Cultures Of Cardiomyocytes Research Articles

Biomaterials with Functionally Graded Stiffness

There are many different tissues in the body, each possessing different mechanical properties and varying amounts of stiffness that are needed for cells to function properly and survive. A challenge to recreating in vitro tissues is to accurately recapitulate the mechanical properties of the native tissue. The focus of this research was to produce a substrate with a functionally graded stiffness for use with in vitro culture of cardiomyocytes, the cells of the heart responsible for contraction.Young's modulus, a measure of stiffness of materials, is a key characteristic parameter for tissues. Currently, there is a lack of substrate systems that span the range of Young's modulus needed to represent both healthy and diseased myocardium. This is problematic when trying to model cardiovascular disease. For example, after myocardial infarction, there are regions of soft and stiff underlying tissue that impact the behavior of cardiomyocytes differently.The goal of this research was to create a substrate system with a stiffness gradient spanning from 5 to 50 kPa upon which the cardiomyocytes derived from stem cell could be cultured. This in vitro substrate system was designed to mimic the range of physiological environments in which cardiomyocytes exist in vivo. This substrate model is specifically being designed to aid in better understanding and reducing cardiovascular disease.The method investigated that showed the most promise was UV curing of polyacrylamide hydrogels. A gradient photomask was designed to allow different amounts of UV light to penetrate over the span of the substrate. This mask was then placed over the prepolymer gel during UV exposure. This method produced a stiffness gradient of 55 to 75 kPa over a 1 cm distance. Further work is needed to optimize the photomasks and gel reagents to produce the lower end of modulus representative of healthy myocardium.Support or Funding InformationSupported with funding from the Karen Thompson Medhi Professor at the University of Wisconsin‐Madison and the National Science Foundation through the University of Wisconsin‐Madison Materials Research Science and Engineering Center (DMR‐0520527) and Nanoscale Science and Engineering Center (DMR‐0425880). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin‐(1‐7) Prevents Thyroid Hormone‐induced Cardiomyocyte Hypertrophy by FOXO3 Signaling Pathway

It is well‐known that high levels of thyroid hormone promote cardiomyocyte hypertrophy, which in part is mediated by activation of Renin‐Angiotensin System (RAS). Recent study of our group demonstrated that the contralateral RAS axis (ACE2/Angiotensin‐(1–7)/Mas receptor) is also involved in this cardiac hypertrophy model and that transgenic rats overexpressing circulating levels of Angiotensin‐(1‐7) (Ang‐(1‐7)) present an improvement of cardiac function and attenuation of cardiac hypertrophy. However, the molecular mechanisms involved in these Ang‐(1‐7) actions have not been fully elucidated. The aim of this study was to investigate if the anti‐hypertrophic effect of Ang‐(1‐7) also occurs in isolated cardiomyocytes, as well as the involvement of the Foxo3 signaling pathway for these effects. All experiments were approved by the Ethics Committee on Animal Experimentation of ICB‐USP (n030/12/CEUA). Primary cardiomyocytes cultures obtained from neonatal rats were treated with triiodothyronine (T3) (15nM) and/or Angiotensin‐(1‐7) (Ang‐(1‐7)) (100nM). All data were analyzed by ANOVA followed by Tukey's post hoc test, and values of P<0.05 were considered statistically significant. The Ang‐(1‐7) treatment prevented the cardiomyocyte hypertrophy induced by T3, which was confirmed by reduction cell surface area, L‐[3,4,5‐3H] leucine incorporation and BNP gene expression. Combined treatment with the MAS receptor antagonist (A779, 100nM) impaired the anti‐hypertrophic effect of Ang‐(1‐7). Regarding molecular mechanisms, although Ang‐(1‐7) treatment did not impair the rapid activation of Akt promoted by T3, Ang‐(1‐7) treatment reduced the phosphorylation of Foxo3 (inactive form) and promoted the nuclear accumulation of Foxo3 in cardiomyocytes, indicating the activation of this protein. Collectively, these findings indicate an important cardioprotective effect of Ang‐(1‐7) in isolated cardiomycoytes treated with T3, occurring via MAS receptor and with possible participation of Foxo3.Support or Funding InformationFoundation for the Support of Research in the State of São Paulo (FAPESP) and National Council of Technological and Scientific Development (CNPq).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prostaglandin E2 Inhibits LPS‐Induced Monocyte Chemoattractant Protein 5 in Adult Cardiac Fibroblasts Through its EP4 Receptor via Reduced Akt and NF‐κB Signaling

Prostaglandin E2 (PGE2) signals through 4 G‐protein coupled receptor sub‐types (EP1–EP4) to elicit a variety of pathophysiological effects. We previously reported that mice lacking the EP4 receptor in cardiomyocytes (EP4 KO) develop heart failure with a phenotype of dilated cardiomyopathy. These mice also have increased levels of monocyte chemoattractant protein‐5 (MCP‐5), in their left ventricles. We recently published that overexpression of the EP4 receptor improves cardiac function in a model of myocardial infarction; associated with anti‐inflammatory effects. Moreover, conditioned media from EP4 KO cardiomyocyte cultures increased macrophage migration in vitro, which was attenuated with a MCP‐5 neutralizing antibody. In a variety of cell types, PGE2 antagonizes lipopolysaccharide (LPS)‐induced secretion of chemokines/cytokines. We therefore hypothesized that PGE2 inhibits LPS‐induced MCP‐5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. Our hypothesis was tested using 16–20‐week old mouse adult ventricular fibroblasts treated with LPS (10 μg/ml). Media and cells were harvested after 1, 2, 4, and 24 hrs of LPS treatment. MCP‐5 secretion was determined by ELISA. LPS treatment for 4 hrs stimulated MCP‐5 secretion 2.3‐fold compared to vehicle (p<0.05) and this stimulation persisted for at least 24 hrs (p < 0.001). Pre‐treatment with PGE2 or an EP4 agonist significantly reduced LPS‐stimulated MCP‐5 by 24 hours (p< 0.01). Both the LPS‐stimulation of MCP‐5 and its inhibition by PGE2‐EP4 takes 4 hours to be detectable, suggesting that PGE2 via EP4 signaling may regulate MCP‐5 transcription. To test this, we examined MCP‐5 mRNA levels after 1, 2, 4, and 24 hours post LPS stimulation. After 1 hour of LPS stimulation, MCP‐5 mRNA significantly increased compared to vehicle (4.49 ± 0.71 vs. 1.00, respectively; p < 0.005). Treatment with the EP4 agonist significantly blunted the response to LPS (2.09 ± 0.82 vs. 4.49 ± 0.71 in LPS‐treated cells; p < 0.005). PGE2 treatment also attenuated MCP‐5 mRNA after LPS stimulation, although this failed to reach statistical significance (2.68 ± 1.04 vs. 4.49 ± 0.71 in LPS‐treated cells; p=0.186). PGE2 and the EP4 agonist only reduced LPS‐stimulated MCP‐5 mRNA at the 1 hr time point, suggesting that EP4 receptor activation leads to downstream signaling that quickly downregulates MCP‐5. Western blot showed PGE2‐EP4 reduced LPS stimulated phosphorylation of Akt and IκBα, indicating reduced NF‐κB activation. Furthermore, blocking NF‐κB with Cardamonin (10 μM) reduced LPS‐stimulated MCP‐5 after 24 hours of treatment (1.71 ± 0.41 vs. 9.11 ± 3.09 in LPS treated cells; p<0.005). Our data also show that the inhibitory effects of PGE2 or the EP4 agonist are independent of cAMP, JNK, p‐44/42, or p38 pathways. Therapeutically, our study may help shed light on the mechanism(s) regulating chemokine production in cardiac fibroblasts.Support or Funding InformationPH: NIH grant [5P01HL028982] (sub‐project 2). TB: NIH T32 Detroit Cardiovascular Training Grant (5T32HL12082205)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anticancer therapy induced cardiotoxicity: Involvement of EPAC1 in cardioprotection and anticancer therapy

Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation and topoisomerase II inhibition, bioenergetics alterations leading to cardiomyocyte death. Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). In vivo, Dox-treated mice developed a DCM associated with Ca2 + homeostasis dysfunction (increase of Ca2 + waves and Ca2 + leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damage and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death.

Cardioprotective effects of a synthetic peptide targeting the extrinsic apoptotic pathway in a mouse model of ischemia-reperfusion

Apoptosis is a main contributor of myocardial reperfusion injury during acute infarction. In a previous study, we showed that reperfusion injury was mediated by the FAS-dependent apoptotic signal mobilizing the DAXX (death-associated protein) adaptor protein. To evaluate the cardioprotective effects of a synthetic peptide that uncouples FAS to the DAXX downstream pathway. The SPOT technology was used to design a synthetic peptide interfering with FAS:DAXX interaction. This peptide was coupled to the Tat cell penetrating peptide (Tat-DAXXp). Infarct size (TTC staining) and apoptosis (DNA fragmentation) were evaluated in the left ventricle of mice subjected to a surgical protocol of reversible coronary artery ligation and treated by the peptide at the onset of reperfusion. Cellular internalization of the peptide was measured using a fluorescent peptide both in primary cultures of cardiomyocytes or in left ventricles after surgery. Anti-apoptotic properties of the synthetic peptide were demonstrated in primary cardiomyocytes. In vivo, one bolus of Tat-DAXXp (1 mg/kg), injected intravenously 5 min before reperfusion in a murine myocardial ischemia-reperfusion model decreased infarct size by 48% after 24 hours of reperfusion. After a 30-min delayed administration, Tat-DAXXp was still able to protect against reperfusion-induced apoptosis and was completely degraded and eliminated within 24 hours thereby reducing risks of potential side effects. Importantly, post-infarction mortality was reduced by 67% by Tat-DAXXp treatment. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting ischemia-reperfusion injury.

Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

Fetal cardiomyocytes shift from glycolysis to oxidative phosphorylation around the time of birth. Myeloid ecotropic viral integration site 1 (MEIS1) is a transcription factor that promotes glycolysis in hematopoietic stem cells. We reasoned that MEIS1 could have a similar role in the developing heart. We hypothesized that suppression of MEIS1 expression in fetal sheep cardiomyocytes leads to a metabolic switch as found at birth. Expression of MEIS1 was assayed in left ventricular cardiac tissue and primary cultures of cardiomyocytes from fetal (100- and 135-d gestation, term = 145 d), neonatal, and adult sheep. Cultured cells were treated with short interfering RNA (siRNA) to suppress MEIS1. Oxygen consumption rate was assessed with the Seahorse metabolic flux analyzer, and mitochondrial activity was assessed by staining cells with MitoTracker Orange. Cardiomyocyte respiratory capacity increased with advancing age concurrently with decreased expression of MEIS1. MEIS1 suppression with siRNA increased maximal oxygen consumption in fetal cells but not in postnatal cells. Mitochondrial activity was increased and expression of glycolytic genes decreased when MEIS1 expression was suppressed. Thus, we conclude that MEIS1 is a key regulator of cardiomyocyte metabolism and that the normal down-regulation of MEIS1 with age underlies a gradual switch to oxidative metabolism.-Lindgren, I. M., Drake, R. R., Chattergoon, N. N., Thornburg, K. L. Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s−1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s−1 (linear relaxation rate), and 10.8 ± 1.3 s−1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

Active uptake of hydrophilic copper complex Cu(ii)-TETA in primary cultures of neonatal rat cardiomyocytes.

Myocardial ischemia leads to copper efflux from the heart. The ischemic tissue with a low copper content fails to take up copper from the circulation even under the conditions of serum copper elevation. Cardiac copper repletion thus requires other available forms of this element than those currently known to bind to copper transport proteins. The copper complex of triethylenetetramine (TETA) is a metabolite of TETA, which has the potential to increase cardiac copper content in vivo. In the present study, we synthesized Cu(ii)-TETA, analyzed its crystal structure, and demonstrated the role of this compound in facilitating copper accumulation in primary cultures of neonatal rat cardiomyocytes. The Cu(ii)-TETA compound formed a square pyramidal chloride salt [Cu(TETA)Cl]Cl structure, which dissociates from chloride in aqueous solution to yield the four-coordinate dication Cu(ii)-TETA. Cu(ii)-TETA was accumulated as an intact compound in cardiomyocytes. Analysis from time-dependent copper accumulation in cardiomyocytes defined a different dynamic process in copper uptake between Cu(ii)-TETA and CuCl2 exposure. An additive copper accumulation in cardiomyocytes was found when the cells were exposed to both CuCl2 and Cu(ii)-TETA. Gene silencing of copper transport 1 (CTR1) did not affect cross-membrane transportation of Cu(ii)-TETA, but inhibited copper cellular accumulation from CuCl2. Furthermore, the uptake of Cu(ii)-TETA by cardiomyocytes was ATP-dependent. It is thus concluded that the formation of Cu(ii)-TETA facilitates copper accumulation in cardiomyocytes through an active CTR1-independent transportation process.

Correction: Therapeutic effect of a novel Wnt pathway inhibitor on cardiac regeneration after myocardial infarction.

After myocardial infarction (MI), the heart is difficult to repair because of great loss of cardiomyoctyes and lack of cardiac regeneration. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable. In the present study, we identified if and how a novel porcupine inhibitor CGX1321 influenced MI and cardiac regeneration. Permanent ligation of left anterior descending (LAD) coronary artery was performed in mice to induce MI injury. Cardiac function was measured by echocardiography, infarct size was examined by TTC staining. Fibrosis was evaluated with Masson's trichrome staining and vimentin staining. As a result, CGX1321 administration blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX1321 improved cardiac function, reduced myocardial infarct size, and fibrosis of post-MI hearts. CGX1321 significantly increased newly formed cardiomyocytes in infarct border zone of post-MI hearts, evidenced by the increased EdU+ cardiomyocytes. Meanwhile, CGX1321 increased Ki67+ and phosphohistone H3 (PH3+) cardiomyocytes in culture, indicating enhanced cardiomyocyte proliferation. The mRNA microarray showed that CGX1321 up-regulated cell cycle regulating genes such as Ccnb1 and Ccne1 CGX1321 did not alter YAP protein phosphorylation and nuclear translocation in cardiomyocytes. In conclusion, porcupine inhibitor CGX1321 reduces MI injury by limiting fibrosis and promoting regeneration. It promotes cardiomyocyte proliferation by stimulating cell cycle regulating genes with a Hippo/YAP-independent pathway.

Microelectrode Array Screening of Different Cardiomyocyte Cultures Reveals Inherent Disparities in Cardiac Cell Electrophysiology

Numerous treatment strategies are available for atrial and ventricular arrhythmias, though the risk of side effects and incomplete effectiveness limit their clinical use. Cardiomyocyte culture models provide a high-throughput platform for screening novel compounds for anti-arrhythmic properties, but characterisation of their basic electrophysiological properties is lacking. This study aimed to directly compare the electrophysiological properties of three commonly used cardiac cultures – neonatal rat ventricular myocytes (NRVM), immortalised atrial HL-1 cells and human iPSC derived cardiomyocytes (hiPSC-CM). Cells were cultured on multi-electrode arrays (MEA) for 48–120 hrs. Extracellular field potentials (FP) were recorded and conduction velocity (CV) mapped in the presence or absence of 1 μM isoproterenol to assess β-adrenergic responsiveness. Spontaneous beating rate was significantly faster in hiPSC-CMs (NRVM vs HL-1 vs hiPSC-CM, bpm: 81.5 ± 6.9 vs 49.7 ± 5.6 vs 162.5 ± 38.0; p < 0.05), and FP morphology differed between all cell types. FP amplitude was greatest in NRVMs (mV: 2.95 ± 0.58 vs 0.26 ± 0.02 vs 0.40 ± 0.09; p < 0.05) and FP duration significantly shorter in HL-1 cells (ms: 40.2 ± 1.4 vs 184.0 ± 10.5 vs 146.9 ± 23.2; p < 0.0001). HL-1 cells also exhibited slower conduction velocities (NRVM vs HL-1, cm/s: 24.25 ± 1.91 vs 0.83 ± 0.03; p < 0.0001) and did not respond to isoproterenol, in contrast to NRVM and hiPSC-CM. This is the first study to quantify the inherent disparities in cardiac cell culture electrophysiology and the necessity to factor in these cellular properties when selecting culture models. These findings are an important step towards the standardisation of cultured cardiomyocyte MEA that will be essential to its application as a high-throughput platform for anti-arrhythmic drug development.

Inhibition of heat shock protein 70 blocks the development of cardiac hypertrophy by modulating the phosphorylation of histone deacetylase 2.

Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70). Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2. These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.

Effect of Gpx3 gene silencing by siRNA on apoptosis and autophagy in chicken cardiomyocytes.

Glutathione peroxidase 3 (Gpx3), as an important selenoprotein, is the most crucial antioxidant defense in cardiomyocytes. However, the role of Gpx3 in Se-deficient cardiomyocyte damage still less reported. Here, we developed Gpx3 silence cardiomyocytes culture model (small interfering RNA; siRNA) for research the crosstalk between autophagy and apoptosis. Quantitative real-time PCR and western blot analysis are performed to detect the expression of apoptosis and autophagy-related genes. MDC stain, flow cytometry, AO/EB stain, and electron microscope were performed to observe the changes of cell morphology. Our results reveal that Gpx3 suppression can significant increases in ROS (p < 0.05) levels, which further induced apoptosis through upregulated the expression of Caspase-3 in cardiomyocytes. Meanwhile, we also found that the whole process is accompanied by the occurrence of autophagy, which are promoted by inhibiting the mTOR, and increasing the expression of ATG-7, ATG-10, and ATG-12. Altogether, we conclude that the apoptotic and autophagic response machineries share antagonistic function in Gpx3 knockdown cardiomyocytes.

Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes.

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.

Comprehensive human stem cell differentiation in a 2D and 3D mode to cardiomyocytes for long-term cultivation and multiparametric monitoring on a multimodal microelectrode array setup

Human pluripotent stem cell derived cardiomyocytes are a promising cell source for research and clinical applications like investigation of cardiomyopathies and therefore, identification and testing of novel therapeutics as well as for cell based therapy approaches. However, actually it´s a challenge to generate matured adult cardiomyocyte-like phenotype in a reasonable time. Moreover, there is a lack of applicable non-invasive label-free monitoring techniques providing quantitative parameters for analysing the culture stability and maturation status. In this context, we established an efficient protocol based on a combined differentiation of hiPSC in 2D cultures followed by a forced reaggregation step that leads to highly enriched (>90% cardiomyocytes) cardiomyocyte clusters. Interestingly, 3D cultures revealed an accelerated maturation as well as phenotype switch from atrial to ventricular cardiomyocytes. More strikingly using combined impedimetric and electrophysiological monitoring the high functionality and long-term stability of 3D cardiomyocyte cultures, especially in comparison to 2D cultures could be demonstrated. Additionally, chronotropic as well as QT-prolongation causing reference compounds were used for validating the cardio specific and sensitive reaction over the monitored time range of more than 100 days. Thus, the approach of multiparametric bioelectronic monitoring offers capabilities for the long-term quantitative analysis of hiPS derived cardiomyocyte culture functionality and long-term stability. Moreover, the same multiparametric bioelectronic platform can be used in combination with validated long-term stable cardiomyocyte cultures for the quantitative detection of compound induced effects. This could pave the way for more predictive in vitro chronic/repeated dose cardiotoxicity testing assays.

Quantification of Cardiomyocyte Beating Frequency Using Fourier Transform Analysis

Pacemaker cardiomyocytes of the sinoatrial node (SAN) beat more rapidly than cells of the working myocardium. Beating in SAN cells responds to β-adrenergic and cholinergic signaling by speeding up or slowing, respectively. Beat rate has traditionally been assessed using voltage or calcium sensitive dyes, however these may not reflect the true rate of beating because they sequester calcium. Finally, in vitro differentiated cardiomyocytes sometimes briefly pause during imaging giving inaccurate beat rates. We have developed a MATLAB automation to calculate cardiac beat rates directly from video clips based on changes in pixel density at the edges of beating areas. These data are normalized to minimize the effects of secondary movement and are converted to frequency data using a fast Fourier transform (FFT). We find that this gives accurate beat rates even when there are brief pauses in beating. This technique can be used to rapidly assess beating of cardiomyocytes in organoid culture. This technique could also be combined with field scanning techniques to automatically and accurately assess beating within a complex cardiac organoid.

Regulation of C2C12 Differentiation and Control of the Beating Dynamics of Contractile Cells for a Muscle-Driven Biosyncretic Crawler by Electrical Stimulation

Biosyncretic robots have potential advantages associated with both living organisms and electromechanical systems. Skeletal muscle tissue is a candidate as bioactuators for biosyncretic robots because of its excellent contraction force and controllability. However, the low quality of myoblast (C2C12) differentiation into contractile myotubes and the lack of control research on biosyncretic robots are two of the main challenges in the development of biosyncretic robots. In this study, an approach with circularly distributed multiple electrodes (CEs) is proposed to improve C2C12 differentiation and to control the movement of a myotube-based biosyncretic crawling robot. To analyze the advantages of the proposed CEs, the electrical characteristics of CEs and a pair of traditional parallel stimulation electrodes (PEs) were simulated and compared with each other. Then, to determine the optimal electrical stimulation parameters and demonstrate the superiorities of the proposed CEs, electrical pulses with different parameters were used to stimulate two-dimensional and three-dimensional cells during culture with the proposed CEs and PEs. After this the control characteristics of the muscle tissue by the CEs were investigated from the relevance of pulse width-threshold voltage, voltage-contractility, frequency-contractility, and electric field direction-contractility by measuring the real-time responses of myotubes to different electrical stimulations. Moreover, to demonstrate the control of biosyncretic robots by the CEs, a biomimetic biosyncretic crawler actuated by myotubes was designed, fabricated, and controlled to move at different speeds by varying directions of electric field. This study not only provides a potential tool for the development and control of biosyncretic robots but is also informative for muscle tissue engineering and cardiomyocyte culture.

2-phenyl-1-(3-pyrrolidin-1-il-propyl)-1H-indole hydrochloride (SS-68): Antiarrhythmic and Cardioprotective Activity and Its Molecular Mechanisms of Action (Part II)

Introduction. In previous studies on different animal models, it was shown that compound N-(N-butylpyrrolidine)-2-phenylindole hydrochloride (SS-68) has a broad antiarrhythmic activity. The molecular mechanisms of the pharmacological action of SS-68 were chosen as the focus for this study.&#x0D; Materials and methods. The study of the molecular basis of the pharmacological action of SS-68 was based on 1) molecular docking with the determination of the affinity constant for κ1-opioid receptors; 2) recording the fluorescence of a culture of cardiomyocytes with the determination of the effect of SS-68 on ionic homeostasis; 3) determining the negative chronotropic action in vitro; 4) studying the effect of SS-68 on the transmembrane ion currents of isolated unidentified neurons of the large pond snail (Lymnaeastagnalis), orb snail (Planorbariuscorneus) and rat hippocampal neuron cultures.&#x0D; Results. 1) In experiments using molecular docking, the affinity of SS-68 for κ1-opioid receptors is significantly higher than that of butorphanol, but lower than that of (-)-U-50.488; 2) In spontaneously excited preparations of the right atrium, SS-68 causes an irreversible negative chronotropic effect. In experiments on atrial myocardium in rats, SS-68 is capable of demonstrating the ability to block M2 and M3-cholinergic receptors; 3) When studying the effects on cardiac myocyte ion currents, it was shown that SS-68 has moderate Na+, K+ and Ca2+ – blocking activity; 4) In the study of isolated neurons, it was shown that SS-68 influences the electrophysiology of neurocytes in a dose-dependent manner.&#x0D; Discussion. The study of the molecular basis of the action of SS-68 showed that this compound has a pleiotropic multitarget effect, which consists of, at least, the effect on Na+, Ca2+ and K+-homeostasis of cardiomyocytes and neurons, M2-, M3-cholinergic receptors, and κ1-opioid receptors.&#x0D; Conclusion. From the point of view of molecular pharmacology, SS-68 can be attributed to an antiarrhythmic drug with a mixed type of action.

Role of Mcl-1 in regulation of cell death in human induced pluripotent stem cell-derived cardiomyocytes in vitro

Targeting the anti-apoptotic protein Mcl-1 holds a promise to improve therapy of multiple types of Mcl-1 dependent cancers but raises concerns of on-target cardiotoxicity due to the presence and reported role of Mcl-1 in heart. Herein, we investigated the importance of Mcl-1 in the survival and contractile function of human pluripotent stem cell-derived cardiomyocytes in culture. Effective knockdown of Mcl-1 with siRNAs reproducibly resulted in early (measured at Day 3) marginal alterations in caspase 3/7 activity, LDH leakage, ATP content and cellular impedance. After 14 days of Mcl-1 knockdown, loss of mitochondrial membrane potential, deteriorating effects on mitochondrial ultrastructure, and alterations in beat rate and amplitude were revealed. Inhibition of Bcl-xL by siRNA-mediated knockdown or selective inhibitors did not cause any overt cellular responses except for a minimal increase in caspase 3/7 activity; however, loss of Mcl-1 concomitant with down-regulated Bcl-xL activated apoptosis and caused extensive cell death as reflected by an 80% loss in cell index, activation of caspase-3 with associated PARP cleavage, and a decrease in beat amplitude and mitochondrial membrane potential after 3 days of Mcl-1/Bcl-xL knockdown., Together, these findings suggest that Mcl-1 and Bcl-xL provide duplicate safeguard measures in maintaining structural and functional integrity of cardiomyocytes. Hence, BH3-mimetic drugs targeting Mcl-1 may be well tolerated in the presence of intact Bcl-xL.

Detection and quantitation of iron in ferritin, transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter 55Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox–active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000 Da) in the neonatal cardiomyocytes.

The effects of antibiotics on culture of human induced pluripotent stem cell-derived cardiomyocytes for recording electrophysiological activity by multi-electrode array

The effects of antibiotics on culture of human induced pluripotent stem cell-derived cardiomyocytes for recording electrophysiological activity by multi-electrode array