Cellular hypertrophy in cardiomyopathic patients is associated with lower creatine-stimulated mitochondrial respiration

The mitochondrial respiration rate and morphometric indices in endomyocardial biopsy samples were measured in 43 patients with dilated cardiomyopathy selected in accordance to WHO criteria by endomyocardial biopsy studies after excluding of various forms of myocarditis, alcoholic cardiomyopathy and other specific diseases of the heart. A group of 13 patients with unusually high mean myocyte diameter, 30±4 m, and nuclear size, 57±5 m, was selected. The remainder of patients (n=30) had significantly lower mean myocyte diameter and nuclear size, 23±3 and 42±6 m, respectively, (p<0.01). Creatine-stimulated elevation in mitochondrial respiration rate as measured in saponin-skinned was found in the former group to be much lower (36±4%) as compared with the remainder (90±12%). Also, the former group of patients had higher left ventricular enddiastolic pressure and volume index with concomitantly decreased ejection fraction. The results indicate that marked nuclear and cellular hypertrophy is associated with lower creatine-stimulated mitochondrial respiration rate and more severe cardiac failure. They suggest that disorders in energy supply to myofibrils may be related to disturbances in cellular genetic apparatus.     

Changes in the calcium current among different transmural regions contributes to action potential heterogeneity in rat heart

To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca²⁺ current (I_Ca,L) by voltage and Ca²⁺-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak I_Ca,L was significantly greater in EPI than in other cells. The inactivation of I_Ca,L by Ca²⁺-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca²⁺ sensitive, with Ca²⁺ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I_Ca,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca²⁺ influx.     

Improvement in pump function with endocardial biventricular pacing increases with activation time at the left ventricular pacing site in failing canine hearts

Recently, attention has been focused on comparing left ventricular (LV) endocardial (ENDO) with epicardial (EPI) pacing for cardiac resynchronization therapy. However, the effects of ENDO and EPI lead placement at multiple sites have not been studied in failing hearts. We hypothesized that differences in the improvement of ventricular function due to ENDO vs. EPI pacing in dyssynchronous (DYSS) heart failure may depend on the position of the LV lead in relation to the original activation pattern. In six nonfailing and six failing dogs, electrical DYSS was created by atrioventricular sequential pacing of the right ventricular apex. ENDO was compared with EPI biventricular pacing at five LV sites. In failing hearts, increases in the maximum rate of LV pressure change (dP/dt; r = 0.64), ejection fraction (r = 0.49), and minimum dP/dt (r = 0.51), relative to DYSS, were positively correlated (P < 0.01) with activation time at the LV pacing site during ENDO but not EPI pacing. ENDO pacing at sites with longer activation delays led to greater improvements in hemodynamic parameters and was associated with an overall reduction in electrical DYSS compared with EPI pacing (P < 0.05). These findings were qualitatively similar for nonfailing hearts. Improvement in hemodynamic function increased with activation time at the LV pacing site during ENDO but not EPI pacing. At the anterolateral wall, end-systolic transmural function was greater with local ENDO compared with EPI pacing. ENDO pacing and intrinsic activation delay may have important implications for management of DYSS heart failure.     

CaMKII inhibition reduces arrhythmogenic Ca2+ events in subendocardial cryoinjured rat living myocardial slices

Spontaneous Ca²⁺ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca²⁺ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca²⁺ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca²⁺ transients to peak after injury. In ENDO LMS, the Ca²⁺ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation–contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.     

Coronary vessels and cardiac myocytes of middle-aged rats demonstrate regional sex-specific adaptation in response to postmyocardial infarction remodeling

Background

An increasing body of evidence indicates that left ventricular (LV) remodeling, especially the degree of reactive myocardial hypertrophy after myocardial infarction (MI), differs in males and females. Surprisingly, to date, the sex-specific post-MI alterations of the coronary vasculature remain undetermined. Therefore, we tested the hypothesis that adaptive coronary arteriolar and capillary modifications occurring in response to reactive myocyte hypertrophy differ between middle-aged male and female post-MI rats.

Methods

A large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague–Dawley rats by ligation of the left coronary artery. Four weeks after surgery, rats with transmural infarctions, greater than 50% of the LV free wall (FW), were evaluated. Sham-operated male (M-Sham) and female (F-Sham) rats served as an age-matched controls.

Results

F-MI and M-MI rats had similar sized infarcts (61.3%?±?3.9% vs. 61.5%?±?1.2%) and scale of LV remodeling, as indicated analogous remodeling indices (1.41?±?0.11 vs. 1.39?±?0.09). The degree of reactive post-MI myocardial hypertrophy was adequate to normalize LV weight-to-body weight ratio in both sexes; however, the F-MI rats, in contrast to males, showed no myocyte enlargement in the LVFW epimyocardium. At the same time, a greater than 50% expansion of myocyte area in the male epimyocardium and in the female endomyocardium was accompanied by a 23% (P?<?0.05) increase in capillary-to-myocyte ratio, indicative of adaptive angiogenesis. Based on arteriolar length density in post-MI hearts, the resistance vessels grew in the male LVFW as well as the septum by 24% and 29%, respectively. In contrast, in females, a significant (30%) expansion of arteriolar bed was limited only to the LVFW. Moreover, in F-MI rats, the enlargement of the arteriolar bed occurred predominantly in the vessels with diameters <30 μm, whereas in M-MI rats, a substantial (two- to threefold) increase in the density of larger arterioles (30 to 50 μm in diameter) was also documented.

Conclusion

Our data reveal that while both sexes have a relatively similar pattern of global LV remodeling and adaptive angiogenesis in response to a large MI, male and female middle-aged rats differ markedly in the regional scale of reactive cardiac myocyte hypertrophy and adaptive arteriogenesis.     

Increased glycolysis as protective adaptation of energy depleted,degenerating human hibernating myocardium

In the current study on human hibernating myocardium (HHM), we tested the hypothesis that increased glycolysis might exert a positive effect during a supply-demand balance situation by augmentation of myocardial energy formation. In 14 patients HHM was preoperatively detected by clinical methods and validated by the recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed using biochemical and morphologic methods. Metabolite contents were normalized for the degree of fibrosis (control: 9.8 ± 0.5%, HHM 28.1 ± 3.0%; p < 0.05), providing values for cardiomyocytes only. In energy depleted HHM, severe intracellular degeneration, glycogen accumulation and myocyte loss were found. Elevated lactate levels (2.22 ± 0.26 vs. 25.38 ± 3.53 mol/wet wt, p < 0.001) were indicative of an increased anaerobic glycolytic flux. In conclusion the presence of abundant intracellular glycogen and an increased anaerobic glycolysis in HHM is indicative of a protective adaptation of this myocardium, which might balance energy deficit and may limit structural damage.     

Transmural distribution of metabolic abnormalities and glycolytic activity during dobutamine-induced demand ischemia

The heterogeneity across the left ventricular wall is characterized by higher rates of oxygen consumption, systolic thickening fraction, myocardial perfusion, and lower energetic state in the subendocardial layers (ENDO). During dobutamine stimulation-induced demand ischemia, the transmural distribution of energy demand and metabolic markers of ischemia are not known. In this study, hemodynamics, transmural high-energy phosphate (HEP), 2-deoxyglucose-6-phosphate (2-DGP) levels, and myocardial blood flow (MBF) were determined under basal conditions, during dobutamine infusion (DOB: 20 microg x kg(-1) x min(-1) iv), and during coronary stenosis + DOB + 2-deoxyglucose (2-DG) infusion. DOB increased rate pressure products (RPP) and MBF significantly without affecting the subendocardial-to-subepicardial blood flow ratio (ENDO/EPI) or HEP levels. During coronary stenosis + DOB + 2-DG infusion, RPP, ischemic zone (IZ) MBF, and ENDO/EPI decreased significantly. The IZ ratio of creatine phosphate-to-ATP decreased significantly [2.30 +/- 0.14, 2.06 +/- 0.13, and 2.04 +/- 0.11 to 1.77 +/- 0.12, 1.70 +/- 0.11, and 1.72 +/- 0.12 for EPI, midmyocardial (MID), and ENDO, respectively], and 2-DGP accumulated in all layers, as evidenced by the 2-DGP/PCr (0.55 +/- 0.12, 0.52 +/- 0.10, and 0.37 +/- 0.08 for EPI, MID, and ENDO, respectively; P < 0.05, EPI > ENDO). In the IZ the wet weight-to-dry weight ratio was significantly increased compared with the normal zone (5.9 +/- 0.5 vs. 4.4 +/- 0.4; P < 0.05). Thus, in the stenotic perfused bed, during dobutamine-induced high cardiac work state, despite higher blood flow, the subepicardial layers showed the greater metabolic changes characterized by a shift toward higher carbohydrate metabolism, suggesting that a homeostatic response to high-cardiac work state is characterized by more glucose utilization in energy metabolism.     

Qualitative and quantitative analysis of granules in atrial appendage cardiocytes in different physiological states

Summary Atrial appendage cardiocytes of mammals, including man, contain multiple cytoplasmic granules that vary in number in different physiological states. Using morphologic and comprehensive morphometric techniques, these granules were assessed in Sprague-Dawley rats following dehydration for 5 days, volume-loading by substituting 1% NaCl as drinking water for 7 days, unilateral nephrectomy plus volume-loading for 7 days, and in late term pregnant animals (18–20 days; term 21 days). Although principally located in the paranuclear region, granules were observed throughout the sarcoplasm. Cytological features indicative of synthetic activity and granule formation were readily apparent in all groups with the exception of pregnant rats where they were infrequently observed. Granule contents were released by exocytosis and observed in the right appendage of control, dehydrated and nephrectomy/volume-loaded groups and left appendage of volumeloaded animals. Exocytosis was not observed in pregnant animals. By point counting, the proportional volume of cardiocytes occupied by granules (V _v ) in controls was significantly greater for right than for left appendage (2.12±0.22% vs 1.29±0.16%; mean±SEM;p<0.05). A significantly similar difference was found for nephrectomy/volume-loaded animals. There was no significant difference inV _v for right appendage between the control and experimental groups; for left appendage there was a significant increase inV _v to 2.42±0.09% (p<0.05) for volume-loaded animals only. Estimation of the maximum diameter of granule profiles in control animals was 238±9 nm and 230±6 nm for right and left appendages, respectively. The profile diameters in the left appendages of dehydrated (202±9 nm) and pregnant (200±7 nm) animals were significantly (p<0.05) less than those of the control animals. The morphometric findings did not correlate with predictions based upon published biochemical data. In the course of this study, a previously unreported bimembranous, circular to ovoid structure was observed in the cardiocyte sarcoplasm of all animals; the nature and function of this structure is unknown.     

Catheter Ablation of Idiopathic Premature Ventricular Contractions and Ventricular Tachycardias Originating from the Vicinity of Endocardial and Epicardial Mitral Annulus

Background

Several previous reports have revealed that idiopathic ventricular arrhythmias (VAs), including premature ventricular contractions (PVCs) and ventricular tachycardias (IVTs), can originate from endocardial mitral annulus (ENDO MA). However, these data are limited to ENDO MA VAs, and little is known about the electrocardiographic (ECG) characteristics and the efficacy of radiofrequency catheter ablation (RFCA) via the coronary venous system for the VAs arising from the epicardial MA (EPI MA).

Methodology/Principal Findings

Characteristics of body surface electrocardiogram and electrophysiologic recordings were analyzed in 21 patients with symptomatic PVCs/IVTs originating from the vicinity of MA. Among 597 patients with PVCs/IVTs, the incidence of VAs originating from the ENDO and EPI MA was 3.52% (21 cases). Eleven (52%) from the ENDO MA, and 10 (48%) from the EPI MA. There were different characteristics of ECG of PVCs/VT originating from the ENDO and EPI MA. The prolonged pseudodelta wave time and intrinsicoid deflection time in lead V2 and the precordial maximum deflection index reliably differentiated EPI MA VAs from ENDO MA VAs with high sensitivity and specificity. Successful RFCA in 18 patients could be achieved (85.7% acute procedural success).

Conclusions/Significance

ECG characteristics of PVCs/VTs originating from the different portions of the MA are different, and can help regionalize the origin of these arrhythmias. RFCA within the coronary venous system was relatively effective and safe for the PVCs/IVTs and should be seen as an alternative approach, when the MA PVCs/IVTs could not be eliminated by RFCA from the endocardium.     

Analysis of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger (NCX) function and current in murine cardiac myocytes during heart failure

Na⁺/Ca²⁺ exchanger (NCX) plays important roles in cardiac electrical activity and calcium homeostasis. NCX current (I_NCX) shows transmural gradient across left ventricle in many species. Previous studies demonstrated that NCX expression was increased and transmural gradient of I_NCX was disrupted in failing heart, but the mechanisms underlying I_NCX remodeling still remain unknown. In present study, we used patch clamp technique to record I_NCX from subepicardial (EPI) myocytes and subendocardial (ENDO) myocytes isolated from sham operation (SO) mice and heart failure (HF) mice. Our results showed that I_NCX was higher in normal EPI cells compared with that in ENDO, whatever for forward mode or reverse mode. In HF group, I_NCX was significantly up-regulated, but EPI-ENDO difference was disrupted because of a more increase of I_NCX in ENDO myocytes. In order to explore the molecular mechanism underlying remodeling of I_NCX in failing heart, we detected the protein expression of NCX1 and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) by Western blot. We found that CaMKII activity was dramatically enhanced and parallel with the expression of NCX1 in failing heart. Our study demonstrated that transmural gradient of I_NCX existed in murine left ventricle, and increased activity of CaMKII should account for I_NCX remodeling in failing heart.     

Striated myofibrils in anti-myosin stained,isolated chicken gizzard smooth muscle cells

Summary Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM.Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 m. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5±0.2 m. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.     

Kainate receptor modification in the fetal guinea pig brain during hypoxia

The present study tests the hypothesis that hypoxia alters the high-affinity kainate receptors in fetal guinea pig brain. Experiments were conducted in normoxic and hypoxic guinea pig fetus at preterm (45 days of gestation) and term (60 days of gestation). Hypoxia in the guinea pig fetus was induced by exposure to maternal hypoxia (FiO₂=7%) for 60 min. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphorreatine. [³H]-Kainate binding characteristics (Bmax=number of receptors, Kd=dissociation constant) were used as indices of kainate receptor modification. P₂ membrane fractions were prepared from the cortex of normoxic and hypoxic fetuses and were washed six times prior to performing the binding assays. [³H]kainate binding was performed at 0°C for 30 min in a 500 l medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 g protein and varying concentrations of radiolabelled kainate ranging from 1 to 200 nM. Non-specific binding was determined in the presence of 1.0 mM glutamate. During brain development from 45 to 60 days gestation, Bmax value increased from 330±16 to 417±10 fmoles/mg protein; however, the Kd was unchanged (8.2±0.4 vs 8.8±0.5 nM, respectively). During hypoxia at 60 days, the Kd value significantly increased as compared to normoxic control (15.5±0.7 vs 8.8±0.5 nM, respectively), whereas the Bmax was not affected (435±12 vs 417±10 fmol/mg protein, respectively). At 45 days, hypoxia also increased the Kd (11.9±0.6 vs 8.2±0.4 nM) without affecting the Bmax (290±15 vs 330±16 fmol/mg protein, respectively). The results show that the number of kainate receptors increase during gestation without change in affinity and demonstrate that hypoxia modifies the high-affinity kainate receptor sites at both ages; however the effect is much stronger at 60 days (term). The decreased affinity of the site could decrease the kainate receptor-mediated fast kinetics of desensitization and provide a longer period for increased Na⁺-influx, leading to increased accumulation of intracellular Ca²⁺ by reversal of the Na⁺–Ca²⁺ exchange mechanism. In addition, Kd values for kainate-type glutamate receptor sites are 30–40 fold lower (i.e. higher affinity) than those for NMDA-displaceable glutamate sites. The higher affinity suggests that the activation of the kainate-type glutamate receptor during hypoxia could precede initiation of NMDA receptormediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.     

Tumor necrosis factor-α in macrophages of heart,liver, kidney,and in the pituitary gland

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n=54; 9±3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean±S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112±69 vs. 34±21x10³ m³) than fast and slow soleus fibers (40±20 vs. 30±14x10³ m³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 m) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 m) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116±51 vs. 55±22 and 44±23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

Spatial Characterization of Electrogram Morphology from Transmural Recordings in the Intact Normal Heart

Purpose

Unipolar (UE) and bipolar electrograms (BE) are utilized to identify arrhythmogenic substrate. We quantified the effect of increasing distance from the source of propagation on local electrogram amplitude; and determined if transmural electrophysiological gradients exist with respect to propagation and stimulation depth.

Methods

Mapping was performed on 5 sheep. Deployment of >50 quadripolar transmural needles in the LV were located in Cartesian space using Ensite. Contact electrograms from all needles were recorded during multisite bipolar pacing from epicardial then endocardial electrodes. Analysis was performed to determine stimulus distance to local activation time, peak negative amplitude (V_-P), and peak-peak amplitude (V_P-P) for (1) unfiltered UE, and (2) unfiltered and 30 Hz high-pass filtered BEs. Each sheep was analysed using repeated ANOVA.

Results

Increasing distance from the pacing sites led to significant (p<0.01) attenuation of UEs (V_-P = 7.0±0.5%; V_P-P = 5.4±0.3% per cm). Attenuation of BE with distance was insignificant (V_p-p unfiltered  = 2.2±0.5%; filtered  = 1.7±1.4% per cm). Independent of pacing depth, significant (p<0.01) transmural electrophysiological gradients were observed, with highest amplitude occurring at epicardial layers for UE and endocardial layers for BE. Furthermore, during pacing, propagation was earlier at the epicardium than endocardial layer by 1.6±2.0 ms (UE) and 1.4±2.8 ms (BE) (all p>0.01) during endocardial stimulation, and 2.3±2.4 ms (UE) and 1.8±3.7 ms (BE) during epicardal stimulation (all p<0.01).

Conclusions

Electrogram amplitude is inversely proportional to propagation distance for unipolar modalities only, which affected V_-P>V_P-P. Conduction propagates preferentially via the epicardium during stimulation and is believed to contribute to a transmural amplitude gradient.     

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl^– conductance (G_Cl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected G_Cl(CF-G_Cl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though G_Cl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-G_Cl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-G_Cl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G _tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl^–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of G_Cl. cAMP activated both apical and basolateral G_Cl. cAMP hyperpolarized gluconate: Cl^– (lumen: bath) transepithelial bionic potentials (V _t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V _t=–8.8±2.9 mV, n=5). cAMP activated basolateral G_Cl as indicated by increased bi-ionic (gluconate: Cl^–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V _t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of G_Cl in nonresponsive ducts and submaximal activation of G_Cl in responsive ducts. We conclude that cAMP activates CF-G _Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF cystic fibrosis - G _t transepithelial conductance - V _b electrical potential across the basolateral membrane - V _a electrical potential across the apical membrane - V _t transepithelial potential - V _b transepithelial currentinduced voltage deflections across the basolateral membrane - V _a transepithelial current-induced voltage deflections across the apical membrane - V _t transepithelial current-induced voltage deflection across the epithelium - VDR voltage divider ratio - G_Cl transepithelial Cl^– conductance - CF-G_Cl cystic fibrosis-affected Cl^– conductance - EMF electromotive force - IPR isoproterenol - IBMX 3-isobutyl-1-methylxanthine - CPT-cAMP chlorophenylthio-adenosine 3-5 cyclic monophosphate - PGE₂ prostaglandin E₂     

Responses of mouse fast and slow skeletal muscle to streptozotocin diabetes: Myosin isoenzymes and phosphorous metabolites

A condition similar to insulin-dependent diabetes mellitus (IDDM) was induced in male CD-1 mice by injection of streptozotocin (STZ). Five weeks after treatment, the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles were isolated for analysis. Phosphorous metabolites were quantified by³¹P-NMR and HPLC, native myosin was characterized electrophoretically, and activities of metabolic enzymes were measured spectrophotometrically. Relative to control animals, STZ-diabetes resulted in a significant 32% decrease in the FM₁ isoform of myosin in EDL and a 24% decrease in IM myosin of SOL. Mass-specific activities of phosphofructokinase, citrate synthase, and cytochrome oxidase were significantly lower in SOL from STZ-diabetic mice than in controls by 23, 18, and 36%, respectively. Intracellular ATP was significantly lower in SOL from STZ-diabetic mice than in controls (3.44±0.20 mol g^–1 wet weight vs. 4.61±0.20 mol g^–1, respectively), as was creatine phosphate (11.98±0.80 mol g^–1 wet weight vs. 14.22±0.44 mol g^–1). In contrast to results from SOL, there were no significant changes in phosphorus metabolites or enzyme activity in EDL. These results show that the effects of IDDM on levels of phosphorus containing metabolites and maximal activities of key regulatory enzymes in muscle are markedly fibertype specific. It is suggested that the muscle type-specific effects of STZ-diabetes may be a consequence of differential accumulation of intracellular fatty acids.     

Autocrine A2 in the T-System of Ventricular Myocytes Creates Transmural Gradients in Ion Transport: A Mechanism to Match Contraction with Load?

Transmural heterogeneities in Na/K pump current (I_P), transient outward K⁺-current (I_to), and Ca²⁺-current (I_CaL) play an important role in regulating electrical and contractile activities in the ventricular myocardium. Prior studies indicated angiotensin II (A2) may determine the transmural gradient in I_to, but the effects of A2 on I_P and I_CaL were unknown. In this study, myocytes were isolated from five muscle layers between epicardium and endocardium. We found a monotonic gradient in both I_p and I_to, with the lowest currents in ENDO. When AT₁Rs were inhibited, EPI currents were unaffected, but ENDO currents increased, suggesting endogenous extracellular A2 inhibits both currents in ENDO. I_P- and I_to-inhibition by A2 yielded essentially the same K_0.5 values, so they may both be regulated by the same mechanism. A2/AT₁R-mediated inhibition of I_P or I_to or stimulation of I_CaL persisted for hours in isolated myocytes, suggesting continuous autocrine secretion of A2 into a restricted diffusion compartment, like the T-system. Detubulation brought EPI I_P to its low ENDO value and eliminated A2 sensitivity, so the T-system lumen may indeed be the restricted diffusion compartment. These studies showed that 33–50% of I_P, 57–65% of I_to, and a significant fraction of I_CaL reside in T-tubule membranes where they are transmurally regulated by autocrine secretion of A2 into the T-system lumen and activation of AT₁Rs. Increased AT₁R activation regulates each of these currents in a direction expected to increase contractility. Endogenous A2 activation of AT₁Rs increases monotonically from EPI to ENDO in a manner similar to reported increases in passive tension when the ventricular chamber fills with blood. We therefore hypothesize load is the signal that regulates A2-activation of AT₁Rs, which create a contractile gradient that matches the gradient in load.     

Losartan inhibits myosin isoform shift after myocardial infarction in rats

Hypertrophy and heart failure following a myocardial infarction in rodents are accompanied by a switch of myosin isoforms from V₁ to V₃. The angiotensin II receptor blocker, Losartan, has been demonstrated to improve cardiac function and long-term survival after myocardial infarction. In this study we have investigated whether chronic Losartan treatment affects myosin isoform composition in the hearts of rats following a myocardial infarction. Rats were subjected to coronary artery ligation and received either Losartan (1 g/L) in the drinking water or water only. Four months after myocardial infarction, rats were classified as having either congestive heart failure (cMI) or uncomplicated myocardial infarction (uMI) based on their lung weight to body weight ratio (LW/BW). Compared with sham operated rats, uMI rats showed a 68.5% increase in the relative contribution of V₃ and a 33.7% decrease in the relative contribution of V₁ (p < 0.05). Untreated cMI showed 39.7% more V₃ and 38.2% less V₁ when compared with untreated uMI (p < 0.05). Losartan treatment after myocardial infarction reduced the incidence of cMI from 30.4 to 4.5% and scar size from 1.52 ± 0.07 to 0.94 ± 0.11 cm² respectively. The percentage of V₁ in Losartan treated uMI (LuMI) was 25.2% higher than the percentage of V₁ in untreated uMI (p < 0.05), whereas the percentage of V₃ in LuMI was 24.2% lower than that in untreated uMI (p < 0.05). A positive correlation of V₃ myosin and scar area was observed. Our study suggests that expression of V₃ myosin in the left ventricle is associated with scar size and the progress of hemodynamic changes after myocardial infarction. Losartan treatment reduces scar size and wall stress of the heart after the infarct, and therefore inhibits the signals shifting myosin isoform expression from V₁ to V₃ after a myocardial infarction.     

Down regulation of myocardial β1-adrenoceptor signal transduction system in pacing-induced failure in dogs with aortic stenosis-induced left ventricular hypertrophy

We recently demonstrated that rapid ventricular pacing caused cardiac failure (Failure) in dogs with aortic stenosis-induced left ventricular hypertrophy (Hypertrophy) and isoproterenol caused no significant increases in function, O₂ consumption and intracellular cyclic AMP level in the failing hypertrophied hearts. We tested the hypothesis that alterations in the ₁-adrenoceptor-signal transduction pathway would correlate with the reduced functional and metabolic responses to -adrenergic stimulation during the transition from the compensated hypertrophy to failure. Pressure overload-induced left ventricular hypertrophy was created using aortic valve plication in 10 dogs over a 6-month period. Five months after aortic valve plication, congestive heart failure was induced in 5 dogs by rapid ventricular pacing at 240 bpm for 4 weeks. The density of myocardial ₁-adrenoceptors (fmoles/mg membrane protein; fmoles/g wet tissue) was significantly reduced in the Failure dogs (176 ± 19; 755 ± 136) when compared to those of the Control (344 ± 51; 1,551 ± 203) and the Hypertrophy (298 ± 33; 1,721 ± 162) dogs. The receptor affinities were not significantly different among all groups. There was a small but significant decrease in the percentage of ₁-adrenoceptors of the failing hypertrophied hearts (62 ± 3%) when compared to that of the hypertrophied hearts (77 ± 5%). The basal myocardial adenylyl cyclase activity (rmoles/mg protein/min) was significantly lower in the Failure dogs (45 ± 4) than in the Control (116 ± 14) and Hypertrophy (86 ± 6) dogs. The forskolin (0.1 mM)-stimulated adenylyl cyclase activity was also significantly lower in the Failure dogs (158 ± 17) than in the Control dogs (296 ± 35) and slightly lower than in the Hypertrophy dogs (215 ± 10). There were no significant differences in low Km cyclic AMP-phosphodiesterase activities among all groups. We conclude that down regulation of ₁-adrenoceptors and reduced adenylyl cyclase activities contribute to the decreases in myocardial functions and -adrenergic responses in the failing hypertrophied hearts induced by rapid ventricular pacing.