Cultures Of Cardiomyocytes Research Articles

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease

The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.

Disrupting the key circadian regulator CLOCK leads to age-dependent cardiovascular disease

The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in heart weight, hypertrophy, dilation, impaired contractility, and reduced myogenic responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts. Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3β, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3β, S6K1 phosphorylation are increased, consistent with the development of age-dependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in growth and renewal; disrupting Clock leads to age-dependent cardiomyopathy. Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

H-Ras Isoform Mediates Protection Against Pressure Overload-Induced Cardiac Dysfunction in Part Through Activation of AKT.

In general, Ras proteins are thought to promote cardiac hypertrophy, an important risk factor for cardiovascular disease and heart failure. However, the contribution of different Ras isoforms has not been investigated. The objective of this study was to define the role of H- and K-Ras in modulating stress-induced myocardial hypertrophy and failure. We used H- and K-Ras gene knockout mice and subjected them to pressure overload to induce cardiac hypertrophy and dysfunction. We observed a worsened cardiac phenotype in Hras-/- mice, while outcomes were improved in Kras+/- mice. We also used a neonatal rat cardiomyocyte culture system to elucidate the mechanisms underlying these observations. Our findings demonstrate that H-Ras, but not K-Ras, promotes cardiomyocyte hypertrophy both in vivo and in vitro. This response was mediated in part through the phosphoinositide 3-kinase-AKT signaling pathway. Adeno-associated virus-mediated increase in AKT activation improved the cardiac function in pressure overloaded Hras null hearts in vivo. These findings further support engagement of the phosphoinositide 3-kinase-AKT signaling axis by H-Ras. Taken together, these findings indicate that H- and K-Ras have divergent effects on cardiac hypertrophy and heart failure in response to pressure overload stress.

URSODEOXYCHOLIC ACID REGULATES CASPASE-9 AND ROS PRODUCTION IN PROTECTING CARDIOMYOCYTES AGAINST HYPOXIA

Ursodeoxycholic acid (UDCA) is known as a therapeutic agent in treating cholestasis and liver diseases. Recently, UDCA has been suggested as a therapeutic drug for heart related diseases. Cardioprotective effect of UDCA against the development of ischemia has been studied. Yet, the mechanism of UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to elucidate the mechanisms of UDCA cardioprotection against hypoxia by investigating the expression of caspase -3/-9 and ROS generation using an in vitro hypoxic heart model. A newborn (0-2 days old) rat heart was isolated for primary cell culture of cardiomyocytes. Hypoxia was chemically induced by using CoCl2. Cardiomyocytes were then incubated with UDCA. The treated cardiomyocytes were subjected for ROS generation detection assay, QuantiGene Plex assay for caspase-3/-9 gene expression and ELISA for caspase-3/-9 protein expression. The data were analyzed by using sample paired t-test and One-way ANOVA. Our results showed that UDCA abolishes the effects on CoCl2 in ROS production and UDCA downregulates caspase-9 protein expression in CoCl2 treated cardiomyocytes. This study provides an insight of UDCA in protecting cardiomyocytes against hypoxia mediated by anti-apoptosis mechanism.

E2F6 Impairs Glycolysis and Activates BDH1 Expression Prior to Dilated Cardiomyopathy.

RationaleThe E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme β-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker.ObjectiveTo define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1.Methods and ResultsWe previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the Bdh1 transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone β-hydroxybutyrate (β-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to β-OHB.ConclusionsThe data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium through a mechanism involving cyclin B1. We reveal BDH1 expression as an early biomarker of heart failure and its potential impact, through ketone signaling, on CX-43 levels in E2F6-induced DCM.

17β-Estradiol enhances sulforaphane cardioprotection against oxidative stress

The lower incidence of ischemic heart disease in female with respect to male gender suggests the possibility that female sex hormones could have specific effects in cardiovascular protection. 17β-Estradiol is the predominant premenopausal circulating form of estrogen and has a protective role on the cardiovascular system. Recent evidences suggest that gender can influence the response to cardiovascular medications; therefore, we hypothesized that sex hormones could also modulate the cardioprotective effects of nutraceutical compounds, such as the isothiocyanate sulforaphane, present in Brassica vegetables. This study was designed to explore the protective effects of sulforaphane in the presence of 17β-estradiol against H2O2-induced oxidative stress in primary cultures of rat cardiomyocytes. Interestingly, 17β-estradiol enhanced sulforaphane protective activity against H2O2-induced cell death with respect to sulforaphane or 17β-estradiol alone as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide and lactate dehydrogenase assays. Moreover, 17β-estradiol boosted sulforaphane ability to counteract oxidative stress, reducing intracellular reactive oxygen species and 8-hydroxy-2′-deoxyguanosine levels and increasing the expression of phase II enzymes. Using specific antagonists of estrogen receptor α and β, we observed that these effects are not mediated by estrogen receptors. Otherwise, ERK1/2 and Akt signaling pathways seem to be involved, as the presence of specific inhibitors of these kinases reduced the protective effect of sulforaphane in the presence of 17β-estradiol. Sulforaphane and 17β-estradiol co-treatment counteracted cell morphology alterations induced by H2O2 as evidenced by transmission electron microscopy. Our results demonstrated, for the first time, that estrogens could enhance sulforaphane protective effects, suggesting that nutraceutical efficacy might be modulated by sex hormones.

A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces

ObjectiveWe aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro. MethodsKeratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4. ResultsKeratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~106 cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination. ConclusionWe demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Metabolites of Hypoxic Cardiomyocytes Induce the Migration of Cardiac Fibroblasts

Background: The migration of cardiac fibroblasts to the infarct region plays a major role in the repair process after myocardial necrosis or damage. However, few studies investigated whether early hypoxia in cardiomyocytes induces the migration of cardiac fibroblasts. The purpose of this study was to assess the role of metabolites of early hypoxic cardiomyocytes in the induction of cardiac fibroblast migration. Methods: Neonatal rat heart tissue was digested with a mixture of trypsin and collagenase at an appropriate ratio. Cardiomyocytes and cardiac fibroblasts were cultured via differential adhesion. The cardiomyocyte cultures were subjected to hypoxia for 2, 4, 6, 8, 10, and 12 h. The supernatants of the cardiomyocyte cultures were collected to determine the differences in cardiac fibroblast migration induced by hypoxic cardiomyocyte metabolites at various time points using a Transwell apparatus. Meanwhile, ELISA was performed to measure TNF-α, IL-1β and TGF-β expression levels in the cardiomyocyte metabolites at various time points. Results: The metabolites of hypoxic cardiomyocytes significantly induced the migration of cardiac fibroblasts. The induction of cardiac fibroblast migration was significantly enhanced by cardiomyocyte metabolites in comparison to the control after 2, 4, and 6 h of hypoxia, and the effect was most significant after 2 h. The expression levels of TNF-α, IL-1β, IL-6, and TGF-β were substantially increased in the metabolites of cardiomyocytes, and neutralization with anti-TNF-α and anti-IL-1β antibodies markedly reduced the induction of cardiac fibroblast migration by the metabolites of hypoxic cardiomyocytes. Conclusion: The metabolites of early hypoxic cardiomyocytes can induce the migration of cardiac fibroblasts, and TNF-α and IL-1β may act as the initial chemotactic inducers.

Effects of docosahexaenoic or arachidonic acid on contractile function in rat cardiomyocytes in primary culture

Effects of docosahexaenoic or arachidonic acid on contractile function in rat cardiomyocytes in primary culture

On the Problem of Cardiogenic Differentiation: Extracellular Matrix as an Emerging Clue

It is well known that myocardium fails to regenerate due to the lack of adult cardiomyocyte proliferation. In this regard, a lot of methods for cardiogenic differentiation of different cells are currently developed to provide an alternative source of functionally active cardiomyocytes, including embryonic stem cells, cardiac stem cells, induced pluripotent stem cells and fibroblasts. However, the approaches available show quite a poor result in the efficiency as well as estimation criteria of cardiac differentiation. Many studies are intended to reveal the fundamental mechanisms of cardiogenesis to mimic them in vitro, but significant advances in studying of intracellular signaling pathways during heart development are insufficient to produce high efficient methods. Extracellular matrix is another component which is becoming acknowledged as a key player in heart differentiation. However, investigation of its spatiotemporal effects on cardiomyocyte maturation is limited due to the challenges in cell-matrix handling within the heart tissue. We consider that cardiomyocyte primary culture may be a good model for studying the role of extracellular matrix in cardiogenesis, being an easy to manipulate homogenous culture of cardiomyocytes, which synchronically follow rearrangements similar to their embryonic developmental stages. The better understanding of the cell-matrix interactions underlying cardiomyocyte maturation may bring researchers closer to the efficient cardiogenic differentiation.

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber.

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly(l-lactide-co-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5-7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell-cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

Expression of the Cardiac Maintenance and Survival Factor FGF-16 Gene Is Regulated by Csx/Nkx2.5 and Is an Early Target of Doxorubicin Cardiotoxicity.

The fibroblast growth factor (FGF) 16 gene (Fgf-16) is preferentially expressed by neonatal cardiomyocytes after birth, with levels increasing into adulthood. Null mice and isolated heart studies suggest a role for FGF-16 in cardiac maintenance and survival, including increased resistance to doxorubicin (DOX)-induced injury. However, the effect of DOX on endogenous FGF-16 synthesis and specifically regulation of cardiac Fgf-16 expression has not been reported. Here we assess the effect of DOX on FGF-16 RNA levels and stability as well as promoter activity and use sequence analysis, knockdown, and overexpression to investigate the role of cardiac transcription factor(s) implicated in the response. Endogenous FGF-16 RNA levels were reduced >70% in 8-week-old rats treated with 15 mg DOX/kg for 6 h. This was modeled in neonatal rat cardiomyocyte cultures, where an equivalent decrease was also seen within 6 h of 1 μM DOX treatment. Six kilobases of mouse Fgf-16 upstream flanking and promoter DNA was also assessed for DOX responsiveness in transfected cardiomyocytes. A decrease in FGF-16 promoter activity was seen with only 747 base pairs containing the Fgf-16 TATA box that includes a putative and highly conserved binding site for the cardiac transcription factor Csx/Nkx2.5. There was also no effect of DOX on FGF-16 RNA stability, consistent with transcriptional control. Levels and binding of Csx/Nkx2.5 to the FGF-16 promoter were reduced with DOX treatment. Knockdown of Csx/Nkx2.5 specifically decreased endogenous FGF-16 RNA and protein levels, whereas Csx/Nkx2.5 overexpression stimulated levels, and increased resistance to the rapid DOX-induced depletion of FGF-16. These observations indicate that Fgf-16 expression is directly regulated by Csx/Nkx2.5 in neonatal cardiomyocytes, and a negative effect of DOX on Csx/Nkx2.5 and, thus, endogenous FGF-16 synthesis may contribute indirectly to its cardiotoxic effects. Targeting FGF-16 levels could, however, offer increased resistance to cardiac injury.

Gene Transfer in Isolated Adult Cardiomyocytes.

During the past few decades, gene delivery using recombinant virus has made tremendous progress. With a higher than 80 % transduction efficiency, even in non-dividing cells, viral transduction has become the method of choice for efficient gene transfer into cardiomyocytes. However, in vitro gene delivery is dependent on a robust cell isolation protocol, as prolonged cultivation is needed to initiate gene expression and target specific cellular processes. This chapter describes some of the important steps that need to be considered for successful in vitro gene transfer into adult cardiomyocytes. Included are detailed protocols for isolating cells, maintaining rod shaped cardiomyocytes in culture over several days, and employing adenovirus for gene transduction.

Sevoflurane postconditioning attenuates cardiomyocyte hypoxia/reoxygenation injury via restoring mitochondrial morphology.

BackgroundAnesthetic postconditioning is a cellular protective approach whereby exposure to a volatile anesthetic renders a tissue more resistant to subsequent ischemic/reperfusion event. Sevoflurane postconditioning (SPostC) has been shown to exert cardioprotection against ischemia/reperfusion injury, but the underlying mechanism is unclear. We hypothesized that SPostC protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury by maintaining/restoring mitochondrial morphological integrity, a critical determinant of cell fate.MethodsPrimary cultures of neonatal rat cardiomyocytes (NCMs) were subjected to H/R injury (3 h of hypoxia followed by 3 h reoxygenation). Intervention with SPostC (2.4% sevoflurane) was administered for 15 min upon the onset of reoxygenation. Cell viability, Lactate dehydrogenase (LDH) level, cell death, mitochondrial morphology, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were assessed after intervention. Mitochondrial fusion and fission regulating proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) were assessed by immunofluorescence staining and western blotting was performed to determine the level of protein expression.ResultsCardiomyocyte H/R injury resulted in significant increases in LDH release and cell death that were concomitant with reduced cell viability and reduced mitochondrial interconnectivity (mean area/perimeter ratio) and mitochondrial elongation, and with reduced mitochondrial membrane potential and increased mPTP opening. All the above changes were significantly attenuated by SPostC. Furthermore, H/R resulted in significant reductions in mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 and significant enhancement of fission proteins Drp1 and Fis1. SPostC significantly enhanced Mfn2 and Opa1 and reduced Drp1, without significant impact on Mfn1 and Fis1.ConclusionsSevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury (HRI) by restoring mitochondrial fusion/fission balance and morphology.

Stiff matrix induces switch to pure β-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular β-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive β-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-β-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and β-MyHC-protein isoforms. Yet, 35 and 75days of cultivation on laminin-coated glass switched 66 and 87% of all cardiomyocytes to exclusively express β-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only β-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of β-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure β-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

A Natural Model of Mouse Cardiac Myocyte Senescence.

Many cardiac aging studies are performed on mice first and then, due to difficulty in mouse cardiomyocyte culture, applied the rat neonatal cardiomyocytes to further determine the mechanisms in vitro. Now, the technological challenge of mouse cardiomyocyte culture has been overcome and there is an increasing need for the senescence models of mouse cardiomyocytes. In this study, we have demonstrated that the senescence of mouse cardiomyocytes occurred with the extended culture time as shown by the increased β-galactosidase staining, increased p53 expression, decreased telomere activity, shorted telomere length, increased production of ROS, increased cell apoptosis, and impaired mitochondrial ΔΨm. These senescent responses shared similar results in aged mouse heart tissues in vivo. In summary, we have established and characterized a novel senescence model of mouse cardiomyocytes induced by the extended culture time in vitro. The cell model could be useful for the increased cardiac aging studies worldwide.

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2a, MLC-2v, CHRM2, ADRB1, DES, EDRNB, Cx40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD, GATA-4, Cx43, SERCA2, BNP, Tbx5, EDNRA and ACTB. Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time. In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures.

N-3 long chain polyunsaturated fatty acids increase the expression of PPARγ-target genes and resistance of isolated heart and cultured cardiomyocytes to ischemic injury.

The goal of present investigation is to check the hypothesis that cardioprotective effect of polyunsaturated fatty acids (PUFAs) is mediated by influence on mRNA expression level of the FATP, IL-1ra and GJP43 through stimulation of PPARγ. Animals obtained n-3 PUFAs orally during 4 weeks (0.1ml/100g b.w. per day) or during prenatal period. In experiments, isolated perfused hearts were subjected to 20-min regional ischemia and 40min reperfusion. The hearts of newborn rats (2-3days old) were used for isolated cardiomyocytes culture preparing. Culture cells underwent 30min of anoxia followed by 60min of reoxygenation. Using rtPCR the level of FATP, IL-1ra, GJP43 and BCL2 mRNA in isolated cardiomyocyte and hearts was evaluated. The data obtained indicate that in heart tissues from pups with prenatal n-3 PUFAs application the level of LA and DHA acids were increased in 3.6-fold and 2-fold correspondingly comparing to control. In adult hearts the level of DHA was increased in 1.4-fold, and the level of EPA-in 6.9-fold. We observed the increase in mRNA level of PPARγ-target genes: FATP in 2.25 times, and IL-1ra in 8.4 times. At the same time the level of mRNA of antiapoptotic gene BCL2 was increased in 2.13 times and Connexin-43 gene in 2.2 times after n-3 PUFAs application. These effects were accompanied by significantly improved cardiac function, and increase of living cardiomyocytes number at modeling of ischemia-reperfusion. n-3 PUFAs application has cardioprotective effects and increases mRNA level of FATP, IL-1ra, GJP43, and BCL2 genes in culture of neonatal cardiomyocytes and in adult hearts.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.