Preservation of cardiomyocytes from the adult heart

    Preservation of cardiomyocytes from the adult heart Najah Abi-Gerges, Amy Pointon, Georgia F. Pullen, Michael J. Morton, Karen L. Oldman, Duncan Armstrong, Jean-Pierre Valentin, Christopher E. Pollard PII: DOI: Reference:

S0022-2828(13)00267-8 doi: 10.1016/j.yjmcc.2013.09.004 YJMCC 7633

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

1 February 2013 8 August 2013 5 September 2013

Please cite this article as: Abi-Gerges Najah, Pointon Amy, Pullen Georgia F., Morton Michael J., Oldman Karen L., Armstrong Duncan, Valentin Jean-Pierre, Pollard Christopher E., Preservation of cardiomyocytes from the adult heart, Journal of Molecular and Cellular Cardiology (2013), doi: 10.1016/j.yjmcc.2013.09.004

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ACCEPTED MANUSCRIPT Title: Preservation of cardiomyocytes from the adult heart

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Author’s surname and short title: Abi-Gerges et al, Myosin II ATPase and cardiomyocyte preservation

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Najah Abi-Gerges, PhD a,*, Amy Pointon, PhD a, Georgia F. Pullen, BSc a, Michael J. Morton, PhD b, Karen L. Oldman, PhD b, Duncan Armstrong, PhD a, Jean-Pierre Valentin, PhD a, Christopher E. Pollard, PhD a

Affiliations: a

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*Corresponding author: Dr Najah Abi-Gerges AstraZeneca, 23S37-69, Mereside Alderley Park, Macclesfield Cheshire, SK10 4TG, UK Tel: +44 1625 230336 Fax: +44 1625 515783

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Discovery Sciences, Innovative Medicines, AstraZeneca R&D, Macclesfield, UK, SK10 4TG

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Translational Safety, Drug Safety & Metabolism, Innovative Medicines, AstraZeneca R&D, Macclesfield, UK, SK10 4TG

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Email: [email protected]

Word count for main text (without Figure captions and References): 6667 Word count (without References): 7522 Total word count: 8798

ACCEPTED MANUSCRIPT ABSTRACT

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Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and Nbenzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes. This study introduces the concept that the inhibition of myosin II ATPase safeguards the structure and function of fully differentiated adult cardiomyocytes. The fact that these preserved cardiomyocytes can be used for numerous days after preparation makes them a robust and versatile tool in cardiac research and allows the investigation of long-term exposure to novel drugs on cardiomyocyte function.

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Keywords: Adult cardiomyocyte, myosin II ATPase, preservation, long-term exposure, novel drugs.

1. Introduction

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Abbreviations: APD90, action potential duration at 90% repolarization; BDM, 2,3-butanedione monoxime; BTS, N-benzyl-p-toluene sulphonamide; Cav1.2, calcium channel produced from the CACNA1C gene; cTnI, cardiac troponin I; C-E curve, concentration-effect curve; Cx43, connexin-43; FDACM, fully differentiated adult cardiomyocyte; hERG, human ether-a-go-go related gene (KV11.1); hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes; IC50, molar concentration producing 50% inhibition; MHC, myosin heavy chain; RT, room temperature; Sarc. short., sarcomere shortening; SR, sarcoplasmic reticulum; TKI, tyrosine kinase inhibitor.

Viable adult cardiomyocytes were first isolated over 36 years ago [1]. Since then, they have been widely used in laboratories to conduct cardiac research (numerous publications per year). For this purpose, scientists have been thoroughly investigating cardiomyocytes from different species using a wide range of biochemical, physiological, pharmacological and morphological approaches. A single adult heart yields millions of cells, but these cardiomyocytes do not survive for long after isolation [1,2]. To overcome this problem, isolated adult cardiomyocytes maintained in culture have been used as a model of the adult myocardium [3]. Consequently, this leads to remodeled cell models that do not accurately reflect the physiology of the fully differentiated adult cardiomyocyte (FDACM). Therefore, enabling the preservation of FDACMs offers a robust and versatile tool in cardiac research. The aim of the current study was to investigate whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve FDACMs [4,5]. We based our investigation on recent studies. The major three characteristics of these studies were the following: (i) a low-temperature range is required to preserve cultured rat neonatal cardiomyocytes [6], rat and guinea pig cardiac slices [7] and rat whole heart [8], (ii) mechanical uncoupling agents, like cytochalasin D [9], 2,3-butanedione monoxime (BDM) [10] and blebbistatin [11,12,13], were found to prolong the viability of adult cardiomyocytes during culture periods of 3-4 days [14,15,16] and (iii) a

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ACCEPTED MANUSCRIPT cardioplegic solution supplemented with BDM allowed cardiac slice characteristics to be

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preserved for up to 28 hours after preparation from guinea-pig or rat ventricle [17]. Consequently, we speculated that inhibition of myosin II ATPase might preserve FDACMs that were kept in standard myocyte Tyrode solution at a refrigerated temperature. Such preservation would be of particular interest to (i) impact our understanding about the intricate role of myosin II ATPase in cardiomyocytes, (ii) allow FDACMs to be used for numerous days after preparation making them a robust and versatile tool in cardiac research and (iii) enable the investigation of long-term exposure to drugs on cardiomyocyte function.

2. Material and methods

An expanded Methods section is available in the Supplementary Data.

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2.1. Isolation of FDACMs, epifluorescence recordings and contractility measurements

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In brief, FDACMs were isolated from canine hearts [18]. Action potentials (APs) (using a di-4-ANEPPS set-up for epifluorescence recordings) and sarcomere shortening (using a video-based cell geometry system) were recorded from cardiomyocytes at physiological temperature as previously described [18,19]. 2.2. Analysis of cell area, length and width of FDACMs

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In order to segment and determine the primary shape of the FDACM objects, we used a canny edge detection function to enable the outline of the FDACM whilst using a minimal smoothing factor (sigma) to reduce edge artefact and applying a threshold aligned to the edge slope of 10 (from a range of 0-100). Background objects were removed based on size and FDACM boundaries were cleaned up using binary morphology operators. The area of each FDACM was calculated from a calibrated image at the operating objective lens magnification (x65), where the number of pixels included in each object defined the area occupied by individual myocytes. The length and width of each FDACM was not determined at the time of original analysis but can be determined on the same images using the same program with additional mathematics to determine relative proportions. Either maximum and minimum feret measures (i.e., orientation independent calliper measures) or middle axis and minor axis ellipsoid measure (i.e., FDACMs defined by an enclosing quadric surface) would enable relative lengths and widths to be determined. 2.3. Analysis of the transverse tubule (T-tubule) network and the hERG channel To visualize the surface membrane and T-tubular network, FDACMs were incubated with 10 µM di-4ANEPPS (Sigma) and placed directly onto glass coverslips and allowed to settle. Measurements begun approximately 2 mins after adding the indicator and continued for 15 mins at RT. Imaging was performed with a Bio-Rad Radiance 2000 laser scanning confocal microscope, using the Nikon 40x oil S Fluor objective. Due to the varied size of cardiomyocytes together with an only partial attachment, a uniform distribution could not be achieved. To visualize the distribution of the hERG channels, FDACMs were fixed in 3.7% formaldehyde phosphate-buffered saline (PBS; pH 7.4) for 20 mins. Subsequently, FDACMs were permeabilized with 0.1% Triton X-100 (Sigma) in PBS (pH 7.4) for 10 mins and blocked in 10% donkey serum in PBS for 2 hrs. Samples were incubated with primary antibody to the hERG channel protein (1:10, Santa Cruz) in blocking solution overnight at ~ 4 C. FDACMs were washed with 0.1% Triton X-100 in PBS four times. Primary antibody was detected with donkey anti-rat immunoglobulin G conjugated to Alexa Flour 488 3

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(Life Technologies)). Subsequently, FDACMs were cytospun (200 rpm for 7 mins) onto glass slides and mounted using vectashield mounting medium with 4'-6-Diamidino-2-phenylindole (DAPI, Vector Laboratories). To access the degree of non-specific binding, primary antibodies were pre-incubated with their antigenic peptide. Additionally species specificity of secondary antibodies was confirmed by incubating cells probed with one primary antibody with a secondary antibody raised against another species. Secondary alone controls were also used to ensure the signal was specific. Fluorescent images were captured on a Zeiss imager Z1 microscope fitted with an AxioCam MRm version 3, using the 40x objective.

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To analyse either the T-tubule or hERG channel data, an application of a dynamic segmentation using adaptive thresholding with a lowpass filter size 7 and offset of either -12 (for T-tubule) or -1 (for hERG channel) was utilized to ensure fluorescence levels were determined above localized background. The FDACM cell boundary was determined (isohypses), so that area, length (feret maximum) and width (feret minimum) could be measured. The cell boundary or perimeter was removed to enable remaining pixel area of the T-tubules or the hERG channel to be measured. Finally, the ratio of the T-tubules or the hERG channel was calculated relative to cell area.

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2.4. Cell counting

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With the use of 10x objective of the Leica DM1RB inverted microscope (Leica Microsystems), five fields within a set of FDACMs taken from 2 hearts (inter-dog viability) were counted using a fuchs rosenthal haemocytometer and averaged. FDACMs which were stored in a glass vial containing myocyte Tyrode solution either in the absence (kept at ~ 37 C, RT or at ~ 4 C) or the presence of BTS at 30 µM were counted at day 1, 2, 3, 4, 8, 9, 10, 11, 12, 15, 16 and 17 time points. The 0 time point was counted 3 hours after completing the isolation of FDACMs. Rod-shaped morphology was the criteria for identifying viable FDACMs, whereas the round-shaped appearance was taken as the criteria for nonviable FDACMs. Viability was defined as a ratio of rod-shaped and total FDACMs. 2.5. Measurements of cellular ATP content, LDH and protein content At the appropriate time point FDACMs were lyzed with ATP somatic cell releasing agent (Sigma; FLSAR) and used to determine cellular ATP content, LDH release and protein concentration. Total protein concentrations were assessed in FDACM lysis using BCA protein assay kit (Pierce; 23225) as per the manufacturer’s instructions. Data was extrapolated from the standard curve to obtain mg protein/ml sample. Cellular ATP concentrations were assessed in FDACM lysis using ATP bioluminescent assay kit (Sigma; FLAA) as per the manufacturer’s instructions. Data was normalized for protein content and represents the mean ATP measurements of triplicate samples on separate plates. LDH was determined in both FDACMs and tyrodes buffer. Lactate dehydrogenase (LDH) concentrations were assessed by using the Cytotoxicity Detection Kit (Roche) as per the manufacturer’s instructions. Data represents the mean LDH measurements of triplicate samples on separate plates. 2.6. RNA isolation At the appropriate time point FDACMs were lysed with RLT buffer (Qiagen) and stored at - 80 ºC until processing. To avoid possible genomic DNA contamination, lysates were subjected to DNase I (Life Technologies) treatment for 15 mins at RT. Total RNA was extracted using an RNeasy Mini Kit (Qiagen). RNA quality was confirmed using RNA6000 nanochips on an Agilent 2100 Bioanalyzer, a RIN score of ≥ 8 was considered acceptable. A NanoDrop spectrophotometer (NanoDrop Technologies) was used to quantify RNA samples.

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2.7. Transcription and quantitative real time PCR (qRT-PCR)

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For qRT-PCR primer pairs were designed to cross exon-intron boundaries to eliminate the detection of any contaminating genomic DNA using primer blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) (Supplementary Table 1). RNA samples were diluted to 300 ng and reverse transcribed using the SuperScript® III first-strand synthesis supermix (Life technologies) containing 50 ng/μl randox hexamers. qRT-PCR was performed using 1 μl of sample from the reverse transcription reaction and syber green (Life Technologies) to monitor amplification on an Applied Biosystens 7900HT under standard cycling conditions. GAPDH was used as an endogenous control. Relative quantification of gene expression was performed using the ΔΔCt relative quantitation method. The mean Ct value was calculated for each gene and normalized to the endogenous control and expressed as a fold change. 2.8. Solutions, drugs and statistics

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3. Results

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Solutions, drugs and statistics are detailed in an expanded Methods section that is provided in the Supplementary Data. Blebbistatin and N-benzyl-p-toluene sulphonamide (BTS) were purchased from Sigma-Aldrich. All other compounds were obtained either from Sigma-Aldrich or the AstraZeneca compound collection. Compounds were initially formulated in DMSO as a 1000x stock solution. All statistical tests were performed at the 5% level (reported as statistically significant if the P-value was less than 0.05) with the exclusion of gene expression data for which the statistical test was performed at the 1% level.

3.1. Myosin ATPase II inhibitors preserve FDACMs Following isolation, FDACMs were left in a glass beaker containing physiological standard myocyte Tyrode solution for 2 - 3 hours at room temperature (RT). Subsequently, cells were placed into glass vials containing myocyte Tyrode solution or N-benzyl-p-toluene sulphonamide (BTS) (concentration range tested 0.1 to 100 M), a selective inhibitor of myosin ATPase II [20], and were stored at RT, ~ 37 C or ~ 4 C in a refrigerator. Compared to those stored at either RT or ~ 37 C, we found that FDACMs which were stored in a glass vial containing standard myocyte Tyrode solution at ~ 4 C maintained a better rate of survival (Fig. 1A) [6]. Additionally, we found that FDACMs that were stored at a refrigerated temperature maintained a better rate of survival in the presence of BTS at an optimal concentration of 30 µM (Fig.1A). As it offered a better rate of survival, this concentration was used from this point as the relevant concentration to assess preservation of FDACMs in this study. Specifically, our data show that the addition of 30 µM BTS (n = 20 dogs) to the FDACMs stored at ~ 4 C preserved the rod-shaped morphology of most FDACMs for 2 weeks with a survival rate of ~ 70% (Fig. 1A). This was not the case after BTS or blebbistatin was added to FDACMs stored at RT or ~ 37 C (Fig. 1A). To further assess the viability of preserved FDACMs, the cellular ATP content and release of LDH were monitored over time as markers for cell health and membrane integrity, respectively. Prior to all experiments, BTS-preserved FDACMs were washed at the start of each experimental day as follows: (i) aliquots of BTS-preserved FDACMs were taken out of the refrigerator and allowed to reach RT for at least 1 hr, (ii) supernatant was discarded, preserved FDACMs were rinsed with normal myocyte Tyrode solution and then allowed to settle for ∼5 min. This step was repeated twice to ensure a good washout was achieved.

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Compared to FDACMs kept at either RT or ~ 4 C, we found that cellular ATP content or LDH activity was not modulated dramatically in BTS-preserved FDACMs for 2 weeks (Fig. 1B-C). Finally, cell area, length and width were also monitored over time as a marker for cardiomyocyte growth. Compared to those freshly isolated, FDACMs stored at ~ 4 C had not displayed a cellular growth in response to the addition of 30 µM BTS (Fig. 1D-F) for the 2-weeks period. 3.2. Relative gene and protein expression levels of BTS-preserved FDACMs are comparable to those in FDACMs

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Before confirming that BTS-preserved FDACMs are optimal for functional investigations, we assessed that the BTS-treatment truly maintains preservation of FDACMs in many important aspects of structural, molecular and signal transduction characteristics that would make them truly useful as a long term model in vitro system. We first performed qRT-PCR analysis against FDACMs and BTS-preserved FDACMs. Comparison of gene expression related to gene categories, namely, apoptosis (2 genes: CASP3 and TP53), cardiomyocyte integrity (9 genes: ACTN1, MYH10, MYH6, MYH7, MYH9, TPM1, TPM2, TPM3 and TPM4), cell signaling (14 genes: AKT1, AKT2, ATP2A2, CAMK2A, CKM, GATA4, GJA1, MAPK1, PDE1A, PLCB4, PLN, PRKACA, PRKCA and PRKCB), ion channels (7 genes: CACNA1C, KCNA4, KCNH2, KCNN4, KCNQ1, SCN5A and SLC8A1), metabolic enzymes (6 genes: ACADM, FABP3, FBP1, HK1, PKM and PPARA), seven-transmembrane-domain receptors (13 genes: ADORA1, ADORA3, AGTR1, BDKRB1, BDKRB2, CHRM2, EDNRA, EDNRB, HTR1A, HTR2A, HTR4, INSR and RYR2) and signal transduction (4 genes: ADRB1, ADRB2, TNNI3 and TNNT2) is shown as a heat map in Fig. 2. The levels of expression of many cardiac-specific genes at all stages of preservation were close to those in the FDACMs. Although the levels of expression of the adrenoceptor beta 2 (ADRB2) and shaker-related voltage-gated potassium channel (KCNA4) at day 14 of preservation were found to be significantly (P<0.01 versus difference from FDACM) increased by 1.30 and 1.86 fold respectively, these changes were still close to the level of expression in FDACMs. These data suggest that the loss of viability seen with BTS-preserved FDACMs after 10 days (Fig. 1A) was not due to instability of the expression of these genes over time in BTS-preserved FDACMs. Next, we used immunofluorescence to confirm that the protein expression of some of these genes was not affected by the BTS treatment. Similarly to FDACMs, immunostainings showing BTS-preserved FDACMs retained the expression of classical cardiomyocyte markers: -actinin protein that plays a key role in the formation and maintenance of Z-lines, mitochondria that are responsible for energy production, hERG channel protein that forms the major portion of the rapid repolarizing potassium channel (see also Fig. 5G-I), Cav1.2 channel protein, an - subunit of L-type voltage-dependent calcium channel, cardiac troponin I, a key regulatory protein in cardiac muscle contraction and relaxation, myosin heavy chain, a cardiac-specific gene, sarcoplasmic reticulum (SR) that functions as a storage and release area for calcium, connexin-43 that is the constitutive protein for the formation of cardiac gap junctions and sodium/calcium exchanger, an antiporter membrane protein that removes calcium from cells. Next, we used fluorescence confocal microscopy to confirm that the highly organized T-tubule network that is essential for rapid electrical stimulation, initiation and synchronous triggering of SR calcium release was not affected by the BTS treatment. Imaging of myocytes stained with di-4-ANEPPS demonstrated no loss of T-tubule staining in BTS-preserved FDACMs compared to the regular array of T-tubules seen in FDACMs (Fig. 3A-D). Moreover, Fig. 3E shows that the T-index for di-4-ANEPPS staining was not significantly changed in BTS-preserved FDACMs compared to control FDACMs. Overall, these data strongly suggest that BTSpreserved FDACMs remain stable and structurally intact. 3.3. BTS-preserved FDACMs are optimal for functional investigations Because blebbistatin was shown to inhibit sarcomere shortening of single rat ventricular myocytes [21], we next asked whether BTS acts as a mechanical uncoupling agent in canine FDACMs. BTS was found to significantly inhibit FDACM contractility with an IC50 (concentration producing 50% inhibition of the 6

ACCEPTED MANUSCRIPT contractility) value of 19 µM (Fig. 4A). The negative inotropic effect of BTS was found to be completely reversible (Fig. 4B). Next we assessed the impact of this finding on the ability of preserved FDACMs to evoke contractility upon washout of BTS and application of field stimulation. Preserved FDACMs that had undergone washing as described in section 3.1. were added to the FHD microscope chamber

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system, perfused with vehicle control and heated to ~ 37 C. Following 2 mins acclimatization, preserved FDACMs were stimulated (4 to 6 V) at 1000 ms cycle length for 3ms duration allowing selection of beating cells and contractility measurements were made. Furthermore, we noted that preserved cardiomyocytes do behave like FDACMs after the removal of BTS (more than 1750 BTSpreserved cardiomyocytes were used in this investigation) and there was no problem in carrying out long experimental protocols. Compared to freshly isolated FDACMs, we found that BTS-preserved FDACMs evoked suitable contractions upon application of field stimulation (Fig. 4C) and similar effects on Sarc. short. upon changes in pacing cycle length (Fig. 4D) indicating a complete reversibility after BTS removal.

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Next, we showed the feasibility of optically recording AP signals from BTS-preserved FDACMs. Optically measured AP duration (APD) in BTS-preserved cardiomyocytes was not affected by sequential additions of vehicle (DMSO 0.1%, Fig. 5A), thus illustrating they can be used to generate meaningful four-point concentration-effect (C-E) curves. Additionally, the APD prolonging-effect of dofetilide, a selective blocker of the rapid delayed rectifier potassium current (IKr), was found to be comparable to that recorded in FDACMs (Fig. 5A and C). Additionally, the positive inotropic effect of either dofetilide or E4031 (another selective blocker of the IKr current) was found to be comparable to that recorded in FDACMs and dependent on cycle length (Fig. 5 F). These APD and contractility data suggest no change in the function of the IKr current in BTS-preserved FDACMs. Imaging of BTS-preserved FDACMs shows that they can be selectively labelled with a primary antibody to the hERG channel (Fig. 5H) and the hERG channel index was not significantly changed in BTS-preserved FDACMs compared to control FDACMs (Fig. 5I). Overall, these data demonstrate the utility of BTS-preserved FDACMs to assess IKrrelated effects on cardiomyocyte function. Furthermore, based on time-control contractility data (Fig. 4E), we concluded these BTS-preserved FDACMs were suitable for further pharmacological examination. We used the L-type calcium channel blocker, verapamil, as a “typical” negative inotrope. Verapamil’s potency was similar for the BTS-preserved FDACMs to those obtained in fresh cells (Fig. 4F). Next, we demonstrated that calcium transients could be recorded from BTS-preserved FDACMs loaded with Fura2 under vehicle conditions and were inhibited by verapamil. Having demonstrated that the BTS-preserved FDACMs were able to generate good quality C-E curves to verapamil with an acceptable degree of variability (Fig. 4F), we tested a diverse range of 18 reference compounds (Fig. 6) that would determine the ability of the BTS-preserved FDACMs to detect different pharmacological mechanisms that affect cardiac contractility and determine how well data generated using the BTS-preserved FDACMs translate to expected contractility effects in FDACMs. Based on their high rate of survival (Fig. 1A), the compounds were tested in day 8 BTS-preserved FDACMs. C-E curves from BTS-preserved FDACMs were found to be comparable to those obtained in FDACMs for inactive compounds (Fig. 6A-E) and for both negative (Fig. 6F-N) and positive (Fig. 6O-R) inotropes. 3.4. BTS-preserved FDACMs are optimal for detecting long-term effects of tyrosine kinase inhibitors To support the development of efficacious and safe tyrosine kinase inhibitors (TKIs) for the treatment of cancer, new in vitro pre-clinical models are required to predict the cardiotoxic potential of new TKIs early in the drug discovery process. Next, we tested whether BTS-preserved FDACMs could be used to reveal the potential of TKIs to induce cardiotoxicity. FDACMs were co-administered with TKI along with 30 M BTS and stored at ~ 4 C, in order to form a non-cumulative C-E curve over the nominal concentration range shown in Fig. 7. Prior to experimentation, the same washout protocol was used as described in section 3.1., but treated BTS-preserved FDACMs with TKIs were washed with the myocyte 7

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4. Discussion

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Tyrode solution containing different concentrations of each TKI. Additionally, contractility experiments were initially started with perfusing the TKI and not the vehicle control. Since BTS-prserved FDACMs were shown to evoke suitable contractions (Fig. 4C) with no sign of ATP depletion (Fig. 1B), we used these two parameters to monitor the manifestation of cardiotoxicity in BTS-preserved FDACMs treated with TKIs. Sunitinib malate, when applied acutely, inhibited fractional sarcomere shortening of FDACMs in a concentration-dependent manner (Fig. 7A), but no loss of cellular ATP was observed (Fig. 7B). Chronic treatment in BTS-preserved FDACMs following 2, 4 and 8 days exposure to sunitinib induced a progressive shift in potency to the left (Fig. 7A). For example, a 10 fold shift in potency was seen at day 8 when compared to acute condition. Additionally, sunitinib malate treatment of BTS-preserved FDACMs depleted ATP in a concentration-dependent manner and a progressive shift to the left in ATP depletion CE curves was seen following 1, 2, 4 and 8 days exposure to sunitinib (Fig. 7B). Next, we assessed the effects of dasatinib and found that neither Sarc. short. (Fig. 7C) nor cellular ATP (Fig. 7D) were affected following acute application of dasatinib at any of the concentrations tested. Taken together, these data may suggest that dasatinib might not induce cardiotoxicity. However, chronic treatment in BTS-preserved FDACMs following exposure to dasatinib inhibited Sarc. short. (Fig. 7C) and depleted ATP (Fig. 7D) in a concentration-dependent manner and resulted in shifting the potency to the left. These data suggest that chronic exposure of BTS-preserved FDACMs revealed the cardiotoxic potential of dasatinib. To further validate the utility of BTS-preserved FDACMs in investigating the cardiotoxic potential of TKIs, we assessed the effects of sorafenib tosylate and imatinib mesylate on cellular ATP. Although sorafenib is associated with low incidence of cardiotoxicity, imatinib-induced cardiotoxocity occurs but it is not common [22]. Like sunitinib malate and dasatinib, neither sorafenib tosylate (Fig. 7E) nor imatinib mesylate (Fig. 7F) depleted ATP acutely. However, both sorafenib tosylate (Fig. 7E) and imatinib mesylate (Fig. 7F) depleted ATP in a concentration-dependent manner following chronic exposure, although sorafenib tosylate led to enhanced ATP loss (Fig. 7E). Overall, our results suggest that BTSpreserved FDACMs might be used to detect long-term effects to novel therapeutics.

4.1. Preservation of FDACMs

Our starting point was based on reports in which a low-temperature range and mechanical uncoupling were required to prolong the viability of cultured cardiomyocytes, cardiac slices or whole heart for only a short-term preservation of approximately 24 to 96 hours. However, the unwanted effects of long-term culture (remodeled cardiomyocytes) and uncoupling agents, like BDM, on cardiomyocytes [23,24] limited the value of their use. We show for the first time that these unwanted effects could be avoided by using uncoupling agents (Fig. 4A-B, Online Supplement Fig. 21A-B, Online Supplement Fig. 22A-B, [21]) that are specific myosin ATPase II inhibitors (blebbistatin that is widely used in cardiac preparations[25] and BTS [20]). When added to FDACMs stored in a standard myocyte Tyrode solution at a refrigerated temperature [6,8], both agents preserved FDACMs for a 2-week period (Fig. 1A, Online Supplement Figs. 3, 19, 20A). Additionally, no loss of cellular ATP content was seen in preserved FDACMs (Fig. 1B, Online Supplement Fig. 20B). This maintenance of ATP level is in agreement with the perception that the safeguarding of ATP correlates with minimal changes to cell structure and function [26] and could be attributed to the absence of gross mitochondrial damage (Online Supplement Fig. 5). Moreover, LDH activity was not increased in preserved FDACMs (Fig. 1C, Online Supplement Fig. 20C). This low activity of LDH enzyme indicates the absence of injury in preserved FDACMs. Furthermore, the absence of cell growth in preserved FDACMs (Fig. 1D-F, Online

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Blebbistatin was shown to inhibit sarcomere shortening of single rat ventricular myocytes [21]. This is in agreement with our canine FDACM data (Online Supplement Fig. 21A). BTS, like blebbistatin, also induced a negative inotropic effect in FDACMs (Fig. 4A). BTS-induced inhibition of Sarc. short. is actually opposed to data shown in the original investigation describing BTS [20]. BTS in that study was shown to be specific for fast skeletal muscle with much less efficiency in suppressing contraction in rat myocardial muscle. However our data not only show that BTS (Fig. 1A) and blebbistatin (Online Supplement Fig. 20A) are equally good at preserving FDACMs, but strongly suggest that BTS and blebbistatin interact with the same molecular target (i.e., myosin ATPase II, Online Supplement Fig. 22) and the only difference in molecular mechanism by which BTS and blebbistatin preserve FDACMs is that blebbistatin binds irreversibly (Online Supplement Figs. 21B, 22B) and BTS binds reversibly (Fig. 4B, Online Supplement Fig. 22A). This non-reversibility of blebbistatin was reported in recent studies [13,21]. Moreover, the effect of blebbistatin was shown to be completely reversible by simultaneous washout and photobleaching by ultraviolet (UV) light [21] or after exposure of the cells to blue light [27]. Given (i) this photochemical reaction is toxic in cells, rapidly stopping cell movements and irreversibly inducing cell death [27,28] and (ii) the difference in binding, we regarded BTS solely on the basis of practicality and usability as the unproblematic preserving agent to use with no impact on cardiomyocyte health.

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BTS and blebbistatin were found to inhibit the ATPase activity of myosin II and do not compete with ADP binding to myosin [20,29]. Additionally, high temperature was shown to weaken BDM suppression of the myosin II ATPase activity [30]. Moreover, myosin II ATPase has a reduced activity at a refrigerated temperature [31]. Our data show that BTS-inhibition of the myosin II ATPase alone is insufficient to preserve FDACMs. We have shown that it is necessary to store FDACMs at 4 °C (and not at RT or 37 °C, Fig. 1A), in addition to exposure to BTS in order to optimally preserve the FDACMs. It may be that the concentration of BTS used was insufficient to achieve sufficient inhibition of myosin II ATPase for the preserving effect at the higher temperatures, while a combination of inherently reduced activity of the enzyme at lower temperature combined with BTS inhibition was sufficient to achieve the preserving effect. Furthermore, since BTS or blebbistatin binding weakens the affinity of myosin for actin with some myosin heads may remain uninhibited [20,29], low temperature might facilitate the preservation of FDACMs by altering the binding so most of the heads are inhibited. Alternatively the temperature and BTS mechanisms may be independent. Although the exact underlying subcellular mechanism that is responsible for preserving FDACMs remains to be determined, our data clearly show optimal conditions to achieve the preserving effect. The gradual decrease in survival rate after 10 days (Fig. 1A, Online Supplement Fig. 20A) might not be explained by a weak binding of myosin to actin in BTS- or blebbistatin-treated FDACMs. Moreover, this decrease in survival rate could not be explained by the poor health of FDACMs, loss of membrane integrity or displaying cellular growth when considering the ATP (Fig. 1B, Online Supplement Fig. 20B), LDH (Fig. 1C, Online Supplement Fig. 20C), cell surface area (Fig. 1D, Online Supplement Fig. 20D) or cell length and width (Fig. 1E-F, Online Supplement Fig. 20E-F) data, respectively. Additionally, a non-stable gene expression might play a role in the decrease of survival rate. Our data on the relative expression of 55 specific cardiac genes we measured at all stages of preservation were found to be comparable to those in the FDACMs (Fig. 2). Specifically, although our qRT-PCR data showed significant changes in expression of 2 genes (ADRB2 and KCNA4) at day 14 of preservation , these changes were still comparable to the levels of expression seen in FDACMs. Despite that the 55 genes we measured were stable, this is not a complete assessment of the genome. Therefore, other genes may be responsible for the decrease in the survival rate. Phase contrast images (Online Supplement Figs. 3, 19), immunofluorescence data for 10 different cardiac markers (Fig. 3, Online Supplement Figs. 4-12) and the requirement to use field stimulation when carrying out the experiments of the AP and Sarc. shor. clearly indicate that no de novo spontaneous 9

ACCEPTED MANUSCRIPT automaticity was developed. Taken together, all these data strongly suggest that BTS-preserved FDACMs remain stable and structurally intact as a consequence of the absence of dedifferentiation. 4.2. Functional characterization of preserved FDACMs

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Another major finding of this investigation is that preserved FDACMs reproduced established electrophysiological characteristics, i.e. normal biophysical characteristics for sodium and L-type calcium currents (Online Supplement Fig. 23), stability of APD (Fig. 5A), normal electrically evoked calcium transients (Online Supplement Fig. 15) [14], suitable stable contractions (Fig. 4C, E, Online Supplement Fig. 21E) [14] and normal reverse-rate dependence (Fig. 4D). Furthermore, in the present study we evaluated the potential of BTS-preserved FDACMs for pharmacological responses and found that they responded to drugs in a similar manner to FDACMs. Dofetilie-induced AP prolongation (Fig. 5A and C) and dofetilide- and E-4031-induced Sarc. short. increase (Fig. 5F) are in agreement with previous works using canine FDACMs [32] and cardiac papillary muscles (cat [33] and guinea pig [34,35]), respectively. Additionally, these IKr blockade-induced effects in BTS-preserved cells were found to be comparable to that in FDACMs (Figs. 5A, C and F). By analogy with our BTS-preserved APD data, it was recently shown that E-4031 increased APD in guinea pig cardiac ventricular slices 24-28 hours after preservation with BDM [17]. The fact that the positive inotropic effect on Sarc. short. seen in our study was frequency dependent and since IKr blockade-induced APD increase is also frequency dependent, our data suggest that IKr blockade affects inotropy via prolongation of the AP. This is not surprising since we would expect a longer time for release of activator calcium from intracellular stores as a consequence of the prolonged AP duration. Moreover, protein expression of the hERG gene was not affected by the BTS treatment (Fig. 5H-I, Online Supplement Fig. 6). Overall our data strongly point to an intact IKr current in BTS-preserved FDACMs, although a detailed electrophysiological characterization of cardiac ionic currents (including IKr) in BTS-preserved cells has to be investigated in future studies. To provide further pharmacological evidence, we demonstrated that the BTS-preserved FDACMs were able to generate good quality C-E curves to verapamil with an acceptable degree of variability (Fig. 4F). These verapamil data combined with no change seen in the protein expression of the CACNA1C gene during the BTS treatment (Online Supplement Fig. 7) indicate that the L-type calcium current is also intact. Subsequent to the generation of verapamil data, a robust pharmacological assessment was done with a diverse range of 18 reference drugs (Fig. 6), which previously were shown to be inactive (5), negative (9) and positive (4) inotropes in FDACMs [19]. Data shown in Fig. 6 imply no alterations in sodium/potassium ATPase pump and phosphodiesterase type 3 enzyme activities, -adrenergic receptor signaling, calcium sensitivity (Fig. 6Q) and ion channels (Fig. 6F-N) which maintain a robust function. For example, our data (Fig. 6P, Online Supplement Table 1) suggest that activity of the key components of the adrenergic pathway (i.e., adrenergic receptor, G protein system, adenylate cyclase, production of cAMP, phosphorylation pathways, L-type channels (Online Supplement Fig. 7)), sarcoplasmic reticulum (Online Supplement Fig. 10), phospholamban (increase in cardiomyocyte relaxation, Online Supplement Fig. 16L) and phosphodiesterase type 3 enzyme (Fig. 6R)) was not affected by the BTS treatment for 8 days when compared to FDACMs. When tested at 0.01 M, we found that isoproterenol-induced increase in Sarc. short. in BTS-preserved FDACMs at days 10, 11, 12 and 15 (data not shown) was also comparable to that seen in FDACMs, supporting the hypothesis that the signaling pathways in preserved cardiomyocytes may remain intact during the 2-week period of preservation. We have demonstrated that some important preserved cardiomyocyte properties and functions were found to be similar to those of fresh cells, although we have not comprehensively examined all properties of preserved cardiomyocytes. Rodent cardiomyocytes are unstable after isolation when compared to mammal cardiomyocytes and this is because of the calcium entry via the sodium/calcium exchanger which overloads the SR and leads to lower viability/higher cell death. It will thus be interesting to not only extend the concept of the canine FDACM preservation described in the current report employing the widely used rodent FDACMs, but to other mammals too. Finally, we anticipate that scientists would further characterize preserved FDACMss so they become widely used as a cellular model of choice. 10

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4.3. Preserved FDACMs as a tool to detect long-term effects of drugs

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The use of the new anticancer TKIs has revolutionized the treatment of certain cancers. However, TKIs used to treat cancers are associated with clinical cardiotoxicity (decrease in left ventricular ejection fraction (LVEF), heart failure, dilated cardiomyopathy and structural changes) [36], although this adverse cardiac event was not anticipated based on their non-clinical safety assessments. As a result, cardiac cellular models that do not have adult properties of native FDACMs, like the myoblastic cell line H9c2 [37], neonatal rat cardiac myocytes [38] and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs [39]), were assessed for their predictivity to identify the cardiotoxic potential of TKIs. Based on our ATP depletion and Sarc.short. data (Fig. 7, both parameters were used to mimic effects of TKIs on the structure and the LVEF), following 8 days exposure to TKIs, BTS-preserved FDACMs offered an improved model over H9c2 cells and hiPSC-CMs for the detection of cardiotoxicity at therapeutically relevant concentrations for all four TKIs [36] (sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate) that were assessed in our study. For example, IC50 values for sunitinib malate-induced ATP depletion were found to be 14.2 and 16.7 µM in H9c2 cells [34] and hiPSC-CMs [39], respectively. In contrast, sunitinib malate depleted ATP in BTS-preserved FDACMs with an IC50 value (0.004 µM) below its therapeutically relevant concentration (0.27 µM) (Online Supplement Table 2). Explanations for these divergent ATP depletion results with these TKIs are likely to involve: (i) usage of serum to maintain the cells [37,38,39], physiological standard myocyte Tyrode solution was used to maintain preserved FDACMs), (ii) exposure time to a TKI (24 hours [37,39] and 24 to 72 hours [37,38,39] versus numerous days with preserved FDACMs), (iii) immature phenotypes of the cardiac cell models [38,39] (preserved FDACMs are fully differentiated adult cells) and (iv) species differences. Since (i) no ATP depletion in preserved FDACMs was seen in the absence of drugs (Fig. 1B), (ii) Sarc. short. of preserved FDACMs were not different from those of FDACMs in the absence of drugs (Fig. 4C), (iii) changes in ATP depletion (Figs. 7B, D-F), Sarc. short. (Figs. 7A, C) and release of cardiac troponin I (cTnI, a biomarker of cardiac toxicity, Online Supplement Fig. 18) in the presence of drugs are time- and concentration-dependent, the ability of preserved FDACMs to detect cardiotoxicity could not be explained by a combination of subtle changes to preserved FDACMs and exposure to drugs. It is possible that our experimental condition for detection of cardiotoxicity may not faithfully mimic the in vivo heart condition (BTS-preserved FDACMs are in stasis and exposure to TKIs in our study may not be comparable to exposure in patients), although our data show that preserved FDACMs not only reveal the cardiotoxic potential of TKIs, but allow a correct classification of TKIs into their rate and grade for inducing cardiotoxicity clinically [38]. Although these preserved FDACMs enable the investigation of long-term exposure to four TKIs on cardiomyocyte function, the accuracy of preserved FDACMs in revealing cardiotoxicity of cancer drugs needs to be thoroughly assessed. Therefore, an additional validation set of cancer therapeutics, including cardiotoxins and non-cardiotoxins, has to be assessed to determine how well data generated using the preserved FDACMs translate to expected clinical cardiotoxic effects. In the case the resulting data suggest that the preserved FDACMs are able to predict the clinical outcome with a high degree of predictivity, ATP depletion, release of cTnI and cardiomyocyte contractility described here could be used in order to (i) facilitate a good throughput of testing for potential cardiotoxins early in the drug discovery process and (ii) provide an aid to prioritization of early-stage compounds for progression along the screening cascade. Furthermore, preserved FDACMs may present an appropriate model to obtain insights into cardiomyocyte cell biology following long exposure to high levels of physiological modulators over a time course that is not amenable with freshly prepared cardiomyocytes and study heart pathologies at the cellular level [22]. This will certainly require future investigations for validating these hypotheses. 4.4. Would the addition of BTS enhance the efficacy of current graft preservation solutions in heart transplantation? 11

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Heart transplantation remains the gold standard in the treatment of end-stage congestive heart failure, although donor heart shortages have now become a critical issue worldwide [40]. Generally, three things have to be in place for potentially successful heart transplantation: (i) the transplant team must always be available at short notice, (ii) proximity of the donor and recipient teams and (iii) heart preservation for transplantation is limited to 4-6 hrs of cold ischaemic storage as the permissible ischaemic time for the heart is short [41]. Developing better preserving solutions that can preserve heart-grafts well for longer time (for example more than 24 hrs) may ensure that all of those hearts that do become available are used. Therefore, use of mechanical uncoupling agents like BTS that safeguards the structure and function of cardiac cells may greatly (i) help to improve/develop current heart preservation solutions by minimizing viability loss and (ii) lead to improved post-transplantation long-term outcome [42]. 4.5. Conclusions

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In summary, our data show that myosin II ATPase inhibiting-agents preserve FDACMs for a 2-week period without affecting their morphology or function. Furthermore, preserved FDACMs offer a robust and versatile model of the adult cardiomyocyte without any remodelling that is normally seen in adult cardiomyocytes maintained in long-term cell culture [43]. Moreover, the current investigation also impacts our understanding about the intricate role of myosin II ATPase in cardiomyocytes and anticipation of adverse cardiac events early in the drug discovery process. Preserved FDACMs have the potential to offer scientists conducting cardiomyocyte cell biology research significant benefits including optimizing resources, reducing experimental costs, offering the flexibility in timing of experiments, improving the relevance of the data generated by allowing cells to be more widely used and developing high-throughput cardiomyocyte screens. Given that global animal use for cardiomyocyte research purposes is considered high, our preserved FDACMs would potentially have significant impact on reducing animal usage globally, not only of dog but also of rodents and other mammals.

Fig. 1. Cell viability of BTS-preserved FDACMs. A. 30 µM BTS resulted in a marked and significant improvement (up to approximately 14 days) of the cell viability of FDACMs that were stored at a refrigerated temperature. Results are from 3 independent isolations and statistical significance is only shown for 3 conditions. £P<0.05, #P<0.01 and *P<0.001 versus values from RT. $P<0.001 and P<0.01 versus values from RT and ~ 4C. ≈P<0.05 versus values from ~ 4C. B-C. ATP content and LDH activity for FDACM stored at RT, ~ 4 C or ~ 4 C + 30 µM BTS, respectively. Results represent the mean measurement of quadriplicate wells. For ATP: #P<0.05 and *P<0.001 versus values from RT. P<0.05 and $P<0.001 versus values from RT. £P<0.01 versus values from ~ 4 C. For LDH: *P<0.001 versus values from RT. $P<0.001 versus values from RT and ~ 4 C. D-F. show the area, length and width of individualized FDACMs per condition that was determined by computer-assisted planimetry (n=76, 111, 110, 138 and 175 cells for fresh, ~4 C, BTS-preserved day 1, BTS-preserved day 8 and BTS-preserved day 14, respectively). Data are mean±SEM and where error bars are not seen, they are smaller than the size of the symbol. Fig. 2. Gene expression of BTS-preserved FDACMs. Relative levels of expression of many cardiacspecific genes related to apoptosis, cardiomyocyte integrity, cell signaling, ion channel, metabolic enzymes, receptor and signal transduction in BTS-preserved FDACMs at days 1, 8 and 14 were compared with those in FDACMs. Description of genes and their corresponding categories are shown in Online Supplement Table 1. Fig. 3. Comparison of FDACMs and BTS-preserved FDACMs for T-tubule membrane network. FDACMs (A) and those preserved with BTS for 1 (B), 8 (C) and 14 (D) days were stained with 10 µM di4-ANEPPS, a lipophilic dye that cannot enter the FDACM and that stains the FDACM surface 12

ACCEPTED MANUSCRIPT membrane. Confocal microscopy images of representative FDACMs show that T-tubule network still exhibited a highly organized striated pattern in BTS-preserved FDACMs. Scale bar, 10 µm. E. shows the average T-index for FDACMs (n=14 cells, 3 dogs) and those preserved with BTS for 1 (n=26 cells, 3 dogs), 8 (n=17 cells, 3 dogs) and 14 (n=11 cells, 1 dog) days.

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Fig. 4. Characterization of BTS-preserved FDACMs. A. BTS inhibited contractility with an immediate recovery (B). C. BTS-preserved FDACMs evoked suitable contractions upon application of field stimulation. D. No difference was seen upon changes in the pacing cycle length between FDACMs and BTS-preserved FDACMs. E. BTS-preserved FDACMs provided stable contractility transients. F. No difference to negative inotropy was present in C-E curves of verapamil from BTS-preserved FDACMs compared to FDACM cells. A complete legend for this figure can be found in the Online Supplement.

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Fig. 5. Rapid delayed rectifier potassium current (IKr) from BTS-preserved FDACMs. A. shows that optically measured APD was not affected by sequential additions of vehicle in BTS-preserved FDACM. B. shows representative optical APs recorded using di-4-ANEPPS-based method with vehicle solution (DMSO 0.1%) and in the presence of 1 μM dofetilide in FDACMs preserved with BTS for 9 days, as well as the corresponding smoothed traces. Note that AP traces shown were obtained from the same FDACM. C. shows that AP prolonging-effect of dofetilide was found to be comparable to that recorded in FDACMs. D-F. Effects of IKr blockers on BTS-preserved FDACM contractility. Typical contractility transients recorded from FDACMs (D) and day 8 BTS-preserved FDACMs (E) exposed to the vehicle and 3 µM E-4031 at a cycle length of 2000 ms. Note that contractility transients shown in each figure were obtained from the same FDACM. F. shows that positive inotropic effect of either dofetilide (n=1836 cells) or E-4031 (n=24-32 cells) on Sarc. short. was found to be comparable to that recorded in FDACMs. G-I. Comparison of FDACMs and BTS-preserved cells for hERG channel protein expression. G. To access the degree of non-specific binding, hERG antibody was pre-incubated with the relevant antigentic peptide, prior to immunofluorescence . Note that the DAPI staining (blue) was used to visualize the nucleus of a FDACM. H. shows that hERG channel labeling is principally in transverse striations in a typical FDACM preserved with BTS for 8 days. Scale bar, 10 µm. I. shows the average hERG-index for FDACMs and those preserved with BTS for 1, 8 and 14 days. A complete legend for this figure can be found in the Online Supplement. Fig. 6. Potency information generated from BTS-preserved FDACMs. Typical non-cumulative C-E curves generated by the canine cardiomyocyte contractility measurements with 18 selected reference compounds are shown. Compounds selected for testing were negative (F-N) and positive inotropes (O-R) as well as inactive (A-E) compounds. A complete legend for this figure can be found in the Online Supplement. Fig. 7. Detection of the chronic effects of TKIs using BTS-preserved FDACMs. Typical non-cumulative C-E curve plots show the effects of sunitinib malate (A) and dasatinib (C) on fractional Sarc. short. and those of sunitinib malate (B), dasatinib (D), sorafenib (E) and imatinib mesylate (F) on cellular ATP content following acute (freshly prepared cells) and long-term (BTS-preserved FDACMs) exposures. IC50 values are shown in Online Supplement Table 2. Disclosures There are no disclosures. Acknowledgments We thank Alex R. Harmer, John R. Foster, Alison L. Bigley, Ann T. Doherty, Jennifer Molloy, Deborah Summerfield, Mandy Wood, Jackie Moors, Ann J. Woods, Harry Holkham, Louise Kelly, Ryan Elkins, Derrick Morgan and Sue J Bickerton for their excellent technical support. The authors acknowledge the 13

ACCEPTED MANUSCRIPT useful discussions with Alex R. Harmer, Caroline Cros, James E. Sidaway, David M. Rock and Kirk S. Schroeder. Sources of funding

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Supplementary data

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Supplementary data to this article can be found online. References

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[38] Hasinoff BB. The cardiotoxicity and Myocyte damage caused by small molecule anticancer tyrosine kinase inhibitors is correlated with lack of target specificity. Toxicol Appl Pharmacol 2010;249(2):132139. [39] Cohen JD, Babiarz JE, Abrams LM, Guo L, Kameoka S, Chiao E, Tauton J, Kolaja KL. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations. Toxicol Appl Pharmacol 2011;257:74-83. [40] Boucek MM, Mashburn C, Dunn SM, Frizell R, Edwards L, Pietra B et al. Pediatric heart transplantation after declaration of cardiocirculatory death. N Engl J Med 2008;359(7):709-14. [41] Nakatani T. Heart transplantation. Circ J 2009;73 Suppl A:A55-60. [42] Thatte HS, Rousou L, Hussaini BE, Lu XG, Treanor PR, Khuri SF. Development and evaluation of a novel solution, somah, for the procurement and preservation of beating and nonbeating donor hearts for transplantation. Circulation 2009;120:1704-1713. [43] Banyasz T, Lozinskiy I, Payne CE, Edelmann S, Norton B, Chen B, Chen-Izu Y, Izu LT, Balke CW. Transformation of adult rat cardiac myocytes in primary culture. Exp Physiol 2008;93(3):370-382.

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ACCEPTED MANUSCRIPT Highlights Cardiomyocytes represent one of the most useful models to conduct cardiac research.

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A heart yields millions of cells, but they do not survive for long after isolation.

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Inhibiting-agents for myosin II preserved adult cardiomyocytes that were stored at a refrigerated temperature for a 2-week period.

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Morphology and function of preserved cardiomyocytes were safeguarded.

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Long-term drug-induced cardiotoxicity was detected with preserved cardiomyocytes.

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