Primary Cardiomyocyte Cultures Research Articles

Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells

Cell therapy represents a promising new paradigm for treatment of heart disease, a major cause of death in the industrialized world. The recent discovery of tissue resident c-Kit+ cardiac progenitor cells (CPCs) has fueled scientific efforts to exploit these cells therapeutically for regenerative interventions, and primary culture of cardiomyocytes is a common in-vitro model to investigate basic molecular mechanisms underlying cardiac degeneration and regeneration. Current protocols for cardiomyocyte isolation frequently result in low cell yield and insufficient depletion of fibroblasts, which then overgrow the cardiomyocytes in culture. In this protocol we describe an improved method for the isolation of neonatal rat cardiomyocytes that also enables enhanced yields of CPCs. Gentle techniques of enzymatic and mechanical tissue processing ensure high cell numbers and viability, while subsequent Percoll density gradient centrifugation minimizes fibroblasts. We compared the advantages of different enzymes and found that Collagenase 2 alone leads to very high yields of cardiomyocytes, whereas the application of Matrase™ enzyme blend increases the relative yield of c-Kit+ CPCs to up to 35%. Cardiomyocytes and CPCs isolated with this protocol may constitute an important cell source for investigating heart disease as well as cell based therapeutic approaches.

Exploring the potential activity spectrum of two 5-nitroindazolinone prototypes on different Trypanosoma cruzi strains

SUMMARYIn the present study, the potential activity of two 5-nitroindazole derivatives previously proposed as suitable antichagasic prototypes was further evaluated on diverseTrypanosoma cruzistrains belonging to two discrete typing units (DTUs) frequently associated with human infection (i.e. DTUs TcII and TcVI). The trypanocidal profile that both 2-benzyl-1-propyl (22) and 2-benzyl-1-butyl (24) derivatives achieved on Tulahuen amastigotes (IC50 = 3·56 ± 0·99 and 6·31 ± 1·04 µm, respectively) correlates with that of formerly obtained on CL Brener, corroborating an outstanding activity on DTU TcVI parasites. Moreover, a sequential screening on extracellular and intracellular stages ofT. cruziY (DTU TcII) demonstrated also the effectiveness of 22 and 24 over this strain on a similar range of activity (IC50epimastigotes = 3·55 ± 0·47 and 7·92 ± 1·63 µm, IC50amastigotes = 2·80 ± 0·46 and 9·02 ± 5·26 µm, respectively). These results, supported by a lack of toxicity registered over either L929 fibroblasts or primary cultures of cardiomyocytes, confirm that 5-nitroindazolinones 22 and 24 display great selectivity on both drug-sensitive (CL and Tulahuen) and drug-moderately resistant (Y)T. cruzistrains, and therefore, represent an important outcome in the research of Chagas disease chemotherapy.

Enrichment of cardiomyocytes in primary cultures of murine neonatal hearts.

In vitro culture of neonatal murine cardiomyocytes is vital for understanding the functions of the heart. Cardiomyocyte cultures are difficult to maintain because they do not proliferate after birth. The maintenance of primary cultures of viable and functional cardiomyocytes is considerably affected by the yield from initial steps of isolation procedures. This protocol describes an efficient and rapid method for isolation and maintenance of long-term cultures of neonatal murine cardiomyocytes by effectively shortening the trypsin enzyme digestion period and the cardiomyocyte enrichment step.

Ischemia and reperfusion induce differential expression of calpastatin and its homologue high molecular weight calmodulin-binding protein in murine cardiomyocytes.

In the heart, calpastatin (Calp) and its homologue high molecular weight calmodulin-binding protein (HMWCaMBP) regulate calpains (Calpn) by inhibition. A rise in intracellular myocardial Ca2+ during cardiac ischemia activates Calpn thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. The present study aims to elucidate expression of Calp and HMWCaMBP with respect to Calpn during induced ischemia and reperfusion in primary murine cardiomyocyte cultures. Ischemia and subsequently reperfusion was induced in ∼80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. Confocal fluorescent microscopy was used to observe protein localization. We observed that ischemia induces increased expression of Calp, HMWCaMBP and Calpn. Calpn expressing NMCC on co-expressing Calp survived ischemic induction compared to NMCC co-expressing HMWCaMBP. Similarly, living cells expressed Calp in contrast to dead cells which expressed HMWCaMBP following reperfusion. A significant difference in the expression of Calp and its homologue HMWCaMBP was observed in localization studies during ischemia. The current study adds to the existing knowledge that HMWCaMBP could be a putative isoform of Calp. NMCC on co-expressing Calp and Calpn-1 survived ischemic and reperfusion inductions compared to NMCC co-expressing HMWCaMBP and Calpn-1. A significant difference in expression of Calp and HMWCaMBP was observed in localization studies during ischemia.

Comparison of docosahexaenoic acid uptake in murine cardiomyocyte culture and tissue: Significance to physiologically relevant studies

Long-chain n−3 (or omega 3) fatty acids, namely docosahexaenoic acid (DHA, 22:6n−3) and eicosapentaenoic acid (EPA, 20:5n−3) have been attributed cardioprotective properties.In this study, we evaluated the incorporation of DHA into cardiomyocytes and the shift in the omega 3/omega 6 ratio after supplementation of primary cardiomyocyte culture. Results are compared with atrial tissue concentrations attained after prolonged feeding of rats. The major difference between in vitro vs. in vivo supplementation is the paradoxical accumulation of arachidonic acid in cultured cardiomyocyte. However, this increase does not give rise to a higher PGE2 production after cellular stimulation, as compared with controls, possibly because of the associated inhibition of sPLA2 by DHA. Notably, in vitro supplementations with DHA 10 to 25μM approximate in vivo pharmacological treatments.

FGF10 promotes regional foetal cardiomyocyte proliferation and adult cardiomyocyte cell-cycle re-entry.

Cardiomyocyte proliferation gradually declines during embryogenesis resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell-cycle activity of cardiomyocytes in the adult heart. This study aims to determine the mechanism by which fibroblast growth factor 10 (FGF10) controls foetal cardiomyocyte proliferation and to test the hypothesis that FGF10 promotes the proliferative capacity of adult cardiomyocytes. Analysis of Fgf10(-/-) hearts and primary cardiomyocyte cultures reveals that altered ventricular morphology is associated with impaired proliferation of right but not left-ventricular myocytes. Decreased FOXO3 phosphorylation associated with up-regulated p27(kip) (1) levels was observed specifically in the right ventricle of Fgf10(-/-) hearts. In addition, cell-type-specific expression analysis revealed that Fgf10 and its receptor, Fgfr2b, are expressed in cardiomyocytes and not cardiac fibroblasts, consistent with a cell-type autonomous role of FGF10 in regulating regional specific myocyte proliferation in the foetal heart. Furthermore, we demonstrate that in vivo overexpression of Fgf10 in adult mice promotes cardiomyocyte but not cardiac fibroblast cell-cycle re-entry. FGF10 regulates regional cardiomyocyte proliferation in the foetal heart through a FOXO3/p27(kip1) pathway. In addition, FGF10 triggers cell-cycle re-entry of adult cardiomyocytes and is thus a potential target for cardiac repair.

Non-genomic effects of spironolactone and eplerenone in cardiomyocytes of neonatal Wistar rats: do they evoke cardioprotective pathways?

Mineralocorticoid receptor (MR) antagonists of aldosterone (spironolactone and eplerenone) display beneficial effects in the treatment of cardiopathies; however, many of these responses are independent of this antagonism. The mechanisms of action of these drugs are not well known; few studies have comparatively evaluated whether eplerenone as well as spironolactone display cardioprotective effects independent of the blockade of aldosterone. To study these mechanisms, which lead to cardioprotective responses, and to evaluate comparatively their effects in vitro, we have evaluated the proliferative effect of spironolactone and eplerenone in primary culture of cardiomyocytes and fibroblasts of neonatal Wistar rats in the presence and absence of aldosterone. Spironolactone and eplerenone promoted proliferation of cardiomyocyte even in the absence of aldosterone, suggesting a signaling pathway independent of the antagonism over aldosterone. Spironolactone was able to reduce the proliferation of fibroblasts and to reverse the proliferation promoted by aldosterone, which was also displayed by eplerenone. To elucidate the biochemical pathways evoked by these drugs, we sought to analyze Ca(2+), cAMP, and cGMP, and the activity of PKC and ERK1/2. Spironolactone and eplerenone increased the levels of Ca(2+), cGMP and activity of ERK 1/2, and reversed the action of aldosterone on the activity of PKC and ERK1/2. Interestingly, only spironolactone increased the levels of cAMP. Our data support the fact that in addition to aldosterone, both spironolactone and eplerenone display rapid responses (non-genomic) such as an increase on cAMP, Ca(2+), and cGMP by spironolactone, and Ca(2+) and cGMP by eplerenone. We have observed a more consistent cardioprotection promoted by spironolactone; however, these effects have yet to be tested clinically. Therefore, our data show that these drugs do not only act as an antagonist of MR, but could lead to a new pharmacological classification of these drugs.

Thymbra capitata essential oil prevents cell death induced by 4-hydroxy-2-nonenal in neonatal rat cardiac myocytes.

An interdisciplinary experimental investigation on the antioxidant activity of Thymbra capitata essential oil was made. This plant is a Mediterranean culinary herb, whose essential oil antioxidant power has recently been demonstrated in vitro as one of the highest in nature. We tested if this in vitro antioxidant capacity was reproducible on biological systems using as model system primary cultures of neonatal rat cardiomyocytes treated with the lipid peroxidation product 4-hydroxy-2-nonenal. The composition and the in vitro antioxidant activity of the T. capitata essential oil were also assessed. Cell viability, mitochondrial membrane potential, and reactive oxygen species level were measured in cells treated with pathophysiologic doses of 4-hydroxy-2-nonenal (< 10 µM) or vehicle after being pre-incubated with small concentrations of the T. capitata essential oil, and the ability of small doses (< 40 ppm) to prevent the death of neonatal rat cardiomyocytes proved very remarkable. Long-term pre-incubation (12 h) with 20 ppm prevented 4-hydroxy-2-nonenal-induced cell death and avoided mitochondrial membrane potential loss and reactive oxygen species generation caused by 4-hydroxy-2-nonenal. A deleterious effect was shown at doses higher than 40 ppm. The results of this study pave the way to further analysis in animal models to achieve a deeper understanding of the in vivo antioxidant power of T. capitata essential oil.

Triiodothyronine prevents cardiac ischemia/reperfusion mitochondrial impairment and cell loss by regulating miR30a/p53 axis.

Mitochondrial dysfunctions critically affect cardiomyocyte survival during ischemia/reperfusion (I/R) injury. In this scenario p53 activates multiple signaling pathways that impair cardiac mitochondria and promote cell death. p53 is a validated target of miR-30 whose levels fall under ischemic conditions. Although triiodothyronine (T3) rescues post-ischemic mitochondrial activity and cell viability, no data are available on its role in the modulation of p53 signaling in I/R. Here we test the hypothesis that early T3 supplementation in rats inhibits the post I/R activation of p53 pro-death cascade through the maintenance of miRNA 30a expression. In our model, T3 infusion improves the recovery of post-ischemic cardiac performance. At the molecular level, the beneficial effect of T3 is associated with restored levels of miR-30a expression in the area at risk (AAR) that correspond to p53 mRNA downregulation. The concomitant decrease in p53 protein content reduces Bax expression and limits mitochondrial membrane depolarization resulting in preserved mitochondrial function and decreased apoptosis and necrosis extent in the AAR. Also in primary cardiomyocyte culture of neonatal rats, T3 prevents both miR-30a downregulation and p53 raise induced by hypoxia. The regulatory effect of T3 is greatly suppressed by miR-30a knockdown. Overall these data suggest a new mechanism of T3-mediated cardioprotection that is targeted to mitochondria and acts, at least in part, through the regulation of miR-30a/p53 axis.

The Formin FHOD1 in Cardiomyocytes

Members of the formin family are known to be involved in the regulation of the actin cytoskeleton. We have recently identified a muscle specific splice variant of the formin FHOD3 and demonstrated its role in the maintenance of the contractile filaments of cardiomyocytes. Here, we characterize the expression and subcellular localization of FHOD3's closest relative, FHOD1, in the heart. Confocal microscopy shows that FHOD1 is mainly located at the intercalated disc, the special type of cell-cell contact between cardiomyocytes, but also partially associated with the myofibrils. Subcellular targeting of FHOD1 is probably mediated by its N-terminal domain, since expression constructs lacking this domain show aberrant localization in primary cultures of neonatal rat cardiomyocytes. Finally, we show that in contrast to FHOD3, FHOD1 shows increased expression levels in dilated cardiomyopathy, suggesting that the two formins play distinct roles and are differentially regulated in cardiomyocytes.

Vascular endothelial growth factor recovers suppressed cytochrome c oxidase activity by restoring copper availability in hypertrophic cardiomyocytes.

Cardiomyocyte hypertrophy induced by phenylepherine (PE) is accompanied by depression of cytochrome c oxidase (COX) activity. Vascular endothelial growth factor (VEGF) recovers the suppressed COX activity and reverses cardiomyocyte hypertrophy. Because PE causes intracellular copper (Cu) depletion and COX activity is Cu-dependent, the present study was undertaken to test the hypothesis that VEGF recovers suppressed COX activity by restoring Cu availability. Primary cultures of neonatal rat cardiomyocytes were treated with PE at a final concentration of 100 µmol/L in cultures for 48 h to induce cell hypertrophy. The hypertrophic cardiomyocytes were exposed to VEGF at a final concentration of 20 ng/mL in cultures for 24 h. Atomic absorption spectrometry analysis revealed that VEGF restored PE-depleted Cu concentrations in hypertrophic cardiomyocytes along with the recovery of COX activity. Western blot analysis showed that protein contents of COX subunit COX-IV and Cu chaperones for COX (COX17, COX11, and SCO2) were decreased in response to PE treatment, and recovered after VEGF treatment. In addition, VEGF treatment suppressed PE-induced accumulation of reactive oxygen species (ROS) and the relevant elevation of homocysteine, which has been shown to form complexes with Cu to restrict Cu availability. This study thus demonstrates that VEGF recovers PE-suppressed COX activity by restoring Cu availability and VEGF suppression of ROS accumulation and homocysteine elevation would contribute to the increased Cu availability.

Alterations of field potentials in isotropic cardiomyocyte cell layers induced by multiple endogenous pacemakers under normal and hypothermal conditions.

The use of autonomous contracting randomly grown cardiomyocyte monolayers cultivated on microelectrode arrays (MEAs) represents an accepted experimental setting for preclinical experimental research in the field of cardiac electrophysiology. A dominant pacemaker forces a monolayer to adhere to a regular and synchronized contraction. Randomly distributed multiple pacemakers interfere with this dominant center, resulting in more or less frequent changes of propagation direction. This study aims to characterize the impact of changing propagation directions at single electrodes of the MEA on the four intrinsic parameters of registered field potentials (FPs) FPrise, FPMIN, FPpre, and FPdur and conduction velocity (CV) under normal and hypothermal conditions. Primary cultures of chicken cardiomyocytes (n = 18) were plated directly onto MEAs and FPs were recorded in a temperature range between 37 and 29°C. The number and spatiotemporal distribution of biological and artificial pacemakers of each cell layer inside and outside of the MEA registration area were evaluated using an algorithm developed in-house. In almost every second myocardial cell layer, interfering autonomous pacemakers were detected at stable temperatures, showing random spatial distributions with similar beating rates. Additionally, a temperature-dependent change of the dominant pacemaker center was observed in n = 16 experiments. A significant spread-direction-dependent variation of CV, FPrise, FPMIN, and FPpre up to 14% could be measured between different endogenous pacemakers. In conclusion, based on our results, disregarding the spatial origin of excitation may lead to misinterpretations and erroneous conclusions of FP parameters in the verification of research hypotheses in cellular electrocardiology.

Effects and mechanism of action of ligustrazine on isoprenaline-induced cardiomyocyte hypertrophy.

The aim of the study is to explore the effects and mechanism of the action of ligustrazine on isoprenaline-induced cardiomyocyte hypertrophy. Primary culture of neonatal rat cardiomyocytes was used as the model, and isoprenaline was used to induce cardiomyocyte hypertrophy. Effects of different dosages of ligustrazine polysaccharide on the cardiomyocyte were observed. RT-PCR was used to detect the expression of atrial natriuretic factor (ANP) mRNA, and Western blot analysis was used to detect the CaN protein level in cardiomyocytes. After treating with ligustrazine, the significant increase of MDA content and decrease of SOD activity were inhibited in supernatant. Compared to the control group, ANP mRNA in isoprenaline-treated cardiomyocytes was significantly increased (P < 0.05); compared to the isoprenaline group, ANP mRNA was significantly decreased in all ligustrazine groups (P < 0.01). In all ligustrazine groups, the CaN expression was inhibited in isoprenaline-treated cardiomyocytes in a dose-dependent manner. In conclusion, ligustrazine has protective effects on isoprenaline-induced neonatal rat cardiomyocyte, which may be related to the decrease of CaN expression.

P148Cardiac myocytes are protected by small leucine rich proteoglycans against simulated ischemia/reperfusion injury

It was previously shown by our group that the overexpression of biglycan in a transgenic mouse model enhances the induction of cardioprotective genes. Ischemia induced cell death was decreased by bigylcan treatment in neonatal cardiomyocytes. In this study we aimed to test whether the biglycan core protein or the glycosaminoglycan is responsible for the cardioprotective effect of biglycan. We also investigated whether decorin, (which has a similar structure to biglycan), has any cardio-protective effects on cardiomyocytes during simulated ischemia/reperfusion (SI/R) injury. Primary cardiomyocyte cultures were isolated from neonatal rat hearts. The 2 day-old cultures were pretreated with 0, 1, 3, 10, 30, and 100 nM decorin, biglycan, biglycan core protein or chondroitin sulfate A for 20 hours followed by an SI/R protocol. The treatment was maintained throughout SI , 240 min, hypoxic solution, hypoxic chamber gassed with 95% N2 es 5% CO2) and reperfusion (120 min, normoxic conditions, medium). At the end of the protocol, a viability assay was performed using calcein staining. Compared to the normoxic untreated control, approximately 40% of the cardiomyocytes died during the simulated ischemia/reperfusion injury. Doses of 10 and 30 nM biglycan increased cell viability significantly, compared to SI/R control (29±4 and 18±5%). The biglycan core protein showed similar cytoprotective effects at 3 and 10 nM doses (21±4 and 24±5%), however, the chondroitin sulfate had no influence on the viability of cardiomyocytes. In the case of decorin, the 3 and 10 nM doses significantly increased the survival of the cells (28±3 and 20±4%). Based on our experiments, we concluded that both decorin and biglycan exert a cyto-protective effect on cardiomyocytes against SI/R injury and this protective effect of biglycan is likely due to the biglycan core protein. Grants: NKTM-OTKA-PD-106001, South-East European Foundation Lykeon Scholarship, GA holds Bolyai Janos Fellowship

Brief Communication: Copper suppression of vascular endothelial growth factor receptor-2 is involved in the regression of cardiomyocyte hypertrophy.

Previous studies revealed that copper (Cu)-induced regression of cardiomyocyte hypertrophy is associated with enhanced activity in the vascular endothelial growth factor receptor-1 (VEGFR-1) signaling pathway. The mechanism by which Cu enhances the activity of VEGFR-1 pathway remains to be defined. The present study was undertaken to test the hypothesis that Cu enhances the VEGFR-1 signaling pathway via suppression of the VEGFR-2 signaling pathway. Primary cultures of neonatal rat cardiomyocytes were exposed to phenylephrine (PE) at a final concentration of 100 µM in cultures for 48 h to induce cell hypertrophy. The hypertrophic cardiomyocytes were exposed to copper sulfate at a final concentration of 5 µM Cu in cultures for 24 h. Western blot analysis showed that PE increased the protein levels of both VEGFR-1 and VEGFR-2. Cu supplementation significantly reduced the increase in VEGFR-2, but had no effect on the elevation of VEGFR-1. Real-time polymerase chain reaction analysis found no difference in the mRNA levels between the VEGFR-1 and VEGFR-2 under the conditions defined above. This study thus demonstrated that Cu selectively suppressed PE-elevated VEGFR-2 levels likely via post-translational regulation, leading to the VEGFR-1 signaling pathway becoming dominant and thereby regressing cardiomyocyte hypertrophy.

Prevention of export of anoxia/reoxygenation injury from ischemic to nonischemic cardiomyocytes via inhibition of endocytosis.

Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin-1. Anoxia and reoxygenation caused collapse of clathrin-mediated endocytosis and stimulation of macropinocytosis, whereas in cultures exposed to conditioned media, the activity of endocytosis was uniformly higher. Conditioned media also significantly aggravated cytotoxic effects of TNF-α and angiotensin II, and suppression of endocytosis reversed these trends, resulting in an overall increase of metabolic activity. Moreover, inhibition of endocytosis prevented binding of oxidized cellular fragments with greater efficiency than targeted neutralization of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1. Many of the observations in HL-1 cardiomyocytes were confirmed in primary cardiomyocyte cultures. Our data suggest that endocytosis is upregulated in border zone cardiomyocytes, and inhibition of endocytosis may be an effective approach to prevent export of injury signals from the infarct zone.

Thyroid hormone upregulates beta‐arrestin 2 (βARR2) expression on cardiomyocytes (LB702)

Changes in thyroid hormone (TH) levels are accompanied by alterations in responsiveness and signaling of G‐protein coupled receptors (GPCRs) in several tissues. β‐arrestins (βARRs) are scaffold proteins primarily involved in GPCRs desensitization. Furthermore, recent studies described that βARRs induce the endocytosis of activated GPCRs and this formed complex may act as a citoplasmatic signaling platform in a G‐protein independent manner. In cardiac tissue, this non‐canonical kind of signaling has been cogitated to be cardioprotective. Considering the beneficial actions of TH on myocardium, we investigated the effect of TH on βARRs isoforms (1 and 2) levels. For this purpose, primary cultures of cardiomyocytes (n=5) were obtained from hearts neonatal Wistar rats by enzymatic disaggregation. The cardiomyocytes were treated for 5 min, 10 min, 15 min, 30 min or 24 h with serum‐free medium (control cells) or with serum‐free medium containing T3 (triiodothyronine, 15ηM, Sigma Aldrich). The βARRs isoforms were evaluated by Western Blot. The data were analyzed by One‐way ANOVA followed by Tukey’s post hoc test. Our data indicate that TH rapidly increased the βARR2 protein levels in cardiomyocytes but no effect was observed after 24h of TH treatment. This preliminary evidence demonstrates for the first time that βARRs signaling is triggered by TH and suggests a possible involvement of this hormone with a new signaling pathway.Grant Funding Source: Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP ‐ grant number: 2013/16142‐9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

0338: Glucagon-like peptide 1 or GLP-1 counteracts NADPH oxidase activation during hyperglycemia through an AMPK-dependent pathway in adult cardiomyocytes

Exposure to high glucose (HG) stimulates NADPH oxidase (NOX2) dependent-ROS production in cardiomyocytes. NOX2 activation is not triggered by an increased glucose metabolism but results from a glucose transport through a sodium-glucose co-transporter (SGLT). The aim of this work is to identify potential therapeutic approaches to counteract glucotoxicity. Primary cultures of adult rat cardiomyocytes were exposed to high glucose concentration (HG, 21mM). AMP-activated protein kinase (AMPK) activation by A769662 or Phenformin nearly suppressed ROS production under hyperglycemia. GLP-1, a new anti-diabetic drug, exerted a similar effect, blocking hyperglycemia-mediated ROS production. Interestingly, GLP-1 treatment induced a AMPK activation, the maximal activation being observed at 100 nM. α2AMPK, the major isoform expressed in cardiomyocytes (but not α1AMPK) was activated in response to GLP-1. Anti-ROS properties of AMPK were not related to change in glucose uptake or glycolysis. Using in situ proximity ligation assay technology, we demonstrated that AMPK activation prevented p47phox translocation to the caveolar structure after high glucose concentration exposure, whatever the AMPK activators used. NOX2 activation by α-methyl-D-glucopyranoside, a glucose analog exclusively transported through SGLT, or by Angiotensin 2 was also counteracted by GLP-1. The crucial role of AMPK in limiting glucotoxicity was demonstrated by overexpressing a constitutively active form of AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG. Finally, in mice cardiomyocytes, GLP-1 did not exert protective action in the absence of α2AMPK. GLP-1 induces α2AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, limiting glucotoxicity.

Primary human cardiomyocyte culture: a model for assessment of cardiovascular disorders (652.12)

The purpose of this research is to establish an in vitro cardiac system using primary human cardiomyocyte cultures (PHCC). We hypothesized that norepinephrine (NE) and endothelin‐1 (ET‐1) will contribute to human cardiomyocyte growth. PHCC were obtained from Celprogen, San Pedro, CA and grown with varied concentrations of NE or ET‐1 for 72h. Protein expressions of contractile proteins were analyzed via Immunoblot and immunocytochemistry. Analysis of confocal images showed a significant decrease in nuclear area in all NE treatment groups. NE (10 µmol) stimulated significant increases in fluorescent intensities of the contractile proteins, f‐actin, troponin and tropomyosin. The protein expression of myosin light chain was significantly lower with increasing concentrations of NE. In addition, the average cell area increased in all groups of ET‐1. ET‐1 (1, 10, 100 nmol) exhibited a significant decrease of fluorescent intensities of f‐actin, troponin and tropomyosin. PHCC were also grown in the presence of LPS (1, 10, 100µg/ml) in a 6‐well plate for 72h. Viability data showed the cells recovered from the endotoxin treatment and maintain steady growth in each concentration of LPS. In contrast to NE, ET‐1 exhibited a decrease on the expression of contractile proteins in PHCC.Grant Funding Source: Office of Chief Science Officer, PCOM

Mitochondrially targeted nitro‐linoleate: a new tool for the study of cardioprotection

Cardiac ischaemia-reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO2 ), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP(+) ) moiety would enhance its protective properties. TPP(+) -LNO2 was synthesized from aminopropyl-TPP(+) and LNO2 , and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP(+) . TPP(+) -LNO2 protected cardiomyocytes from SIR injury more potently than the parent compound LNO2 . In addition, TPP(+) -LNO2 modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP(+) -LNO2 . Together, these results emphasize the importance of ANT1 as a target for the protective effects of LNO2 , and suggest that TPP(+) -conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.