This study aims to elucidate the mechanisms by which tumor necrosis factor alpha (TNFα) provides protection from hypoxic damage to neonatal rat cardiomyocyte cultures. We show that when intracellular ...Ca
2+
(Ca
2+
i
) levels are elevated by extracellular Ca
2+
(Ca
2+
o
) or by hypoxia, then TNFα decreased Ca
2+
i
in individual cardiomyocytes. However, TNFα did not reduce Ca
2+
i
after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNFα attenuates Ca
2+
overload through Ca
2+
uptake by SERCA2a. TNFα did not reduce Ca
2+
i
, following its elevation when Ca
2+
o
levels were elevated in TNFα receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNFα when the cardiomyoctyes were subjected to hypoxia, as determined by lactate dehydrogenase (LDH) and creatine kinase (CK) released and from the cardiomyocytes. Moreover, when the levels of Ca
2+
i
were increased by hypoxia, H-89, but not KN93, (a calmodulin kinase II inhibitor), prevented the reduction in Ca
2+
i
by TNFα. TNFα increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNFα against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNFα attenuated this decrease following hypoxia. It is suggested that TNFα activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca
2+
overload.
Celotno besedilo
Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background: Sudden cardiac death of a child is a devastating event for the family and an enormous challenge for the attending physician.
Methods and Results: We report a family with repeat events of ...sudden cardiac death and recurrent ventricular fibrillation in a teenage girl, where autopsy data and clinical investigations were inconclusive. The diagnosis of catecholaminergic polymorphic ventricular tachycardia (CPVT) was established only following finding a gene mutation in the cardiac ryanodine receptor.
Conclusions: Interpretation of autopsy data, provocation testing and genetic testing in victims of sudden death and family members are discussed to correctly identify the cause and properly manage asymptomatic carriers in such families.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value.
This study presents a CPVT ...family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers.
The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2.
Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes.
M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.
Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ ...rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic–reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca2+ homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage.
We demonstrate here that rapamycin protects rat heart cultures from hypoxic–reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels (Ca2+i) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of Ca2+i. Rapamycin also attenuated 45Ca2+ uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the “reverse” mode of Na+/Ca2+ exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased Ca2+i following its elevation by extracellular Ca2+ (Ca2+o) influx, thapsigargin treatment, or depolarization with KCl.
We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca2+ outside the cardiomyocytes.
According to our findings, rapamycin preserves Ca2+ homeostasis and prevents Ca2+ overload via extrusion of Ca2+ surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress.
Humans and genetically engineered mice with recessively inherited CPVT develop arrhythmia which may arise due to malfunction or degradation of calsequestrin (CASQ2). We investigated the relation ...between protein level and arrhythmia severity in CASQ2D307H/D307H (D307H), compared to CASQ2Δ/Δ (KO) and wild type (WT) mice. CASQ2 expression and Ca2+ transients were recorded in cardiomyocytes from neonatal or adult mice. Arrhythmia was studied in vivo using heart rhythm telemetry at rest, exercise and after epinephrine injection. CASQ2 protein was absent in KO heart. Neonatal D307H and WT hearts expressed significantly less CASQ2 protein than the level found in the adult WT. Adult D307H expressed only 20% of CASQ2 protein found in WT. Spontaneous Ca2+ release was more prevalent in neonatal KO cardiomyocytes (89%) compared to 33–36% of either WT or D307H, respectively, p<0.001. Adult cardiomyocytes from both mutant mice had more Ca2+ abnormalities compared to control (KO: 82%, D307H 63%, WT 12%, p<0.01). Calcium oscillations were most common in KO cardiomyocytes. We then treated mice with bortezomib to inhibit CASQ2D307H degradation. Bortezomib increased CASQ2 expression in D307H hearts by ∼50% (p<0.05). Bortezomib-treated D307H mice had lower CPVT prevalence and less premature ventricular beats during peak exercise. No benefit against arrhythmia was observed in bortezomib treated KO mice. These results indicate that the mutant CASQ2D307H protein retains some of its physiological function. Its expression decreases with age and is inversely related to arrhythmia severity. Preventing the degradation of mutant protein should be explored as a possible therapeutic strategy in appropriate CPVT2 patients.
When cardiomyocytes were subjected to hypoxia, tumor necrosis factor-α (TNF-α; 3–50 ng/ml) or adenosine (1–100 μM), decreased hypoxic damage as was detected by lactate dehydrogenase (LDH) release, ...MTT (3-4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) absorbance, ROS (reactive oxygen species) measurement or desmin immunostaining. This cardioprotection was not prevented in TNF-α-treated cultures by 5-hydroxydecanoic acid (5-HD). Our aim was to elucidate whether adenosine and TNF-α mediate a similar protective mechanism against hypoxia in primary heart cultures and in H9c2 cardiomyocytes. Adenosine and TNF-α are known for their negative inotropic effects on the heart. We have suggested that deoxyglucose uptake reflects heart contractility in cell cultures; therefore, we assayed its accumulation under various conditions. Treatment for 20 min with adenosine, R-PIA (−)-
N(6)-phenylisopropyladenosine (10 μM), or TNF-α reduced
3H-deoxyglucose uptake in primary heart cultures and also in H9c2 cardiomyocytes by 30–50%. Isoproterenol accelerated
3H-deoxyglucose uptake by 50%. Adenosine, R-PIA, or TNF-α attenuated the stimulatory effect of isoproterenol on
3H-deoxyglucose uptake to control levels. Hypoxia reduced
3H-deoxyglucose uptake by 50%, as in the treatment of the hypoxic cultures with TNF-α or adenosine. Glibenclamide (2 μM), 5-HD (300 μM), or diazoxide (50 μM) increased
3H-deoxyglucose uptake by 50–80%. Adenosine (100 μM) and TNF-α (50 ng/ml) stimulated
86Rb efflux. Glibenclamide attenuated this effect. We demonstrate that TNF-α, like adenosine, accelerated Ca
2+ uptake into the sarcoplasmic reticulum (SR) by 50–100% and therefore prevented cardiomyocyte Ca
2+ overload. Our findings further suggest that TNF-α, as well as adenosine, may mediate an adaptive effect in the heart by preventing Ca
2+ overload via activation of SR Ca-ATPase (SERCA
2a).
Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A1 adenosine receptor radioligand 3H-8-cyclopentyl-1,3-dipropylxanthine ...(3HCPX), to monitor the level of the receptors on intact cardiocytes. The binding showed high affinity (Kd = 0.13 nM) and the number of 3HCPX binding sites (Bmax) was 23.1 fmol/dish (21 fmol/mg protein). The Ki of the agonists R-N6-(2-phenylisopropyl)-adenosine (R-PIA) and S-N6-(2-phenylisopropyl)-adenosine (S-PIA), and of the antagonists CPX and theophylline were 3.57, 49.0, 1.63 and 4880 nM, respectively. The number of adenosine receptors was very low during the first days in cultures (5 fmol/dish) and increased gradually until it reached a plateau on days 8-10. Treatment with norepinephrine or isoproterenol which accelerated the rate of contractions, induced up regulation of the receptors. Bmax increased 2-3-fold by application of norepinephrine for 4 days, while receptor affinity to the radioligand was unaffected. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity increased only by 22 and 38%, respectively. Similarly, 3 days treatment with triiodothyronine (T3, 10(-8) M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to 3HCPX. Carbamylcholine (5 x 10(-6) M), which reduced the rate of heart contractions, caused 26% down regulation while the affinity of the receptors remained unchanged. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, the level of adenosine receptors may respond to drug-induced chronic changes in cardiac contractile activity so as to restore conditions to normal (basal) contractions.
Molecular area/surface pressure Langmuir isotherms of amphiphilic dipalmitoylphosphatidylcholine (DPPC) monolayers indicated that abscise acid (ABA) has a marked rigidifying effect, expressed as ...reduction of molecular area and increase of monolayer collapse point. Moreover, ABA markedly increased aqueous droplet hydrophobicity, as indicated by a concentration-dependent increase of contact angle when placed on a hydrocarbon chain surface; no such effects were obtained on either amphiphilic or octadecyltrichlorosilane surfaces. A combination of TLC and mass spectometry revealed the presence of DPPC in Vicia faba and Commelina communis guard-cell protoplast membranes. ABA also increased plasma membrane rigidity as evidenced by probing with lipid specific membrane probes, namely diphenylhexatriene and its trimethyl derivative. Regarded together the results suggest a specific site of ABA binding to DPPC. The linkage between senescence and stomatal closure is discussed in the light of the new data presented here. It is suggested that DPPC in guard-cell membranes may have a physical role in preventing collapse and/or bursting. In this connection an analogy is drawn with pulmonary mechanisms.
Considering the therapeutic effect of statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and simvastatin in patients with coronary heart disease, our first hypothesis was that ...simvastatin should inhibit apoptosis (programmed cell death) in angiotensin II-treated cultured myocytes. But after realizing that simvastatin stimulates apoptosis, we changed our hypothesis and began to study its apoptotic effect in primary cultured rat cardiomyocytes. We found that simvastatin induced apoptosis in a dose-dependent manner (0.1 to 3 micromol/L), as evidenced by the appearance of increased DNA fragmentation in agarose gels and characteristic apoptotic patterns in nuclei labeled with Hoechst 33342, as well as increased activity of caspase 3. FACS analysis of simvastatin-treated cardiomyocytes showing annexin V binding and propidium iodide exclusion ruled out the possibility of necrosis. Increased intracellular enzymatic activity of creatine phosphokinase, aldolase, and lactic dehydrogenase, markers for normal cell function, could reflect the hypertrophic effect of simvastatin. The results indicate that simvastatin-induced apoptosis in cultured heart cells is concentration-dependent and additive to the apoptotic effect of angiotensin II.
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