Heavy Chain mRNA Expression Research Articles

Age exacerbates chronic catecholamine-induced impairments in contractile reserve in the rat

Contractile reserve decreases with advancing age and chronic isoproterenol (ISO) administration is a well-characterized model of cardiac hypertrophy known to impair cardiovascular function. This study evaluated whether nonsenescent, mature adult rats are more susceptible to detrimental effects of chronic ISO administration than younger adult rats. Rats received daily injections of ISO (0.1 mg/kg sc) or vehicle for 3 wk. ISO induced a greater impairment in contractile reserve [maximum of left ventricular pressure development (Δ+dP/dt(max))] in mature adult ISO-treated (MA-ISO) than in young adult ISO-treated rats (YA-ISO) in response to infusions of mechanistically distinct inotropes (digoxin, milrinone; 20-200 μl·kg(-1)·min(-1)), while basal and agonist-induced changes in heart rate and systolic arterial pressure (SAP) were not different across groups. ISO decreased expression of the calcium handling protein, sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a, in MA-ISO compared with YA, YA-ISO, and MA rats. Chronic ISO also induced greater increases in cardiac hypertrophy [left ventricular (LV) index: 33 ± 3 vs. 22 ± 5%] and caspase-3 activity (34 vs. 5%) in MA-ISO relative to YA-ISO rats. Moreover, β-myosin heavy chain (β-MHC) and atrial natriuretic factor (ANF) mRNA expression was significantly elevated in MA-ISO. These results demonstrate that adult rats develop greater impairments in systolic performance than younger rats when exposed to chronic catecholamine excess. Reduced contractile reserve may result from calcium dysregulation, increased caspase-3 activity, or increased β-MHC and ANF expression. Although several studies report age-related declines in systolic performance in older and senescent animals, the present study demonstrates that catecholamine excess induces reductions in systolic performance significantly earlier in life.

G.P.2.06 Diaphragm muscle weakness in an experimental intensive care unit model

Diaphragm muscle dysfunction contributing to weaning failure in critically ill patients remains unclear. It may not fully depend on ventilator-induced modifications. Sepsis and administration of pharmacological agents such as corticosteroids and neuromuscular blocking agents may be involved. The objectives of this study were to examine to which extent sepsis, corticosteroid treatment and neuromuscular blocking agent administration aggravate ventilator-related diaphragm dysfunction using a unique piglet intensive care unit model. Piglets were randomly sham-operated, or for five days, mechanically ventilated, mechanically ventilated together with an endotoxin-induced sepsis, mechanically ventilated together with corticosteroid treatment, mechanically ventilated together with neuromuscular blocking agent administration, or mechanically ventilated together with sepsis, corticosteroid and neuromuscular blocking agent. On day 5, muscle fibers were isolated from diaphragm biopsies and frozen or membrane-permeabilized. The cross-sectional area and maximal force normalized to cross-sectional area were evaluated at the single muscle fiber level. Myosin heavy chain and actin protein mRNA expression were assessed in diaphragm muscle samples. Results show (i) a decrease in fiber maximal force normalized to cross-sectional area coupled with a reduction in myosin heavy chain mRNA level in all mechanically ventilated piglets; (ii) a decrease in fiber size in piglets that were mechanically ventilated in combination with sepsis, corticosteroid and neuromuscular blocking agent. In conclusion, five days mechanical ventilation had a significant negative effect on the force generating capacity of individual diaphragm muscle fibers. Sepsis, corticosteroid or neuromuscular blocking agent had no additional on force generation or muscle size, but both force normalized to fiber cross-sectional area and fiber size decreased significantly when all were combined with mechanical ventilation.

MyoD, Myogenin and Myosin Heavy Chain mRNA Expression in Rat Skeletal Muscle after a Single Session of Low-Intensity Treadmill Exercise

[Purpose] We examined the time course of changes in the activation of muscle satellite cells and muscle protein synthesis after a single session of low-intensity treadmill exercise. [Subjects] A total of 25 male Wistar rats aged 11 weeks were used. [Methods] Rats were run on a treadmill with a 16° decline for 30 min at 24 m/min, except for the control group (CON). Blood was collected to measure creatine phosphokinase (CPK) and both soleus muscles were removed to analyze histological and muscle gene expression at 24, 48, 72 and 96 h post-exercise. [Results] CPK levels in exercised rats at 24 and 96 h were significantly higher than in CON, and CPK levels at 96 h were higher than those at 48 h. Infiltration of macrophages and gathering of small myofibers were detected in the exercise group, but there were no significant differences in MyoD, myogenin, myosin heavy chain (MHC) -1 and MHC-2a mRNA levels. [Conclusion] These results suggest that muscle satellite cells and muscle protein synthesis are not significantly activated, despite the occurrence of myotrauma, in the acute phase (24 h to 96 h) after a single session of low-intensity exercise.

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

PYK2 is a Ca 2+-dependent, nonreceptor protein tyrosine kinase that is involved in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. We and others have previously investigated PYK2's function in vitro using cultured neonatal and adult rat ventricular myocytes as model systems. However, the function of PYK2 in the in vivo adult heart remains unclear. Here we evaluate the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. First we demonstrate that CRNK functions as a dominant-negative inhibitor of PYK2-dependent signaling, presumably by displacing PYK2 from focal adhesions and costameres. Then, male Sprague–Dawley rats (~ 300 g) underwent permanent left anterior descending coronary artery ligation. One wk post-MI, either Adv-GFP ( n = 34) or Adv-CRNK ( n = 28) was administered (10 10 pfu, 0.1 ml) via catheter-based, Optison ®-mediated gene transfer. LV structure and function were evaluated by echocardiography 1 and 3 wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK overexpression was readily detected by Western blotting 1 wk following gene transfer. Adv-CRNK improved overall survival ( P = 0.03; Logrank Test) and LV fractional shortening (23 ± 2% vs. 31 ± 2% for Adv-GFP vs. Adv-CRNK infected animals, respectively; P < 0.05). Whereas MI hearts exhibited increased β-, and decreased α-myosin heavy chain (MHC) mRNA expression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3 ± 1.4 fold increase in αMHC; 0.4 ± 0.1 fold decrease in βMHC; P < 0.05 for both). In summary, CRNK gene transfer improves survival, increases LV function, and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI.

Point:Counterpoint: Satellite cell addition is/is not obligatory for skeletal muscle hypertrophy

Myofiber size is dynamically regulated, increasing and decreasing depending on muscle use. Hypertrophy is defined by increases in myofiber cross-sectional area and mass as well as myofibrillar protein content. Myofibers contain many hundreds of nuclei, each of which has a nuclear domain. A nuclear

Effects of stretching stimulation with different rates on the expression of MyHC mRNA in mouse cultured myoblasts

In vivo studies have shown that changes in the characteristics of skeletal muscle fiber are determined by type of exercise or training. These earlier studies on mechanical stimulation, however, have all employed stimulation applied at a constant intensity, and no studies appear to have investigated change with variation of intensity of stimulation. In this study, we investigated the characteristics and differentiation of myoblasts stretched at different rates. Myoblasts were stimulated at 3 different rates, and the numbers of cells and nuclei on days 1, 3, and 5 were compared. The myosin heavy chain (MyHC) mRNA expression level was also compared. We investigated expression of MyHC-perinatal to determine speed of differentiation of myoblasts, and expression of MyHC-2b, 2d, and 2a to ascertain muscle cell characteristics. Counting cells and nuclei of myoblasts revealed clear promotion of differentiation with stretching. With rapid stretching, expression of MyHC-perinatal was high at first, but then showed a decrease. In terms of effect on muscle fiber characteristics, MyHC-2b, MyHC-2d, and MyHC-2a were high with rapid, medium, and slow stretching, respectively. This indicated that myoblast differentiation was promoted regardless of difference in stretching speed, with the myoblasts acquiring the muscle-fiber characteristics appropriate to each rate of stretching.

Molecular analysis of successful cell line selection in transfected GS‐NS0 myeloma cells

The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.

Animal age affects soleus myosin heavy chain (MHC) mRNA expression and myostatin protein following hindlimb unweighting (HU)

Aim to determine the effect of age (maturity) on myosin heavy chain (MHC) expression in the soleus muscle following HU. We hypothesized a similar shift in soleus MHC expression and myostatin protein following HU in growing and mature rats. Growing (2 mos, n=12) and mature (8 mos, n=12) male rats were ambulatory or HU. Soleus total protein (BCA), myosin (SDS-PAGE), MHC isoforms (RT-PCR), and myostatin (immunoblot) were determined using standard techniques. Protein content and total myosin decreased similarly with HU in growing and mature animals. IIa expression was unchanged with HU in growing animals but significantly upregulated in mature rats. With HU a greater than 500-fold increase in IIb MHC expression was observed in growing rats whereas IIb expression in mature animals was down-regulated 40%. Unweighting resulted in a 0.5-fold myostatin protein increase in young rats, and a 2.5-fold increase in mature animals, compared to control values. It may be concluded that the age of the animal will not affect total protein or total myosin following HU. However, maturity of the animal will influence soleus MHC expression and myostatin protein, with HU. This information may be useful in comparing across studies relating fiber type and inactivity/activity. NA65-12300, AG15796, NAA04CC62G

Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCε phosphorylation

We have previously reported a transgenic mouse that over-expresses constitutively active PKCepsilon in the myocardium and exhibits a steady progression to heart failure. Associated with the decline in function was an increased phosphorylation of sarcomeric proteins including cardiac troponin I (cTnI). To determine whether PKCepsilon phosphorylation of cTnI is sufficient to induce cardiac maladaptation, we have generated a double transgenic mouse (DbTG) that expresses constitutively active PKCepsilon and cTnI harboring non-phosphorylatable mutations in the putative PKC phosphorylation sites (S43A, S45A). We compared the hemodynamic and biochemical properties of the hearts from the DbTG mice to the non-transgenic and single transgenic lines at both 3 and 12 months of age. While no significant differences in LV function were noted in 3-month groups, the depression of function in the PKCepsilon mice was attenuated in the double transgenic mice at 12 months. The improvement in cardiac function was correlated with decreased beta-myosin heavy chain and ANF mRNA expression in the 12m DbTG mice. The extent of cTnI phosphorylation was determined using a novel one-dimensional, non-equilibrium isoelectric focusing technique. At 3 months the migration of cTnI phospho-species was different in the PKCepsilon mice and to a lesser degree in the DbTG compared to all other groups. At 12 months additional phospho-species were observed in both the PKCepsilon and DbTG samples, along with an overall shift in the distribution of phospho-species in all groups due to age. These results suggest that phosphorylation of cTnI by PKCepsilon is associated with contractile dysfunction and partial replacement of serines 43/45 improves cardiac performance. Therefore, we conclude that phosphorylation of cTnI at Ser 43 and 45 may contribute to the progression of failure.

In vivo inhibition of nitric oxide synthase impairs upregulation of contractile protein mRNA in overloaded plantaris muscle

Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth following 5 or 12 days of functional overload. Male Sprague-Dawley rats (approximately 350 g) were randomly divided into three treatment groups: control, N(G)-nitro-L-arginine methyl ester (L-NAME; 90 mg.kg(-1).day(-1)), and 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg.kg(-1).day(-1)). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris. Sham surgery performed on the left hindlimb served as a normally loaded control. L-NAME and TRIM treatments prevented OL-induced skeletal alpha-actin and type I (slow) myosin heavy chain mRNA expression at 5 days. Conversely, neither L-NAME nor TRIM affected hepatocyte growth factor or VEGF mRNA responses to OL at 5 days. However, OL induction of IGF-I and mechanogrowth factor mRNA was greater (P < 0.05) in the TRIM group compared with the controls. Furthermore, the phosphorylated-to-total p70 S6 kinase ratio was higher in OL muscle from NOS-inhibited groups, compared with control OL. At 12 days of OL, the cumulative proliferation of plantaris satellite cells was assessed by subcutaneous implantation of time release 5'-bromo-2'-deoxyuridine pellets during the OL-inducing surgeries. Although OL caused a fivefold increase in the number of mitotically active (5'-bromo-2'-deoxyuridine positive) sublaminar nuclei, this was unaffected by concurrent NOS inhibition. Therefore, NOS activity may provide negative feedback control of IGF-I/p70 S6 kinase signaling during muscle growth. Moreover, NOS activity may be involved in transcriptional regulation of skeletal alpha-actin and type I (slow) myosin heavy chain during functional overload.

Myosin heavy chain mRNA expression correlates higher with muscle protein accretion than growth in Atlantic salmon, Salmo salar

In order to link growth and protein accretion in Atlantic salmon the mRNA expression of muscle myosin heavy chain ( MyHC) was analysed. MyHC gene is expressed throughout muscle development and is consistent with the hypertropic growth in fish. Total RNA was isolated from white muscle tissues ( N = 32) from salmon fed a fish meal based diet with three levels of solubilised protein incorporated as fish protein hydrolysate (FPH) control (0 g kg − 1 ), medium (180 g kg − 1 ) and high (300 g kg − 1 ) FPH inclusion. The salmon were PIT-tagged and fed the experimental diets for a period of 68 days. A high level of dietary FPH resulted in significant up-regulation of MyHC by a factor of 2.4 compared to fish fed the medium FPH inclusion. The fish with highest MyHC expression also contained significant higher levels of muscle protein. MyHC expression correlated higher with protein accretion than individual specific growth rate (SGR) in salmon. These observations indicate that MyHC mRNA expression can be a useful marker for understanding of growth and protein accretion in Atlantic salmon.

Changes in myosin heavy chain mRNA and protein expression in human skeletal muscle with age and endurance exercise training

Aging is associated with reduced muscle strength and atrophy of type II muscle fibers. Muscle fiber type and contractile function are primarily determined by myosin heavy chain (MHC) isoforms. There are few data available on the effects of aging on MHC isoform expression in humans. In the present study, we tested the hypothesis that MHC isoform protein composition and mRNA abundance would favor a fast-to-slow isoform shift with aging and in response to endurance exercise training. Muscle biopsies were obtained from previously sedentary, healthy men and women, aged 21-87 yr before (n = 77) and after (n = 65) 16 wk of bicycle training (up to 45 min at 80% peak heart rate, 3-4 days/wk). At baseline, MHC I mRNA was unchanged with age, whereas IIa and IIx declined by 14 and 10% per decade, respectively (P < 0.001). MHC IIa and IIx protein declined by 3 and 1% per decade with a reciprocal increase in MHC I (P < 0.05). After training, MHC I and IIa mRNA increased by 61 and 99%, respectively, and IIx decreased by 50% (all P < 0.001). The increase in MHC I mRNA was positively associated with age, whereas the changes in MHC IIa and IIx mRNA were similar across age. MHC I protein increased by 6% and was positively related to age, whereas IIx decreased by 5% and was inversely related to age. These results suggest that the altered expression of MHC isoforms with aging is transcriptionally regulated. In response to endurance exercise, regulation of MHC isoform transcripts remains robust in older muscle, but this did not result in corresponding changes in MHC protein expression.

Changes in detrusor smooth muscle myosin heavy chain mRNA expression following spinal cord injury in the mouse.

Smooth muscle myosin heavy chain (SMMHC) isoform composition has been shown to be developmentally regulated and to be associated with functional changes in smooth muscle activity. In this study, we sought to determine expression patterns of SMMHC isoforms in a murine model of spinal cord injury (SCI) and to compare these expression patterns to neurologic, cytometric, and morphometric findings. Baseline cystometry was performed on adult, female mice followed by either thoracic spinal cord transection (SCI) or sham operation (Sham). At 1, 3, or 6 weeks postoperatively neurologic evaluation and cystometry were performed, bladders were harvested, and expression patterns of SMMHC isoforms (SM1 vs. SM2 and SMA vs. SMB) were assessed by RT-PCR. Morphometrics utilizing computer-assisted color image analysis was also performed on all bladders. There was a significant increase in bladder weight and capacity 1 week following SCI which normalized over time, however, morphometric analysis did not reveal an alteration in tissue composition amongst the three groups. One week following SCI, SM1 was predominantly expressed over SM2 and began to normalize at 3 weeks. This coincided with the emergence of reflex voiding and detrusor overactivity. SMA was expressed following SCI only, and the number of bladders found to express SMA decreased with increasing duration since SCI. Smooth muscle myosin heavy chain mRNA expression patterns appear to be affected by SCI. We believe the induction of SMA may be a factor in altered bladder function following injury.

B-Type Natriuretic Peptide Prevents Acute Hypertrophic Responses in the Diabetic Rat Heart

Stimulation of cardiomyocyte guanosine 3',5'-cyclic monophosphate (cyclic GMP) via endothelial-derived nitric oxide (NO) is an important mechanism by which bradykinin and ACE inhibitors prevent hypertrophy. Endothelial NO dysfunction and cardiac hypertrophy are morbid features of diabetes not entirely prevented by ACE inhibitors. In cardiomyocyte/endothelial cell cocultures, bradykinin efficacy is abolished by high-glucose-induced endothelial NO dysfunction. We now demonstrate that antihypertrophic actions of natriuretic peptides, which stimulate cyclic GMP independently of NO, are preserved in cardiomyocytes despite high-glucose-induced endothelial dysfunction. Further, streptozotocin-induced diabetes significantly impairs the effectiveness of acute antihypertrophic strategies in isolated rat hearts. In hearts from citrate-treated control rats, angiotensin II-stimulated [(3)H]phenylalanine incorporation and atrial natriuretic peptide and beta-myosin heavy chain mRNA expression were prevented by B-type natriuretic peptide (BNP), bradykinin, the ACE inhibitor ramiprilat, and the neutral endopeptidase inhibitor candoxatrilat. These antihypertrophic effects were accompanied by increased left ventricular cyclic GMP. In age-matched diabetic hearts, the antihypertrophic and cyclic GMP stimulatory actions of bradykinin, ramiprilat, and candoxatrilat were absent. However, the blunting of hypertrophic markers and accompanying increases in cyclic GMP stimulated by BNP were preserved in diabetes. Thus BNP, which increases cyclic GMP independently of NO, is an important approach to prevent growth in the diabetic myocardium, where endothelium-dependent mechanisms are compromised.

EFFECTS OF ORAL CREATINE AND HEAVY RESISTANCE TRAINING ON MYOGENIC REGULATORY FACTOR AND CREATINE KINASE EXPRESSION

We have recently shown 12 wks of oral creatine (Cr) supplementation and heavy resistance training to increase myosin heavy chain mRNA and protein expression (MSSE, 33(10): 1674–1681, 2001). Using muscle samples from our previous study the purpose of this study was to further determine the effects of oral Cr and heavy resistance training on skeletal muscle creatine kinase (M-CK) mRNA expression and the mRNA and protein expression of the myogenic regulatory factors Myo-D, myogenin, MFR-4, and Myf5. Twenty-two untrained males were randomly assigned to either a control (CON), placebo (PLC), or Cr (CRT) group in a double blind fashion. Muscle biopsies were obtained before and after 12 wks of resistance training. PLC and CRT trained thrice weekly using 3 sets of 6–8 repetitions at 85%-90% 1-RM on the leg press, knee extension, and knee curl exercises. CRT ingested 6 g/d of Cr for 12 wks white PLC consumed the equal concentration of dextrose placebo. After training, M-CK mRNA expression, as well as myogenin and MRF-4 mRNA and protein expression, were found to be significantly greater for CRT compared to PLC and CON, while PLC was also significantly different from CON (P <0.05). For Myo-D mRNA and protein, both CRT and PLC were significantly different from CON (P <0.05) but CRT and PLC were not different from one another. No significant differences were located for Myf5 mRNA or protein (P >0.05). M-CK mRNA was correlated with myogenin and MRF-4 protein (P <0.05). Long-term Cr supplementation increases M-CK mRNA expression, likely due to concomitant increases in the protein expression of the transcription factors myogenin and MRF-4. The increase in myogenin and MRF-4 mRNA and protein may have also played a rote in the increases in myosin heavy chain expression that we have previously shown to occur with Cr supplementation. Supported in part by NutraSense, Inc., Shawnee Mission, KS

Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di- ortho-substituted polychlorinated biphenyls

Halogenated aromatic hydrocarbons (HAHs) are ubiquitous environment contaminants that produce many of their toxic effects by binding to the aryl hydrocarbon receptor (AhR). However, several investigations have demonstrated that certain polychlorinated biphenyl (PCB) congeners, principally di- ortho-chlorinated PCB congeners, or mixtures containing multiple di- ortho-chlorinated PCBs, inhibit AhR-mediated responses induced by other toxic HAHs. Most relevant to the present study are past reports demonstrating antagonism by these uniquely acting PCB congeners on AhR agonist-mediated inhibition of humoral immune responses. The mechanism responsible for antagonism of AhR agonists by certain PCBs is presently unknown. The present study evaluated the antagonist activity of several di- ortho-substituted PCB congeners [PCB47 (2,2′,4,4′), PCB52 (2,2′,5,5′), PCB128 (2,2′,3,3′,4,4′), and PCB153 (2,2′,4,4′,5,5′)] when present in combination with AhR agonists [TCDD (2,3,7,8,-tetrachlorodibenzo- p-dioxin), PCB126 (3,3′,4,4′,5), and PCB77 (3,3′,4,4′)] on CYP1A1 induction and inhibition of lipopolysaccharide (LPS)-induced immunoglobulin production in the CH12.LX B cell line. In contrast to non- ortho-substituted PCB (PCB77), which showed additive effects on CYP1A1 induction in combination with TCDD, all of the di- ortho-substituted PCBs examined produced antagonism. Di- ortho-substituted PCB (PCB52) also antagonized TCDD- or PCB126- mediated inhibition of IgM secretion and immunoglobulin heavy chain mRNA expression in the LPS-activated B cells. In addition, PCB52 inhibited TCDD-induced AhR DNA binding to a dioxin-responsive element. Collectively, these results suggest that the mechanism responsible for antagonism by di- ortho-substituted PCB congeners of AhR agonist-mediated CYP1A1 induction and inhibition of antibody responses in B cells occurs through interference with agonist activation of the cytosolic AhR complex.

Coordinate Changes in Myosin Heavy Chain Isoform Gene Expression Are Selectively Associated With Alterations in Dilated Cardiomyopathy Phenotype

The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.

Oestrogen action on the myocardium in vivo: specific and permissive for angiotensin-converting enzyme inhibition.

In contrast to the vasculature, it remains unclear whether oestrogens also directly affect the myocardium. In this study, we addressed basic questions regarding oestrogen effects on the myocardium, including specificity, pathophysiological relevance and potential clinical implications, with a special focus on interactions between oestrogen and angiotensin-converting enzyme (ACE) inhibitors in an established in-vivo model of cardiac hypertrophy. Female spontaneously hypertensive rats (SHR) were ovarectomized (OVX) or sham-operated and treated with 17beta-oestradiol (2 microg/kg per day subcutaneously), the oestrogen receptor antagonist ZM-182780 (250 microg/kg per day subcutaneously) and the ACE-inhibitor moexipril (10 mg/kg per day orally) alone or in combination for 3 months. Hormone replacement restored physiological oestradiol serum levels and prevented uterus atrophy. Whereas moexipril alone was ineffective in OVX rats, substitution of oestradiol restored the beneficial effect of moexipril on systolic blood pressure (-30 +/- 5 mmHg) and relative heart weight (-11 +/- 3%) in OVX rats. Oestradiol upregulated alpha-myosin heavy chain (MHC) mRNA (+37 +/- 7%) and protein expression (+43 +/- 6%) in spite of increased blood pressure in OVX rats. Simultaneous treatment with oestradiol plus moexipril most effectively shifted the ratio of alpha-/beta-MHC mRNA and protein expression towards alpha-MHC in OVX animals. Oestradiol (10 nmol/l) also upregulated alpha-MHC mRNA and protein in cultured cardiac myocytes. The oestrogen receptor antagonist ZM-182780 significantly inhibited the observed oestrogen effects. Oestrogen replacement is permissive for the beneficial effects of ACE-inhibition in female SHR rats. Oestrogen effects on the myocardium in vivo are specific (i.e. oestrogen receptor mediated) because they are inhibited by a pure oestrogen receptor antagonist and occur at physiological hormone levels.

Pivotal Role of a gp91 phox -Containing NADPH Oxidase in Angiotensin II-Induced Cardiac Hypertrophy in Mice

Angiotensin II induces both cardiac and vascular smooth muscle (VSM) hypertrophy. Recent studies suggest a central role for a phagocyte-type NADPH oxidase in angiotensin II-induced VSM hypertrophy. The possible involvement of an NADPH oxidase in the development of cardiac hypertrophy has not been studied. Methods and Results- Mice with targeted disruption of the NADPH oxidase subunit gp91(phox) (gp91(phox-/-)) and matched wild-type mice were subjected to subcutaneous angiotensin II infusion at a subpressor dose (0.3 mg/kg/day) for 2 weeks. Systolic blood pressure was unaltered by angiotensin II in either group. Angiotensin II significantly increased heart/body weight ratio, atrial natriuretic factor and beta-myosin heavy chain mRNA expression, myocyte area, and cardiac collagen content in wild-type but not gp91(phox-/-) mice. Angiotensin II treatment increased myocardial NADPH oxidase activity in wild-type but not gp91(phox-/-) mice. A gp91(phox)-containing NADPH oxidase plays an important role in the development of angiotensin II-induced cardiac hypertrophy, independent of changes in blood pressure.

Reduced muscle redox capacity after endurance training in patients with chronic obstructive pulmonary disease.

The present study was undertaken to test whether endurance training in patients with COPD, along with enhancement of muscle bioenergetics, decreases muscle redox capacity as a result of recurrent episodes of cell hypoxia induced by high intensity exercise sessions. Seventeen patients with COPD (FEV(1), 38 +/- 4% pred; PaO2), 69 +/- 2.7 mm Hg; PaCO2, 42 +/- 1.7 mm Hg) and five age-matched control subjects (C) were studied pretraining and post-training. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and gamma-glutamyl cysteine synthase heavy subunit chain mRNA expression (gammaGCS-HS mRNA) were measured in the vastus lateralis. Pretraining redox status at rest and after moderate (40% Wpeak) constant-work rate exercise were similar between groups. After training (DeltaWpeak, 27 +/- 7% and 37 +/- 18%, COPD and C, respectively) (p < 0.05 each), GSSG levels increased only in patients with COPD (from 0.7 +/- 0.08 to 1.0 +/- 0.15 nmol/ mg protein, p < 0.05) with maintenance of GSH levels, whereas GSH markedly increased in C (from 4.6 +/- 1.03 to 8.7 +/- 0.41 nmol/ mg protein, p < 0.01). Post-training gammaGCS-HS mRNA levels increased after submaximal exercise in patients with COPD. No evidence of lipid peroxidation was observed. We conclude that although endurance training increased muscle redox potential in healthy subjects, patients with COPD showed a reduced ability to adapt to endurance training reflected in lower capacity to synthesize GSH.