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Isolated rat papillary muscles of the right ventricle were used to discover the origin of reduced myocardial efficiency in chronic heart failure. Right ventricular hypertrophy was induced by monocrotaline injection, causing pulmonary hypertension. Control (n = 7) and hypertrophied (n = 11) papillary muscles were subjected to sinusoidal length changes at 37[degrees]C and 5 Hz with a peak-to-peak amplitude of 15% of the length giving maximum force ([L.sub.max]) after being stretched to 92.5% of [L.sub.max]. Isometric tension at [L.sub.max] was similar in control and hypertrophied muscles. Work was assessed from the area encompassed by force-length loops. Work per loop was 0.93 [+ or -] 0.11 and 0.84 [+ or -] 0.11 [mu]J/[mm.sup.3] (means [+ or -] SE) for control and hypertrophied muscles, respectively (P = 0.591). Suprabasal [O.sub.2] uptake per work loop was 5.7 [+ or -] 0.7 pmol/[mm.sup.3] in control muscles and 8.7 [+ or -] 1.7 pmol/[mm.sup.3] in hypertrophied muscles (P = 0.133). Net mechanical efficiency was calculated from the ratio of work output and suprabasal [O.sub.2] uptake. The efficiency of hypertrophied muscles was 29.1 [+ or -] 3.7% and was smaller than in control muscles (43.7 [+ or -] 2.2%, P = 0.016). The right ventricular cardiomyocyte cross-sectional area increased from 272 [+ or -] 17 [micro][m.sup.2] in control muscles to 396 [+ or -] 31[micro][m.sup.2.] in hypertrophied muscles (P < 0.003). Mechanical efficiency correlated negatively with right ventricular wall thickness and cardiomyocyte cross-sectional area [Spearman rank correlation coefficients of -0.50 (P = 0.039) and -0.53 (P = 0.024), respectively]. We conclude that efficiency decreases with increasing cardiomyocyte hypertrophy. Thus, the reduced efficiency of diseased whole hearts can be at least partly explained by reduced efficiency at the cardiomyocyte level. oxygen consumption; work output; monocrotaline; pulmonary hypertension; right ventricular hypertrophy doi: 10.1152/ajpheart.00773.2009.

Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A ([ET.sub.A])/type B ([ET.sub.B]) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart. Bosentan; Sildenafil; mitochondria; oxidative capacity; chronic heart failure

We have previously shown that high-sugar diets increase mortality and left ventricular (LV) dysfunction during pressure overload. The mechanisms behind these diet-induced alterations are unclear but may involve increased oxidative stress in the myocardium. The present study examined whether high-fructose feeding increased myocardial oxidative damage and exacerbated systolic dysfunction after transverse aortic constriction (TAC) and if this effect could be attenuated by treatment with the antioxidant tempol. Immediately after surgery, TAC and sham mice were assigned to a high-starch diet (58% of total energy intake as cornstarch and 10% fat) or high-fructose diet (61% fructose and 10% fat) with or without the addition of tempol [0.1% (wt/wt) in the chow] and maintained on the treatment for 8 wk. In response to TAC, fructose-fed mice had greater cardiac hypertrophy (55.1% increase in the heart weight-to-tibia length ratio) than starch-fed mice (22.3% increase in the heart weight-to-tibia length ratio). Treatment with tempol significantly attenuated cardiac hypertrophy in fructose-fed TAC mice (18.3% increase in the heart weight-to-tibia ratio). Similarly, fructose-fed TAC mice had a decreased LV area of fractional shortening (from 38 [+ or -] 2% in sham to 22 [+ or -] 4% in TAC), which was prevented by tempol treatment (33 [+ or -] 3%). Markers of lipid peroxidation in fructose-fed TAC hearts were also blunted by tempol. In conclusion, tempol significantly blunted markers of cardiac hypertrophy, LV remodeling, contractile dysfunction, and oxidative stress in fructose-fed TAC mice. antioxidants; myocardial oxidative damage; systolic dysfunction

The study aim was to assess three-dimensional electrocardiogram (ECG) changes during development of pulmonary arterial hypertension (PAH). PAH was induced in male Wistar rats (n = 23) using monocrotaline (MCT; 40 mg/kg sc). Untreated healthy rats served as controls (n = 5). ECGs were recorded with an orthogonal three-lead system on days O, 14, and 25 and analyzed with dedicated computer software. In addition, left ventricular (LV)-to-right ventricular (RV) fractional shortening ratio was determined using echocardiography. Invasively measured RV systolic pressure was 49 (SD 10) mmHg on day 14 and 64 (SD 10) mmHg on day 25 vs. 25 (SD 2) mmHg in controls (both P < 0.001). Baseline ECGs of controls and MCT rats were similar, and ECGs of controls did not change over time. In MCT rats, ECG changes were already present on day 14 but more explicit on day 25: increased RV electromotive forces decreased mean QRS-vector magnitude and changed QRS-axis orientation. Important changes in action potential duration distribution and repolarization sequence were reflected by a decreased spatial ventricular gradient magnitude and increased QRS-T spatial angle. On day 25, LV-to-RV fractional shortening ratio was increased, and RV hypertrophy was found, but not on day 14. In conclusion, developing PAH is characterized by early ECG changes preceding RV hypertrophy, whereas severe PAH is marked by profound ECG changes associated with anatomical and functional changes in the RV. Three-dimensional ECG analysis appears to be very sensitive to early changes in RV afterload. fight ventricular hypertrophy; monocrotaline; electrocardiogram

A study was conducted to examine how protein kinase C with phorbol 12-myristate 13-acetate (PMA) influence the time-dependent changes of apical membrane epithelial Na+ channels. Isolated epithelial of frog skin and connective tissue of the corium were determined from the abdominal skins of R. pipiens pipiens. Results indicated that the effects of time of exposure on transport and time course were not reversible by omission of PMA from the bathing solution.

The probability of open channels and the total number of sodium channels were determined using a modified method of blocker-induced noise analysis to investigate the response of early time-dependent response of blocker-sensitive epithelial sodium channels (ENaCs) to stimulation using aldosterone. Results reveal that the early response of these channels to aldosterone stimulation is to increase the density of apical ENaCs.

Aldosterone and corticosterone stimulates transport of Na+ in tissue-cultured A6 epithelia by increasing the density of functional channels. In studying a variety of passages of A6 cells from different laboratories, it has been found that channel open probability of amiloride-sensitive Na+ channels remain constant. This outcome is retained regardless of substrate, serum, growth medium or baseline expression of transport.

The interaction between 3,5,3'-triiodo-L-thyronine (T3) and the coordinated regulation of SERCA sarcoplasmic reticulum isoforms and myosin heavy chains was analyzed during T3-dependent slow- to fast-twitch fiber transition. The coordinated regulation of fast isoforms of SERCA and MHC in soleus skeletal muscle fibers was detected during the induction of T3. T3 also stimulated the expression of the SERCA1 and SERCA2a isoforms at the pretranslational level in specific fibers. High levels of SERCA1 expression and sarcoplasmic reticulum Ca2+-adenosine triphosphatase activity were also stimulated by T3.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged [Alpha]- or [Beta]-subunits of the epithelial [Na.sup.+] channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in [Na.sup.+] transport. The basal level of [Na.sup.+] transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated [Na.sup.+] transport was 64% greater than control in both the [Alpha]- and [Beta]-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated [Na.sup.+] transport. sodium transport; amiloride; aldosterone; insulin; channel trafficking; adenosine 3',5'-cyclic monophosphate, signal transduction; epithelial sodium channel; green fluorescent protein

Butterworth, Michael B., Sandy I. Helman, and Willem J. Els. cAMP-sensitive endocytic trafficking in A6 epithelia. Am J Physiol Cell Physiol 280: C752-C762, 2001.--Blocker-induced noise analysis and laser scanning confocal microscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial [Na.sup.+] channels (ENaCs) at apical membranes of A6 epithelia. After forskolin stimulation of [Na.sup.+] transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities ([N.sub.T]) decreased exponentially (time constant ~20 min) from mean values of 320 to 98 channels/ cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/ cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to [is greater than] 5 [micro]m below the apical membrane. Because steady states require identical rates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at the apical membranes, but also principally to increase exocytic rates. These observations are consistent with and support, but do not prove, that vesicle trafficking is a mechanism for cAMP-mediated regulation of apical membrane channel densities in A6 epithelia. epithelial sodium channel; noise analysis; confocal microscopy; channel trafficking; vesicles; forskolin; kidney; cortical collecting ducts; endocytosis

Angiogenesis induced by growth factor-releasing microspheres can be an off-the-shelf and immediate alternative to stem cell therapy for acute myocardial infarction (AMI), independent of stem cell yield and comorbidity-induced dysfunction. Reliable and prolonged local delivery of intact proteins such as VEGF is, however, notoriously difficult. Our objective was to create a platform for local angiogenesis in human-sized hearts, using polyethylene-glycol/polybutylene-terephthalate (PEG-PBT) microsphere-based VEGF165A delivery. PEG-PBT microspheres were biocompatible, distribution was size dependent, and a regimen of 10 × 106 15-μm microspheres at 0.5 × 106/min did not induce cardiac necrosis. Efficacy, studied in a porcine model of AMI with reperfusion rather than chronic ischemia used for most reported VEGF studies, shows that microspheres were retained for at least 35 days. Acute VEGF165A release attenuated early cytokine release upon reperfusion and produced a dose-dependent increase in microvascular density at 5 wk following AMI. However, it did not improve major variables for global cardiac function, left ventricular dimensions, infarct size, or scar composition (collagen and myocyte content). Taken together, controlled VEGF165A delivery is safe, attenuates early cytokine release, and leads to a dose-dependent increase in microvascular density in the infarct zone but does not translate into changes in global or regional cardiac function and scar composition. [ABSTRACT FROM AUTHOR]

Isolated rat papillary muscles of the right ventricle were used to discover the origin of reduced myocardial efficiency in chronic heart failure. Right ventricular hypertrophy was induced by monocrotaline injection, causing pulmonary hypertension. Control (n = 7) and hypertrophied (n = 11) papillary muscles were subjected to sinusoidal length changes at 37°C and 5 Hz with a peak-to-peak amplitude of 15% of the length giving maximum force (L max) after being stretched to 92.5% of L max Isometric tension at L max was similar in control and hypertrophied muscles. Work was assessed from the area encompassed by force-length 1oops. Work per 1oop was 0.93 ± 0.11 and 0.84 ± 0.11 μJ/mm3 (means ± SE) for control and hypertrophied muscles, respectively (P = 0.591). Suprabasal O2 uptake per work loop was 5.7 ± 0.7 pmol/mm3 in control muscles and 8.7 ± 1.7 pmol/mm3 in hypertrophied muscles (P = 0.133). Net mechanical efficiency was calculated from the ratio of work output and suprabasal O2 uptake. The efficiency of hypertrophied muscles was 29.1 ± 3.7% and was smaller than in control muscles (43.7 ± 2.2%, P = 0.016). The right ventricular cardiomyocyte cross-sectional area increased from 272 ± 17 μm2 in control muscles to 396 ± 31 μm2 in hypertrophied muscles (P < 0.003). Mechanical efficiency correlated negatively with right ventricular wall thickness and cardiomyocyte cross-sectional area [Spearman rank correlation coefficients of -0.50 (P 0.039) and -0.53 (P = 0.024), respectively]. We conclude that efficiency decreases with increasing cardiomyocyte hypertrophy. Thus, the reduced efficiency of diseased whole hearts can be at least partly explained by reduced efficiency at the cardiomyocyte level. [ABSTRACT FROM AUTHOR]

Tissue-specific knockouts generated through Cre-loxP recombination have become an important tool to manipulate the mouse genome. Normally, two successive rounds of breeding are performed to generate mice carrying two floxed target-gene alleles and a transgene expressing Cre-recombinase tissue-specifically. We show herein that two promoters commonly used to generate endothelium-specific (Tie2) and smooth muscle-specific [smooth muscle myosin heavy chain (Smmhc)] knockout mice exhibit activity in the female and male germ lines, respectively. This can result in the inheritance of a null allele in the second generation that is not tissue specific. Careful experimental design is required therefore to ensure that tissue-specific knockouts are indeed tissue specific and that appropriate controls are used to compare strains. [ABSTRACT FROM AUTHOR]

The value of the diffusion coefficient for oxygen in muscle is uncertain. The diffusion coefficient is important because it is a determinant of the extracellular oxygen tension at which the core of muscle fibers becomes anoxic (Po2crit). Anoxic cores in muscle fibers impair muscular function and may limit adaptation of muscle cells to increased load and/or activity. We used Hill's diffusion equations to determine Krogh's diffusion coefficient (Dα) for oxygen in single skeletal muscle fibers from Xenopus laevis at 20°C (n = 6) and in myocardial trabeculae from the rat at 37°C (n = 9). The trabeculae were dissected from the right ventricular myocardium of control (n = 4) and 'monocrotaline-treated, pulmonary hypertensive rats (n = 5). The cross-sectional area of the preparations, the maximum rate of oxygen consumption (Vo2 max), and Po2crit were determined. Dα increased in the following order: Xenopus muscle fibers Dα = 1.23 nM·mm²·mmHg-1·s-1 (SD 0.12), control rat trabeculae Dα = 2.29 nM·mm²·mmHg-1·s-1 (SD 0.24) (P = 0.0012 vs. Xenopus), and hypertrophied rat trabeculae Dα = 6.0 nM·mm²·mmHg-1·s-1 (SD 2.8) (P = 0.039 vs. control rat trabeculae). Dα increased with extracellular space in the preparation (Spearman's rank correlation coefficient = 0.92, P < 0.001). The values for Dα indicate that Xenopus muscle fibers cannot reach Vo2 max in vivo because Po2crit can be higher than arterial Po2 and that hypertrophied rat cardiomyocytes can become hypoxic at the maximum heart rate. [ABSTRACT FROM AUTHOR]