Tumor necrosis factor alpha protects heart cultures against hypoxic damage via activation of PKA and phospholamban to prevent calcium overload.

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.

The role of mutant protein level in autosomal recessive catecholamine dependent polymorphic ventricular tachycardia (CPVT2).

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 CASQ2(D307H/D307H) (D307H), compared to CASQ2(Δ/Δ) (KO) and wild type (WT) mice. CASQ2 expression and Ca²âº 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 Ca²âº 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 Ca²âº 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 CASQ2(D307H) 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 CASQ2(D307H) 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.

A family with recurrent sudden death and no clinical clue.

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.

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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) protects against hypoxic damage in primary heart cultures via Na+/Ca2+ exchanger activation.

AIMS: 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 Ca(2+) homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. MAIN METHODS: 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 ([Ca(2+)](i)) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca(2+)](i). Rapamycin also attenuated (45)Ca(2+) 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(+)/Ca(2+) exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca(2+)](i) following its elevation by extracellular Ca(2+) ([Ca(2+)](o)) influx, thapsigargin treatment, or depolarization with KCl. KEY FINDINGS: We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca(2+) outside the cardiomyocytes. SIGNIFICANCE: According to our findings, rapamycin preserves Ca(2+) homeostasis and prevents Ca(2+) overload via extrusion of Ca(2+) surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress.

Increase of neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy.

BACKGROUND AND OBJECTIVES: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated. MATERIALS AND METHODS: Whole brains were dissected from 16 days Sprague-Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4-14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVR-Gel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW. RESULTS: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P<0.01), which formed dense branched interconnected networks of thick neuronal fibers. CONCLUSIONS: 780 nm laser phototherapy of embryonic rat brain cultures embedded in hyaluronic acid-laminin gel and attached to positively charged cylindrical MCs, stimulated migration and fiber sprouting of neuronal cells aggregates, developed large size neurons with dense branched interconnected network of neuronal fibers and, therefore, can be considered as potential procedure for cell therapy of neuronal injury or disease.

Adenosine and TNF-alpha exert similar inotropic effect on heart cultures, suggesting a cardioprotective mechanism against hypoxia.

When cardiomyocytes were subjected to hypoxia, tumor necrosis factor-alpha (TNF-alpha; 3-50 ng/ml) or adenosine (1-100 microM), 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-alpha-treated cultures by 5-hydroxydecanoic acid (5-HD). Our aim was to elucidate whether adenosine and TNF-alpha mediate a similar protective mechanism against hypoxia in primary heart cultures and in H9c2 cardiomyocytes. Adenosine and TNF-alpha 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 microM), or TNF-alpha reduced (3)H-deoxyglucose uptake in primary heart cultures and also in H9c2 cardiomyocytes by 30-50%. Isoproterenol accelerated (3)H-deoxyglucose uptake by 50%. Adenosine, R-PIA, or TNF-alpha attenuated the stimulatory effect of isoproterenol on (3)H-deoxyglucose uptake to control levels. Hypoxia reduced (3)H-deoxyglucose uptake by 50%, as in the treatment of the hypoxic cultures with TNF-alpha or adenosine. Glibenclamide (2 microM), 5-HD (300 microM), or diazoxide (50 microM) increased (3)H-deoxyglucose uptake by 50-80%. Adenosine (100 microM) and TNF-alpha (50 ng/ml) stimulated (86)Rb efflux. Glibenclamide attenuated this effect. We demonstrate that TNF-alpha, 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-alpha, as well as adenosine, may mediate an adaptive effect in the heart by preventing Ca(2+) overload via activation of SR Ca-ATPase (SERCA(2)a).

Further development of reconstructive and cell tissue-engineering technology for treatment of complete peripheral nerve injury in rats.

UNLABELLED: In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve. IN CONCLUSION: (1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.

A biophysical study of abscisic acid interaction with membrane phospholipid components.

Molecular area/surface pressure Langmuir isotherms of amphiphilic dipalmitoylphosphatidylcholine (DPPC) monolayers indicated that abscisic 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.