Rat Neonatal Myocytes Research Articles

Efficient dual crosslinking of protein–in–polysaccharide bioink for biofabrication of cardiac tissue constructs

Myocardial infarction (MI) is a lethal cardiac disease that causes most of the mortality across the world. MI is a consequence of plaque in the arterial walls of heart, which eventually result in occlusion and ischemia to the myocardial tissues due to inadequate nutrient and oxygen supply. As an efficient alternative to the existing treatment strategies for MI, 3D bioprinting has evolved as an advanced tissue fabrication technique where the cell–laden bioinks are printed layer–by–layer to create functional cardiac patches. In this study, a dual crosslinking strategy has been utilized towards 3D bioprinting of myocardial constructs by using a combination of alginate and fibrinogen. Herein, pre-crosslinking of the physically blended alginate–fibrinogen bioinks with CaCl2 enhanced the shape fidelity and printability of the printed structures. Physicochemical properties of the bioinks such as rheology, fibrin distribution, swelling ratio and degradation behaviour, were determined post-printing for only ionically crosslinked & dual crosslinked constructs and found to be ideal for bioprinting of cardiac constructs. Human ventricular cardiomyocytes (AC 16) exhibited a significant increase in cell proliferation on day 7 and 14 in AF–DMEM–20 mM CaCl2 bioink when compared to A–DMEM–20 mM CaCl2 (p < 0.05). Furthermore, myocardial patches containing neonatal ventricular rat myocytes (NVRM) showed >80 % viability and also expressed sarcomeric alpha actinin & connexin 43. These results indicate that the dual crosslinking strategy was cytocompatible and also possess the potential to be used for biofabrication of thick myocardial constructs for regenerative medicine applications.

Calcineurin Aβ gene knockdown inhibits transient outward potassium current ion channel remodeling in hypertrophic ventricular myocyte.

It has been shown that the activation of calcineurin is involved in regulating ion channel remodeling in hypertrophic cardiomyocytes. But the precise role of calcineurin in the regulation of transient outward potassium current (I to), an ion channel associated with fatal arrhythmia, remains controversial. This study aimed to examine the effects of calcineurin Aβ (CnAβ) gene knockdown on I to channel remodeling and action potential duration (APD) in the hypertrophic ventricular myocytes of neonatal rats. Results showed that phenylephrine stimulation caused hypertrophy of ventricular myocytes, upregulation of CnAβ protein expression, downregulation of Kv4.2 mRNA and protein expression, a decrease in I to current density, and prolongation of APD. CnAβ gene knockdown significantly inhibited the effects of phenylephrine stimulation. Our data indicate that CnAβ gene knockdown can inhibit I to channel remodeling and APD prolongation in hypertrophic neonatal rat ventricular myocytes. This finding suggests that calcineurin may be a potential target for the prevention of malignant ventricular arrhythmia in a hypertrophic heart.

A QSAR study that compares the ability of bisdioxopiperazine analogs of the doxorubicin cardioprotective agent dexrazoxane (ICRF-187) to protect myocytes with DNA topoisomerase II inhibition

The cardiotoxicity caused by doxorubicin and extravasation injury caused by anthracyclines is reduced by the clinically approved bisdioxopiperazine drug dexrazoxane. Dexrazoxane is a rings-closed analog of EDTA and is hydrolyzed in vivo to a form that strongly binds iron. Its protective effects were originally thought to be due to the ability of its metabolite to remove iron from the iron-doxorubicin complex, thereby preventing oxygen radical damage to cellular components. More recently it has been suggested that dexrazoxane may exert its protective effects by inhibiting topoisomerase IIβ in the heart and inducing a reduction in its protein levels through induction of proteasomal degradation. The ability of dexrazoxane, other bisdioxopiperazines, and mitindomide to protect against doxorubicin-induced damage was determined in primary neonatal rat myocytes. This QSAR study showed that the protection that a series of bisdioxopiperazine analogs of dexrazoxane and the bisimide mitindomide offered against doxorubicin-induced myocyte damage was highly correlated with the ability of these compounds to catalytically inhibit the decatenation activity of topoisomerase II. The structural features of the dexrazoxane analogs that contribute to the binding and inhibition of topoisomerase II have been identified. These results suggest that the inhibition of topoisomerase II in myocytes by dexrazoxane is central to its role in its activity as an anthracycline cardioprotective agent. Additionally, sequence identity analysis of the amino acids surrounding the dexrazoxane binding site showed extremely high identity, not only between both invertebrate topoisomerase II isoforms, but also with yeast topoisomerase II as well.

The Role of Topoisomerase IIβ in the Mechanisms of Action of the Doxorubicin Cardioprotective Agent Dexrazoxane.

Dexrazoxane is clinically used to reduce doxorubicin cardiotoxicity and anthracycline-induced extravasation injury. Dexrazoxane is a strong catalytic inhibitor of topoisomerase II. It can also undergo metabolism to form an iron-binding analog of EDTA. Dexrazoxane was originally thought to act by reducing iron-dependent doxorubicin-based oxidative stress. However, a competing hypothesis posits that dexrazoxane may be protective through its ability to inhibit and reduce topoisomerase IIβ protein levels in the heart. A primary neonatal rat myocyte model was used to study the mechanism by which dexrazoxane protects against doxorubicin-induced myocyte damage. This study characterized the kinetics of the rapid and nearly complete dexrazoxane-induced loss of topoisomerase IIβ protein from neonatal rat cardiac myocytes. Immunofluorescent staining of attached myocytes for topoisomerase IIβ revealed that most of the topoisomerase IIβ was localized to the nucleus, although it was also present in the cytoplasm. Dexrazoxane treatment resulted in an almost complete reduction of topoisomerase IIβ in the nucleus and a lesser reduction in the cytoplasm. The recovery of topoisomerase IIβ levels after a pulse topoisomerase IIβ inhibitory concentration of dexrazoxane occurred slowly, with partial recovery only occurring after 24h. The ability of dexrazoxane to reduce doxorubicin-induced damage to myocytes was greatest when topoisomerase IIβ levels were at their lowest.

P5725Identification of novel cardiomyogenic factors by transcriptome analysis of conditionally immortalized atrial myocytes

Abstract Background Cardiac development involves the properly timed expression of cardiomyogenic differentiation factors (CDFs). CDFs have mainly been discovered using animal models and, more recently, pluripotent stem cell-derived cardiomyocytes (PSC-CMCs). These models are, however, laborious, time-consuming and costly. Also, cardiomyogenic differentiation of CMCs is heterogeneous and yields phenotypically immature CMCs. Recently, our research group generated a monoclonal line of conditionally immortalized atrial myocytes, called iAM-1. After removal of the proliferation stimulus these cells spontaneously and synchronously differentiate into mature atrial myocytes, making them ideally suited for transcriptome analysis and the discovery of novels factors involved in cardiomyogenic differentiation. Methods Whole transcriptome analysis of iAM-1 cells was performed at 9 different time points during cardiomyogenic differentiation and subsequent dedifferentiation by RNA sequencing. Six genes upregulated during cardiomyogenic differentiation were selected for knockdown studies in differentiating iAM-1 cells. Each of these genes was targeted by a bicistronic lentiviral vector (LV) driving expression of a specific short hairpin RNA (shRNA) and of enhanced green fluorescent protein (eGFP). Knockdown effects during cardiomyogenic differentiation were studied by immunocytology. The LVs were also used in primary neonatal atrial and ventricular rat cardiomyocytes to study the role of the selected genes in cardiomyocyte homeostasis. Results Whole transcriptome analysis of differentiating iAM-1 cells identified the dynamic expression levels of ± 13.000 genes, including the expected profile for genes known to play a role in atrial myocyte differentiation, like Nkx2–5, Tbx3, Tbx5 and Nppa. Six genes with an unknown role in cardiomyocyte differentiation and homeostasis were selected based on significant upregulation during iAM-1 differentiation, substantial mRNA levels and selective expression in cardiac tissue. Inhibiting gene expression by lentiviral RNA interference resulted for Nkx2–5 as well as for 3 out of 6 target genes in disturbed iAM-1 differentiation, as evinced by loss of sarcomeric cross-striations. Similar effects were observed in shRNA-expressing (i.e. eGFP-positive) primary atrial and ventricular neonatal rat myocytes. Taken together, these results highlight the importance of these novel genes during cardiomyogenic differentiation and homeostasis in atrial as well as ventricular myocytes. Conclusions Transcriptome analysis of cardiomyogenic differentiation in conditionally immortalized atrial myocytes combined with genetic knockdown experiments led to the identification of several novel factors involved in the differentiation and homeostasis of atrial and ventricular myocytes. These results highlight the suitability of iAM-1 as model for fundamental research of cardiomyogenic differentiation. Acknowledgement/Funding ZonMW

Abstract 409: Regulation of Cardiomyocyte Maturation by an RNA Splicing Regulator Rbfox1

The maturation of stem cell derived cardiomyocytes (iCMs) is incomplete relative to the fully matured adult myocytes. Lack of maturation represents a major limitation to the applications of iCMs as heart disease models or heart failure therapies. Current attempts to promote iCMs maturation, such as prolonged culture, mechanical stretching and electronic pacing, are often based on empirical methods with poor reproducibility or little mechanistic basis. In order to better understand the molecular mechanisms driving cardiomyocyte maturation, we performed extensive transcriptome analyses in neonatal vs. adult hearts. In addition to metabolic and cell cycle regulatory pathways, Gene Ontology analysis revealed RNA splicing regulation is significantly enriched in the transcriptome reprogramming during postnatal maturation in heart. Specifically, we find a cardiomyocyte enriched RNA splicing factor Rbfox1 is dramatically induced in the perinatal maturating mouse hearts. Ectopic expression of Rbfox1 in neonatal cardiomyocytes markedly promotes the cellular and molecular features of adult cardiomyocyte, including contractility, calcium handling, sarcomere organization, morphology, electrophysiology and gene expression. Most remarkably, expression of RBFox1 in human iPSC derived cardiomyocytes promotes similar maturation process as observed in the neonatal rat myocytes. At mechanistic level, RBFox1 expression in the iCMs enhances transcriptome maturation as indicated by targeted RNA splicing in genes involved in muscle contraction, gene expression, RNA processing and sarcomere organization. In summary, we have uncovered a novel molecular path towards neonatal myocyte maturation in perinatal murine hearts by targeted modulation of cardiomyocyte transcriptome via RNA-splicing. This approach has potential to be employed as a molecular approach to promote human iCMs maturation.

Regulation of inward rectifier potassium current ionic channel remodeling by AT1 -Calcineurin-NFAT signaling pathway in stretch-induced hypertrophic atrial myocytes.

Previous studies have shown that the activation of angiotensin II receptor type I (AT1 ) is attributed to cardiac remodeling stimulated by increased heart load, and that it is followed by the activation of the calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway. Additionally, AT1 has been found to be a regulator of cardiocyte ionic channel remodeling, and calcineurin-NFAT signals participate in the regulation of cardiocyte ionic channel expression. A hypothesis therefore follows that stretch stimulation may regulate cardiocyte ionic channel remodeling by activating the AT1 -calcineurin-NFAT pathway. Here, we investigated the role of the AT1 -calcineurin-NFAT pathway in the remodeling of inward rectifier potassium (Ik1 ) channel, in addition to its role in changing action potential, in stretch-induced hypertrophic atrial myocytes of neonatal rats. Our results showed that increased stretch significantly led to atrial myocytes hypertrophy; it also increased the activity of calcineurin enzymatic activity, which was subsequently attenuated by telmisartan or cyclosporine-A. The level of NFAT3 protein in nuclear extracts, the mRNA and protein expression of Kir2.1 in whole cell extracts, and the density of Ik1 were noticeably increased in stretched samples. Stretch stimulation significantly shortened the action potential duration (APD) of repolarization at the 50% and 90% level. Telmisartan, cyclosporine-A, and 11R-VIVIT attenuated stretch-induced alterations in the levels of NFAT3 , mRNA and protein expression of Kir2.1, the density of Ik1 , and the APD. Our findings suggest that the AT1 -calcineurin-NFAT signaling pathway played an important role in regulating Ik1 channel remodeling and APD change in stretch-induced hypertrophic atrial myocytes of neonatal rats.

Abstract 1878: Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib

Abstract Bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib are anticancer drugs that target the proteasomal system. Bortezomib, carfilzomib, and ixazomib, which are approved for clinical use, have shown some cardiotoxicity. Carfilzomib and oprozomib are epoxyketones that form an irreversible bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. A primary neonatal rat myocyte model was used to study these cardiotoxic mechanisms. Using LDH release as a measure of damage in the myocyte model, after drug treatments of either 1, 4 and 72 hr all these drugs induced myocyte damage at low- to sub-micromolar concentrations. Oprozomib induced significantly less myocyte damage than any of the other drugs. Utilizing a fluorogenic substrate the kinetics of the slow-binding inhibition of the chymotrypsin-like proteasomal activity of human 20S proteasome were also determined in order to see if differences in the binding kinetics could explain the degree of myocyte damage. Oprozomib inhibited the 20S proteasome with a second-order binding “on” rate constant that was 60-fold slower than for ixazomib, the fastest binding drug. The first-order dissociation “off” rate constants for the three boronic acids were also determined. Delanzomib dissociated from its complex with the 20S proteasome with a half-time that was more than 20-fold slower than for ixazomib, the fastest dissociating drug. In conclusion, while all these drugs were potently toxic to myocytes at submicromolar concentrations, oprozomib was considerably less toxic than any of the other drugs. As a group the boronic acid drugs were not significantly more toxic than the epoxyketone drugs. The dissociation half-times of the boronic acid complexes were also not correlated with myocyte damage. The fact that oprozomib bound to the 20S proteasome more slowly than any of the other drugs may be the reason, in part, that it is less toxic. Support: CIHR; a Canada Research Chair in Drug Development. Citation Format: Brian B. Hasinoff. Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1878.

Phosphodiesterase‐5 inhibitors “Sildenafil and Ordonafil” suppress/reverse L‐NAME induced cardiac hypertrophy in isolated rabbit heart and in phenylephrine induced hypertrophy in isolated neonatal rat myocytes

Phosphodiesterase 5 inhibitors (PDE5Is) have been shown to suppress and reverse myocardial hypertrophy induced by pressure overload. Calcineurin, a protein phosphatase, has been suggested to function as an important hypertrophic factor in cardiac myocytes and its signaling regulates the cardiac hypertrophic response in coordination with mitogen‐activated protein kinases (MAPKs). The present study investigated the effects of two PDE5Is i.e. Sildenafil (SILD) ‘ Viagra’ and Ordonafil (ORD) on N‐nitro‐L‐arginine methyl ester (L‐NAME) induced cardiac hypertrophy and assessed their ability to attenuate the activity of calcineurin, p38 and protein synthesis to exert their effect on hypertrophied cardiac myocytes.MethodsL‐NAME (10 mg/kg/day) was used to induce cardiac hypertrophy in rabbits' hearts and left ventricular thicknesses (LVTs) were measured in L‐NAME and PDE5Is treated hearts. Then, in cultured neonatal rat ventricular myocytes, Western Blot technique was used to assess the ability of these PDE5Is (different doses) to attenuate the activation of calcineurin, p38 and protein synthesis in phenylphrine‐induced ventricular hypertrophy as indicated by the cell surface area of cultured neonatal rat cardiomyocytes.ResultsL‐NAME significantly increased LVT to 6.1± 0.18 mm vs. 4.6±0.13 mm in control rabbit hearts, (p&lt;0.05). SILD and ORD significantly regressed LVT to 4.8 ± 0.2 mm and 5.1 ± 0.1 mm, respectively (p&lt;0.05). Phenylphrine significantly increased neonatal rat ventricular myocyte cell surface area to 131% of control values (p&lt;0.05). This hypertrophic effect of phenylphrine was associated with significant increment in protein synthesis to 143+5% of control values, (p&lt;0.05). Both drugs (in a dose dependent, 0.001–1 mg/L) decreased cell surface area to 95% and 90% of control value respectively, and both decreased protein synthesis to 88% of control values. Phosphatase activity of calcineurin significantly decreased from 1.93±0.09 IU to 1.15±0.05 IU after one hour and to 1.2±0.06 IU after 24 hours incubation with ORD (0.1 mg/l) incubation, (p&lt;0.05). Whereas this activity significantly decreased after 24 hours (1.22±0.04 IU, (p&lt;0.05)), but not after one hour (1.84±0.05 IU) of SILD (0.1 mg/l) incubation. Protein synthesis and p38 activation were significantly attenuated by both drugs (0.1 mg/L).ConclusionsThese results suggest that both SILD and ORD have the ability to reverse L‐NAME induced cardiac hypertrophy in rabbits and suppress phenylphrine induce hypertrophic myocytes. The results indicate these effects are mediated through inhibiting protein synthesis and the attenuation of calcineurin and p38 activity signaling pathways in neonatal rat ventricular myocytes which is reported in this study for the first time.Support or Funding InformationDeanship of Scientific research ‐ Jordan University of Science &amp; TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of Oxidative Stress and Apoptosis in Electrically Stimulated Neonatal Rat Cardiomyocytes by Resveratrol and Underlying Mechanisms.

We explored the effects of resveratrol on oxidative stress in cardiomyocytes subjected to rapid electrical stimulation (RES) and also investigated the underlying mechanisms. Cultured ventricular myocytes of neonatal rat were subjected to RES at 4.0 Hz, with or without resveratrol, an NADPH oxidase inhibitor apocyanin (APO) or a Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor autocamtide-2-inhibitory peptide (AIP). Cell counts, to optimize resveratrol concentration, and angiotensin II content were evaluated. Reactive oxygen species (ROS), intracellular Ca in cardiomyocytes, and cardiomyocyte apoptosis were also assessed. Levels of methionine sulfoxide reductase A (MsrA), Nox, oxidative CaMKII (OX-CaMKII), and cleaved caspase-3 in cardiomyocytes were examined. Resveratrol treatment, as compared with APO and AIP, significantly decreased ROS levels, improved Ca amplitudes, and intracellular Ca transient decay rates, and inhibited cardiomyocyte apoptosis. Resveratrol also increased MsrA protein levels. In cardiomyocytes subjected to RES, after pretreatment with resveratrol or APO, protein levels of Nox4, Nox2, OX-CaMKII, and cleaved caspase-3 were decreased. In comparison, with AIP pretreatment, only Nox2, OX-CaMKII, and cleaved caspase-3 were decreased. However, in the presence of dimethyl sulfoxide, a competitive inhibitor of MsrA function, a decrease in cleaved caspase-3 did not occur. Resveratrol decreased ROS, partially through the inhibition of NADPH oxidase activity and upregulation of MsrA expression.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering.

Cardiac tissue engineering (CTE) is currently a prime focus of research because of an enormous clinical need. In the present work, a novel functional material, poly(3-hydroxyoctanoate), P(3HO), a medium chain-length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of neat P(3HO) patches, assessed using neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated into the patches. In addition, no deleterious effect was observed on adult cardiomyocyte contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO)-based multifunctional cardiac patches are promising constructs for efficient CTE. This work will have a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials. Copyright © 2016 John Wiley & Sons, Ltd.

The exon junction complex senses energetic stress and regulates contractility and cell architecture in cardiac myocytes.

The exon junction complex (EJC) is the main mechanism by which cells select specific mRNAs for translation into protein. We hypothesized that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes, with implications for the failing heart. In cultured rat neonatal myocytes, we examined the cellular distribution of two EJC components eukaryotic translation initiation factor 4A isoform 3 (eIF4A3) and mago nashi homologue (Mago) in response to metabolic stress. There was significant relocalization of eIF4A3 and Mago from the nucleus to cytoplasm following 18 h of hypoxia. Treating myocytes with 50 mM NaN3 for 4 h to mimic the metabolic stress induced by hypoxia also resulted in significant relocalization of eIF4A3 and Mago to the cytoplasm. To examine whether the effects of metabolic stress on the EJC proteins were dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1 μM dorsomorphin (DM) in combination with NaN3. DM augmented the translocation of Mago and eIF4A3 from the nucleus to the cytoplasm. Knockdown of eIF4A3 resulted in cessation of cell contractility 96 h post-treatment and a significant reduction in the number of intact sarcomeres. Cell area was significantly reduced by both hypoxia and eIF4A3 knockdown, whilst eIF4A3 knockdown also significantly reduced nuclear size. The reduction in nuclear size is unlikely to be related to apoptosis as it was reversed in combination with hypoxia. These data suggest for the first time that eIF4A3 and potentially other EJC members play an important role in the myocyte stress response, cell contractility and morphology.

Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10−7 to 2.10−5M) and verapamil (7 concentrations from 10−11 to 10−5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10−6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10−6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10−6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10−6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10−8M and prolonged the APD with EC50 of 2.5·10−8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10−7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.

Amelioration of Chronic Intermittent Hypobaric Hypoxia on DecreasedL-type Ca^(2+) Currents by Ischemia/Reperfusion in Cardiomyocytes ofDeveloping Rat

The aim of the present study was to observe the effect of chronic intermittent hypobaric hypoxia (CIHH) on L-type calcium channel current (I_(CaL)) in developing rat in order to investigate the ionic mechanism of the car-dioprotective effect of CIHH. Age- and body weight-matched postnatal male Sprague-Dawleyrats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). CIHH neonatal rats were experienced a CIHH treatment simulating 3000-m altitude (P_B = 525 mmHg, P_(O2) = 108.8 mmHg, 5 h/day) in a hypobaric chamber for 28 and 42 days, respectively. Whole-cell patch clamp technique was used to record I_(CaL) in ventricular myocytes of neonatal rats. The results showed: There were no differences of peak current, current-voltage (I-V) relationship curve, and dynamics of I_(CaL) between CON and CIHH cardiomyocytes under normal external solution (P ＞ 0.05). The peak I_(CaL) was decreased and I-V relationship curve was shifted upward during simulated ischemia/reperfusion (I/R) (P ＜ 0.05), but the change of I_(CaL) was much smaller in CIHH cardiomyocytes than in CON cardiomyocytes (P ＜ 0.05). The steady-state inactivation curve of I_(CaL) was shifted to the left during I/R in CON28 and CON42 cardiomyocytes (P ＜ 0.05), but not in CIHH28 and CIHH42 cardiomyocytes (P ＞ 0.05). In conclusion, CIHH has no effect on I_(CaL) in ventricular myocytes of developing rat under basic condition, but effectively antagonizes the inhibition of simulated I/R on I_(CaL), which might be one of the ionic mechanisms for CIHH cardiac protection against I/R injury in developing rats.

The Myocyte-Damaging Effects of the BCR-ABL1-Targeted Tyrosine Kinase Inhibitors Increase with Potency and Decrease with Specificity.

Five clinically approved BCR-ABL1-targeted tyrosine kinase inhibitors (bosutinib, dasatinib, imatinib, nilotinib, and ponatinib) used for treating chronic myelogenous leukemia have been studied in a neonatal rat myocyte model for their relative ability to induce myocyte damage. This was done in order to determine if kinase inhibitor-induced myocyte damage was a consequence of inhibiting ABL1 (on-target effects), or due to a lack of kinase selectivity (off-target effects) since previous studies have come up with conflicting conclusions about whether imatinib-induced cardiotoxicity results directly from inhibition of ABL1. The most specific and least potent inhibitors, imatinib and nilotinib, induced the least myocyte damage, while the least specific and most potent inhibitors, ponatinib and dasatinib, induced the most damage. Inhibitor-induced myocyte damage also correlated with clinically observed cardiovascular toxicity. Growth inhibition of the erythroleukemic K562 human cell line with a constitutively active BCR-ABL1 kinase was negatively correlated with inhibitor-induced myocyte damage, which suggests that inhibition of ABL1 causes myocyte damage. Myocyte damage was also negatively correlated with inhibitor dissociation binding constants and with inhibition of enzymatic ABL1 kinase activity. Myocyte damage was also positively correlated with two measures of inhibitor selectivity, which suggests that a lack of inhibitor selectivity is responsible for myocyte damage. In conclusion, myocyte damage, and thus the cardiovascular toxicity of the BCR-ABL1-targeted tyrosine kinase inhibitors, is due to direct inhibition of ABL1 and/or their lack of inhibitor selectivity.

Abstract 3060: Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib

Abstract Bortezomib and carfilzomib are anticancer drugs that target the proteasomal system. Case reports indicate that the use of these drugs can result in cardiotoxicity. These drugs may also be used in combination with doxorubicin, which is itself cardiotoxic. Thus, in order to determine the mechanism of this cardiotoxicity studies were undertaken using a neonatal rat myocyte primary cell model. Using LDH release as a measure of damage in the myocyte model we showed that even brief exposure (6 h) to submicromolar concentrations of bortezomib resulted in significant myocyte damage. Carfilzomib was slightly less toxic and displayed myocyte damage in the low micromolar concentration range. Utilizing a fluorogenic substrate both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the submicromolar concentration range. The inhibition kinetics were consistent with formation of an essentially irreversible covalent complex at the active site. Bortezomib irreversibly inhibited proteasomal activity about four-fold faster than carfilzomib. Thus, the reduced toxicity of carfilzomib may be due to its slower inhibition kinetics. A brief pre-exposure (6 h) of myocytes to low non-toxic nanomolar concentrations of bortezomib greatly increased doxorubicin-mediated damage. The doxorubicin cardioprotective agent dexrazoxane partially protected the myocytes from doxorubicin treatment and from bortezomib plus doxorubicin treatment under these conditions. However, at slightly toxic bortezomib concentrations dexrazoxane offered little protection against bortezomib plus doxorubicin treatment. Bortezomib, at least over short times, did not induce oxidative damage in myocytes as measured with a fluorogenic DCF assay. Likewise, a 6 h bortezomib treatment did not affect the mitochondrial membrane potential of myocytes as measured in a JC-1 ratiometric assay. In conclusion both bortezomib and carfilzomib are potently toxic to myocytes at submicromolar concentrations, likely through their ability to irreversibly inhibit myocyte proteasomal activity. Support: CIHR; a Canada Research Chair in Drug Development. Citation Format: Brian B. Hasinoff, Xing Wu, Daywin Patel. Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3060.

Molecular Mechanisms of the Cardiotoxicity of the Proteasomal-Targeted Drugs Bortezomib and Carfilzomib.

Bortezomib and carfilzomib are anticancer drugs that target the proteasome. However, these agents have been shown to exhibit some specific cardiac toxicities by as yet unknown mechanisms. Bortezomib and carfilzomib are also being used clinically in combination with doxorubicin, which is also cardiotoxic. A primary neonatal rat myocyte model was used to study these cardiotoxic mechanisms. Exposure to submicromolar concentrations of bortezomib and carfilzomib resulted in significant myocyte damage and induced apoptosis. Both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar concentration range and exhibited time-dependent inhibition kinetics. The high sensitivity of myocytes, which were determined to contain high specific levels of chymotrypsin-like proteasomal activity, to the damaging effects of bortezomib and carfilzomib was likely due to the inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. A brief preexposure of myocytes to non-toxic nanomolar concentrations of bortezomib or carfilzomib greatly increased doxorubicin-mediated damage, which suggests that the combination of doxorubicin with either bortezomib or carfilzomib may produce more than additive cardiotoxicity. The doxorubicin cardioprotective agent dexrazoxane partially protected myocytes from doxorubicin plus bortezomib or carfilzomib treatment, in spite of the fact that bortezomib and carfilzomib inhibited the dexrazoxane-induced decreases in topoisomerase IIβ protein levels in myocytes. These latter results suggest that the doxorubicin cardioprotective effects of dexrazoxane and the doxorubicin-mediated cardiotoxicity were not exclusively due to targeting of topoisomerase IIβ.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure.

The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase-1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea-pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1Hz, or electrically paced myocytes at 6.8V (1Hz) for 48h. The MLP ratio decreased by 50% (P<0.05, n=4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10μm blebbistatin resulted in an ∼3-fold increase in the MLP ratio (n=8, P<0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO-1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea-pig model of angiotensin II infusion (400ngkg(-1) min(-1) ) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n=4, P<0.01) during compensated hypertrophy but decreased significantly during heart failure (P<0.001, n=4). The MLP ratio correlated significantly with the E/A ratio (r=0.71, P<0.01, n=12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signalling.

The Exon Junction Complex proteins are non‐AMP kinase dependent sensors of energetic stress and regulate contractility and sarcomere structure in cardiac myocytes

The heart is known to undergo significant alterations in gene expression during stress and sometimes this adaptation can lead to heart failure. The mechanism by which cells select specific mRNAs for translation is not fully understood, but the Exon Junction Complex (EJC) plays a major role in this process in eukaryotes. The EJC selects the mRNA isoforms to be exported to the cytoplasm and determines whether they are then translated into protein. In this study we hypothesised that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes. Using cultured rat neonatal myocytes and subcellular fractionation we examined the cellular distribution of two major components of the EJC in response to metabolic stress. In cardiac myocytes we found that there was a significant relocalisation of eIF4A3 and mago from the nucleus to cytoplasm following 18 hours of hypoxia. To examine the effect of metabolic stress on the EJC in more detail we treated cardiac myocytes with 50mM NaN3 for 4 hours to mimic the metabolic stress induced by hypoxia. Following NaN3 treatment there was a significant relocalisation of eIF4A3 and mago to the cytoplasm and this was associated with the formation of cytoplasmic stress granules. However, immunocytochemical staining with the stress granule marker PABP1 showed that cytoplasmic mago did not colocalise with stress granules. To examine whether the effects of metabolic stress on the EJC proteins was dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1μM dorsomorphin in combination with NaN3. Dorsomorphin augmented the translocation of mago and eIF4A3 from the nucleus to the cytoplasm. This indicates that the EJC proteins respond to metabolic stress independently of AMPK activity. Moreover, the inhibition of AMPK was associated with increased metabolic cellular stress resulting in the augmented subcellular translocation of EJC proteins. Constructs of eIF4A3 tagged with green fluorescence protein on the N terminus resulted in a predominantly cytosolic protein distribution that did not relocalise in response to NaN3 treatment. This suggests that the N‐terminus of eIF4A3 is necessary for subcellular localisation. Finally, to determine the function of eIF4A3 in cardiac myocytes we knocked down the protein with siRNA. Protein knockdown of eIF4A3 resulted in cessation of cell contractility 96 hours post treatment. There was also a significant reduction in the number of intact sarcomeres suggesting that the contractile filaments were degrading in response to reduced eIF4A3. Taken together, these data suggest that eIF4A3 and other EJC members play an important role in the myocyte stress response, cell contractility and morphology.Support or Funding InformationThis work was supported by the British Heart Foundation project grant PG/10/64/28520 and PG/10/97/28666

Dynamics of PKA phosphorylation and gain of function in cardiac pacemaker cells: a computational model analysis.

Cardiac pacemaker cell function is regulated by a coupled-clock system that integrates molecular cues on the cell-membrane surface (i.e., membrane clock) and on the sarcoplasmic reticulum (SR) (i.e., Ca(2+) clock). A recent study has shown that cotransfection of spontaneous beating cells (HEK293 cells and neonatal rat myocytes) with R524Q-mutant human hyperpolarization-activated cyclic nucleotide-gated molecules (the dominant component of funny channels) increases the funny channel's sensitivity to cAMP and leads to a decrease in spontaneous action potential (AP) cycle length (i.e., tachycardia). We hypothesize that in rabbit pacemaker cells, the same behavior is expected, and because of the coupled-clock system, the resultant steady-state decrease in AP cycle length will embody contributions from both clocks: the initial decrease in the spontaneous AP beating interval, arising from increased sensitivity of the f-channel to cAMP, will be accompanied by an increase in the adenylyl cyclase (AC)-cAMP-PKA-dependent phosphorylation activity, which will further decrease this interval. To test our hypothesis, we used the recently developed Yaniv-Lakatta pacemaker cell numerical model. This model predicts the cAMP signaling dynamics, as well as the kinetics and magnitude of protein phosphorylation in both normal and mutant pacemaker cells. We found that R524Q-mutant pacemaker cells have a shorter AP firing rate than that of wild-type cells and that gain in pacemaker function is the net effect of the R514Q mutation on the functioning of the coupled-clock system. Specifically, our results directly support the hypothesis that changes in Ca(2+)-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells.