Influence of phospholipid long chain polyunsaturated fatty acid composition on neonatal rat cardiomyocyte function in physiological conditions and during glucose-free hypoxia-reoxygenation

There is evidence that dietary polyunsaturated fatty acids (PUFA) may protect against cardiovascular diseases, but the involvement of the cardiac muscle cell in this beneficial action remain largely unknown. The present study compared the respective influence of n-3 and n-6 PUFA on the function of cultured neonatal rat cardiomyocytes (CM). Cells were grown for 4 days in media enriched either n-3 (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) or n-6 (arachidonic acid, AA) PUFA. The PUFA n-6/n-3 ratio in the phospholipids was close to 1 and 20 in the n-3 and n-6 cells, respectively. The transmembrane potentials were recorded using microelectrodes and the contractions were monitored with a photoelectric device. In physiological conditions, the increase of n-6 PUFA level in the phospholipids resulted in a significant decrease in the maximal rate of initial depolarization (–16%). In opposition, the action potential amplitude and duration were not altered, and the cell contractio n outline was not affected. Ischemia was simulated in vitro using a substrate-free, hypoxia-reoxygenation procedure in a specially designed gas-flow chamber. The progressive loss of electrical activity induced by the substrate-free, hypoxic treatment was affected by the n-6/n-3 ratio, since the n-6 rich CM displayed a slower depression of the AP amplitude and duration parameters. Conversely, the recovery of the resting potential (MDP) during reoxygenation was faster in n-3 CM, whereas the recovery of the contraction parameters was unaffected by the fatty acid composition of the cells. These results suggested that, in physiological conditions, the modification of long chain PUFA balance in the phospholipids of cardiac muscle cells may modulate the initial AP upstroke, which is governed by sodium channels. Moreover, the presence of n-3 PUFA appeared to accelerate the electrical depression during substrate-free hypoxia but in turn to allow a faster recovery upon reoxygenation. (Mol Cell Biochem 175: 253–262, 1997)     

In vivo gene delivery of HSP70i by adenovirus and adeno-associated virus preserves contractile function in mouse heart following ischemia-reperfusion

Inducible heat shock protein 70 (HSP70i) has been shown to exert a protective effect in hearts subjected to ischemia-reperfusion. Although studied in heat-shocked animals and in transgenic mice that constitutively overexpress the protein, the therapeutic application of the protein in the form of a viral vector-mediated HSP70i expression has not been widely examined. Accordingly, we have examined the effects of HSP70i delivered in vivo to the left ventricular free wall of the heart via viral gene therapy in mice. The affect of virally mediated HSP70i expression in preserving cardiac function following ischemia-reperfusion was examined after short-term expression (5-day adenovirus mediated) and long-term expression (8-mo adeno-associated virus mediated) in mice by subjecting ex vivo Langendorff perfused hearts to a regime of ischemia-reperfusion. Both vectors were capable of increasing HSP70i expression in the heart, and neither vector had any effect on cardiac function during aerobic (preischemic) perfusion when compared with corresponding controls. In contrast, both adenovirus-mediated and adeno-associated virus-mediated expression of HSP70i improved the contractile recovery of the heart after 120 min of reperfusion following ischemia. This study demonstrates the feasibility of using both short- and long-term expression of virally mediated HSP70i as a therapeutic intervention against cardiac ischemia-reperfusion injury.     

Substrate dependence of the postischemic cardiomyocyte recovery: Dissociation between functional, metabolic and injury markers

Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.     

p70s6 kinase is a functional target of insulin activated Akt cell-survival signaling

Insulin administration attenuates cardiac ischemia-reperfusion apoptosis via activation of Akt-mediated cell-survival signaling. As p70s6 kinase is a cognate Akt-mediated phosphorylation target we evaluated whether p70s6 kinase activation is a functional requirement in insulin-mediated cell survival program during post-ischemic reoxygenation. Human cardiac-derived girardi cells were subjected to 6h of simulated ischemia and 2h of reoxygenation+/-insulin treatment [0.3mU/ml]. Concurrently, cells were pre-treated with anti-sense oligodeoxynucleotides (ODNs) corresponding to the initiation start-site of human p70s6 kinase mRNA. Sense ODN and scrambled ODN were used as controls. Cell viability was measured using lactate dehydrogenase (LDH) release and propidium iodide (PI) exclusion. Insulin at reoxygenation enhanced cell viability with attenuated LDH release (>or=50% , p<0.001 vs. ischemic controls) and reduced PI uptake by >or=30% vs. ischemic controls. The protection afforded by insulin was abolished by anti-sense ODN targeting p70s6 kinase, but not by the sense or scrambled ODNs. In parallel, insulin administration at reoxygenation significantly increased p70s6 kinase levels and activity compared with controls. P70s6 kinase activity was abolished by pre-treatment with anti-sense ODNs. Collectively, these data demonstrate that p70s6 kinase activation is a functional target of Akt following insulin-activated cytoprotection during ischemia-reoxygenation-induced injury.     

Relation between enzyme release and metabolic changes in reversible anoxic injury of myocardial cells

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 μmol. ATP/g_WW, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations.     

Hindlimb unloading increases muscle content of cytosolic but not nuclear Id2 and p53 proteins in young adult and aged rats.

This study tested the hypothesis that inhibitor of differentiation-2 (Id2), p53, and heat shock proteins (HSP) are responsive to suspension-induced muscle atrophy. Fourteen days of hindlimb suspension were used to unload the hindlimbs and induce atrophy in gastrocnemius muscles of young adult and aged rats. Following suspension, medial gastrocnemius muscle wet weight was reduced by approximately 30%, and the muscle wet weight normalized to the animal body weight decreased by 11 and 15% in young adult and aged animals, respectively. mRNA abundances of Id2, p53, HSP70-2, and HSP27 did not change with suspension, whereas HSP70-1 mRNA content was lower in the suspended muscle compared with the control muscle in both young adult and aged animals. Our immunoblot analyses indicated that protein expressions of HSP70 and HSP60 were not different between suspended and control muscles in both ages, whereas HSP27 protein content was increased in suspended muscle relative to control muscle only in young adult animals. Id2 and p53 protein contents were elevated in the cytosolic fraction of suspended muscle compared with the control muscle in both young and aged animals, but these changes were not found in the nuclear protein fraction. Furthermore, compared with young adult, aged muscles had a lower HSP70-1 mRNA content but higher HSP70-2 mRNA content and protein contents of Id2, p53, HSP70, and HSP27. These findings are consistent with the hypothesis that Id2 and p53 are responsive to unloading-induced muscle atrophy. Moreover, our data indicate that aging is accompanied with altered abundances of HSP70-1 and HSP70-2 mRNA, in addition to Id2, p53, HSP70, and HSP27 protein in rat gastrocnemius muscle.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.     

Antidepressants inhibit human acetylcholinesterase and butyrylcholinesterase activity

Magnesium deficiency in experimental animals leads to inflammation, exacerbated immune stress response and a decrease of specific immune response. It also results in a significant increase in free radical species and subsequent tissue injury. An accelerated thymus involution was observed in Mg-deficient rats in relation to enhanced apoptosis and enhanced susceptibility to oxidative stress. To examine the stress-inducing effects of low Mg status on thymocytes, cDNA arrays were used to evaluate changes in gene expression in weaning rats submitted to Mg deficiency of short duration (2 days). Several genes exhibited changes in their expression caused by Mg deficiency before any perceptible modification in cell integrity and functions. The up-regulated genes included cytochrome c oxidase, glutathione transferase, CuZn superoxide dismutase, genes associated with the stress response (HSP70 and HSP84) and a gene involved in DNA synthesis and repair (GADD45). The down-regulated genes included Na/P cotransporter 1. These findings are consistent with altered cell growth, modifications of ion fluxes and oxidative stress described during Mg deficiency. The observation of induction of genes involved in protection and repair in cells from Mg-deficient animals provides additional evidence of the role of oxidative stress in the pathobiology of this deficiency.     

粉防己碱抑制血管平滑肌细胞增殖及对HSP70和p53表达的影响

目的：观察粉防己碱（Ｔｅｔ）对ＶＳＭＣ增殖的作用及对热应激蛋白７０ｋｄ（ＨＳＰ７０）及其ｍＲＮＡ和抑癌基因ｐ５３ｍＲＮＡ的影响。方法：用内皮素建立培养的血管平滑肌细胞增殖模型。采用氚－胸腺嘧啶核苷（［３Ｈ］ＴｄＲ）掺入法流式细胞术,Ｗｅｓｔｅｒｎ及Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交方法。结果：Ｔｅｔ能逆转内皮素所致的［３Ｈ］ＴｄＲ掺入量增多（Ｐ＜０．０１）,阻止血管平滑肌细胞由静止期（Ｇ０／Ｇ１期）进入ＤＮＡ合成期（Ｓ期）和有丝分裂期（Ｇ２／Ｍ期）,并能逆转内皮素引起的ＨＳＰ７０及ｍＲＮＡ表达增强（Ｐ＜０．０１或Ｐ＜０．０５）,ｐ５３抑癌基因ｍＲＮＡ表达减弱（Ｐ＜０．０５）。结论：Ｔｅｔ能抑制血管平滑肌细胞增殖,与ＨＳＰ７０及ｐ５３的调控有关     

Combined hypoxia and sodium nitrite pretreatment for cardiomyocyte protection in vitro

Methods that increase cardiomyocyte survival upon exposure to ischemia, hypoxia and reoxygenation injuries are required to improve the efficacy of cardiac cell therapy and enhance the viability and function of engineered tissues. We investigated the effect of combined hypoxia/NaNO₂ pretreatment on rat neonatal cardiomyocyte (CM), cardiac fibroblast, and human embryonic stem cell‐derived CM (hESC‐CM) survival upon exposure to hypoxia/reoxygenation (H/R) injury in vitro. Cells were pretreated with and without hypoxia and/or various concentrations of NaNO₂ for 20 min, then incubated for 2 h under hypoxic conditions, followed by 2 h in normoxia. The control cells were maintained under normoxia for 4 h. Pretreatment with either hypoxia or NaNO₂ significantly increased CM viability but had no effect on cardiac fibroblast viability. Combined hypoxia/NaNO₂ pretreatment significantly increased CM viability but significantly decreased cardiac fibroblast viability. In rat neonatal CMs, cell death, as determined by lactate dehydrogenase (LDH) activity, was significantly reduced with hypoxia/NaNO₂ pretreatment; and in hESC‐CMs, hypoxia/NaNO₂ pretreatment increased the BCL‐2/BAX gene expression ratio, suggesting that hypoxia/NaNO₂ pretreatment promotes cell viability by downregulating apoptosis. Additionally, we found a correlation between the prosurvival effect of hypoxia/NaNO₂ pretreatment and the myoglobin content of the cells by comparing neonatal rat ventricular and atrial CMs, which express high and low myoglobin respectively. Functionally, hypoxia/NaNO₂ pretreatment significantly improved the excitation threshold upon H/R injury to the level observed for uninjured cells, whereas pretreatment did not affect the maximum capture rate. Hence, hypoxia/NaNO₂ pretreatment may serve as a strategy to increase CM survival in cardiac regenerative therapy applications and tissue engineering. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:482–492, 2015     

热休克反应中小鼠心脏HSP70表达的免疫组织化学研究

用免疫组织化学方法研究了小鼠心脏在不同温度（40、42、44、46℃）热休克处理后，各恢复期（2、4、8、12、24小时）HSP70 的表达。结果表明，（1）44、46℃热处理能诱导心肌细胞合成 HSP70，以46℃为多（P＜0.01），且于恢复期4～8小时为合成高峰（P＜0.01）；（2）阳性免疫反应定位于心肌细胞质中，核呈阴性反应。提示了心脏有较强的耐热能力。     

The use of transgenic mice to study cytoprotection by the stress proteins

Heat shock or stress proteins (HSPs) have been shown to be able to confer cytoprotection in a diversity of cell types and organisms. We were interested in assessing if HSPs, in particular HSP70, were protective against pathophysiological stresses such as myocardial ischemia. Our approach was to generate a transgenic mouse line that would constitutively express high levels of an inducible rat HSP70 isoform in the heart. The hearts of the transgenic mice were then used in an isolated perfused mouse heart model to assess whether increased expression of HSP70 alone was protective against ischemia-reperfusion injury. Our study showed that there was a significant improvement in contractile recovery, less cellular damage, and a reduction in infarct size in the hearts of transgenic mice as compared to non-transgenic mice following global ischemia in our isolated perfused mouse heart model. Additional studies have since shown that increased expression of HSP70 as well as other stress proteins in transgenic mice protects against different forms of pathological stresses. We present here the methods we used to generate HSP70 transgenic mice and assess their increased tolerance to ischemia-reperfusion injury.     

Acidic reoxygenation protects against endothelial dysfunction in rat aortic rings submitted to simulated ischemia

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.     

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.     

Localization of constitutive heat shock proteins in developing ascidians

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage-stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti-HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.     

Prolonged treadmill training increases HSP70 in skeletal muscle but does not affect age-related functional deficits

Skeletal muscle atrophy and weakness are major causes of frailty in the elderly. Functional deficits in muscles of old humans and rodents are associated with attenuated production of heat shock proteins (HSPs) after exercise, and transgenic overexpression of HSP70 reverses this functional decline. We hypothesized that training would increase HSP70 content of muscle in adult and old wild-type mice and that this would protect against the development of age-related functional deficits. A 10-wk treadmill training protocol at 15 m/min, for 15 min, 3 days/wk resulted in a significant increase in HSP70 content of muscles of adult mice. Muscles of old untrained mice demonstrated a significant increase in HSP70 protein content and a reduction in HSP70 mRNA content compared with adult untrained mice. Training for 12 mo starting at age 12-14 mo old or for 10 wk starting from age 24 mo old resulted in modification of HSP70 protein and mRNA content to levels of adult mice. Training did not change force generation of extensor digitorum longus muscles of old mice or improve recovery after damaging contractions. The twofold increase in HSP70 content in muscles of adult mice after training may have not been sufficient to provide protection in this instance.     

DNA damage sensible engineered promoter for cellular biosensing of cytotoxicity

We have established a cytotoxic sensor cell line by transfecting HepG2 cells with a luciferase protein plasmid derived from the heat shock protein 70B′ (HSP70B′) promoter, which is induced by cytotoxic reagents. HSP70B genes are up‐regulated by a wide‐range of cytotoxic stimulators, in particular, those that denature proteins. However, the HSP70B genes do not respond to DNA damage. We used a PCR array to detect marker genes of DNA damage‐related cytotoxic stimulation and found the BTG2 gene to be one such gene. Analysis of the BTG2 gene functional promoter region by transfection of various deletion constructs into HepG2 cells indicated that the p53 and NFY biding sites on BTG2 are important for the response to DNA damage. We then constructed HepG2 sensor cells using the functional BTG2 promoter, and found that these sensor cells can specifically detect the cytotoxicity accompanied by DNA strand breaks with high sensitivity. Biotechnol. Bioeng. 2009;102: 1460–1465. © 2008 Wiley Periodicals, Inc.