Toward discovering a novel family of peptides targeting neuroinflammatory states of brain microglia and astrocytes.

Microglia are immune-derived cells critical to the development and healthy function of the brain and spinal cord, yet are implicated in the active pathology of many neuropsychiatric disorders. A range of functional phenotypes associated with the healthy brain or disease states has been suggested from in vivo work and were modeled in vitro as surveying, reactive, and primed sub-types of primary rat microglia and mixed microglia/astrocytes. It was hypothesized that the biomolecular profile of these cells undergoes a phenotypical change as well, and these functional phenotypes were explored for potential novel peptide binders using a custom 7 amino acid-presenting M13 phage library (SX7) to identify unique peptides that bind differentially to these respective cell types. Surveying glia were untreated, reactive were induced with a lipopolysaccharide treatment, recovery was modeled with a potent anti-inflammatory treatment dexamethasone, and priming was determined by subsequently challenging the cells with interferon gamma. Microglial function was profiled by determining the secretion of cytokines and nitric oxide, and expression of inducible nitric oxide synthase. After incubation with the SX7 phage library, populations of SX7-positive microglia and/or astrocytes were collected using fluorescence-activated cell sorting, SX7 phage was amplified in Escherichia coli culture, and phage DNA was sequenced via next-generation sequencing. Binding validation was done with synthesized peptides via in-cell westerns. Fifty-eight unique peptides were discovered, and their potential functions were assessed using a basic local alignment search tool. Peptides potentially originated from proteins ranging in function from a variety of supportive glial roles, including synapse support and pruning, to inflammatory incitement including cytokine and interleukin activation, and potential regulation in neurodegenerative and neuropsychiatric disorders.

Biomaterials and glia: Progress on designs to modulate neuroinflammation.

Microglia are multi-functional cells that play a vital role in establishing and maintaining the function of the nervous system and determining the fate of neurons following injury or neuropathology. The roles of microglia are diverse and essential to the capacity of the nervous system to recover from injury, however sustained inflammation can limit recovery and drive chronic disease processes such as neurodegenerative disorders. When assessing implantable therapeutic devices in the central nervous system, an improved lifetime of the implant is considered achievable through the attenuation of microglial inflammation. Consequently, there is a tremendous underexplored potential in biomaterial and engineered design to modulate neuroinflammation for therapeutic benefit. Several strategies for improving device compatibility reviewed here include: biocompatible coatings, improved designs in finer and flexible shapes to reduce tissue shear-related scarring, and loading of anti-inflammatory drugs. Studies about microglial cell cultures in 3D hydrogels and nanoscaffolds to assess various injuries and disorders are also discussed. A variety of other microglia-targeting treatments are also reviewed, including nanoparticulate systems, cellular backpacks, and gold plinths, with the intention of delivering anti-inflammatory drugs by targeting the phagocytic nature of microglia. Overall, this review highlights recent advances in biomaterials targeting microglia and inflammatory function with the potential for improving implant rejection and biocompatibility studies. STATEMENT OF SIGNIFICANCE: Microglia are the resident immune cells of the central nervous system, and thus play a central role in the neuroinflammatory response against conditions than span acute injuries, neuropsychiatric disorders, and neurodegenerative disorders. This review article presents a summary of biomaterials research that target microglia and other glial cells in order to attenuate neuroinflammation, including but not limited to: design of mechanically compliant and biocompatible stimulation electrodes, hydrogels for high-throughput 3D modelling of nervous tissue, and uptake of nanoparticle drug delivery systems. The goal of this paper is to identify strengths and gaps in the relevant literature, and to promote further consideration of microglia behaviour and neuroinflammation in biomaterial design.

Induced Neural Differentiation of MMP-2 Cleaved (RADA)4 Drug Delivery Systems.

(RADA)4 self-assembling peptides (SAPs) are promising for neural nanoscaffolds with on-demand drug delivery capabilities due to their automated synthesis, in-situ assembly, and potential for interaction with and release of biomolecules. Neuroinflammation cued on-demand drug release, due to up-regulated proteases, may well be vital in the treatment of several neurological diseases. In these conditions, releasing neurotrophic growth factors (NTFs) could potentially lead to neuroprotection and neurogenesis. As such, (RADA)4 was made with the high and low activity matrix metalloproteinase 2 (MMP-2) cleaved sequences, GPQG+IASQ (CP1) and GPQG+PAGQ (CP2), the brain-derived NTF secretion stimulating peptide MVG (DP1) and the ciliary NTF analogue DGGL (DP2). PC-12 cell culture was performed to assess bioactive substrate cell adhesion and NTF specific neuronal differentiation. The laminin-derived IKVAV peptide, known for neural cell attachment and interaction, was tethered to (RADA)4-IKVAV and mixed in increasing increments with (RADA)4 for this purpose. With 1 nanomolar MMP-2 treatment, product formation was observed to increase over a three day period, with (RADA)4/(RADA)4-CP1/CP2 mixture, however there was little difference between groups. Smaller CP1/CP2 concentrations displayed comparable (RADA)4 nanoscale morphology to higher concentrations. Acetylcholine esterase and neural differentiation was observed over 3 days with 1 nM MMP-2 treatment according to the following makeup: 8/1/1 (RADA)4/(RADA)4-IKVAV/(RADA)4-CP1/CP2-DP1/DP2. Signalling gradually increased in all groups, and neurite outgrowth was visible after three days.

Neural tissue engineering: Bioresponsive nanoscaffolds using engineered self-assembling peptides.

UNLABELLED: Rescuing or repairing neural tissues is of utmost importance to the patient's quality of life after an injury. To remedy this, many novel biomaterials are being developed that are, ideally, non-invasive and directly facilitate neural wound healing. As such, this review surveys the recent approaches and applications of self-assembling peptides and peptide amphiphiles, for building multi-faceted nanoscaffolds for direct application to neural injury. Specifically, methods enabling cellular interactions with the nanoscaffold and controlling the release of bioactive molecules from the nanoscaffold for the express purpose of directing endogenous cells in damaged or diseased neural tissues is presented. An extensive overview of recently derived self-assembling peptide-based materials and their use as neural nanoscaffolds is presented. In addition, an overview of potential bioactive peptides and ligands that could be used to direct behaviour of endogenous cells are categorized with their biological effects. Finally, a number of neurotrophic and anti-inflammatory drugs are described and discussed. Smaller therapeutic molecules are emphasized, as they are thought to be able to have less potential effect on the overall peptide self-assembly mechanism. Options for potential nanoscaffolds and drug delivery systems are suggested. STATEMENT OF SIGNIFICANCE: Self-assembling nanoscaffolds have many inherent properties making them amenable to tissue engineering applications: ease of synthesis, ease of customization with bioactive moieties, and amenable for in situ nanoscaffold formation. The combination of the existing knowledge on bioactive motifs for neural engineering and the self-assembling propensity of peptides is discussed in specific reference to neural tissue engineering.

Brain biocompatibility and microglia response towards engineered self-assembling (RADA)4 nanoscaffolds.

(RADA)4-based nanoscaffolds have many inherent properties making them amenable to tissue engineering applications: ease of synthesis, ease of customization with bioactive moieties, and amenable for in situ nanoscaffold formation. There is a dearth in the literature on their biocompatibility in brain tissues; where the glia response is key to regulating the local host response. Herein, nanoscaffolds composed of (RADA)4 and (RADA)4-IKVAV mixtures were evaluated in terms of their effect on primary microglia in culture and general tissue (in vivo) biocompatibility (astrocyte and migroglia). Laminin-derived IKVAV peptide was chosen to promote beneficial cell interaction and attenuate deleterious glial responses. Microglia remained ramified when cultured with these nanoscaffolds, as observed using TNF-α and IL-1ß, NO, and proliferation assays. Evidence suggests that cultured microglia phagocytise the matrix whilst remaining ramified and viable, as shown visually and metabolically (MTT). Nanoscaffold intracerebral injection did not lead to microglia migration or proliferation, nor were glial scarring and axonal injury observed over the course of this study. IKVAV had no affect on microglia activation and astrogliosis. (RADA)4 should be advantageous for localized injection as a tuneable-platform device, which may be readily cleared without deleterious effects on tissue-resident microglia. STATEMENT OF SIGNIFICANCE: Self-assembling nanoscaffolds have many inherent properties making them amenable to tissue engineering applications: ease of synthesis, ease of customization with bioactive moieties, and amenable for in situ nanoscaffold formation. A dearth of literature exists on their biocompatibility in brain tissues; where the glia response is key to regulating the local host response. Herein, nanoscaffolds composed of the peptides (RADA)4 and (RADA)4-IKVAV mixtures were evaluated in terms of their effect on microglia cells in culture and general tissue (in vivo) biocompatibility (astrocyte and migroglia). Laminin-derived IKVAV peptide was chosen to promote beneficial cell interaction and attenuate deleterious glial responses. (RADA)4 nanoscaffolds showed no adverse effect from these cell types and should be advantageous for localized injection as a tuneable-platform device.

Faecal dimeric M2 pyruvate kinase in colorectal cancer and polyps correlates with tumour staging and surgical intervention.

OBJECTIVE & METHOD: A dimeric form of pyruvate kinase isoenzyme (tumour M2-PK) is predominantly found in highly proliferating cells. Sandwich ELISA with monoclonal antibodies against dimeric (tumour) M2-PK was used to measure faecal tumour M2-PK in; 13 controls, 10 patients with colonic polyps and 32 patients with colorectal cancer. RESULTS: Levels of faecal tumour M2-PK were higher in patients with colorectal cancer (median 11.72 U/ml; range 0.9-146.95 U/ml, P = 0.0001) and polyps greater than 10 mm (median 2.54 U/ml; range 0.9-29.46 U/ml, P = 0.041) when compared with controls (median 1.75 U/ml; range 0.9-3.41 U/ml). Furthermore, levels were higher in stages Duke's B (P = 0.013) and Duke's C (P = 0.43) than in Duke's A. Six months postsurgery faecal tumour M2-PK levels fell significantly to 3.46 U/ml (range 1.03-9.05 U/ml, P = 0.001). The sensitivity of a positive faecal tumour M2-PK test, defined as a level above 3.33 U/ml, was 91% for colorectal cancer, 60% for >10 mm and 20% for <10 mm polyps, with a specificity of 92%. CONCLUSION: Faecal tumour M2-PK is a highly sensitive marker for colorectal cancer and larger polyps. It also correlates with more advanced stages of colorectal cancer and its reduction is associated with successful surgical intervention.

The outcome of surgery in fulminant Clostridium difficile colitis.

BACKGROUND: The clinical presentation of Clostridium difficile infection ranges from asymptomatic carriage, colitis with or without pseudomembranes, to fulminant colitis. Although not common, fulminant C. difficile colitis can result in bowel perforation and peritonitis with a high mortality rate. Colectomy is often indicated in these cases. METHODS: We retrospectively analysed the outcome of 14 patients who underwent surgery for fulminant C. difficile colitis in the period 1996-2003 in our Unit. RESULTS: The indications for surgery were systemic toxicity and peritonitis (n = 10), radiological and clinical evidence of progressive toxic colonic dilatation (n = 3) and progressive colonic dilatation with bowel perforation (n = 1). C. difficile infection as the cause of colitis was diagnosed pre-operatively in seven (50%) patients, six of whom underwent a total colectomy and one a right hemicolectomy. Overall mortality in our series was 35.7%. Total colectomy was associated with a lower mortality rate of 11.1% (1/9) when compared with left hemicolectomy was 100% (4/4) (P = 0.01). One patient who underwent a right hemicolectomy (on the basis of deceptively normal external appearance of the rest of the colon intra-operatively) survived after a prolonged hospital stay. CONCLUSIONS: Early or pre-operative microbiological diagnosis of C. difficile infection can be difficult in patients with a fulminant presentation. Those patients with C. difficile colitis, who develop signs of toxicity, peritonitis or perforation, should undergo a total colectomy as the operation of choice.

The metabolic marker tumour pyruvate kinase type M2 (tumour M2-PK) shows increased expression along the metaplasia-dysplasia-adenocarcinoma sequence in Barrett's oesophagus.

BACKGROUND: Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK). In tumours cells, M2-PK usually exists in dimeric form (tumour M2-PK), causing the accumulation of glycolytic phosphometabolites, which allows cells to invade areas with low oxygen and glucose concentrations. AIMS: To investigate the expression of tumour M2-PK during the metaplasia-dysplasia-adenocarcinoma sequence of Barrett's oesophagus, and to assess the prognostic usefulness of tumour M2-PK in oesophageal cancer. MATERIALS/METHODS: One hundred and ninety cases selected from the histopathology archives as follows: 17 reflux oesophagitis, 37 Barrett's oesophagus, 21 high grade dysplasia, 112 adenocarcinomas, and three control tumours. Sections were stained immunohistochemically with antibody to tumour M2-PK. RESULTS: Tumour M2-PK was expressed in all cases, and increased cytoplasmic expression was seen with progression along the metaplasia-dysplasia-adenocarcinoma sequence. All cases of adenocarcinoma showed 100% staining so that tumour M2-PK was not a useful prognostic marker. CONCLUSIONS: Tumour M2-PK is not a specific marker of Barrett's adenocarcinoma, but may be important as a marker of transformed and highly proliferating clones during progression along the metaplasia-dysplasia-adenocarcinoma sequence.

High-dose diclofenac for postoperative analgesia after elective caesarean section in regional anaesthesia.

Although the use of non-steroidal anti-inflammatory drugs (NSAIDs) is well established in the postoperative setting, their use after caesarean sections is still controversial. In a randomised, double-blinded, placebo controlled study we have estimated the opioid-sparing effect of diclofenac suppositories after elective caesarean sections in spinal anaesthesia. Eighty-two women ASA class I or II scheduled for caesarean section were randomised to receive either diclofenac suppositories 100 mg or placebo every 12 h after the operation. The diclofenac group (n = 40) consumed significantly less morphine in the postoperative period (14.0 +/- 1.5 mg in 32 h) compared with the placebo group (21.5 +/- 1.6 mg in 32 h, P < 0.05). The average level of postoperative pain as estimated by a visual analogue scale (VAS) and a verbal scale tended to be lower in the diclofenac group, but this was not significant. There were no differences in demographic data, perioperative bleeding, side-effects or discharge time between the groups. Diclofenac suppositories 100 mg given twice daily after caesarean section are opioid sparing.

Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3' mismatches in forward and reverse primers.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system using primers with mismatches at the 3' ends was developed to determine polymorphisms in IL-10 promoter region. Three previously described biallelic polymorphisms in IL-10 were linked in a 12 reaction PCR-SSP system and the method used to provide genotype data on 233 UK and 166 Polish controls. There are eight possible polymorphic combinations in IL-10 promoter gene but only three were observed in both control groups. Population frequencies of IL-10 genotypes show, in contrast to HLA, that UK and Polish frequencies are remarkably similar.

Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies.

The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF alpha), lymphotoxin-alpha (LT alpha) and interleukin-10 (IL-10) genes on stimulated TNF alpha and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn's disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF alpha, LT alpha and IL-10 genes. Production of the TNF alpha and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF alpha gene, three haplotypes of LT alpha and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT alpha-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT alpha-2 haplotypes were associated with higher TNF alpha production in CD patients, and the TNF-4 haplotype was associated with lower TNF alpha production in UC patients. The A allele in the IL-10 promoter region at position -1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF alpha, LT alpha and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls.

Is interleukin-6 important in inflammatory bowel disease?

The IL-6 gene maps to an area of chromosome 7 known to be significant for susceptibility to inflammatory bowel disease. The functional effects of interleukin-6 (IL-6) polymorphisms in the 4th intron and in the 3' flanking region of IL-6 gene were studied in 192 inflammatory bowel disease patients and healthy subjects. A polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to determine a G to A polymorphism (* at position 4470 in intron 4 of IL-6 gene). Four alleles in the 3' flanking region were studied using a variable number of tandem repeats PCR (VNTR-PCR) amplification. Production of IL-6 was measured in lipopolysaccharide (LPS) stimulated whole blood samples by an enzyme-linked immunosorbent assay (ELISA). A modest increase in the frequency of the IL-6*G allele was noted in Crohn's disease (CD) patients (50%) and ulcerative colitis (UC) patients (46.1%) as compared to controls (39.8%, P = 0.025). We were unable to find any significant functional effect of the IL-6 polymorphisms tested on IL-6 protein production. We postulate that the IL-6 polymorphisms investigated here may be in linkage disequilibrium with a susceptibility gene and that they may be utilised as genetic markers.

Diagnosis and treatment of a fractured third phalanx in a Masai giraffe (Giraffe camelopardalis tippelskirchi).

A 10-yr-old male Masai giraffe (Giraffe camelopardalis tippelskirchi) presented with acute right forelimb lameness. Radiographs revealed a fracture of the medial claw of the distal phalanx penetrating into the distal interphalangeal joint. The giraffe was sedated while it was standing in a chute, and a wooden "hoof block" was applied to the lateral claw of the same limb. The animal was no longer lame 3 days after the procedure. Subsequent treatments included vitamin E, phenylbutazone, and glycosaminoglycans. For 7 wk it was maintained in a small holding yard on packed sand during the day and on deep sand during the night. The hoof block slowly wore down, and at 7 wk, it was placed back on concrete in the evening. At 8 wk, the block had completely worn off and the animal was no longer lame, but radiographs indicated minimal fracture healing. Radiographs performed at 7 mo indicated that there was still a radiolucent zone at the fracture line but calcification was evident at the margins of the fracture.

Bupivacaine 2.5 mg/ml versus bupivacaine 0.625 mg/ml and sufentanil l microg/ml with or without epinephrine 1 microg/ml for epidural analgesia in labour.

We have compared three different methods of epidural analgesia in labour, bupivacaine 2.5 mg/ml (group B), bupivacaine 0.625 mg/ml + sufentanil 1 microg/ml (group BS) and bupivacaine 0.625 mg/ml + sufentanil 1 microg/ml + epinephrine 1 microg/ml (group BSE). One hundred and forty parturients with a singleton fetus with cephalic presentation were randomly allocated to one of the three groups. Group BSE had significantly less pain than groups B and BS. Group B had a significantly higher degree of motor blockade assessed on the Bromage scale. Significantly, more women in group B required urinary bladder catheterization than in the two other groups and they also had significantly less urge to push during active delivery. The incidence of mild pruritus was 18% in group BS and 36% in group BSE. The frequency of instrumental delivery and caesarean section was low (12% and 6.4%, respectively) with no significant differences between the groups. All women were highly satisfied with the method of analgesia and 97% would prefer the same kind of pain alleviation at the next delivery. We conclude that epidural analgesia with low-dose bupivacaine and sufentanil is as good an analgesic method as high-dose bupivacaine. Addition of low-dose epinephrine improves the analgesia.

Phospholamban modulates murine atrial contractile parameters and responses to beta-adrenergic agonists.

Phospholamban gene transcript levels are much lower in murine atria as compared to murine ventricles and this reduced phospholamban expression has been suggested to result in enhanced atrial contractile parameters. To delineate the functional role of phospholamban in murine atrium, the contractile parameters of isolated muscles from phospholamban knockout and cardiac-specific phospholamban overexpression mice along with their isogenic wild-type controls were evaluated. Assessment of the times (ms) to peak tension development and to half-relaxation of developed tension, as well as the rates (mg/s) of tension development and relaxation in paced atrial muscles, revealed that phospholamban ablation was associated with enhanced rates of relaxation with no significant effect on contraction rate, while phospholamban overexpression (three-fold) was associated with depressed rates of both contraction and relaxation. Isoproterenol stimulation resulted in significant increases in the rates of developed tension and relaxation in both phospholamban deficient and phospholamban overexpression atria, indicating that the beta-adrenergic pathway was functional in these muscles. These findings suggest that phospholamban is an important modulator of atrial contractility and its responses to beta-adrenergic agonists.

The presence of anti-neutrophil antibodies reflects clinical and genetic heterogeneity within inflammatory bowel disease.

A detailed investigation of the relationship between anti-neutrophil cytoplasmic antibodies (ANCA) status, HLA genotype, and clinical patterns of inflammatory bowel disease was carried out, involving 236 European patients resident in the United Kingdom [120 had ulcerative colitis (UC), 116 had Crohn's disease (CD)]. ANCA status was determined on coded plasma samples in Los Angeles using a two-stage assay [fixed neutrophil enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence], and HLA genotyping was carried out by polymerase chain reaction. The results provide evidence that ANCA reflect clinical and genetic heterogeneity within the inflammatory bowel diseases. In the UC patients, 78.3% were ANCA positive [64.2 perinuclear (pANCA)], but only 46.5% CD patients were ANCA positive (19.3% pANCA). Furthermore, mean ELISA binding was significantly lower in CD (14.5% +/- 18.8% versus 40.5% +/- 41.0% in UC, p = 2.31 x 10(-9)). Only 15 CD samples, all from patients with colonic disease, displayed ELISA > 20%; and the six CD patients with highest ELISA binding had clinical features very similar to ulcerative colitis. Moreover, in UC, significant relationships between ANCA status and genotype were noted. Thus, 92.7% of patients with the DR3 DQ2 TNF2 haplotype were ANCA positive [p = 0.03 versus DR3 DQ2 TNF2-negative patients (73.9%)]. ELISA binding was increased in DR3 DQ2 TNF2-positive patients (56.0 versus 35.7%, p = 0.02). In this population of UC, ANCA was not associated with DR2, DR4, or clinical pattern. These data emphasize the many factors that need to be considered in genetic marker studies in inflammatory bowel disease. Extensive disease heterogeneity, ethnicity, and methodological differences in ANCA detection are all pertinent.

The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility.

Phospholamban is a regulatory phosphoprotein which modulates the active transport of Ca2+ by the cardiac sarcoplasmic reticular Ca(2+)-ATPase enzyme (SERCA2) into the lumen of the sarcoplasmic reticulum. Phospholamban, which is a reversible inhibitor of SERCA2, represses the enzyme's activity, and this inhibition is relieved upon phosphorylation of phospholamban in response to beta-adrenergic stimulation. In this way, phospholamban is an important regulator of SERCA2-mediated myocardial relaxation during diastole. This report centers on the hypothesis that the relative levels of phospholamban: SERCA2 in cardiac muscle plays an important role in the muscle's overall contractility status. This hypothesis was tested by comparing the contractile parameters of: a) murine atrial and ventricular muscles, which differentially express phospholamban, and b) murine wild-type and phospholamban knock-out hearts. These comparisons revealed that atrial muscles, which have a 4.2-fold lower phospholamban: SERCA2 ratio than ventricular muscles, exhibited rates of force development and relaxation of tension, which were three-fold faster that these parameters for ventricular muscles. Similar comparisons were made via analyses of left-ventricular pressure development recorded for isolated, work-performing hearts from wild-type and phospholamban knock-out mice. In these studies, hearts from phospholamban knock-out mice, which were devoid of phospholamban, exhibited enhanced parameters of left-ventricular contractility in comparison to wild-type hearts. These results suggest that the relative phospholamban: SERCA2 ratio is critical in the regulation of myocardial contractility and alterations in this ratio may contribute to the functional deterioration observed during heart failure.

In vitro and in vivo promoter analyses of the mouse phospholamban gene.

To determine the mechanisms responsible for regulation of the phospholamban (PLB) gene expression, a critical regulatory phosphoprotein in cardiac muscle, the mouse PLB gene was isolated and promoter analysis was performed in vitro and in vivo. The PLB gene consists of two exons separated by a single large intron. Deletion analysis revealed that a 7-kb 5' flanking fragment (including exon 1, the entire intron and part of exon 2) was necessary for maximal transcriptional activity in H9c2 and L6 cell lines. Interestingly, deletion of a 2.4-kb intronic region, which contained repetitive elements, caused a dramatic increase in CAT activity in both these cell lines. In vivo analysis indicated that the PLB fusion gene containing 7 kb of the 5'-flanking region was capable of cardiac specific gene expression in transgenic mice. Furthermore, these mice exhibited 3-fold higher levels of CAT activity in the ventricles compared with the atria, mimicking endogenous PLB mRNA expression. Our findings suggest that: (a) PLB gene expression may be regulated by the interplay of cis-acting regulatory elements located within the 5' flanking and intronic regions; and (b) the 7-kb upstream region is capable of directing cardiac-specific and compartment-specific expression in vivo.

Phospholamban: a prominent regulator of myocardial contractility.

Our understanding of the role of phospholamban in cardiac physiology has evolved over the past two decades to the point where this protein is now understood to be a critical repressor of myocardial contractility. Phospholamban, through its inhibitory effects on the affinity of the cardiac sarcoplasmic reticulum Ca2+ pump for Ca2+, represses both the rates of relaxation and contraction in the mammalian heart. These inhibitory effects can be relieved through (1) phospholamban phosphorylation, (2) down-regulation of phospholamban gene expression, and (3) disruption of the phospholamban-Ca(2+)-ATPase interaction. Thus, genetic approaches and pharmacological interventions, designed to relieve the phospholamban inhibitory action on the cardiac sarcoplasmic reticulum Ca2+ pump and myocardial relaxation, may prove valuable in reversing the effects of several diseases in the mammalian heart. Such interventions could be designed to inhibit the phospholamban phosphatase, stabilize the phosphorylated state of phospholamban, interrupt the phospholamban-Ca(2+)-ATPase interaction, decrease phospholamban transcription, or disrupt phospholamban mRNA stability. Development of such therapeutic strategies to target phospholamban will be an important future goal for the clinical improvement of contractility in the failing heart.

Differential phospholamban gene expression in murine cardiac compartments. Molecular and physiological analyses.

Phospholamban, the regulator of the Ca2+ pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to alpha-myosin heavy chain (alpha-MHC) revealed a 3-fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca(2+)-ATPase (ratio of sarcoplasmic reticulum Ca(2+)-ATPase [SERCA2] to alpha-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40 +/- 0.71 milliseconds for atrium versus 30.58 +/- 2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36 +/- 0.82 milliseconds) compared with ventricular muscle (44.60 +/- 2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments.