Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavß2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gßγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Gißγ-AKT-Cavß2 pathway, suggesting a new mechanism for regulation of cardiac function.

Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Maple syrup urine disease (MSUD)--clinical profile of 47 Filipino patients.

Maple syrup urine disease (MSUD) is a very rare disorder of branched-chain amino acid metabolism. However, it is the most common inborn error of metabolism in the Philippines. We present a retrospective review of 21 patients diagnosed with MSUD between 1999 and 2004. The patients presented clinically between 2 and 14 days of life (mean 5 days) and the diagnosis of MSUD was established between 6 days and 11 months of age (mean 39 days). The classical burnt sugar odour was noted in the majority of patients (81%). The diagnosis of MSUD was initially based on clinical suspicion and confirmed biochemically by measurement of leucine/isoleucine levels by thin-layer chromatography. The acute management included removal of accumulated branched-chain amino acids by peritoneal dialysis in 62% of the patients. Mortality rate of this group of patients was 24% and follow-up rate was 87%. We compared this series with a previously reported series of 26 patients to determine whether diagnosis and the management of MSUD improved over the two periods. Four cases have been diagnosed early since 1992, the majority of whom had the classic form of MSUD with the onset of symptoms in the first two weeks of life. A small subset of patients with early nonspecific symptoms was diagnosed much later owing to a low-level clinical suspicion among clinicians. Overall, however, there appears to be a small but general trend towards earlier diagnosis, reduced mortality and long-term follow up in the later series. Although we are able to diagnose and manage MSUD in the Philippines, we recognize that the clinical outcome remains poor and is due mainly to late referral of cases and inadequate long-term management. In the Philippines, we recommend that all newborns who are considered to be septic, have feeding difficulties, fail to regain their birth weight or present with any other symptoms suggestive of MSUD be evaluated in the first instance by analysis of urine for ketones and if they are positive have blood collected and sent to our laboratory for leucine/isoleucine measurement.

The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content.

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker® Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.

Low citrulline may not be diagnostic of ornithine transcarbamylase deficiency: a case report.

A newborn boy with family history of severe ornithine transcarbamylase (OTC) deficiency was investigated prospectively and managed aggressively at birth based on an existing protocol for at risk neonates. Undetectable citrulline levels at birth suggested that the infant was affected; however, normal plasma glutamine and urine orotic acid levels confused the diagnosis to some extent. Mutation testing confirmed that the patient did not have OTC deficiency. Thus the low plasma citrulline level did not validate our initial biochemical suspicion of OTC deficiency, and this highlights the importance of considering all available clinical, biochemical and molecular evidence in determining disease status.

Lesch-Nyhan disease in a 20-year- old man incorrectly described as developing 'cerebral palsy' after general anaesthesia in infancy.

Lesch-Nyhan disease (LND) is a rare X-linked recessive genetic disorder caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The classic clinical condition is characterized by cognitive impairment, hypotonia at rest, choreoathetosis, hyperuricaemia and the hallmark symptom of severe and involuntary self-mutilation. We describe a man with LND who was initially thought to have suffered from a dyskinetic cerebral palsy after an uncomplicated inguinal herniorrhaphy under general anaesthesia at 5 1/2 months of age. In the absence of overt self-injurious behaviour, the diagnosis was not considered for nearly two decades. The diagnosis of LND was established at 20 years of age through clinical review, biochemical examinations and molecular analysis. HPRT haemolysate activity was 7.6% of the normal control, suggesting that he had a milder variant of the disease. Mutation analysis of the HPRT gene revealed a novel missense mutation, c.449T > G in exon 6 (p.V150G). Cascade testing of family members revealed that the mother was heterozygous for the mutation but two siblings (a brother and a sister) did not carry the sequence mutation. Whether the onset of neurological abnormalities in this particular case can be attributed to the general anaesthesia is discussed.

Effects of propranolol on the hemodynamic, coronary sinus blood flow and myocardial metabolic response to atrial pacing.

The hemodynamic, coronary sinus blood flow and myocardial metabolic effects of 0.15 mg/kg body weight of intravenously administered propranolol were studied in 19 patients with coronary artery disease and 6 normal patients. Atrial pacing was performed in all patients and produced angina in 15 of the 19 patients with coronary artery disease. In these patients propranolol reduced heart rate from 78 to 69 beats/min, cardiac index from 3.0 to 2.6 liters/min per m2 and left ventricular stroke work index from 47 to 43 g-m/m2; it increased total peripheral resistance from 24 to 28 units and lactate extraction from 16.3 to 22.5%. There was no significant change in mean arterial pressure, left ventricular end-diastolic pressure, coronary sinus blood flow or myocardial oxygen consumption. During a second pacing stress propranolol produced clinical improvement in 9 of the 15 patients who experienced angina initially. The improvement was associated with less severe abnormalities in S-T depression and left ventricular end-diastolic pressure, increased lactate extraction and no significant change in coronary sinus blood flow or myocardial oxygen consumption. Thus, propranolol appears to be capable of modifying the anginal threshold as determined with atrial pacing, and the clinical response appears to be independent of global changes in coronary sinus blood flow and myocardial oxygen consumption.

The spectrum of unstable angina: prognostic role of serum creatine kinase determination.

A prospective study of 199 patients with unstable angina pectoris was undertaken to assess whether frequent serial sampling of serum creatine kinase (CK) was useful in predicting prognosis. Nineteen percent of the patients had transient CK elevations suggestive of a small myocardial infarct that was outside the detective ability of conventional electrocardiographic and enzymatic determinations. These patients had a 1 year mortality rate of 16 percent, which was significantly higher than that in the remaining patients (Fisher's exact test p = 0.05). Furthermore, the recurrence rate of myocardial infarction (14 percent) in the patients who had transient CK elevation was significantly greater than that (2 percent) in those who did not have CK elevation (Fisher's exact test p = 0/01). These data suggest that frequent serum CK sampling in the first 48 hours after admission for unstable angina has prognostic value and that persons with CK elevation may warrant a more aggressive approach to investigation and management.

Cardioplegic arrest in pigs. Effects of glucose-containing solutions.

A clinically used cardioplegic solution was evaluated in the laboratory in an attempt to elucidate the value of glucose in maintaining the glycogen and energy stores of the myocardium during aortic cross-clamping. In one group of animals the cardioplegic solution contained glucose; in the other group it did not. Energy stores were determined in full-thickness biopsies of left ventricular myocardium taken prior to bypass, at 60 minutes of cardioplegic arrest, and after 20 minutes of reperfusion. At the end of cardioplegic arrest, glycogen levels were slightly higher than control values, with no differences between the groups. Creatine phosphate (CP) and adenosine triphosphate (ATP) fell by 69% to 73% and 43% to 55%, respectively, from the control values, but there were no statistical differences between these groups. At the end of reperfusion, glycogen and CP stores had returned to control but ATP concentration remained below control values in both groups. These results indicate that glucose is of no value either to maintain myocardial glycogen or to influence the changes in CP and ATP during cardioplegic arrest. The dissociation between the complete and partial restoration of CP and ATP stores to control levels during reperfusion suggests either that ATP consumption is greater than the energy transfer between CP and ADP:ATP systems or that the energy transfer is defective under these experimental conditions.

Ventricular fibrillation induced prior to cardioplegic arrest in hypertrophied pig hearts.

We hypothesized that by inducing ventricular fibrillation (VF) prior to cardioplegic arrest in nonvented hypertrophied hearts of pigs, the metabolic characteristics of the epicardial and endocardial regions would be compromised compared with animals in which cardioplegic solution was infused while the hearts were in normal sinus rhythm (NSR). These abnormalities would be reflected not only in greater deterioration of myocardial metabolism after reperfusion in the VF group, but they would also be more pronounced in the subendocardial layers of hypertrophied left ventricles. Results obtained in hypothermic hearts (28 degrees C) maintained at 8 degrees to 12 degrees C during cardioplegic arrest demonstrated no major consistent differences in the stores of glycogen, creatine phosphate, adenine nucleotides, and lactate in both groups of hearts, for either layer of the left ventricular myocardium. The only significant difference was slightly lower creatine kinase content in the VF hearts than in the NSR group. It is concluded that induction of VF in hypothermic (28 degrees C), nonvented, hypertrophied hearts prior to infusion of cardioplegic solution does not affect myocardial energy stores compared with hearts in NSR, provided that the period of VF prior to clamping is short (3 minutes) and that the myocardial temperature is lowered to 28 degrees C prior to VF and is maintained at 8 degrees to 12 degrees C during cardioplegic arrest.

The hemodynamic and metabolic effects of cardioplegic rearrest in the pig.

The hemodynamic and metabolic effects of two consecutive 1-hour periods of cardioplegic arrest with a 20-minute interval of reperfusion or cardioplegic rearrest were evaluated in pig hearts. This model was designed to recreate in the laboratory a situation occasionally encountered during open-heart operation. Results indicate that at the end of 40 minutes of reperfusion following cardioplegic rearrest and 20 minutes after cardiopulmonary bypass (CPB), the stores of glycogen, adenosine triphosphate and total adenine nucleotides were lower than those found in hearts beating under CPB for an identical period of time. These stores were, however, sufficient to permit hemodynamic recovery, and they compared favorably with those found in hearts subjected to a single hour of cardioplegic arrest and reperfusion. The laboratory data and our previous clinical experience suggest that cardioplegic rearrest is a feasible alternative when surgical difficulties demand a second period of aortic cross-clamping after an initial period of cardioplegic arrest and reperfusion.

Should ventricular fibrillation be induced prior to the infusion of cardioplegic solution?

The present study evaluates the metabolic effects on the left ventricular energy stores of a clinically used cardioplegic solution that was infused into the ascending aorta of pigs while the heart was either fibrillating (induced ventricular fibrillation) or in normal sinus rhythm prior to aortic clamping. Fibrillating hearts had lower stores of glycogen in the epicardium and endocardium compared with hearts in normal sinus rhythm. There was no difference in the stores of creatine phosphate between the hearts for both the epicardium and endocardium, but stores of adenosine triphosphate (ATP) in both layers were lower in fibrillating hearts. These results indicate that for ideal myocardial protection the cardioplegic solution should be infused while the heart is beating under cardiopulmonary bypass, and that ventricular fibrillation induced and maintained prior to cross-clamping may cause myocardial damage.

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.

Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli.

Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.

Omeprazole, a specific gastric secretion inhibitor on oxynticopeptic cells, reduces gizzard erosion in broiler chicks fed with toxic fish meals.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

[Biochemical characterization of proteins in the crab Homalaspis plana]. / Caracterización bioquímica de las proteínas de jaiba mora (Homalaspis plana).

Homalaspis plana is a crab found in the Pacific Ocean coasts from Guayaquil (Ecuador) all the way down to the Strait of Magellan, including all of the Chilean coast. This crustacean is an important resource because it provides jobs for artisanal fishermen. In Chile there is no product diversification from this crab and it has been poorly studied from the scientific point of view. The biochemical characterization of its proteins was initiated to be able to develop several products from its meat. We report the proteolytic activity from freshly extracted crab meat and the electrophoretical characterization of its proteins. No proteolytic activity was detected in freshly extracted crab meat, under any of the assay conditions used. SDS Polyacrylamide gel electrophoresis (PAGE) and native PAGE were carried out with proteins extracted from crab meat. Using extraction solutions at two different ionic strengths (0.05 and 0.5), myofibrillar and sarcoplasmic were separated, each showing different electrophoretical patterns in SDS-PAGE.

Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

Cardiomyocyte death: mechanisms and translational implications.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.