activation of AT2. K currents were stimulated by Ang II and evoked mainly a current consistent with the activation of K channels.

Genetic and clinical study of myeloperoxidase's association with coronary artery disease.

BACKGROUND: Unraveling myeloperoxidase's (MPO) correlation with coronary artery disease (CAD) and genetic variations, this study seeks to enhance diagnostic precision and therapeutic strategies. RESULTS: CAD patients were found to be older and more male than controls. Several clinical parameters, including glucose, total bilirubin, alkaline phosphatase, creatinine, and troponin levels, showed significant variations. Moreover, CAD patients had lower red cell distribution width (RDW%) and mean platelet volume (MPV) than controls. Serum MPO levels did not differ significantly between CAD patients and controls, and no correlation was found with other clinical parameters except for glucose, creatinine, and total bilirubin. CONCLUSIONS: The data suggest that serum MPO levels are not substantially related to CAD patients, as indicated by lower MPO levels in CAD patients compared to controls. While highlighting the potential of MPV and RDW% as predictors of severe atherosclerosis in CAD. Further research is needed to validate the diagnostic and prognostic value of RDW%, MPV, and MPO levels in CAD. TRIAL REGISTRATION: 15092021-9-12. Registered 15 September 2021.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex.

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100%) and specificity (100%) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection.

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.

Transient prenatal ruxolitinib treatment suppresses astrogenesis during development and improves learning and memory in adult mice.

Ruxolitinib is the first janus kinase 1 (JAK1) and JAK2 inhibitor that was approved by the United States Food and Drug Administration (FDA) agency for the treatment of myeloproliferative neoplasms. The drug targets the JAK/STAT signalling pathway, which is critical in regulating the gliogenesis process during nervous system development. In the study, we assessed the effect of non-maternal toxic dosages of ruxolitinib (0-30 mg/kg/day between E7.5-E20.5) on the brain of the developing mouse embryos. While the pregnant mice did not show any apparent adverse effects, the Gfap protein marker for glial cells and S100ß mRNA marker for astrocytes were reduced in the postnatal day (P) 1.5 pups' brains. Gfap expression and Gfap+ cells were also suppressed in the differentiating neurospheres culture treated with ruxolitinib. Compared to the control group, adult mice treated with ruxolitinib prenatally showed no changes in motor coordination, locomotor function, and recognition memory. However, increased explorative behaviour within an open field and improved spatial learning and long-term memory retention were observed in the treated group. We demonstrated transplacental effects of ruxolitinib on astrogenesis, suggesting the potential use of ruxolitinib to revert pathological conditions caused by gliogenic-shift in early brain development such as Down and Noonan syndromes.

Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment.

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.

Deletion lengthening at chromosomes 6q and 16q targets multiple tumor suppressor genes and is associated with an increasingly poor prognosis in prostate cancer.

Prostate cancer is characterized by recurrent deletions that can considerably vary in size. We hypothesized that large deletions develop from small deletions and that this "deletion lengthening" might have a "per se" carcinogenic role through a combinatorial effect of multiple down regulated genes. In vitro knockdown of 37 genes located inside the 6q12-q22 deletion region identified 4 genes with additive tumor suppressive effects, further supporting a role of the deletion size for cancer aggressiveness. Employing fluorescence in-situ hybridization analysis on prostate cancer tissue microarrays, we determined the deletion size at 6q and 16q in more than 3,000 tumors. 16q and 6q deletion length was strongly linked to poor clinical outcome and this effect was even stronger if the length of both deletions was combined. To study deletion lengthening in cancer progression we eventually analyzed the entire cancers from 317 patients for 6q and 16q deletion length heterogeneity and found that the deletion expanded within 50-60% of 6q and 16q deleted cancers. Taken together, these data suggest continuous "deletion lengthening" as a key mechanism for prostate cancer progression leading to parallel down regulation of genes with tumor suppressive properties, some of which act cooperatively.

Angiogenic and wound healing potency of fermented virgin coconut oil: in vitro and in vivo studies.

OBJECTIVE: The process of wound healing involves activation of keratinocytes, fibroblasts, endothelial cells, etc. Angiogenesis is crucial during the process of wound healing. Virgin coconut oil is widely utilized in South Asia for various purposes including food, medicinal and industrial applications. This study aimed to evaluate the potency of fermented virgin coconut oil (FVCO) in angiogenesis and wound healing via both in vitro and in vivo assays. METHODS: Human umbilical vein endothelial (HUVEC), fibroblast (CCD-18) and retinal ganglion (RGC-5) cells were cultured in medium containing different concentrations of FVCO. The proliferation, migration and morphological changes of cells were determined. The angiogenic effect of FVCO was evaluated by rat aortic assay. The therapeutic effect of FVCO on wound healing was further assessed in a wound excision model in Sprague Dawley rats. The expression of phospho-VEGFR2 (vascular endothelial growth factor receptor 2) in HUVECs was detected by Western blot. RESULTS: FVCO (6 and 12 µg/mL) significantly improved the proliferation of HUVEC, CCD-18 and RGC-5 cells (P < 0.05 or 0.01). FVCO (25 µg/mL) markedly increased the migration ability of CCD-18 and RGC-5 cells (P < 0.05). FVCO did not affect cell morphology as indicated by fluorescein diacetate (FDA), rhodamine 123 and Hoechst staining. FVCO (25, 50 and 100 µg/mL) significantly stimulated the ex vivo blood vessel formation as compared with negative control (P < 0.05). Rats in FVCO group had significantly smaller wound size, higher wound healing percentage, and shorter wound closure time when compared with control group since day 8 (P < 0.05), suggesting that oral FVCO administration notably promoted the wound healing process. FVCO treatment (6 and 12 µg/mL) significantly enhanced the phospho-VEGFR2 expression in HUVECs (P = 0.006 and 0.000, respectively). CONCLUSION: Our study confirms a high angiogenic and wound healing potency of FVCO that might be mediated by the regulation of VEGF signing pathway.

A New Ursane-type Triterpenoid and Other Constituents from the Leaves of Crataegus azarolus var. aronia.

A new ursane-type triterpene acid, named azarolic acid (1), along with four known phenolic compounds and four known triterpene acids, was isolated from the crude EtOAc extract of the leaves of Crataegus azarolus var. aronia L. The structure of 1 was determined from 1D and 2D NMR spectroscopic data. Euscaphic acid showed high anti-vasoconstriction effects on aortic rings, supporting the use of this medicinal plant in cardiovascular disease.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100 percent) and specificity (100 percent) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts.

Type 1 protein phosphatase (PP1) is a negative regulator of cardiac function. However, studies on the status and regulation of sarcoplasmic reticulum (SR)-associated PP1 activity in failing hearts are limited. We studied PP1 activity and protein and mRNA expression of the catalytic subunit of PP1 (PP1C) and protein levels of PP1-specific inhibitors [inhibitor 1 (Inh-1) and inhibitor 2 (Inh-2)] in the left ventricular (LV) myocardium of 6 dogs with heart failure (HF; LV ejection fraction, 23 +/- 2%) and 6 normal dogs. In failing LV tissue, PP1 activity values (expressed as pmol 32P. min-1. mg of noncollagen protein-1) in the homogenate, crude membranes, cytosol, and purified SR were increased by 52, 54, 55, and 72%, respectively. Trypsin treatment released PP1 but not type 2A protein phosphatase from the SR. In the supernatant of trypsin-treated SR, PP1 activity was approximately 24% higher in failing hearts than in normal control hearts. A similar increase in protein expression of PP1C was observed in the nontrypsinized SR. Heat-denatured phosphorylated SR inhibited PP1 activity by 30%, which suggests the presence of Inh-1 or -2 or both in the SR. With the use of a specific antibody, both Inh-1 and -2 proteins were found in the SR; the former was decreased by 56% in the failing SR, whereas the latter did not change. These results suggest that protein phosphatase activity bound to the SR is increased and is predominantly type 1. Increased SR-associated PP1 activity in failing hearts appears to be due partly to increased expression of PP1C and partly to reduced levels of Inh-1 but not Inh-2 protein. Thus inhibition of PP1 activity in the SR appears to be a potential therapeutic target for improving LV function in failing hearts, because it may lead to increased SR Ca2+ uptake, which is impaired in failing hearts.