Lamers, J.M.J.

Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction (Article)

2009-05-01T00:00:00Z

It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (Fmax). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+ homeostasis, as myofilament Fmax actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and L-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."

Prevention of myofilament dysfunction by β-blocker therapy in postinfarct remodeling (Article)

2009-05-01T00:00:00Z

Background-Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether β-blocker therapy could restore myofilament contractility. Methods and Results-In pigs with a MI induced by ligation of the left circumflex coronary artery, β-blocker therapy (bisoprolol, MI+β) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (Fmax) was lower, whereas Ca2+sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca2+sensitivity was abolished by treatment of cells with the β-adrenergic kinase, protein kinase A. β-blocker therapy partially reversed Fmaxand Ca2+sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca2+sensitivity in MI+β compared with untreated myocardium, the protein kinase A induced reduction in Ca2+sensitivity was largest in cardiomyocytes from myocardium treated with β-blockers. Phosphorylation of β-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. β-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions-β-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of β-blockade on global left ventricular function after MI.

Cardiac myosin-binding protein C mutations and hypertrophic ardiomyopathy haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction (Article)

2009-03-24T00:00:00Z

Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3mut).Methods and Results- Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864-2865delCT, n=4) and nonfailing donors (n= 13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mutby 33 ±5%. Cardiac MyBP-C phosphorylation in MYBPC3mutsamples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84 ±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the β-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross- sectional area of the myocytes in MYBPC3mut(20.2±2.7 kN/m2) compared with donor (34.5± 1.1 kN/m2). Moreover, Ca2+sensitivity was higher in MYBPC3mut(pCa50=5.62±0.04) than in donor (pCa50=5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic β-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca sensitivity between MYBPC3mut(pCa50=5.46±0.03) and donor (pCa50=5.48±0. 02).Conclusions- Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+sensitivity in MYBPC3mutis due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Identification by a differential proteomic approach of the induced stress and redox proteins by resveratrol in the normal and diabetic rat heart (Article)

2008-09-01T00:00:00Z

A recent study showed cardioprotective effects of resveratrol on the diabetic heart. The present study sought to compare the protein profiles of the normal versus diabetic hearts after resveratrol treatment using differential proteomic analysis. Rats were randomly divided into two groups: control and diabetic. Both groups of rats were fed resveratrol (2.5 mg/kg/day) for 7 days, and then the rats were sacrificed, hearts were isolated and cytoplasmic fraction from left ventricular tissue was collected to carry out proteomic profiling as well as immunoblotting. Compared to normal hearts, diabetic hearts show increased myocardial infarct size and cardiomy-ocyte apoptosis upon ex vivo global ischaemia of 30 min. followed by 2 hrs of reperfusion. Resveratrol reduced infarct size and apop-totic cell death for both the groups, but the extent of infarct size and apoptosis remained higher for the diabetic group compared to the normal group. The left ventricular cytoplasmic proteins were analysed by 2D-DIGE and differentially displayed bands were further analysed by nano Liquid Chromatography-Mass Spectroscopy (LC-MS/MS). The results showed differential regulation of normal versus diabetic hearts treated with resveratrol of many proteins related to energy metabolism of which several were identified as mitochondrial proteins. Of particular interest is the increased expression of several chaperone proteins and oxidative stress and redox proteins in the diabetic group including Hsc70, HSPp6, GRP75, peroxiredoxin (Prdx)-1 and Prdx-3 whose expression was reversed by resveratrol. Western blot analysis was performed to validate the up- or down-regulation of these stress proteins. The results indicate the differential regulation by resveratrol of stress proteins in diabetic versus normal hearts, which may explain in part the beneficial effects of resveratrol in diabetic induced cardiovascular complications.

Integrative control of coronary resistance vessel tone by endothelin and angiotensin II is altered in swine with a recent myocardial infarction (Article)

2008-05-01T00:00:00Z

Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT1) and ETA/ETBreceptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ETA/ETBreceptor blockade in the presence of AT1 blockade was similar to vasodilation produced by ETA/ETBblockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT1and ETA/ETBreceptor blockade were virtually abolished, despite similar coronary arteriolar AT1and ETAreceptor expression compared with normal swine. Unexpectedly, in the presence of AT1blockade (which had no effect on circulating ET levels), ETA/ETBreceptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone. Copyright

Quantitative analysis of myofilament protein phosphorylation in small cardiac biopsies (Article)

2007-10-01T00:00:00Z

Phosphorylation of cardiac myofilament proteins represents one of the main post-translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time- (or dose-) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2-DE and Pro-Q® Diamond stained gradient gels using minor amounts (-0.5 mg dry weight) of human and pig cardiac tissue.

Time dependent changes in cytoplasmic proteins of the right ventricle during prolonged pressure overload (Article)

2007-08-01T00:00:00Z

In many forms of congenital heart disease, the right ventricle (RV) is subject to abnormal loading conditions resulting in RV hypertrophy and remodeling. We determined the alterations in RV cytoplasmic proteomic phenotype that occur during prolonged periods of RV pressure overload. We performed a differential proteomic profiling study on RV hypertrophy using an animal model of various durations of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. This hemodynamic evaluation showed that after 6, 12 and 20 weeks of PAB, the RV is in a compensated state of hypertrophy. Overall, the majority of protein changes were metabolism related indicating a shift towards the glycolytic pathway at the expense of β-oxidation in the RV of the PAB animals. The changes in proteins related to the glycolytic pathway, exemplified by enolase and creatine kinase B-chain, tended to precede changes in β-oxidation. In parallel, increases in stress chaperones, exemplified by several phosphorylated HSP-27 species, are present from the 6 week time point, whereas increases in antioxidant proteins, exemplified by peroxiredoxin 2 and 6, appear to be restricted to the 12 week time point. The p38 MAPK signal transduction pathway appears not to be activated. Observed protein changes are likely part of a protective mechanism against the development of RV failure.

Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction (Article)

2007-04-01T00:00:00Z

The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca-handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8±1 to 12±1%, and LVdP/dtP30 from 5295±207 to 5794±207 mm Hg/s (both P<0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca-sensitivity (ΔpCa50=0.037). This effect of exercise was PKA-mediated and likely because of improved β1-adrenergic signaling, as suggested by the increased β1-adrenoceptor protein (48%) and cAMP levels (36%; all P<0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (Fmax increased from 14.3±0.7 to 18.3±0.8 kN/mP<0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1±0.3 to 7.0±0.6%; P<0.05). Furthermore, exercise reduced diastolic Ca-concentrations (by ∼30%, P<0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca-transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.

Cardiac effects of postconditioning depend critically on the duration of index ischemia (Article)

2007-03-01T00:00:00Z

Postconditioning (POC) is known as the phenomenon whereby brief intermittent ischemia applied at the onset of reperfusion following index ischemia limits myocardial infarct size. Whereas there is evidence that the algorithm of the POC stimulus is an important determinant of the protective efficacy, the importance of the duration of index ischemia on the outcome of the effects of POC has received little attention. Pentobarbital sodium-anesthetized Wistar rats were therefore subjected to index ischemia produced by coronary artery occlusions (CAO) of varying duration (15-120 min) followed by reperfusion, without or with postconditioning produced by three cycles of 30-s reperfusion and reocclusion (3POC30). 3POC30 limited infarct size produced by 45-min CAO (CAO45) from 45 ± 3% to 31 ± 5%, and CAO60 from 60 ± 3% to 47 ± 6% (both P ≤ 0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 ± 1% to 19 ± 6% and CAO30 from 36 ± 6 to 48 ± 4% (both P ≤ 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH- kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production. Copyright

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban (Article)

2007-02-01T00:00:00Z

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca2+pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca2+sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable >99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7±2% left), whereas SERCA, Na+/Ca2+exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca2+uptake at 0.34μM of free Ca2+, and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca2+uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca2+sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Genomic and nongenomic effects of aldosterone in the rat heart: why is spironolactone cardioprotective? (Article)

2005-07-01T00:00:00Z

1. Mineralocorticoid receptor (MR) antagonism with spironolactone reduces mortality in heart failure on top of ACE inhibition. To investigate the underlying mechanism, we compared the actions of both aldosterone and spironolactone to those of angiotensin (Ang) II in the rat heart. 2. Hearts of male Wistar rats were perfused according to Langendorff. Ang II and aldosterone increased left ventricular pressure (LVP) by maximally 11+/-4 and 9+/-2%, and decreased coronary flow (CF) by maximally 36+/-7 and 20+/-4%, respectively. Spironolactone did not significantly affect LVP or CF. 3. In hearts that were exposed to a 45-min coronary artery occlusion and 3 h of reperfusion, a 15-min exposure to spironolactone prior to occlusion reduced infarct size (% of risk area) from 68+/-2 to 45+/-3%, similar to the reduction (34+/-2%) observed following 'preconditioning' (15 min occlusion followed by 10 min reperfusion) prior to the 45-min occlusion. Aldosterone exposure did not affect infarct size (71+/-5%). 4. In cardiomyocytes, aldosterone decreased [(3)H]thymidine incorporation maximally by 73+/-3%, whereas in cardiac fibroblasts it decreased [(3)H]proline incorporation by 33+/-7%. Spironolactone inhibited both effects. Ang II increased DNA and collagen synthesis, and these effects were reversed by aldosterone. 5. In conclusion, aldosterone induces positive inotropic and vasoconstrictor effects in a nongenomic manner, and these effects are comparable to those of Ang II. Aldosterone reduces DNA and collagen synthesis via MR activation, and counteracts the Ang II-induced increases in these parameters. MR blockade reduces infarct size and increases LVP recovery following coronary artery occlusion. The MR-related phenomena may underlie, at least in part, the beneficial actions of spironolactone in heart failure.

Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II (Article)

2002-03-04T00:00:00Z

Cardiomyocytes bind, internalize, and activate prorenin, the inactive precursor of renin, via a mannose 6-phosphate receptor (M6PR)--dependent mechanism. M6PRs couple directly to G-proteins. To investigate whether prorenin binding to cardiomyocytes elicits a response, and if so, whether this response depends on angiotensin (Ang) II, we incubated neonatal rat cardiomyocytes with 2 nmol/L prorenin and/or 150 nmol/L angiotensinogen, with or without 10 mmol/L M6P, 1 micromol/L eprosartan, or 1 micromol/L PD123319 to block M6P and AT(1) and AT(2) receptors, respectively. Protein and DNA synthesis were studied by quantifying [(3)H]-leucine and [(3)H]-thymidine incorporation. For comparison, studies with 100 nmol/L Ang II were also performed. Neither prorenin alone, nor angiotensinogen alone, affected protein or DNA synthesis. Prorenin plus angiotensinogen increased [(3)H]-leucine incorporation (+21 +/- 5%, mean +/- SEM, P<0.01), [(3)H]-thymidine incorporation (+29 +/- 6%, P<0.01), and total cellular protein (+14 +/- 3%, P<0.01), whereas Ang II increased DNA synthesis only (+34 +/- 7%, P<0.01). Eprosartan, but not PD123319 or M6P, blocked the effects of prorenin plus angiotensinogen as well as the effects of Ang II. Medium Ang II levels during prorenin and angiotensinogen incubation were <1 nmol/L. In conclusion, prorenin binding to M6PRs on cardiomyocytes per se does not result in enhanced protein or DNA synthesis. However, through Ang II generation, prorenin is capable of inducing myocyte hypertrophy and proliferation. Because this generation occurs independently of M6PRs, it most likely depends on the catalytic activity of intact prorenin in the medium (because of temporal prosegment unfolding) rather than its intracellular activation. Taken together, our results do not support the concept of Ang II generation in cardiomyocytes following intracellular prorenin activation.

Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs (Article)

2002-01-01T00:00:00Z

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors (Article)

2001-01-01T00:00:00Z

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin (Article)

2001-01-01T00:00:00Z

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes (Article)

2001-01-01T00:00:00Z

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.

Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun. (Article)

2000-09-14T00:00:00Z

The transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75-1.50x10(5)cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- beta-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10(-8)M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.

Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter (Article)

2000-01-01T00:00:00Z

BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

Biochemie van hartfalen: de compensatie reeds decompensatie? (Inaugural Lecture)

1994-12-08T00:00:00Z

Myocardial malondialdehyde and uric acid release after short-lasting coronary occlusion during coronary angioplasty: potential mechanisms for free radical generation (Article)

1991-01-01T00:00:00Z

Pyruvate metabolism and transport in intestinal epithelium (Doctoral Thesis)

1975-10-01T00:00:00Z

The small intestinal mucosa is known to have a high rate of aerobic glycolysis. The absence of a Pasteur effect in the small intestine is related to this observation. It was questioned whether this is an artefact. The knowledge of the rate-limiting factors of glycolysis is therefore important. The possibility that hexokinase or phosphofructokinase is rate-limiting is discussed. For phosphofructokinase activity, the energy charge is of critical importance. The energy charge in the usual in vitro preparations, such as the everted sac or isolated epithelial cells, is very low. Oxygen availability in vitro might be limited. Therefore the vascularly perfused small intestine was chosen as a model system. It was found that increasing the oxygen availability by the addition of fluorocarbons to the perfusion medium, raised the energy charge up to in vivo values. Under these conditions a clearcut Pasteur effect could be demonstrated.