Effects of docosahexaenoic acid on calcium pathway in adult rat cardiomyocytes

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.     

Host and parasite apoptosis following<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> infection in in vitro and in vivo models

The mechanism of cell death which occurs during Chagas' cardiopathy is disputed. To address this issue we analyzed the molecular pathways implicated in the death of cardiomyocytes during T. cruzi invasion and found that they undergo apoptosis during both in vitro and in vivo infections. However, the death rates and onset were related to the parasite stocks belonging to different biodemes, which can be correlated to the different histological inflammation findings that have already been reported. Our in vitro data provide additional support for this hypothesis since higher levels and earlier apoptosis induction were noted during the interaction with the Dm28c (type I) as compared to the Y and CL stocks (type II). Modifications of the surface carbohydrates of the infected cardiomyocytes were observed and these molecular events may be acting as "eat me" tags for their final engulfment by macrophages and/or other non-professional phagocytes. The analysis of other host cell types showed that the in vitro infection of fibroblasts did not result in host apoptosis even when a highly infective stock was used. Conversely, infected macrophages undergo apoptosis but at a higher degree than cardiomyocytes. Apoptotic intracellular parasites were observed to varied extents depending on the T. cruzi stock, which was related to the parasite invasion and proliferation. In summary, our results show that during T. cruzi infection, the extent of apoptosis varies according to the host cell type and the parasite stocks. The apoptosis of both host and T. cruzi can contribute to the silent spreading and persistence of the parasite without triggering an exacerbated inflammatory response.The present study was supported by grants from the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), INSERM and PAPESIII/FIOCRUZ. Support by the "INSERM-FIOCRUZ convention" is also acknowledged.     

Parallel development of cardiomyocytes and neurons in embryonic stem cell culture

Recent studies suggest that there are strong parallels between development and patterning of the vertebrate vascular system and the nervous system. While previous observations reported generation of vascular and neuronal progenitors from embryonic stem (ES) cells, the question of parallel development of vascular and neuronal cells in the same culture has not yet been investigated. Mouse D3 ES cells were cultured for 4 days in differentiation medium IMDM with 15% FBS in 100 mm non-adhesive Petri dishes to allow cells to aggregate and form embryoid bodies. At day 5, fibronectin or all-trans retinoic acid with fibronectin was added to the culture. On day 9, the embryoid bodies were seeded on poly-L-ornithine/fibronectin-coated plates. After plating, half of the plates were treated with laminin for 3 days and maintained for 1 week in Neurobasal media with B27. Here we show that ES cells differentiate into interconnected rhythmically contracting aggregates of functional cardiomyocytes and neurons. Double immunofluorescence with anti-phospholamban, anti-SERCA2 antibodies to detect cardiomyocytes and with anti-MAP2 antibodies to detect neurons revealed the cell aggregates consisting entirely of cardiomyocytes with neuronal cells located on the periphery or covering the aggregate's surface. The observed concurrent development of cardiomyocytes and neurons suggests bidirectional communication between both cell types. We propose that crosstalk between cardiovascular and neuronal progenitors is an important mechanism for the development of both systems.     

Tonic component of myocardial contraction

Calcium transients and contractions of cardiac myocytes consist of phasic component, relaxing spontaneously independently of membrane voltage and of the tonic component (TC) relaxing only upon repolarization. Experimental data reviewed in this article suggest that most Ca(2+) activating TC is released from sarcoplasmic reticulum (SR) via the ryanodine receptors (RyRs). Most likely these RyRs are activated by sustained Ca(2+) influx. However, its route may differ depending on species and state of the cells. It seems that in rat RyRs responsible for TC are activated by the sustained Ca(2+) current. In guinea-pig the blockers of Ca(2+) current or reverse mode Na(+)/Ca(2+) exchange do not inhibit TC, so these routes seem unlikely. In myocytes of the failing human hearts TC is activated mostly via the reverse mode Na(+)/Ca(2+) exchange and contribution of SR is negligible. The mechanism of TC in the normal human cardiomyocytes has not been investigated. Thus, despite investigation of TC for half a century many problems concerning the mechanism of its activation and maintenance as well as its physiological meaning remain unsolved.     

Downregulation of Akt2 attenuates ER stress-induced cytotoxicity through JNK-Wnt pathway in cardiomyocytes

Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that promotes survival and growth in response to extracellular signals. Akt1 has been demonstrated to play vital roles in cardiovascular diseases, but the role of Akt2 in cardiomyocytes is not fully understood. This study investigated the effect of Akt2 knockdown on tunicamycin (TM)-induced cytotoxicity in cardiomyocytes and the underlying mechanisms with a focus on the JNK-Wnt pathway. TM treatment significantly increased the expression of Akt2 at both mRNA and protein levels, which was shown to be mediated by the induction of reactive oxygen species (ROS). Knockdown of Akt2 expression via siRNA transfection markedly increased cell viability, decreased lactate dehydrogenase (LDH) release and reduced cell apoptosis after TM exposure. The results of western blot showed that downregulation of Akt2 also attenuated the TM-induced activation of the unfolded protein response (UPR) factors and ER stress associated pro-apoptotic proteins. In addition, Si-Akt2 transfection partially prevented the TM-induced decrease in nuclear localization of β-catenin. By using the selective inhibitor SP-600,125 to inhibit JNK phosphorylation, we found that knockdown of Akt2-induced protection and inhibition of ER stress was mediated by reversing TM-induced decrease of Wnt through the JNK pathway. In summary, these data suggested that Akt2 play a pivotal role in regulating cardiomyocyte survival during ER stress by modulating the JNK-Wnt pathway.     

Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.     

Role of dystrophin in acute Trypanosoma cruzi infection

Previous studies have demonstrated loss/reduction of dystrophin in cardiomyocytes in both acute and chronic stages of experimental Trypanosoma cruzi (T. cruzi) infection in mice. The mechanisms responsible for dystrophin disruption in the hearts of mice acutely infected with T. cruzi are not completely understood. The present in vivo and in vitro studies were undertaken to evaluate the role of inflammation in dystrophin disruption and its correlation with the high mortality rate during acute infection. C57BL/6 mice were infected with T. cruzi and killed 14, 20 and 26 days post infection (dpi). The intensity of inflammation, cardiac expression of dystrophin, calpain-1, NF-κB, TNF-α, and sarcolemmal permeability were evaluated. Cultured neonatal murine cardiomyocytes were incubated with serum, collected at the peak of cytokine production and free of parasites, from T. cruzi-infected mice and dystrophin, calpain-1, and NF-κB expression analyzed. Dystrophin disruption occurs at the peak of mortality and inflammation and is associated with increased expression of calpain-1, TNF-α, NF-κB, and increased sarcolemmal permeability in the heart of T. cruzi-infected mice at 20 dpi confirmed by in vitro studies. The peak of mortality occurred only when significant loss of dystrophin in the hearts of infected animals occurred, highlighting the correlation between inflammation, dystrophin loss and mortality.     

低硒心肌损伤与钾离子通道蛋白的关系研究

目的：心肌上的离子通道蛋白与心肌损伤有很大的关系,本研究通过低硒喂养对C57BL／6小鼠心肌组织损伤的影响及其对钾通道蛋白的改变。方法：将实验小鼠分为4组：对照组,低硒30天组,低硒90天组和低硒180天组。采用低硒饲料（硒含量0．0045μg／g）喂养的方法建立低硒小鼠模型,对照组给予正常饲料（硒含量0．256μg/g）,与低硒组同时喂养;硒含量的测定和HE染色方法观察心肌损伤情况,WesternBlotting方法检测其钾通道蛋白的表达。结果：低硒饲料喂养小鼠的心脏硒含量与正常饲料喂养的硒含量相比明显降低（P〈0．01）;并出现轻微的心肌损伤,钾通道蛋白的表达量在低硒30天组,低硒90天组和低硒180天组下调（P〈0．01）。结论：成功建立低硒小鼠模型,低硒能引起小鼠心肌损伤,这种改变可能有心脏的钾通道蛋白的表达水平有关。     

丹参酮ⅡA调节microRNA-1保护缺氧心肌细胞的作用研究

目的:研究丹参酮Ⅱ A(TanshinoneⅡA)通过调节microRNA-1抗心肌细胞缺氧损伤的作用。方法:原代培养新生大鼠心肌细胞,建立心肌细胞缺氧模型。MTT法检测心肌细胞存活率(%);TUNEL、流式细胞术测心肌细胞凋亡率;激光共聚焦检测心肌细胞内钙离子[Ca2+]i浓度的变化情况。结果:MTT结果显示丹参酮ⅡA对缺氧心肌细胞及过表达miR-1引起心肌细胞损伤具有保护作用。丹参酮ⅡA增加了缺氧心肌细胞的存活率(P0.05),同时给予丹参酮ⅡA和miR-1组与单独miR-1损伤组相比较,存活率也明显升高,呈现剂量依赖性。TUNEL结果显示丹参酮ⅡA可以抑制缺氧诱导的细胞凋亡,丹参酮ⅡA可以明显降低由缺氧导致的细胞凋亡率(P0.05)。共聚焦检测结果显示,缺氧损伤的心肌细胞内[Ca2+]i显著升高1322.72±5.16(vs正常对照组,P0.05),丹参酮ⅡA则有效抑制由缺氧引起过高的[Ca2+]i。miR-1诱导的细胞内[Ca2+]i升高至1349.33±62.63,约为正常对照组的1.96倍,而丹参酮ⅡA则有效抑制胞内过高的[Ca2+]i,从而发挥心肌保护作用。结论:丹参酮ⅡA可能是通过抑制胞内miR-1的表达,参与对钙离子浓度的调控,发挥其对心肌细胞的保护作用。     

Oxygen consumption of human heart cells in monolayer culture

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71 ± 0.38 pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77 ± 0.32 pmol/h/cell and 1.61 ± 0.70 pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.