Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10µmol/L) and the Akt inhibitor V (20µmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

Daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), for the treatment of moderately to severely active ulcerative colitis: a randomised, double blind, placebo controlled, dose ranging trial.

BACKGROUND: An uncontrolled pilot study demonstrated that daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), might be effective for the treatment of active ulcerative colitis. METHODS: A randomised, double blind, placebo controlled trial was conducted to evaluate the efficacy of daclizumab induction therapy in patients with active ulcerative colitis. A total of 159 patients with moderate ulcerative colitis were randomised to receive induction therapy with daclizumab 1 mg/kg intravenously at weeks 0 and 4, or 2 mg/kg intravenously at weeks 0, 2, 4, and 6, or placebo. The primary end point was induction of remission at week 8. Remission was defined as a Mayo score of 0 on both endoscopy and rectal bleeding components and a score of 0 or 1 on stool frequency and physician's global assessment components. Response was defined as a decrease from baseline in the Mayo score of at least 3 points. RESULTS: Two per cent of patients receiving daclizumab 1 mg/kg (p = 0.11 v placebo) and 7% of patients receiving 2 mg/kg (p = 0.73) were in remission at week 8, compared with 10% of those who received placebo. Response occurred at week 8 in 25% of patients receiving daclizumab 1 mg/kg (p = 0.04) and in 33% of patients receiving 2 mg/kg (p = 0.30) versus 44% of those receiving placebo. Daclizumab was well tolerated. The most frequently reported adverse events in daclizumab treated patients compared with placebo treated patients were nasopharyngitis (14.6%) and pyrexia (10.7%). CONCLUSION: Patients with moderate ulcerative colitis who are treated with daclizumab are not more likely to be in remission or response at eight weeks than patients treated with placebo.

Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitor improves left ventricular function and alters the pattern of left ventricular dilatation after myocardial infarction.

Background- Left ventricular (LV) remodeling after myocardial infarction (MI) is associated with fibrosis, dilatation, and dysfunction. We postulated that prevention of fibrosis after MI with a prolyl 4-hydroxylase inhibitor (P4HI) would preserve LV function and attenuate LV enlargement. Methods and Results- Adult female rats (200 to 250 g) had experimental MI and were then randomized to treatment with P4HI (MI-FG041, n=29) or vehicle (MI-control, n=29) 48 hours after MI for 4 weeks in 2 phases. Echocardiograms were performed weekly with a 15-MHz linear transducer, and at 4 weeks, collagen isoform determinations and in vivo hemodynamics were performed. At randomization, the infarct size and LV function and size were similar in MI-FG041 and MI-control but significantly different from shams (n=9). At week 4, the LV function in MI-FG041 was significantly better than in MI-controls (fractional shortening 21% versus 16%, P=0.01; fractional area change 30% versus 19%, P=0.002; ejection fraction 35% versus 23%, P=0.001). In the FG041 group, LV area in systole was less (P<0.05), the dP/dt(max) after isoproterenol was higher (P<0.05), and types I and III collagen in noninfarcted LV were less than in MI-control. The hydroxyproline/proline ratio was increased by 64% in MI-control and reduced to the sham value in MI-FG041 rats. In the scar tissue, it was reduced by 24% in MI-FG041. Conclusions- This study demonstrates that prevention of interstitial fibrosis with a P4H inhibitor alters the pattern of LV enlargement and produces partial recovery of LV function after MI.

Effects of a motilin receptor agonist (ABT-229) on upper gastrointestinal symptoms in type 1 diabetes mellitus: a randomised, double blind, placebo controlled trial.

INTRODUCTION: Erythromycin, a motilin agonist, is a potent prokinetic. ABT-229 is a specific motilin agonist that dose dependently accelerates gastric emptying. Dyspepsia and gastroparesis are common problems in type 1 diabetes mellitus. We aimed to evaluate the efficacy of ABT-229 in symptomatic diabetic patients with and without delayed gastric emptying. METHODS: Patients with type 1 diabetes and postprandial symptoms were randomised (n=270). Based on a validated C(13) octanoic acid breath test, patients were assigned to either the delayed or normal gastric emptying strata. Patients received one of four doses of ABT-229 (1.25, 2.5, 5, or 10 mg twice daily before breakfast and dinner) or placebo for four weeks following a two week baseline. A self report questionnaire measured symptoms on visual analogue scales; the primary outcome was assessment of change in the total upper abdominal symptom severity score (range 0-800 mm) from baseline to the final visit. RESULTS: The treatment arms were similar regarding baseline characteristics. There was symptom improvement on placebo and a similar level of improvement on active therapy for the upper abdominal discomfort severity score (mean change from baseline -169, -101, -155, -143, and -138 mm for placebo, and 1.25, 2.5, 5, and 10 mg ABT-229, respectively, at four weeks by intent to treat). The results were not significantly different in those with and without delayed gastric emptying. The severity of bloating, postprandial nausea, epigastric discomfort, heartburn, and acid regurgitation worsened dose dependently in a greater number of patients receiving ABT-229 than placebo. Overall, 63% of patients on placebo reported a good or excellent global response, and this was not different from the active treatment arms. CONCLUSIONS: The motilin agonist ABT-229 was not efficacious in the relief of postprandial symptoms in diabetes mellitus in the presence or absence of delayed gastric emptying.

Prevention of pancreatitis in patients with idiopathic recurrent pancreatitis: a prospective nonblinded randomized study using endoscopic stents.

BACKGROUND AND STUDY AIMS: Currently there is no available therapy to prevent attacks of acute pancreatitis in patients with idiopathic recurrent pancreatitis (IRP). This randomized, nonblinded prospective, controlled trial was undertaken to evaluate the effectiveness of pancreatic duct stents in preventing attacks of pancreatitis in IRP. PATIENTS AND METHODS: During a 5-year period 34 patients met the diagnostic criteria for IRP. Patients were randomly assigned to one of two groups; 19 patients (14 women, 5 men, mean age 44) to the pancreatic stent group; and 15 patients (10 women, five men, mean age 47) to the control group. The stent group received three stents over a period of 1 year and the control group had selective pancreatograms but no stent. Mean follow-up was 33 months (range 13-77) and 35 months (range 10-78) in the stent and control groups, respectively. Episodes of pancreatitis, frequency and intensity of pain requiring emergency room visits, and hospitalizations were recorded. RESULTS: Recurrence of pancreatitis occurred in eight out of 15 patients (53%) in the control group, but in only two our of 19 patients (11%) in the stent group (P<0.02). Two patients in the control group who had recurrences of pancreatitis crossed over to stent therapy and had no further pancreatitis thereafter. Six patients each, 32% and 40% in the stent and control groups respectively, continued to have pancreatic type pain. In the study period 17 stents were occluded and 14 migrated out. CONCLUSION: The results of this study suggest that pancreatic duct stenting may prevent recurrent attacks of pancreatitis in IRP patients. Intermittent pancreatic duct sphincter dysfunction or relative outlet obstruction may be the underlying cause for the recurrent attacks of pancreatitis.

PKC-beta is not necessary for cardiac hypertrophy.

Studies in human and rodent models have shown that activation of protein kinase C-beta (PKC-beta) is associated with the development of pathological hypertrophy, suggesting that ablation of the PKC-beta pathway might prevent or reverse cardiac hypertrophy. To explore this, we studied mice with targeted disruption of the PKC-beta gene (knockout, KO). There were no detectable differences in expression or distribution of other PKC isoforms between the KO and control hearts as determined by Western blot analysis. Baseline hemodynamics were measured using a closed-chest preparation and there were no differences in heart rate and arterial or left ventricular pressure. Mice were subjected to two independent hypertrophic stimuli: phenylephrine (Phe) at 20 mg x kg(-1) x day(-1) sq infusion for 3 days, and aortic banding (AoB) for 7 days. KO animals demonstrated an increase in heart weight-to-body weight ratio (Phe, 4.3 +/- 0.6 to 6.1 +/- 0.4; AoB, 4.0 +/- 0.1 to 5.8 +/- 0.7) as well as ventricular upregulation of atrial natriuretic factor mRNA analogous to those seen in control animals. These results demonstrate that PKC-beta expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response.

Angiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats.

Recently, the calcium-calmodulin-dependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and betaMHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in AngII treated rats, suggesting that the calcium-calmodulin-dependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts.

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.

Collagenase degrades collagen in vivo in the ischemic heart.

Previously, we showed that ischemic rat heart contains an activated procollagenase capable of degrading collagen in vitro. We now demonstrate that the collagen resident in such hearts (in vivo) also becomes degraded, producing characteristic fragments implicating the action of an activated collagenase. The evidence is the appearance of amino-terminal dansyl-Ile (+dansyl-Leu) residues in pepsin digests of re-oxygenated rat hearts and immunoblots showing 3/4 length (alphaA) fragments from type I collagen. Also, in ischemic rat myocardium, alphaA(I) and alphaA(III) fragments were detected in pepsin digests. The time periods required for the cleavage and degradation of collagen suggest the participation of a procollagenase that becomes activated. Results demonstrate for the first time that an interstitial collagenase in such hearts initiates in vivo degradation of types I and III collagens.

Clinical presentation and short-term outcome of endoscopic therapy of patients with symptomatic incomplete pancreas divisum.

BACKGROUND: The clinical significance of incomplete pancreas divisum (IPD) has not been fully described. In this study we report the clinical presentation and results of endoscopic treatment of the 32 (0.6%) patients with IPD seen at our center over a 10-year period. METHODS: The study population consisted of 24 women and 8 men (mean age 42 years, range 13 to 82 years). Ten (31%) patients presented with acute recurrent pancreatitis, 5 (16%) with chronic pancreatitis, and 3 (9%) with pancreatic type pain. Detailed history, laboratory tests, US, CT, and ERCP excluded other etiologies for their symptoms. The remaining 14 (44%) presented with biliary problems. The 18 symptomatic patients with IPD were treated as follows: 8 received dorsal duct stents, 3 underwent minor papilla endoscopic sphincterotomy and dorsal duct stent placement, 4 had minor papilla dilatation only, and 3 had ventral duct stents placed. RESULTS: Patients were then followed for recurrence of pancreatitis and pancreatic-type pain. Mean follow-up was 15.5 months (range 3 to 30 months). Six (60%) of the patients with acute recurrent pancreatitis and 4 (80%) with chronic pancreatitis benefitted from the endoscopic therapy. However, only 1 (33%) of the patients with pancreatic-type pain benefitted. CONCLUSION: The clinical presentation and response to endoscopic therapy of patients with ICP appeared to be similar to that of patients with complete pancreas divisum.

beta-adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy.

To establish whether catecholamines per se in the absence of significant increases in systolic load induce myocardial damage via apoptosis, rats were treated with vehicle or isoproterenol (400 microg . kg-1 . h-1). Apoptotic cardiocytes (Apo) were identified in paraffin-embedded sections using terminal deoxynucleotide transferase-mediated dUTP nick end labeling. Results were confirmed using an independent ligase assay. Systolic blood pressures were comparable in isoproterenol-treated and control rats. Twenty-four hours of treatment with isoproterenol resulted in significant numbers of Apo compared with control [7.9 +/- 2.5 vs. 0.3 +/- 0.3 (SE) cm-2, P < 0.05]. A cohort of animals was subjected to ventricular pacing to induce a tachycardia equivalent to that induced by isoproterenol, and these animals did not show an increase in Apo. The left ventricular hypertrophy induced by 2 wk of abdominal aortic banding also increased Apo ( approximately 7. 2-fold); however, 24 h of isoproterenol infusion did not induce additional Apo in these rats. Thus catecholamines, in the absence of altered systolic load, induce Apo which is not mediated solely by tachycardia. Left ventricular hypertrophy secondary to abdominal aortic banding is associated with Apo, but this does not increase sensitivity to isoproterenol-induced Apo.

The role of surveillance endoscopic retrograde cholangiopancreatography in preventing episodic cholangitis in patients with recurrent common bile duct stones.

BACKGROUND AND STUDY AIMS: Approximately 2-7% of patients who have undergone previous removal of bile duct stones have recurrence often presenting as ascending cholangitis. The aim of this study was to identify the incidence, clinical presentation, and objective findings in this group of patients. Additionally, the effect of surveillance endoscopic retrograde cholangiopancreatography (ERCP) in preventing cholangitis, was studied. PATIENTS AND METHODS: Two thousand and ninety-six patients who underwent ERCP for cholelithiasis were studied with 45 of these patients being identified as having recurrent common bile duct stones. Of the 45, 13 had two or more recurrences without having any obvious predisposing factors. The mean age of the 13 patients was 57 years. The characteristics of 13 patients were reviewed, including sphincterotomy size, liver function tests, and contrast drainage time. RESULTS: All 13 patients with recurrent stones presented with ascending cholangitis. Stones were found to be soft, brown and accompanied by a large amount of sludge. The common bile duct in all 13 patients was noted to be dilated and had notable, widely patent sphincterotomes. There was significant delayed drainage in 77% of these patients. Yearly surveillance ERCPs were performed in the 13 patients, the incidence of acute cholangitis episodes per patient decreased from 2 to 0.6 with a four-year follow-up. CONCLUSION: In a subgroup of patients with multiple common bile duct stone recurrences, annual surveillance ERCP with stone removal decreases the incidence of recurrent episodes of ascending cholangitis as well as its associated morbidity and mortality.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade.

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Repeated catecholamine surges alter cardiac isomyosin expression but not protein synthesis in the rat heart.

To assess the role of intermittent beta-adrenergic stimulation on alpha-myosin heavy chain expression and cellular hypertrophy, we studied the effect of intermittent dobutamine on myosin heavy chain isoform distribution and protein synthesis in the heterotopic rat heart preparation. This model allows the analysis of a pharmocologic stimulus in isolation from the mechanical load on the myocardium induced by the drug. Intermittent administration of dobutamine resulted in elevated alpha-MHC levels (75 +/- 12%) compared to control (55 +/- 10%; X +/- s.e.; P<0.05) transplanted hearts. This effect was not altered by alpha-receptor blockade with terazosin (72 +/- 8%). Intermittently pacing the transplanted hearts at the same rate as observed with dobutamine alone, also elevated alpha-MHC levels (70 +/- 5%). In contrast, total protein synthesis in the transplanted hearts was not altered with any of the drug or pacing interventions compared to control hearts. These data suggest that intermittent beta-receptor stimulation and/or intermittent increased heart rate contribute to altered patterns of myosin heavy chain expression. However, increases in cardiac mass and protein synthesis are probably mediated by hemodynamic factors rather than catecholamine stimulation.

GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes.

GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction.

Angiotensin receptor 1 blockade does not prevent physiological cardiac hypertrophy in the adult rat.

The renin-angiotensin system has been implicated in the hypertrophic adaptation of the heart to exogenous pathological loads, such as hypertension and aortic stenosis; however, the role of this hormonal system in the cardiac adaptations to physiological loads, such as chronic exercise conditioning, has not been established. We therefore studied the effect of angiotensin receptor 1 (AT1) blockade on the chronic cardiac responses of rats subjected to an 8-wk swimming program. Compared with matched sedentary controls, untreated swimmers increased their left ventricular weights by 13%, and swimmers treated with the AT1 antagonist L-158809 increased their left ventricular weights by 11% (both P < 0.05 vs. sedentary controls). The incorporation of labeled amino acids into the heart at the time of death was unchanged in all groups, and therefore the increase in heart weight in both swim-conditioned groups appeared to reflect a decrease in the rate of protein degradation in the heart. Hearts from both swim-conditioned groups manifested an increase in the V1-predominant myosin isoform pattern but not an increase in atrial natriuretic factor mRNA expression or protein kinase C translocation. The fact that these patterns of adaptation are preserved in exercised conditioned animals treated with an AT1 antagonist suggests that the chronic hypertrophic response of the heart to physiological loads is not influenced by the renin-angiotensin system.