A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes.

Abstract

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.

Full text

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Cell Calcium 43 (2008) 59–71
A primary culture system for sustained expression of a calcium
sensor in preserved adult rat ventricular myocytes
Cedric Viero, Udo Kraushaar∗,1, Sandra Ruppenthal, Lars Kaestner, Peter Lipp∗
Institute for Molecular Cell Biology, Medical Faculty, Saarland University, Building 61, 66421 Homburg/Saar, Germany
Received 6 February 2007; received in revised form 27 March 2007
Available online 5 September 2007
Abstract
Forstudyingheartpathologiesonthecellularlevel,culturedadultcardiacmyocytesrepresentanimportantapproach.Weaimedtoexplorea
novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells
such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and
electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv)
Ca2+transients,and(v)theirtolerancetowardsadenovirus-mediatedexpressionoftheCa2+sensor“inversepericam”.Conventionally,ineither
serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were
impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin–transferrin–selenite mixture on substrates
coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes
displayed optimised preservation of their contractile behaviour and Ca2+signalling even under conditions of continuous electrical pacing.
SustainedexpressionofinversepericamdidnotaltermyocytefunctionandallowedlonglastinghighspeedCa2+imagingofelectricallydriven
adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Calcium; Cardiac; Culture; Adult ventricular myocyte; Imaging; Contraction; Rat
1. Introduction
The acute isolation of adult cardiac myocytes has been
established decades ago [1] to investigate the cells’ physi-
ological behaviour. In contrast, studies requiring extended
culture periods, e.g. for protein expression or knock-down,
have always been limited to a couple of days in culture
due to extensive morphological and physiological alterations
of the adult myocytes occurring shortly after isolation [2].
This restriction could not be compensated for adequately by
the creation of cardiac cell lines since they do not repre-
sent cardiac myocyte physiology well enough [3]. Currently,
∗Corresponding authors. Tel.: +49 6841 16 26103;
fax: +49 6841 16 26104.
E-mail addresses: Udo.Kraushaar@nmi.de (U. Kraushaar),
peter.lipp@uks.eu (P. Lipp).
1Presentaddress:Dept.Electrophysiology,NaturalandMedicalSciences
Institute at the University of Tuebingen, 72770 Reutlingen/Germany.
neonatal myocytes serve as a limited model for the adult cell,
but it has to be noted, that in comparison to adult myocytes
neonatal cells display a different phenotype and genotype.
Nevertheless,long-termculturingofthesecellseveninlarger
quantities is routine.
In conventional culture, isolated adult rat cardiomyocytes
rapidly change from a “brick-like” structure towards a more
stellated, neonatal-like shape. Moreover, their size increases
considerably [4]. In serum-free culture medium, adult car-
diac myocytes from guinea-pigs, rats, rabbits and mice are
usually quiescent and retain their viability and unique rod-
shaped morphology for at least a couple of days [5–7]. These
cells maintain highly organised membrane and myofibril-
lar structures that support contractions induced by electrical
stimulation. Thus, they appear suitable to short-term (1–3
days) virus-mediated expression of exogenous proteins [8].
For future studies requiring long-term expression of exoge-
nous proteins or vector-based RNA interference (RNAi) to
0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2007.04.001

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
knock-downproteinexpressionitappearsessentialtoemploy
longer culture periods without a loss of morphology and
physiology of the freshly isolated cells. Moreover, experi-
mentalmanoeuvresinducing“slow-onset”cellularresponses
will also entail long-term culturing of the myocytes. Addi-
tionally, molecular biology techniques such as Western
blotting demand large amounts of proteins from homoge-
neous cell populations. Thus, culturing set-ups are needed
that offer the possibility to electrically stimulate large homo-
geneous populations of cells simultaneously. A decade ago,
an adult rat ventricular myocytes culture system was devel-
oped with conditions that allow short-term (3 days) culture
together with the ability to impose arbitrary electrical pulse
protocols [9].
The goal of the present study was to use that basic
approach and refine it to a long-term culture system (1 week)
withdiminishedcellulardedifferentiation.Wetestedthesuit-
abilityofoursysteminmultiplewaysincludingmorphology,
survival rate, contractile behaviour, Ca2+signalling and suc-
cess for adenoviral mediated expression of an exogenous
protein (inverse pericam, a fluorescence calcium indicator
based on calmodulin [10]).
2. Methods
2.1. Isolation and primary culture of adult rat
ventricular myocytes
We adopted a protocol for cell isolation based on estab-
lishedproceduresinrabbitandmouse[11,12]fortheratheart.
Adult male Wistar rats (6–12 weeks old, 200–400g) were
handled and sacrificed in accordance with the Guide for the
Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23,
revised 1996). Animals were anaesthetised by an intraperi-
toneal injection (i.p.) of pentobarbital sodium, 160mg/kg
body weight (Narcoren; Merial, Germany). Directly after-
wards, we injected (i.p.) 0.5–1ml (according to the body
weight) of a citrate (40mM) solution to prevent formation of
bloodclots.Tenminuteslater,theanimalwaskilledbydecap-
itation.Theheartwasflushedwith10mlofice-coldCa2+-free
solution (CFS) containing (in mM): NaCl 134, Glucose 11,
KCl 4, MgSO41.2, Na2HPO41.2, HEPES 10 (Merck, Ger-
many)(pHadjustedto7.35withNaOH).Afterthat,theheart
was removed, attached to a Langendorff apparatus and per-
fused retrogradly with O2saturated CFS containing 200?M
EGTA at a rate of 4ml/min for 5min. The perfusate was then
changed to O2saturated CFS containing collagenase type I
(Worthington, New Jersey, USA) at a final concentration of
1mg/ml for 25min.
The ventricles were removed, minced and placed in O2
saturated CFS containing 1mg/ml collagenase (at 37◦C in
a water bath for 2min). After sedimentation, the result-
ing supernatant was discarded and the pellet was mixed
and resuspended in 20–25ml of O2 saturated CFS and
incubated as above. The supernatant was discarded again
and the pellet was mixed and resuspended in 20–25ml of
O2 saturated low-Ca2+solution (LCS) containing 50% of
CFS and 50% of high-Ca2+solution (HCS) and incubated
as above. HCS is composed of CFS supplemented with
0.09% of DNAse and 200?M of Ca2+. Furthermore, the
supernatant was discarded, the pellet was resuspended in
20–25ml of O2saturated HCS and incubated as above. Then
rat ventricular myocytes were released from the soft tis-
sue by gentle trituration. The cell suspension was plated
into “peel-off” culture flasks (Techno Plastic Products AG,
Switzerland), the internal bottom surface of which were
coated with poly-l-lysine (500?g/ml; Sigma, USA) or with
amixtureofextracellularmatrixproteins(ECM,1.11mg/ml;
Harbor Bio-Products, Norwood, MA, USA). The myocytes
were allowed to settle down for approximately 1h in
mediumM199withEarle’smodifiedsalts,glutamine(Biow-
est; Nuaille, France), 100?g/ml Penicillin/Streptomycin and
50?g/ml Kanamycin (PAA Laboratories, Austria). In addi-
tion to the control condition (pure medium), the medium was
supplemented with either 5% fetal calf serum (FCS sup-
plemented medium) or 870nM insulin, 65nM transferrin
and 29nM Na–selenite (Sigma, USA) (ITS supplemented
medium). Myocytes were cultured in an incubator at 37◦C
with a 5% CO2atmosphere. After plating the medium was
changed at 1h, day in vitro (DIV) 1, 3 and 6 with warm
fresh medium, in order to remove the loosely attached
cells.
For the experiments involving the viral gene transfer the
cells were plated on ECM-coated cover slips, placed in 12-
wellplatesandkeptinM199mediumsupplementedwithITS.
Adenovirus-mediatedgenetransferwasinitiated1haftercell
platingtoallowafastproteinexpression.Themyocyteswere
transfected with a multiplicity of infection (MOI) of 5–20
plaque-forming units/cells. The regime for exchanging the
culture medium was as described above.
The continuous electrical stimulation was performed at
37◦C. All other experiments were carried out at room tem-
perature (20–22◦C).
2.2. Electrical stimulation
For electrical stimulation of entire cell populations we
designed and built Plexiglas lids, resistant to heat sterilisa-
tion, as shown in Fig. 1A with the following features: (i) two
parallel carbon electrodes for electrical field stimulation; (ii)
standardised connectors for external electrical pulses; and
(iii) silicone sealing for taking the culture flask out of the
incubator while maintaining sterile internal conditions. The
set-up for electrical stimulation comprised a custom-made
high-current pulse amplifier (cp. Fig. 1B; Babraham Technix,
Cambridge, UK). The software “Cardiac Stimulator” was
running under LabView software allowing continuous pac-
ing of culture flasks at an adjustable frequency, cp. Fig. 1C.
We used 0.2Hz throughout all culture conditions involving
pacing of cardiac myocytes.

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Fig. 1. Electrical stimulation and prolonged culture of adult cardiomyocytes. Panels (A–C) illustrate components of the optimised culturing system. (A) Shows
the custom-made stimulation lid (top) and “peel-off” flask (bottom), (B) two high-power amplifiers on top of a standard cell incubator connected to several
“peel-off” flasks for electrical stimulation of myocyte populations. Panel (C) depicts the graphical user interface of the LabView based software “Cardiac
stimulator” controlling the electrical pacing of the myocytes in the flasks. Panel (D) displays the relationship of set voltage (x-axis) and output voltage (y-axis)
of a commercial single cell stimulator (dahsed line) and the custom made pulse amplifier (solid line) connected to an individual culture flask filled with medium
as shown in panels (A) and (B). The typically required voltages used during myocyte culture are highlighted.
2.3. Measurements of cell length change
Electrical stimulation induced cell-length changes were
recorded with a fast video camera (sampling rate 240Hz)
from cells maintained in the culture flask. For this we
transferred the flasks from the incubator onto the stage of
an inverted microscope (Eclipse TS100, NIKON, Japan)
equipped with a cell-length measurement system (IonOptix
Corporation, USA). The system directly put out cell-length
changes that were further analysed in Igor Pro software
(Wavemetrics, USA) with custom-made macros.
2.4. Fluorimetric Ca2+recordings
GlobalCa2+transientweremeasuredwitheitherfura-2or
the Ca2+sensitive fluorescent protein inverse pericam [10].
To perform such recordings, cardiac myocytes were seeded
oncoatedglasscoverslipsthatwereplacedintocultureflasks
(for fura-2) or into wells of a 12-well plate (for inverse peri-
cam) before seeding. For fluorescence recordings the cover
slipsweremountedinacustommadechamberonthestageof
an inverted microscope (TE2000U, NIKON; Japan) attached
tovideo-imaginghardware.Imagingwascarriedoutthrough
a 20× oil-immersion objective (Planfluor 0.75 NA, NIKON,
Japan). The system comprised a video camera (for fura-
2: Imago, TILL Photonics, Germany; for inverse pericam:
iXon DV887, Andor Inc., Ireland) and a monochromator for
excitation (Polychrome IV, TILL Photonics, Germany).
For the fura-2 recordings cover slips were loaded with
dye (fura-2-AM, 0.4–0.75?M, from a stock of 1mM in
DMSO/20% pluronic) for 30min at room temperature. Prior
to recording, the loading solution was exchanged with extra-
cellular solution (ES) composed of (in mM): NaCl 135, KCl
5.4,MgCl21,glucose10,CaCl22,HEPES10adjustedtopH
7.35withNaOH.Imagingwasperformedbyexcitingthecells
at the Ca2+-dependent wavelength (380nm) and recording
the fluorescence signal (>440nm; image exposure duration:
15ms). The excitation at 380nm was interrupted every 50th
image by recording a single image at the Ca2+-independent,
isosbestic excitation wavelength of 355nm (see Fig. 4A).
For calculating ratiometric data we linearly interpolated
betweenthe355nm-imagesandratioedthefluorescenceval-
ues against the corresponding 380nm-image to obtain true
F355/F380-fura-2 ratio data at an acquisition frequency of

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
66Hz. This ratioing and further semi-automatic peak detec-
tionwasperformedinIgorProsoftwarerunningcustommade
macros after averaging the fluorescence of regions of interest
in the imaging software.
Inverse pericam is a chimeric protein comprising a circu-
larlypermutedgreenfluorescentproteinandcalmodulin[10].
Imaging of the inverse pericam fluorescence was performed
by exciting the fluorophore at 490nm and recording the
fluorescence through a 510nm long-pass filter (image expo-
sure duration 15–20ms, resulting in an imaging frequency
of 50–66Hz). Single fluorescence images were obtained by
exportingentiremoviesasmulti-pageTIFFfilesandprocess-
ing them in ImageJ (W. Rasband, NIH, USA). For self ratio
traces we calculated the Fo/?F ratio since the emitted flu-
orescence of the inverse pericam decreased with increasing
Ca2+concentrations, thus the term “inverse”.
2.5. Adenovirus construction
Generation of recombinant adenoviruses was accom-
plishedusingtheTranspose-AdTMAdenoviralVectorSystem
(MP Biomedicals, USA) according to the manufacturer’s
instructions. A pCR259 adenovirus transfer vector encod-
ing for the calcium-sensitive fluorescence protein inverse
pericam was transformed in HighQ-1 Transpose-AdTM294
competent cells, a bacterial cell line carrying the Transpose-
AdTM294plasmidandaplasmidencodingatrans-actingTn7
transposase. After a Tn7-based transposition, recombinant
adenoviral genome was purified from bacteria and trans-
fected into the QBI-HEK 293 cell line using Lipofectamine
2000(Invitrogen,Germany).Inthiscellline,therecombinant
adenoviruses were generated and propagated.
ThepcDNA3-inversepericamvectorwaskindlyprovided
byDr.AtsushiMiyawaki(InstituteofPhysicalandChemical
Research (RIKEN), Wako, Saitama, Japan).
2.6. Data analysis
Results were analysed using a Mann–Whitney rank sum
test(SigmaStatsoftware,USA).Effectswereregardedassig-
nificant when p<0.05 (marked with an asterisk). The results
are expressed as mean values±S.E.M.
3. Results
3.1. Electrical field stimulation of cardiac myocytes
In our peel off flask/lid system (see Fig. 1A and Section
2.2) a field voltage of 40–65V at pulse durations of 5ms
(rectangular pulses) was necessary to trigger a visible con-
traction in at least 75% of the isolated myocytes. In order to
generate these pulses, commercially available pulse genera-
tors such as the MyoPacer (IonOptix Corp., USA) were not
sufficient, because (i) their voltage output is limited to 40V
and (ii) the electric current necessary for the peel off flask/lid
system was higher than the limit of the total output power of
the MyoPacer (compare Fig. 1D). This restricted the highest
achievable output voltage to 25V (measured with two inde-
pendent MyoPacers). It has to be mentioned here that such
amplifiers had been designed solely for single cell experi-
ments and our findings might simply indicate design specific
limitation. We thus obtained custom made high power and
fast switching pulse amplifiers (cp. Fig. 1B and Section 2.2),
which delivered enough power to simultaneously drive four
of our peel of flasks (per output channel) at a maximal volt-
age of more than 80V (voltage stability confirmed; voltage
change during the pulse <5%). For this the hardware gener-
ated an electrical current of approximately 2.1A (calculation
based on a specific electrical resistance of culture medium of
approximately 125?cm, an electrode distance of 8cm and
a cylindrical electrode geometry of a length of 7.3cm and a
diameter of 6mm).
The comparison between a single cell stimulator and our
custom made pulse amplifier is depicted in Fig. 1D. For this
we connected a single culture flask filled with medium to the
amplifier and measured the actual output voltage for a range
of set voltages.
During the myocyte culture the voltage was re-adjusted
on a daily basis. With this we ensured to always drive at least
75%ofthemusclecellswhichwasinspectedvisuallythrough
amicroscope.Weobservedthatthevoltagenecessaryforthat
increased from about 40V at DIV0 to approximately 65V at
DIV6. In our studies we applied the pulses (square-shaped;
5ms in duration) at a constant frequency of 0.2Hz for the
entire culture period.
3.2. Long-term culture of cardiomyocytes: morphology
and survival rates
Isolation of adult rat ventricular myocytes yielded more
than 80% living cells of which more than 70% displayed a
rod-shapedmorphology(datanotshown).Initially,weevalu-
atedvariouscultureconditionsbasedonthelightmicroscopic
morphologyofthemyocytesduringaweekofculture.Fig.2A
summarises our findings for five different culture conditions.
Each row of panels depicts the culture conditions while the
columns represent successive DIVs.
The first two rows show typical results when culturing
adult rat ventricular myocytes in a supplement-free medium
(Fig. 2A). Over the time course of 7 days, less than 50% of
the cells were able to largely retain their elongated pheno-
type. We found that after a few days the myocytes developed
numerous small vesicles or vacuoles as indicated by the two
insets in the first row of images, the development of which
were independent of the substrate coating (Fig. 2A 1st and
2nd rows).
When cultured in medium supplemented with 5% FCS,
the myocytes rapidly “de-differentiated” in their morphol-
ogy (Fig. 2A). This process was so fast that from DIV3
onwards, reliable contraction measurements based on edge
detection (c.p. 2.3) were difficult due to massive changes in

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Fig.2. Morphologicalpropertiesandsurvivalratesofcardiomyocytesinlong-termculture.Panel(A)depictstransmissionimagesoftypicaladultratventricular
myocytes under various culturing conditions. The rows represent different culture conditions for the time points shown (columns). For selected combinations
part of the cell body has been redrawn in a magnified inset (the same reference length bar indicates 40?m for the images and 10?m for the magnified insets).
Theblackarrowheadsinthepanelsofthefirsttworowshighlighttheoccurrenceofnumerousvesiclesandvacuoles.Inpanel(B),wehaveplottedthenormalised
yield of living cells (as percentage of the total number of cells, i.e. living and dead) versus the time of culturing. The data were taken from 4 to 8 different rat
heart preparations for the two most promising culture conditions. Rectangular symbols indicate ITS supplemented culture conditions while triangles refer to
media without serum.

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
the cell geometry, i.e. the majority of cells were rounded
up (>65%). Furthermore, at DIV6 most cells started to
develop lamellipodia-like structures and adopted a flattened
“fried egg” shape (Fig. 2A, third row, rightmost image).
In comparison to serum conditions, for myocytes cultured
underserum-freeandITS-supplementedconditions(Fig.2A,
two lower rows) such morphological “de-differentiation”
was significantly reduced regardless of the substrate coating
(poly-l-lysine: Fig. 2A 4th row or ECM-coated substrates:
Fig.2Abottomrow).Evenafter6daysinculture>32%elon-
gated myocytes were present with the poly-l-lysine coating,
without any lamellipodia-like structures. The rate of elon-
gated cells on the flask surfaces coated with ECM was even
exceeding those rates (>42%).
From these results we concluded that the two most
favourable culture conditions so far were either without any
medium supplements on poly-l-lysine coating or with ITS-
supplement on ECM-coated substrates. We thus investigated
those two conditions further to identify the superior one with
respect to cell survival and conservation of the morphology.
Fig.2Bcomparesthesurvivalratesofthemyocytesunder
the two most promising culture conditions, i.e. ITS/ECM
and no serum/poly-l-lysine for pulsed and non-pulsed cells.
From these data it became apparent that the overall survival
rates of ITS/ECM cultured cells were not significantly dif-
ferent compared to the non supplemented culture conditions,
a finding observed for pulsed and non-pulsed conditions.
This obviously indicated that electrical pacing did not exert
a detrimental effect on cell survival.
Interestingly a higher total number of cells was found
on the ECM-coated surfaces after the isolation, plating and
initial washing steps (data not shown) in comparison to
the poly-l-lysine substrate coating. This might indicate a
stronger interaction between the cells and the coating when
seededontoECM-coatings.Moreover,wefoundthatreliable
cell length measurements with poly-l-lysine were difficult
during the first 4h after seeding, because a large proportion
of the plated cells displayed highly increased spontaneous
activity that ceased over the time course of a few hours after
plating (data not shown).
When we visually inspected the myocytes under both cul-
turing conditions at DIV6 we found that in comparison to the
ITS/ECMconditionthecellsinserumfreemediumdisplayed
(i) numerous vesicles and/or vacuoles (see DIV6, first row in
Fig. 2A) and (ii) a loss of apparent cross-striation (compare
DIV6 first and last row in Fig. 2A).
3.3. Long-term culture of cardiomyocytes: analysis of
cross-striation
In order to quantify the presence of cross-striation as an
indicationfortheconservationofhighlyorganisedcontractile
filaments and structures we calculated spatial power spec-
tra from elongated adult rat cardiac myocytes under serum
freeandITS/ECMconditions(Fig.3).Forthis,wegenerated
intensity profiles (Fig. 3Aa and b, black lines) along the lon-
Fig.3. Cross-striationofcardiomyocytesinlong-termculture.Panels(A–B)
compare rat ventricular myocytes at DIV0 and DIV6 under various cultur-
ing conditions. In order to quantify cross striation we recorded intensity
profiles (panel A) of myocyte images taken with a regular phase-contrast
transmissionmicroscopealongtheirlongitudinalaxis.Theleftimagedepicts
a myocyte at DIV6 cultured in serum free medium on poly-l-lysine (Aa)
whilst the right image represents a myocyte at DIV6 cultured in ITS sup-
plemented medium on ECM (Ab). For (B) we performed a powerspectral
analysis of such line profiles taken from myocytes at DIV 0 and DIV 6. The
inset illustrates a magnified view onto the power peak around spatial fre-
quencies of 0.56?m−1. Symbols highlight the particular peak. Details for
the construction of the powerspectra can be found in the Section 2. These
results were typical for all cells analysed (n=9 at DIV0 and n=6 for each
DIV6 condition; cells were taken from three rat hearts).
gitudinalaxisofthemyocytesatDIV0and6andconstructed
power spectra (Fig. 3B). For cells at DIV0 we consistently
found a peak at the spatial frequency of 0.56±0.015?m−1
(n=6 cells, translating to a regular structure with a rep-
etition every 1.78?m) regardless of the particular culture
condition. This value for the spatial frequency was very
close to the one expected for sarcomeric structures (i.e.
1.8?m sarcomeric length; [13]). From this we concluded
that the regular banding visually identified in the myocytes
at DIV0 was indeed caused by the typical cross-striation
generated by the regular arrangement of the contractile fila-
ments and t-tubules. A similar analysis was performed with
cells at DIV6 either in ITS/ECM or in serum-free/poly-l-
lysine conditions. We found that the cells in the former
condition displayed a frequency peak that appeared slightly
shifted towards higher frequencies (0.61±0.027?m−1for

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65
DIV6 versus 0.56±0.018?m−1for DIV0, n=6 for each
DIV, translating into 1.64?m for DIV6 versus 1.78?m for
DIV0). The amplitude in that peak was also reduced to
68.9%±10% (n=6). Even for the cells cultured in serum
free/poly-l-lysine conditions at DIV6 we could identify a
spectral frequency peak in the very same region, although
as described above cross-striation was often absent when
analysedbyvisualinspectiononly.Nevertheless,thesepeaks
were shifted towards higher spatial frequencies even further
(0.66±0.03?m−1, n=5 cells, translating into 1.5?m). In
additiontheamplitudeinthatpeakswassignificantlyreduced
to 24.5±15% (n=5) when compared to the DIV0 condi-
tion (see Fig. 3B, inset; compare open circle with other
symbols).
After this initial analysis of the culture conditions we set
out to comprehensively investigate the physiology of the cul-
turedcells.Forthisweanalysedthefrequencydependenceof
theircontractilityandCa2+transientsduringelectricalpacing
from DIV0 to DIV6 under various culture conditions.
3.4. Shortening-frequency relationship
Fig. 4A exemplifies the stimulation protocol used for the
cell length and for the Ca2+measurements described below.
Fig.4Bdepictsthetimecourseofcelllengthchanges(0.5Hz,
pulse duration 5ms) at DIV0. While the first contraction
was strong, a typical progressive decay in the contraction
amplitudecouldbeobserved,aphenomenontermedpost-rest
potentiation [13]. In Fig. 4C and D traces are exemplified
for two different culture conditions (left: no medium sup-
plement on poly-l-lysine; right: ITS supplemented medium
on ECM coating) at DIV1 (Fig. 4C) and DIV6 (Fig. 4D).
While at DIV1 both cells displayed post-rest potentiation,
the myocyte cultured without supplement showed a greatly
diminishedpotentiationatDIV6whilethecellwithITS/ECM
still revealed post-rest potentiation.
It is noteworthy that the absolute maximal cell length
changes decreased over time from DIV3 onwards in all con-
ditions tested (data not shown). Most likely this reduction of
the absolute amplitudes of cell shortening is attributed to an
increase of the interaction between cells and substrates (see
details in Section 4). Because of this we analysed changes
of the post-rest behaviour of contraction (normalised to
the pre-stimulation contractions) rather than absolute twitch
amplitudes.
In order to quantify the degree of post-rest potentiation
we calculated the relative change of contractility by ratioing
the twitch amplitude under steady-state conditions (mean
value of the last five peaks; Tss) by the initial contraction
amplitude (T1) as depicted in Fig. 4A. Fig. 5 summarises
the frequency dependence of that ratio and its relation
to the culture conditions. We found that under serum
free/poly-l-lysine, FCS/poly-l-lysine and ITS/poly-l-lysine
conditions, the negative frequency dependence of the
Tss/T1ratio was lost between DIV3 and DIV6. In contrast,
myocytes cultured in ITS-supplemented medium on ECM
coated substrates largely retained the negative frequency
dependence (Figs. 5Ad, Bd, Cd, Dd for DIV6 data). We
observed a particular dramatic change for cells cultured
in FCS-supplemented medium. The negative frequency
dependence turned into a positive relationship termed
post-rest decay (Fig. 5Bd, closed symbols).
When we compared data from cells not stimulated during
the culture period with those derived from pulsed cell pop-
Fig. 4. Post-rest behaviour of contraction in cultured adult rat ventricular myocytes. The basic stimulation protocol for characterising the post-rest behaviour
of the cultured myocytes is depicted in panel (A). Panel (B) illustrates the typical time course of cell length changes during such trains of stimulations at DIV0.
In panels (C) and (D) cell length changes were plotted for cells at DIV1 (C) and DIV6 (D) that were cultured in serum free/poly-l-lysine (a) or ITS/ECM (b)
conditions.

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
Fig. 5. Frequency dependence of the post-rest behaviour of contraction in cultured adult rat ventricular myocytes. For panels (A–D) populations of cells (taken
from between 2 and 6 rat hearts) have been analysed and their post-rest behaviour has been quantified as Tss/T1ratios (see Fig. 2A and text for details). This
ratio has been plotted against the stimulation frequencies (0.1–1.0Hz; random order, 5ms duration). Open and closed symbols represent data from myocytes
that had been continuous pulsed (0.2Hz, 5ms duration) or not-pulsed, respectively. The numbers adjacent to each data point gives the number of myocytes
observed. Pairs of data points with a significant difference have been marked with an asterisk.
ulations (Fig. 5 open symbols) we found no major changes
in the contractile behaviour of the myocytes regardless of
their other culture conditions. Nevertheless, we observed a
significant change at DIV3 with a stimulation frequency of
0.2Hz for cells cultured in ITS supplemented medium with
a poly-l-lysine coating (Fig. 5Cc, marked with an aster-
isk). The Tss/T1ratio displayed a 45% decrease in pulsed
myocytes (n=11; non-pulsed cells, n=8). Furthermore, sig-
nificant differences between paced and non-paced cells were
apparent for myocytes cultured under serum free/poly-l-
lysine conditions for DIV3 (Fig. 5Ac). Nevertheless, the
rather flat frequency dependence was still preserved during
pacing.
From these data we concluded that the contractile
behaviour at DIV0 was best preserved in a culture medium
supplemented with ITS when cells were grown on ECM
coated substrates regardless of whether they were continu-
ously paced or not. In the following we conducted a similar
series of experiments analysing global Ca2+transients with
the Ca2+sensitive fluorescent probe fura-2.
3.5. Calcium–frequency relationship
Figs. 6 and 7 summarise experiments performed under
similarexperimentalconditionsasforFigs.4and5usingcells
from the same preparations in order to be able to correlate
the data with each other. Fig. 6A illustrates the method of
fura-2 imaging that we used (for a detailed description see
Section2).SimilartothetwitchdatapresentedinFig.4A,the
global Ca2+transients also displayed post-rest potentiation
when measured in freshly isolated rat ventricular myocytes
(Fig.6BforDIV0).Wecomparedtime-dependentchangesof
the Ca2+transient amplitude under conditions of serum free
mediumandpoly-l-lysinecoatingwiththebehaviourofcells
in ITS supplemented medium and on ECM coating (Fig. 6C
and D, a and b, respectively). As a result we also found a loss
of post-rest potentiation only in the former condition while
underITS/ECMconditionspost-restpotentiationwaslargely
conserved. At DIV6 in the absence of serum the initial post-
rest potentiation even turned into a strong post-rest decay
(Fig. 6Da).

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67
Fig.6. Post-restbehaviourofCa2+transientsinculturedadultratventricularmyocytes.Panel(A)illustratesthemodeoffura-2recording(downwarddeflections
marked by a filled circle correspond to the 355nm excitation images; for further details see text). The typical time course of fura-2 ratio transients during such
trains of stimulations at DIV0 is depicted in panel (B). For the stimulation regime see Fig. 2A. In panels (C) and (D) fura-2 ratio transients were plotted for
cells at DIV1 (C) and DIV6 (D) that were cultured in serum free/poly-l-lysine (a) or ITS/ECM (b) conditions.
Since in almost 50% of all cells tested at DIV0 the
Ca2+transients fused together (relaxation was not com-
plete between the transients leading to a gradual diastolic
build-up of the Ca2+concentration) when stimulation fre-
quencies exceeded 0.5–0.7Hz we omitted the 1Hz data in
the further analysis. All other conditions were similar to
those described in Fig. 5. In contrast to the relationships
of the twitch amplitude the height of the Ca2+transients
only displayed a modest post-rest potentiation with a basi-
cally flat frequency dependence at DIV0 (Fig. 7Aa). This flat
amplitude–frequency relationship was basically preserved
for all DIVs and for all culture conditions with the exception
of a sole set of conditions. Here, the myocytes at DIV6, that
were paced continuously in the absence of any medium sup-
plement on poly-l-lysine coating, displayed a dramatic shift
from modest post-rest potentiation at 0.1Hz to a significant
post-rest decay at 0.5Hz (open symbols in Fig. 7Ad). Simi-
larly to the results we obtained for the twitch measurements
(Fig. 5) continuous pacing did not make any difference to the
frequencyrelationshipsatanyDIVnorunderanyculturecon-
ditionapartfromtheITS/poly-l-lysinecombinationatDIV3
(Fig. 7Cc).
Thus, myocytes cultured in ITS-supplemented medium
andgrowingonECMcoatedsubstratesmostcloselyretained
theirmorphology,contractilityandCa2+handlingwhencom-
pared to their properties at DIV0.
Wethusconductedthefinalseriesofexperimentstoinves-
tigate whether cardiomyocytes cultured under ITS/ECM
conditions were a good system to perform long-term expres-
sion of exogenous proteins. From our fura-2 data we knew
that under our culture conditions, Ca2+handling was largely
conserved through the 1-week period of culturing. We thus
tested expression of a genetically coded Ca2+indicator
by adenoviral gene transfer (inverse pericam as originally
described by Nagai et al. [10]).
3.6. Inverse pericam expression and Ca2+measurements
The freshly isolated ventricular myocytes were infected
with the virus 1h after plating and first fluorescence could
already be recorded within 24h. Typically we started experi-
ments18haftertransfection.Alreadyatthatearlystage,more
than 95% of all viable myocytes displayed sufficient levels
of fluorescence for recording Ca2+-dependent fluorescence
changes (data not shown). Fig. 8A exemplifies such changes
as recorded in response to electrical stimulations. In all cells
analysed at DIV1 we could record a maximal relative fluo-
rescence change of 25±3.1% (n=65). Since excitation of
inverse pericams does not require the application of UV light
we tested the possibility to perform long-term recordings
as depicted in Fig. 8B. We designed an electrical stimula-
tion regime employing constant pulsing at 0.1Hz and optical
recording for 40s periods separated by 6min without light
excitation. For the recordings in Fig. 8B we continued this
stimulation regime for a total of 90min without a detectable
loss in pericam self-ratio amplitude or signal quality. During
the same time period the absolute inverse pericam fluores-
cence was decreased by less then 15%, most probably due to
bleaching. In some experiments we recorded for more than
2h with a similar experimental regime without a noticeable
decrease in signal quality (data not shown).
4. Discussion
The aim of this study was to develop and explore a cul-
ture system for adult rat cardiac myocytes and procedures

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
Fig. 7. Frequency dependence of the post-rest behaviour of Ca2+transients in cultured adult rat ventricular myocytes. For panels (A–D) a population of cells
(taken from 2 to 6 rat hearts) have been analysed and the amplitude ratios Tss/T1have been plotted against the stimulation frequencies (0.1–0.5Hz, random
order, 5ms duration) for various culture conditions as depicted in each panel. Open and closed symbols display mean values for the continuously pulsed and
non-pulsed myocytes respectively (0.2Hz, 5ms pulse length). The numbers adjacent to each value give the number of myocytes analysed. Pairs of data points
with a significant difference have been marked with an asterisk.
that allow extended experimental manipulation of these cells
underconditionsofdiminisheddedifferentiation.Forthatwe
setupanexperimentalsystemforalong-termcultureofadult
cardiac myocytes that largely suppressed de-differentiation,
bestmaintainedthemorphologicalandphysiologicalproper-
ties of freshly isolated cells and allowed (to our knowledge)
for the first time long-term expression of a genetically
encoded Ca2+indicator.
One of the biggest problems with long-term cultures of
adult cardiac myocytes is the rapid development of morpho-
logical and physiological dedifferentiation. The structural
changes occurring during extended culturing times of rat
ventricular myocytes were studied extensively (e.g. [14,15]).
In parallel there were numerous attempts to modulate cul-
ture conditions towards minimising dedifferentiation. Such
approaches included omitting or substituting the medium
supplement FCS (e.g. [11]) and electrical pacing of the
adult cells (e.g. [9]). All of those approaches have provided
progress towards conditions allowing extended culture peri-
ods and reduced dedifferentiation of the adult cells.
We omitted serum from our medium and substituted that
by an ITS mixture. In addition, we coated the substrates
(plastic and glass) with ECM. Both steps revealed major
improvements in both, the number of viable cells (i.e. the
loss of viable cells was greatly reduced between DIV0 and
DIV6) and the preservation of cell morphology, subcellu-
lar microarchitecture (contractile filaments) and physiology
(contractility and Ca2+handling) of the cells over the time
course of culturing.
As described before, also in our hands the presence of
serum in the culture medium appeared to promote dediffer-
entiation resulting in a flattened morphology. Interestingly,
similar shapes are “normal” for cardiac cell lines such as the
H9C2 cell line investigated earlier (e.g. [16]). We found that
simply omitting the serum supplement is slowing down the
dedifferentiation process (see also [11] for rabbit cardiomy-
ocytes) and more cells survive the culture period with an
elongated cell body. Unfortunately, adult rat cells cultured
under these conditions displayed a progressively increasing
numberofsubcellularvacuolesand/orvesicles,agradualloss

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69
Fig. 8. Fluorescence transients of inverse pericam in cultured rat ventricular myocytes after adenoviral gene transfer. Panel (A) depicts typical self-ratio traces
of the inverse pericam fluorescence recorded from cultured myocytes at the time points given. Panel (Ac) illustrates the stimulation regime used (black arrows
indicate electrical impulses, 5ms duration). Cell images are added for each example and the cell used for calculation of the ratio traces has been marked with
a white asterisk. Panel (B) shows ratio traces of adult ventricular myocytes 1 day (Ba) and 6 days (Bb) after virus infection. Dashed arrows depict recording
periods for which exemplified individual ratio transients have been replotted. In the fluorescence images an asterisk marks the myocyte from which the ratio
was calculated. Here, we calculated F0/?F (F0—fluorescence at the beginning of the recording at rest) to obtain positive changes of the ratio, since the
inverse pericam displays a decrease of the fluorescence with increasing Ca2+concentrations. Traces shown here were typical for all cells analysed under these
experimental conditions (n>60 from three rat hearts).

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
of cross-striation and alterations of their post-rest behaviour.
From such findings we concluded that this culture condi-
tion was in fact not optimal. In contrast ITS-supplemented
medium greatly improved the conservation of the cellular
properties found at DIV0 whether the cells were seeded on
poly-l-lysine or ECM coated substrates, whereby the latter
condition gave even better results.
In the vast majority of conditions, there was neither a
beneficial nor a detrimental effect of electrical pacing unlike
the report by Berger et al. [9]. This held true for both
physiological parameters that we analysed, the contractile
performance and Ca2+transients in response to electrical
stimulation as well as the stimulation frequency dependence
of both parameters. Nevertheless, this does not exclude the
possibility that certain culture conditions combined with
particular stimulation protocols might increase or decrease
survival of the cells or their morphological/physiological
state. Interestingly other studies described hypertrophic
responses of myocytes when pacing frequencies were raised
to higher stimulation frequencies in adult myocyte cultures
(3Hz;[17]).Thisispuzzlingsincethesestimulationratesare
rather physiological for rodent hearts (3–10Hz) at 37◦C.
We made the observation that the absolute maximal cell
length change was progressively decreasing over the cul-
turing time. This reduced twitch amplitude can most likely
be attributed to a progressively increasing mechanical inter-
action between the cells and the substrate. Indeed, during
regular washing of our culture flasks relative loss of cells
was reduced at later DIVs, a finding that might hint to a
tightersubstrateinteractionofthemyocytes(datanotshown).
Such an increased mechanical coupling will simply decrease
absolute twitch amplitudes despite a constant contraction
force. A mechanism that might be responsible for this was
recently found and described the production of extracellular
matrix proteins and modulation of the matrix by myocytes
themselves [18]. Therefore, we investigated our cells using
their post-rest behaviour at various stimulation frequencies:
(i) post-rest contractile behaviour of isolated myocytes is
attributed to their ability to adjust Ca2+handling to changes
(frequency, rest) of the excitation–contraction coupling [13],
(ii) the rational behind this protocol was to work with rela-
tive shortening changes and to avoid individual differences
andchangesofcell-substrateinteractionsasdescribedabove,
and(iii)tocompareanentirerangeoffrequencydependences
rather than selected electrical stimulation regimes.
Genetic manipulation of adult cardiac myocytes is often
limited to short term procedures and to generation of genet-
ically modified donor animals. While the latter is a very
elegant way of introducing proteins to or knocking proteins
out from cardiac myocytes, the generation of genetically
modified animals is expensive and tissue specific, inducible
genetic manipulation is not an easy routine work. Thus,
genetic manipulation of isolated cardiac myocytes might
be a feasible intermediate step towards genetically modi-
fied animals. Traditionally, genetic manipulation of cardiac
myocytes has been performed on neonatal ventricular cells
from the rat (e.g. [19]). Neonatal cells can be transfected
with traditional means (i.e. commercially available transfec-
tants [20]) but in adult cells the yield of such an approach is
usually <10%. Better expression or knock-out rates can be
achieved with viral gene transfer systems, e.g. adenovirus.
Unfortunately, results obtained from neonatal systems are
often difficult to transfer to the adult situation, due to signifi-
cant developmental differences in the expression pattern and
signalling. Therefore, it is desirable to perform such exper-
iments on adult cardiac myocytes. Indeed, there are some
examples where genetic manipulation, e.g. with an adenovi-
ral system has been applied successfully (e.g. [21]). Mostly,
such approaches have been limited to short-term expression
(1–2 DIV) and have thus avoided unwanted dedifferentiation
of the cells. Nevertheless, longer expression would be highly
desirable since many effects of genetic manipulations and/or
stimulation regimes will require longer culture periods.
To our knowledge we described for the first time the
expressionofageneticallyencodedCa2+sensor(inverseperi-
cam) in adult rat ventricular myocytes for extended periods
of time. In our hands, the myocytes cultured under ITS/ECM
conditions were not negatively affected by the gene transfer
through an adenovirus. Expression levels were already high
enough for fluorescence recording less than 24h after infec-
tion and throughout our culture period of 7 days expression
levels were steadily increasing. This will certainly require
additional investigations and titration of the best MOI for
rapid onset of fluorescence and stable expression levels. In
less than 24h expression levels allowed extended recording
regimes of Ca2+transients without detrimental effects in the
signal to noise ratio. In some experiments we were able to
record for more than 2h and the recording was not limited
by bleaching or a decrease in the cell quality. Moreover, the
frequency of data acquisition was not reduced in favour of a
prolonged recording time; we collected images at a rate of
50–60frames/s that allowed, e.g. the recoding of Ca2+waves
(data not shown). Thus, extended expression of genetically
encodedCa2+sensorsisapromisingapproachforlong-term,
continuous observation of Ca2+handling in isolated cardiac
myocytes, especially when working on species for which an
appropriatetransgenedoesnotexist.Apotentiallyinteresting
transgenicmouseexpressingaCa2+indicatorwasintroduced
recently [22].
In the present report we have introduced a poten-
tially important approach for culturing rat ventricular
myocytes combined with expression of exogenous proteins
for extended periods of time. Such an approach will allow
further explorations of in vitro models for studying long-
term signal transduction in cardiac myocytes enabling quasi
continuous supervision of physiological and morphologi-
cal parameters such as contractility and Ca2+handling. We
envisage our report to be a foundation for the further devel-
opment of high-content screening systems [23] employing
adult cardiac myocytes because the application of adenoviral
gene transfer allows the application of genetically encoded
reporters such as Ca2+sensors but also sensors of other sig-

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C. Viero et al. / Cell Calcium 43 (2008) 59–71
71
nal transduction processes (e.g. phosphorylation) in cardiac
myocytes.
Acknowledgements
This work was supported by Deutsche Forschungs
Gemeinschaft (SFB 530 TP B6), Graduate Research School
“CellularRegulationandGrowth”(GRK377/3)andSaarland
University (ZFK and HOMFOR). We are grateful to Anne
Vecerdea, Tanja Buhles, Dr. Klaus Neumann and J¨ org Sauer-
baum for their excellent technical support. We also would
like to thank Rod O’Connor (The Babraham Institute, UK)
for the initial version of the software “Cardiac Stimulator”.
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