Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ± 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ± 2.4 and 6.7 ± 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ± 0.97 and 7.5 ± 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves

It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 ± 94 µg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 ± 0.016 and 1.19 ± 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 µM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 ± 58 to 650 ± 100 µg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 µM nifedipine. However, the use of an intracellular calcium chelator (15.0 µM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions

CaMKIIdelta overexpression in hypertrophy and heart failure: cellular consequences for excitation-contraction coupling

Ca/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta) is the predominant isoform in the heart. During excitation-contraction coupling (ECC) CaMKII phosphorylates several Ca-handling proteins including ryanodine receptors (RyR), phospholamban, and L-type Ca channels. CaMKII expression and activity have been shown to correlate positively with impaired ejection fraction in the myocardium of patients with heart failure and CaMKII has been proposed to be a possible compensatory mechanism to keep hearts from complete failure. However, in addition to these acute effects on ECC, CaMKII was shown to be involved in hypertrophic signaling, termed excitation-transcription coupling (ETC). Thus, animal models have shown that overexpression of nuclear isoform CaMKIIdeltaB can induce myocyte hypertrophy. Recent study from our laboratory has suggested that transgenic overexpression of the cytosolic isoform CaMKIIdeltaC in mice causes severe heart failure with altered intracellular Ca handling and protein expression leading to reduced sarcoplasmic reticulum (SR) Ca content. Interestingly, the frequency of diastolic spontaneous SR Ca release events (or opening of RyR) was greatly enhanced, demonstrating increased diastolic SR Ca leak. This was attributed to increased CaMKII-dependent RyR phosphorylation, resulting in increased and prolonged openings of RyR since Ca spark frequency could be reduced back to normal levels by CaMKII inhibition. This review focuses on acute and chronic effects of CaMKII in ECC and ETC. In summary, CaMKII overexpression can lead to heart failure and CaMKII-dependent RyR hyperphosphorylation seems to be a novel and important mechanism in ECC due to SR Ca leak which may be important in the pathogenesis of heart failure.

Multigenerational Brazilian family with malignant hyperthermia and a novel mutation in the RYR1 gene

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1). Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test (CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser, was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2 MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4932Ser variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its location within the protein.

Effect of felodipine on myocardial and renal injury induced by aldosterone-high salt hypertension in uninephrectomized rats

It has been recently shown that calcium channel blockers might have a protective effect on cardiac fibrogenesis induced by aldosterone. The objective of this study was to evaluate the protective effect of felodipine, a dihydropyridine calcium channel blocker, against heart and kidney damage caused by aldosterone-high sodium intake in uninephrectomized rats. Wistar rats were divided into three groups: CNEP (uninephrectomized + 1% NaCl in the drinking water, N = 9); ALDO (same as CNEP group plus continuous infusion of 0.75 µg/h aldosterone, N = 12); ALDOF (same as ALDO group plus 30 mg·kg-1·day-1 felodipine in the drinking water, N = 10). All results were compared with those of age-matched, untreated rats (CTL group, N = 10). After 6 weeks, tail cuff blood pressure was recorded and the rats were killed for histological analysis. Blood pressure (mmHg) was significantly elevated (P < 0.05) in ALDO (180 ± 20) and ALDOF (168 ± 13) compared to CTL (123 ± 12) and CNEP (134 ± 13). Heart damage (lesion scores - median and interquartile range) was 7.0 (5.5-8.0) in ALDO and was fully prevented in ALDOF (1.5; 1.0-2.0). Also, left ventricular collagen volume fraction (%) in ALDOF (2.9 ± 0.5) was similar to CTL (2.9 ± 0.5) and CNEP (3.4 ± 0.4) and decreased compared to ALDO (5.1 ± 1.6). Felodipine partially prevented kidney injury since the damage score for ALDOF (2.0; 2.0-3.0) was significantly decreased compared to ALDO (7.5; 4.0-10.5), although higher than CTL (null score). Felodipine has a protective effect on the myocardium and kidney as evidenced by decreased perivascular inflammation, myocardial necrosis and fibrosis.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Prevalence of metabolic syndrome and its associated factors in renal transplant recipients

INTRODUCTION: The population of patients undergoing renal transplantation is considered at highrisk for developing obesity and changes in lipid and glucose metabolism, due to the use of immunosuppressive drugs and increased food freedom in the post-transplant period. OBJECTIVE: This study was designed to assess the prevalence of metabolic syndrome in renal transplant recipients and to identify factors associated with its occurrence. METHODS: A cross-sectional study was performed in renal transplant patients, with more than six months of follow-up. The metabolic syndrome was diagnosed according to the criteria of the National Cholesterol Education Program Adult Treatment Panel III. RESULTS: Among the 87 pa- tients enrolled, 39 (44.8%) presented the phenotype of metabolic syndrome. The mean age of the patients was 43.5 ± 12.1 years-old, with a predominance of male (69.0%) and white (66.7%). The mean and median times of post transplant follow-up were 64.2 ± 49.4 and 56 months, respectively. All the 12 patients who developed post-transplant diabetes mellitus also met the criteria for metabolic syndrome, which compromised the inclusion of this variable in the logistic regression. In the univariate analysis, patients with metabolic syndrome had higher mean age (p = 0.008), higher median blood level of cyclosporine (p = 0.021), higher prevalence of history of coronary disease (p = 0.023), and they were more frequent users of beta (p = 0.011) and calcium- channel blockers (p = 0.039). In the multivariate analysis, age (HR = 1.06; 95% CI=1.01-1.11, p=0.006) and use of beta-blockers (HR = 4.02; 95% CI = 1.41 - 11.4, p = 0.009) were asso- ciated with increased risk of metabolic syndrome. CONCLUSION: Metabolic syndrome was highly prevalent in the population of renal trans- plant recipients studied, and it was associated with older age, use of beta-blockers, and post-transplant diabetes mellitus.

Do cardiologists at a university hospital adopt the guidelines for the treatment of heart failure?

OBJECTIVE: To verify whether the guidelines for the treatment of heart failure have been adopted at a university hospital. The guidelines recommend the following: use of angiotensin-converting enzyme inhibitors for all patients with systolic ventricular dysfunction, use of digitalis and diuretics for symptomatic patients, use of beta-blockers for patients in functional classes II or III, use of spironolactone for patients in functional classes III or IV. METHODS: We analyzed the prescriptions of 199 patients. All these patients had ejection fraction (EF) <=0.50, their ages ranged from 25 to 86 years, and 142 were males. Cardiomyopathy was the most frequent diagnosis: 67 (33.6%) patients had dilated cardiomyopathy, 65 (32.6%) had ischemic cardiomyopathy. RESULTS: Angiotensin-converting enzyme inhibitors were prescribed for 93% of the patients. 71.8% also had a prescription for digitalis, 86.9% for diuretics, 27.6% for spironolactone, 12% for beta-blockers, 37.2% for acetylsalicylic acid, 6.5% for calcium channel antagonists, and 12.5% for anticoagulants. In regard to vasodilators, 71% of the patients were using captopril (85.2mg/day), 20% enalapril (21.4mg/day), 3% hydralazine and nitrates. In 71.8% of the cases, the dosages prescribed were in accordance with those recommended in the large studies. CONCLUSION: Most patients were prescribed the same doses as those recommended in the large studies. Brazilian patients tolerate well the doses recommended in the studies, and that not using these doses may be a consequence of the physician's fear of prescribing them and not of the patient's intolerance.