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Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 19 recordsLinked to original sources
PubMed2006

Aging aggravates heterogeneities in cell-size and stress-intolerance of cardiac ventricular myocytes.

RATIONALE: Heterogeneity of ventricular myocytes from old murine hearts was tested in regard to cell volume and tolerance to stress induced by ROS generated by photoactivation of tetramethylrhodamine methylester. RESULTS: During repetitive linescans, ROS generated 'scars' of depolarized mitochondria. All young and 60% of old myocytes had type1 scars that were small and remained stable with time. Forty percent cells from old hearts responded with type2 scars that were wide and expanded with time. Depolarization of individual mitochondria was characterized by its half decay time. HDT was 6.9s in young mice (type1 mitochondria). HDT was 5.6s in old mice due to a 30% population of mitochondria with reduced ROS-tolerance (type2, HDT=3.0 s). Type1 and type2 mitochondria did not coexist in the same cell, however, in the same old heart (different myocytes). In a different set of myocytes the volumes were measured to be larger (83+/-29 pl) and more heterogeneous than in young myocytes (49+/-13 pl). CONCLUSION: Hearts of old mice contain a subpopulation of myocytes with reduced mitochondrial stress-tolerance that is attributed to an age-dependent reduction of cellular ROS defence capacity. We discuss how heterogeneities in stress-tolerance and cell volume could be causally linked.

Aging↗
PubMed2005

CREB expression in cardiac fibroblasts and CREM expression in ventricular myocytes.

Activation of gene expression by the cAMP-dependent signaling pathway is regulated by members of the cAMP response element binding protein (CREB) family consisting of CREB, CREM, and ATF-1. It is decisively for the understanding of the heart function as to which type of heart cells expresses CREB and/or CREM. Ventricular myocytes and fibroblasts of young (3 months) and old (24 months) rat hearts were separately investigated to analyse CREB, CREM, and phospho-CREB. Western blot showed CREB expression exclusively in fibroblasts but CREM was predominantly detected in ventricular myocytes. CREB-positive nuclei in heart sections were only revealed in fibroblasts. CREB was activated by forskolin (10 microM), PMA (500 nM), and cyclical mechanical strain (1 Hz, 5% elongation) in fibroblasts. The number of CREB-positive myocytes in old rats was larger than in young rats. But CREB could not be activated by forskolin (10 microM) in all myocytes. Our results suggest that the expression of CREB depends on the cell type and the age of the animal. We discuss that modulation of gene expression as it occurs with a age could be affected by the change within the CREB family members.

Aging↗
PubMed2004

Mechanisms underlying enhanced cardiac excitation contraction coupling observed in the senescent sheep myocardium.

Ageing related stiffening of the vascular system is believed to be in part responsible for a number of clinical outcomes including hypertension and heart failure. In the present study, we sought to determine whether there are alterations in cardiac excitation contraction coupling that may help compensate for the increased vessel stiffness. Experiments were performed on single cardiac myocytes isolated from young (18 months) and aged (>8 years) sheep. Intracellular Ca(2+) concentration, action potentials, L-type Ca(2+) currents and SR Ca(2+) content were measured at 23 degrees C. With ageing, cell capacitance increased by 26% indicating cellular hypertrophy. Action potential duration (APD90) (590 +/- 21 vs. 726 +/- 36 ms), Ca(2+) transient amplitude (112 +/- 15 vs. 202 +/- 25 nmol l(-1)) and fractional cell shortening (by 37%) also increased in the aged hearts (all values P < 0.05). The larger Ca(2+) transient amplitude observed under current clamp conditions was maintained under voltage clamp control; however, SR Ca(2+) content was identical. Both the peak L-type Ca(2+) current (2.8 +/- 0.3 vs. 4.9 +/- 0.5 pA pF(-1)) and integrated Ca(2+) entry (5.1 +/- 0.7 vs. 7.9 +/- 0.8 micromol l(-1), all P < 0.01) were greater in aged cells. In this study we show that in the ageing ovine myocardium, the amplitude of the systolic Ca(2+) transient is increased. The larger Ca(2+) transients cannot simply be explained by changes in APD and we suggest that the greater inward L-type Ca(2+) current provides a more effective trigger for calcium-induced-calcium release from the SR whilst maintaining a stable SR Ca(2+) content. These changes in cardiac excitation contraction coupling may help maintain cardiac output in the face of increased great vessel stiffness.

Action Potentials↗
PubMed2003

Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats.

Mechanical load as stimulus for apoptosis and necrosis could be responsible for the loss of cardiomyocytes. Ventricular myocytes from young (3 mo) and old (14-24 mo) rats underwent cyclical mechanical stretch (CMS; 5% elongation, 1 Hz) for 24 h. Spontaneous apoptosis was in myocytes from young rats 0.33 +/- 0.12% and from old rats 1.05 +/- 0.35% [Tdt-mediated dUTP nick-end labeling (TUNEL) assay]; associated with a decrease of Bcl-2. CMS increased the apoptosis to 0.58 +/- 0.18% in myocytes from young rats. Western blot analysis showed that CMS reduced Bcl-2 and increased p53 (young rats). Bax was not changed by CMS. These were confirmed by cytochrome c release (31 +/- 13%) and by the enrichment of cytosolic nucleosomes (11 +/- 8%). CMS did not influence the apoptosis in myocytes from old rats (TUNEL assay, Bcl-2, Bax, or p53). CMS did not cause necrosis in myocytes from young rats. CMS increased the number of necrotic cells by showing the cell membrane rupture in myocytes from old rats (50 +/- 13% 5-hexadecanoylaminofluorescein-positive and 38 +/- 6% propidium iodide-positive cells) as well as by measuring the lactate dehydrogenase release. The results suggest that CMS-induced apoptosis in myocytes of young rats but necrosis in myocytes from old rats, which could be attributed to more stress sensitivity of cells from old rats.

Aging↗
PubMed2003

Normal and pathological excitation-contraction coupling in the heart -- an overview.

This issue of The Journal of Physiology includes a series of review articles arising from a symposium held at the joint meeting of the UK, German and Scandinavian Physiological Societies. The articles focus on different aspects of the cellular control of contraction. The basic mechanism of cardiac excitation-contraction coupling ('calcium-induced calcium release') is now reasonably well-established. Calcium enters the cell from the extracellular fluid via the voltage-dependent L-type Ca(2+) channel. This results in a 'trigger' increase of [Ca(2+)](i) in the space between the sarcolemma and sarcoplasmic reticulum (SR) and this leads to the opening of the SR Ca(2+) release channel or 'ryanodine receptor' (RyR). As exemplified by the papers from the symposium, much current work is focused on how this mechanism is modified in different circumstances. These include autonomic modulation, but also pathological conditions such as cardiac hypertrophy and failure, a recurrent theme in several of these papers.

Action Potentials↗
PubMed2003

Ca2+ transients of cardiomyocytes from senescent mice peak late and decay slowly.

Ventricular myocytes were isolated from either young (2 months, "young myocytes") or senescent (20-26 months, "senescent myocytes") mice. Ca2+ transients were evoked by 40ms voltage-clamp pulses depolarising at 0.4, 1, 2, 4 or 8Hz. At 8Hz, Ca2+ transients from senescent cells peaked later (39ms versus 23ms) to smaller systolic [Ca2+](c) (667nM versus 1110nM) and decayed at slower rate (16s(-1) versus 33s(-1)) to higher end-diastolic [Ca2+](c) (411nM versus 220nM) than those from young myocytes. These differences were less pronounced at lower frequencies of pulsing and could not be explained by differences of the time integral of Ca2+ inward current. Since concentrations of SERCA2a and SERCA2b proteins were similar in young and senescent cells, slow rate of Ca2+ decay and high diastolic [Ca2+]c are explained on the assumption that the usual Ca2+ stimulation of SERCA2 activity is attenuated in senescent cells. The prolonged time-to-peak [Ca2+]c is discussed to result from insufficient SR Ca2+ filling by SERCA2 and, in context with confocal images, from a shift of the SERCA2b distribution to the subsarcolemmal space. The age-related changes of the Ca2+ transients are discussed to cause systolic and diastolic failure if senescent mouse hearts beat at high frequencies.

Actinin↗
PubMed2001

Cytochalasin D reduces Ca2+ currents via cofilin-activated depolymerization of F-actin in guinea-pig cardiomyocytes.

1. L-type Ca2+ channel currents (I(Ca)) were measured in guinea-pig ventricular myocytes (22 degrees C, 300 ms steps from -45 to +10 mV). Pulsing at 0.5 Hz reduced I(Ca) within 5 min to 92 +/- 3% (mean +/- S.E.M., n = 14) and within 10 min to 83 +/- 4 % ('run-down' with reference to I(Ca) after a 5 min equilibration period). 2. Bath-applied cytochalasin D (cytD, 10 microM) reduced I(Ca) to 75 +/- 4% within 5 min and to 61 +/- 4% within 10 min ('cytD reduction of I(Ca)') by reduction of maximal Ca2+ conductance (suggested by fits of time course and of current-potential (I-V) curves). 3. Preincubation with phalloidin (bath applied, 100 microM, 5 h) prevented the cytD reduction of I(Ca). Since phalloidin specifically blocks F-actin depolymerization, cytD reduction of I(Ca) is linked to depolymerization of F-actin. 4. CytD did not attenuate the beta-adrenergic stimulation of I(Ca) (30 nM isoproterenol), suggesting that A kinase anchoring proteins are unlikely to mediate the cytD reduction of I(Ca). The cytD reduction of I(Ca) was abolished by extra-/intracellular acidosis (pH(o) 6.9), by cell dialysis of 5 mM BAPTA, or by serine/threonine protein phosphatase inhibitors. 5. Actin-depolymerizing factor (ADF)/cofilin are proteins that bind to actin, mediate a pH-sensitive depolymerization of F-actin, and are activated by dephosphorylation. Western blots from hearts perfused with solutions containing zero or 10 microM cytD indicated that cytD reduces the ratio of phosphorylated to total ADF/cofilin content by 50%. 6. The data support the concept that cytD mediates dephosphorylation and activation of ADF/cofilin, leading to depolymerization of F-actin with a subsequent reduction of I(Ca).

Acidosis↗
PubMed2001

Inotropic response to beta-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts.

1. The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti-adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS-/-) mice. 2.eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT-PCR analysis. 3. In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS-/- hearts were not significantly different from those of WT hearts (LVP: 97 +/- 11 mmHg WT vs. 111 +/- 11 mmHg eNOS-/-; dP/dtmax: 3700 +/- 712 mmHg s(-1) WT vs. 4493 +/- 320 mmHg s)-1) eNOS-/-). 4. The dobutamine (10-300 nM)-induced increase in LVP was enhanced in eNOS-/- hearts. In contrast, L-type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS-/- hearts showed no differences after beta-adrenergic stimulation. Dibutyryl-cGMP (50 microM) reduced basal ICa,L in WT cells to 72 +/- 12 % while eNOS-/- ICa,L was insensitive to the drug. The pre-stimulated ICa,L (30 nM isoproterenol) was attenuated by dibutyryl-cGMP in WT and eNOS-/- cells to the same extent. 5. The Ca2+ (1.5-4.5 mM)-induced increase in inotropy was not different between the two experimental groups and beta-adrenergic receptor density was increased by 50% in eNOS-/- hearts. 6. The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti-adrenergic effect of both compounds was identical in WT and eNOS-/- hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7. These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to beta-adrenergic stimulation and with elevated -adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS-/- cardiac myocytes to beta-adrenergic stimulation suggests that endothelium-derived NO is important in mediating the whole-organ effects and (3) eNOS is unimportant for the anti-adrenergic effect of ACh and adenosine.

Acetylcholine↗
PubMed2001

Mechanically induced potentials in rat atrial fibroblasts depend on actin and tubulin polymerisation.

When atrial tissue contracts, mechanically induced potentials (MIPs) are generated in fibroblasts, presumably by activation of a non-selective cation conductance Gns. Non-stimulated atrial fibroblasts had a mean (+/-SD) membrane potential (Em) of -22 +/- 2 mV and an input resistance of 510 +/- 10 MS. MIP amplitude (AMIP) was 38+/-4 mV when current injection had polarised Em to Vm = -50 mV. The slope of the function relating AMIP to Vm can be regarded as a mechanosensitive factor (Xms) that describes the relative increase in Gns during a MIP. Putative involvement of cytoskeletal fibres in activation of Gns was studied by delivering drugs from the intracellular recording microelectrode. Destabilisation of F-actin by 0.2 mM cytochalasin D reduced AMIP from 38 to 16 mV and Xms from 5 to 1.8. Destabilisation of tubulin with 0.2 mM colchicine reduced AMIP to 21 mV and Xms to 2.1. The combination colchicine plus cytochalasin D reduced AMIP to 9 mV and Xms to 1.4. Promoting F-actin stability with exogenous adenosine 5'-triphosphate (ATP) increased AMIP and Xms and attenuated the effects of cytochalasin D. Similarly, facilitation of tubulin stability with guanosine 5'-triphosphate (GTP) or taxol increased AMIP and Xms and attenuated the effects of colchicine. The results suggest that transfer of mechanical energy from the deformed fibroblast surface to the Gns channel protein depends on intact F-actin and tubulin fibres.

Actins↗
PubMed2001

RNA from heart of young and old rats leads to the expression of protein(s) in Xenopus oocytes that alter the transport activity of rat Na+,K+-ATPases differently.

To address the question of whether the function of Na+,K+-ATPases differs in the heart of young and old rats, enzymes formed from the alpha1 or alpha2 isoform with the beta1 subunit of rat were expressed in Xenopus oocytes. In addition to injections of the cRNA coding for the respective subunits, oocytes were co-injected with total RNA from the left ventricle of young or old rats. To assess alterations in transport activity due to the co-injections, ouabain-sensitive 86Rb+ uptake was measured. Co-injection of the RNA from young rats led to 31% inhibition of 86Rb+ uptake into oocytes with the alpha1/beta1 pumps while uptake into oocytes with the alpha2/beta1 pumps was hardly affected. Co-injection of the RNA from old rats, on the other hand, reduced 86Rb+ uptake only in cells with the alpha2/beta1 isoform (to 85%). The steady-state current generated in the absence of external Na+ by the alpha1/beta1 ATPase was significantly reduced by co-injection of RNA only from young rats to 70%, and this inhibition was hardly affected by membrane potential. For the alpha2/beta1 ATPase co-injection of RNA only from old rats also led to a significant reduction of pump-mediated current at potentials more negative than -70 mV to 70-80%. In the presence of Na+, inhibition of the alpha1 isoform by co-injection of RNA from young rats is voltage-dependent, increasing with more negative potentials. For the alpha2/beta1 pump, co-injection of RNA from old rats was no longer effective, but voltage-dependent inhibition by co-injection of RNA from young rats became apparent. The data indicate that changes in protein expression occurring in young and old rat hearts may modulate transport activity of the Na+,K+-ATPase and this modulation depends on membrane potential and the presence of external Na+. We propose that the described mechanisms may play a functional role in working myocardium, and may form a basis for processes involved in heart aging.

Aging↗
PubMed2001

Soluble substances released from postischemic reperfused rat hearts reduce calcium transient and contractility by blocking the L-type calcium channel.

OBJECTIVES: This study was designed to investigate the effects of cardiodepressant substances released from postischemic myocardial tissue on myocardial calcium-regulating pathways. BACKGROUND: We have recently reported that new cardiodepressant substances are released from isolated hearts during reperfusion after myocardial ischemia. METHODS: After 10 min of global ischemia, isolated rat hearts were reperfused, and the coronary effluent was collected for 30 s. We tested the effects of the postischemic coronary effluent on cell contraction, Ca2+ transients and Ca2+ currents of isolated rat cardiomyocytes by applying fluorescence microscopy and the whole-cell, voltage-clamp technique. Changes in intracellular phosphorylation mechanisms were studied by measuring tissue concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), as well as activities of cAMP-dependent protein kinase (cAMP-dPK) and protein kinase C (PKC). RESULTS: The postischemic coronary effluent, diluted with experimental buffer, caused a concentration-dependent reduction of cell shortening and Ca2+ transient in the field-stimulated isolated cardiomyocytes of rats, as well as a reduction in peak L-type Ca2+ current in voltage-clamped cardiomyocytes. The current reduction resulted from reduced maximal conductance--not from changes in voltage- and time-dependent gating of the L-type Ca2+ channel. The postischemic coronary effluent modified neither the tissue concentrations of cAMP or cGMP nor the activities of cAMP-dPK and PKC. However, the effluent completely eliminated the activation of glycogen phosphorylase after beta-adrenergic stimulation. CONCLUSIONS: Negative inotropic substances released from isolated postischemic hearts reduce Ca2+ transient and cell contraction through cAMP-independent and cGMP-independent blockage of L-type Ca2+ channels.

Animals↗
PubMed2001

Prospective evaluation of balloon-sheathed catheter US system.

BACKGROUND: Catheter US probes must rely on luminal water to create images because they do not incorporate a water-filled balloon such as that used with a designated echoendoscope. The purpose of this study is to determine the effectiveness and safety of a balloon sheath for the US catheter system. METHODS: Catheter EUS was performed on 50 patients by using a 2.3 mm 12 MHz or 20 MHz catheter probe. Catheter EUS was used in 47 cases, and a newly developed water-filled balloon sheath was used in 41 cases. Both devices were used in 39 cases. Procedure time, depth of ultrasound penetration, and a subjective assessment of image quality and ease of use were recorded, along with TMN stage as applicable. Catheter EUS findings were confirmed with a standard radial scanning echoendoscopy (S-EUS) in 18 cases. RESULTS: Catheter probe EUS (C-EUS) and catheter probe plus balloon (CB-EUS) imaging was obtained of 25 esophageal, 8 gastric, 4 rectal, 1 biliary, and 1 duodenal lesion. Time required for the ultrasound portion of the examination was identical with C-EUS and CB-EUS. Depth of penetration increased with CB-EUS with both the 12 MHz and 20 MHz probes (p < 0.05). Subjective assessment of image clarity improved when CB-EUS was used in the esophagus. C-EUS failed to identify 2 esophageal cancers and 2 sets of paraesophageal lymph nodes, and understaged 1 esophageal cancer. The remaining 14 cancers were staged identically by both modalities. The catheter probes with and without the balloon sheath were easy to use, even in markedly narrow esophageal strictures. CB-EUS did not significantly improve resolution in the stomach or rectum. S-EUS confirmed findings of CB-EUS in all 18 cases in which both instruments were used. There were no procedure-related complications. CONCLUSIONS: For esophageal lesions, CB-EUS improves images compared with C-EUS, and enhances depth of penetration without prolonging or encumbering the examination. CB-EUS offers no advantage over C-EUS in organs other than the esophagus. S-EUS, when possible, remains the preferred imaging modality for esophageal cancers because of the ability to image the celiac axis and other deep structures.

Catheterization↗
PubMed2001

Wire-guided intraductal US: an adjunct to ERCP in the management of bile duct stones.

BACKGROUND: Endoscopic retrograde cholangiography (ERC) may misdiagnose bile duct stones if air bubbles are introduced during contrast injection, and it may also fail to diagnose stones in the presence of bile duct dilation. METHODS: Our aim was to determine whether intraductal US (IDUS) improves the accuracy of cholangiography and whether it is a useful adjunct in the management of bile duct stones. IDUS with a wire-guided US probe was performed after initial ERC in patients in whom bile duct stones were suspected. The diagnostic accuracy of ERC alone was compared with that of ERC plus IDUS. RESULTS: ERC with IDUS was performed in 62 patients who were suspected to have bile duct stones. Both IDUS and ERC were performed by the same endoscopist, and ERC was performed with a C-arm fluoroscope. The presence of bile duct stones and/or sludge were confirmed after sphincterotomy and extraction in 34 patients. Overall, the accuracy of ERC combined with IDUS in the diagnosis of bile duct stone and/or sludge was higher than that of ERC alone (97% vs. 87%, p < 0.05). With dilated bile ducts, the diagnostic accuracy of ERC combined with IDUS was also higher than that of ERC alone (95.5% vs. 72.7%, p < 0.05). Additional diagnostic information provided by IDUS included identification of cystic duct stones in 5 patients, characterization of bile duct strictures in 2 patients, and choledochal varices in 1 patient. Performance of wire-guided IDUS required 5% of the total procedure time. CONCLUSIONS: IDUS improves diagnostic accuracy of ERC and is a useful adjunct to ERC when bile duct stones are suspected.

Aged↗
PubMed2001

High-resolution endoscopic imaging of the GI tract: a comparative study of optical coherence tomography versus high-frequency catheter probe EUS.

BACKGROUND: Both optical coherence tomography (OCT) and catheter probe EUS (CPEUS) are candidates for high-resolution imaging of the GI wall, but their potential roles in this clinical context have not been investigated. METHODS: OCT and CPEUS were used to image normal-appearing portions of the GI tract at the same sites. CPEUS was performed with a 20-MHz or a new 30-MHz catheter probe. RESULTS: Forty-four histologically confirmed normal sites in 27 patients were evaluated. With OCT, mucosa and muscularis mucosa were clearly seen at all sites. Except for stomach, OCT demonstrated the submucosa in all sites. OCT penetration ranged from 0.7 to 0.9 mm. Microscopic structures such as esophageal glands, intestinal villi, colonic crypts, and blood vessels were easily identified. CPEUS penetration ranged from 10 mm to 20 mm, and 5 to 7 distinct layers were discernible. However, both mucosa and submucosa were seen as thin layers without microscopic detail. CONCLUSION: OCT resolution is superior to high-frequency CPEUS, but depth of penetration is limited to mucosa and submucosa. OCT images the major structural components of the mucosa and submucosa whereas CPEUS does not. Potentially, OCT and high-frequency CPEUS may be complementary for clinical imaging.

Endoscopy, Gastrointestinal↗
PubMed2001

Evaluation of a rapid urine amylase test using post-ERCP hyperamylasemia as a model.

OBJECTIVE: The initial diagnosis of acute pancreatitis is often based on clinical criteria together with elevations of serum amylase and lipase. A reliable bedside urine test could facilitate the early diagnosis of pancreatitis. We evaluated a rapid urine amylase test (Rapignost) by using post-ERCP hyperamylasemia as a human model of acute development of hyperamylasemia suggestive of pancreatitis. METHODS: Seventy-five patients undergoing ERCP were prospectively evaluated. Patients with renal insufficiency, hyperlipidemia, or hyperglycemia were excluded. Before ERCP, patients had serum amylase and lipase measured, and urine amylase tested with the Rapignost test strip. At 4 and 16-24 h post-ERCP, a serum and urine (test strip) amylase were measured again; the adequacy of urine collection was verified by measuring a 2-h creatinine clearance. Patients were clinically assessed for the development of clinical pancreatitis. The concordance of the strip result with post-ERCP hyperamylasemia was assessed. RESULTS: The sensitivity of the test strip for the detection of hyperamylasemia was greatest at 16-24 h post-ERCP (78%). Specificity was uniformally high (100% specificity at 16-24 h post-procedure). The test strip was positive in all cases of clinical pancreatitis. Of three cases of clinically evident ERCP-induced pancreatitis, only one was urine test strip positive by 4 h post-procedure. CONCLUSIONS: Using post-ERCP hyperamylasemia as a model, the Rapignost rapid urine amylase test strip was only marginally sensitive but highly specific for hyperamylasemia. The urine test strip was positive in all cases of clinical pancreatitis and may be a useful bedside test for the diagnosis of acute pancreatitis.

Adolescent↗