A fluorescent investigation of subcellular damage in H9c2 cells caused by pavetamine, a novel polyamine.

Abstract

Gousiekte, which can be translated literally as "quick disease", is one of the six most important plant toxicoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants characterised by the sudden death of animals within a period of 4-8weeks after the initial ingestion of the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study, fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic compound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed abnormalities that were not present in the control cells. The lysosomes of treated cells were more abundant and enlarged than those of the control cells. There was increased activity of cytosolic hexosaminidase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca(2+) channels. Fluorescent staining demonstrated that pavetamine may damage a number of organelles, all of which can influence the proper functioning of the heart.

Full text

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A fluorescent investigation of subcellular damage in H9c2 cells caused
by pavetamine, a novel polyamine
C.E. Ellisa,b,*, D. Naickera,b, K.M. Bassona, C.J. Bothab, R.A. Meintjesc, R.A. Schultza
aFood, Feed and Veterinary Public Health Programme, Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa
bDepartment of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa
cDepartment of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa
a r t i c l e i n f o
Article history:
Received 18 July 2009
Accepted 4 February 2010
Available online 8 February 2010
Keywords:
Acidic vesicles
Cardiotoxicity
Cytoskeleton
Fluorescent staining
Gousiekte
H9c2 cell line
Mitochondria
Pavetamine
Polyamine
Sarcoplasmic reticula
a b s t r a c t
Gousiekte, which can be translated literally as ‘‘quick disease”, is one of the six most important plant tox-
icoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants
characterised by the sudden death of animals within a period of 4–8 weeks after the initial ingestion of
the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study,
fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic com-
pound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed
abnormalities that were not present in the control cells. The lysosomes of treated cells were more abun-
dant and enlarged than those of the control cells. There was increased activity of cytosolic hexosamini-
dase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an
important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence
of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in
the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence
protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca2+channels. Fluorescent
staining demonstrated that pavetamine may damage a number of organelles, all of which can influence
the proper functioning of the heart.
? 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Gousiekte (‘‘quick disease”) is a disease of ruminants character-
ised by acute heart failure without any premonitory signs 4–
8 weeks after the initial ingestion of certain rubiaceous plants
(Theiler et al., 1923; Pretorius and Terblanche, 1967). The toxic
compound pavetamine that causes gousiekte was isolated from
Pavetta harborii (Fourie et al., 1995). Ultrastructural changes ob-
served in sheep hearts, intoxicated with extracts of Pachystigma
pygmaeum and dried plants known to cause gousiekte included a
loss of cardiac myofilaments. Disintegration of the myofibres,
which appeared frayed, was accompanied by replacement fibrosis
(Schutte et al., 1984; Kellerman et al., 2005; Prozesky et al., 2005).
In addition, mitochondria varied in shape and size, with ruptured
swollen cristae, and the sarcoplasmic reticula (SR) were dilated
and proliferated (Prozesky et al., 2005). Schultz et al., (2001) re-
ported that pavetamine, administered intraperitoneally to rats,
inhibits protein synthesis in the heart, but not in the liver, kidney,
spleen, intestine and muscle. Exposure of H9c2 cells (derived from
embryonic rat cardiac cells) to pavetamine caused damage to mito-
chondria and SR, as demonstrated by transmission electron
microscopy (TEM) (Ellis et al., 2010). Eventual death of H9c2 cells
after exposure to 20 lM pavetamine for 72 h was attributed to
necrosis, with membrane blebbing and lactate dehydrogenase
(LDH) release into the medium (Ellis et al., 2010).
Cardiomyocytes, performing vigorous mechanical work for an
entire lifetime, have elegant mechanisms for protein quality
0887-2333/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2010.02.002
Abbreviations: ATP, adenosine triphosphate; Ca2+, calcium; CICR, Ca2+-induced
Ca2+release; CLSM, confocal laser scanning microscope; CytoD, cytochalasin D;
DAPI, 40,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium;
ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein
degradation; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; H9c2 cells, a
clonal myogenic cell line derived from embryonic rat ventricle; HBSS, Hank’s
balanced salt solution; LDH, lactate dehydrogenase; MHC, myosin heavy chain;
DWm, mitochondrial membrane potential; NCX, Na+/Ca2+exchanger; PBS, phos-
phate-buffered saline; PQC, protein quality control; RyR, ryanodine receptor;
SERCA, sarcoplasmic reticulum Ca2+ATPase; SR, sarcoplasmic reticulum; TEM,
transmission electron microscopy; UPR, unfolded protein response; UPS,ubiquitin–
proteasome system.
* Corresponding author. Address: Food, Feed and Veterinary Public Health
Programme, Agricultural Research Council-Onderstepoort Veterinary Institute,
Private Bag X5, Onderstepoort 0110, South Africa. Tel.: +27 125299254; fax: +27
125299249.
E-mail address: ellisc@arc.agric.za (C.E. Ellis).
Toxicology in Vitro 24 (2010) 1258–1265
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Page 2

control (PQC) (Wang et al., 2008). Protein synthesis occurs on cyto-
solic free ribosomes and, depending on cell type, in the rough SR
(Blobel, 2000). In the SR, numerous chaperones, other proteins
and factors ensure efficient protein folding as part of the SR-asso-
ciated PQC. For efficient protein folding during protein synthesis,
the correct redox status is required for protein disulphide bond for-
mation (Glembotski, 2008). Endoplasmic reticulum (ER) stressors,
namely dithiothreitol, thapsigargin and calcium levels, impair
proper folding of proteins and lead to the accumulation of mis-
folded and dysfunctional proteins, thereby triggering the unfolded
protein response (UPR) (Kozutsumi et al., 1988). Cardiomyocytes,
in which the myofibrillar proteins occupy more than 80% of the cell
volume, have an SR-independent PQC, namely the ubiquitin–pro-
teasome system (UPS) (Wang et al., 2008). Target proteins are
ubiquitinated by ubiquitin E3 ligases and degradation occurs on
the 26S proteasome. Proteolysis in cardiomyocytes can also occur
via calpains and caspases.
The cytoskeleton of cardiomyocytes consists of actin microfil-
aments, microtubules and intermediate filaments (Kustermans
et al., 2008). Actin has several essential roles, namely cell motil-
ity, membrane dynamics, endocytosis, exocytosis, vesicular traf-
ficking and cytokinesis (Lanzetti, 2007; Kustermans et al.,
2008). G-actin (globular-actin) exists as a monomer and is bound
to ATP, whereas F-actin (filamentous actin) is a linear polymer
(Kustermans et al., 2008). Many actin-binding proteins regulate
the dynamics of actin polymerization (the coordinated assembly
and disassembly of actin filaments in response to cellular signal-
ing) (Kustermans et al., 2008). Actin is also found in the nucleus
and is an important regulator of transcription, chromatin remod-
elling and transcription factor activity (Vartiainen, 2008). Fur-
thermore, cardiac L-type calcium channel regulation is tightly
controlled by actin filament organization (Lader et al., 1999;
Rueckschloss and Isenberg, 2001).
The purpose of this study was to determine the effect of pave-
tamine on the structure of the mitochondria, sarcoplasmic reticula,
lysosomes and the F-actin cytoskeleton in the H9c2 cell line, a sub-
clone derived from embryonic rat heart tissue, using fluorescent
probes.
2. Materials and methods
2.1. Chemicals
Cytochalasin D (CytoD), digitonin, Dulbecco’s modified Eagle’s
medium (DMEM), fast red violet LB, magnesium chloride (MnCl2),
naphthol phosphate AS-BI, phalloidin-FITC, p-nitrophenyl-N-acet-
yl-b-D-glucosaminide and thapsigargin were purchased from Sig-
ma–Aldrich (St. Louis, MO, USA). Foetal calf serum (FCS) and
penicillin–streptomycin were purchased from Gibco (Grand island,
NY, USA). ER-Tracker Green dye, MitoTracker Green FM dye, Lyso-
sensor Green DND-189 probe and ProLong Gold antifade reagent
were purchased from Invitrogen (Eugene, OR, USA). DAPI (40,6-
diamidino-2-phenylindole) was purchased from Roche Diagnostics
(Mannheim, Germany) and N0-N0-dimethylformamide was ob-
tained from BDH Chemicals Ltd (Poole, England).
2.2. H9c2 cell line
The H9c2 cell line (Kimes and Brandt, 1976) was obtained from
the American Type Culture Collection (cat no: CRL-1446™, Manas-
sas, USA). The cells were grown on sterile glass coverslips in
DMEM, supplemented with 10% FCS and 100 U/ml penicillin-
100 lg/ml streptomycin sulphate.
2.3. Purification of pavetamine
Pavetamine was extracted and purified from the leaves ofP. har-
borii S. Moore, collected near Ellisras (23?320S, 27?420E) in the
Limpopo province, South Africa, by staff of the ARC-OVI Toxicology
department according to the method described by Fourie et al.,
1995. The yield during 2007 to 2008 was 2.7 mg pavetamine per
kg dried material.
2.4. Treatment of H9c2 cells
H9c2 cells were treated with 20 lM pavetamine and untreated
cells served as controls. In addition, they were also exposed to
3 lM thapsigargin, an ER stressor. In a previous study using TEM,
pavetamine was shown to cause damage to the mitochondria
and SR of H9c2 cells after 24 h of exposure, and secondary lyso-
somes only appeared after 48 h of exposure to pavetamine (Ellis
et al., 2010). For this reason, the H9c2 cells were stained after
exposure for 24 and 48 h to detect organelle damage, and only
stained after 48 h to determine lysosomal defects. Cells were incu-
bated in a humidified atmosphere of 5% CO2at 37 ?C.
2.5. Fluorescent staining
Labeling of H9c2 cells was done with probes to stain subcellular
organelles. The images in the figures are derived from three inde-
pendent experiments and the aim being to study qualitative differ-
ences betweencontrol cells
pavetamine.
andH9c2cellstreated with
2.5.1. Staining of the sarcoplasmic reticulum
After a 24 h exposure period, the cells were washed twice for
5 min at a time with Hank’s balanced salt solution (HBSS), contain-
ing 1% FCS. They were then fixed in 100% acetone for 10 min at
?20 ?C, followed by staining with 20 lM ER-Tracker Green dye
for 30 min at 37 ?C and two final 5 min washes with HBSS/1% FCS.
2.5.2. Staining of mitochondria
After a 24 h exposure period, the cells were washed twice for
5 min at a time with DMEM/1% FCS, stained with 0.2 lM MitoTrac-
ker Green FM dye for 45 min at 37 ?C and washed twice for 5 min
with DMEM/1% FCS.
2.5.3. Staining of lysosomes
After a 48 h exposure period, the cells were washed twice for
5 min each time with PBS/1% FCS, stained with 1 lM Lysosensor
Green DND-189 probe for 30 min at 37 ?C, followed by two final
washes for 5 min each time with PBS/1% FCS.
2.5.4. Staining of F-actin cytoskeleton
After exposure to pavetamine for 24 or 48 h, the F-actin of H9c2
cells was stained with phalloidin-FITC. Cytochalasin D (CytoD), a
cell-permeable mycotoxin and an inhibitor of actin polymeriza-
tion, was included as a positive control. H9c2 cells were exposed
to 12 lM CytoD for 10 min. Briefly, the cells were washed with
PBS for 5 min, followed by fixation in 100% ice-cold acetone at
?20 ?C for 10 min. They were then washed twice with PBS for
5 min and stained with phalloidin-FITC (diluted to 1 lg/ml in
PBS) for 30 min at 37 ?C. The washing was repeated and the nuclei
were then stained with 1.3 lg/ml 40,6-diamidino-2-phenylindole
(DAPI) for 15 min at 37 ?C, followed by two 5 min washes in PBS.
2.5.5. Fluorescence microscopy
After staining and washing, the coverslips were mounted on
microscopy glass slides using ProLong Gold antifade reagent.
C.E. Ellis et al./Toxicology in Vitro 24 (2010) 1258–1265
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Fluorescence imaging was performed using a confocal laser scan-
ning microscope (CLSM) (Model LSM 510, ZEISS, Germany).
2.6. Determination of lysosomal hexosaminidase activity
The hexosaminidase activity was determined according to the
method described by Theodossiou et al., 2006, with few modifica-
tions. Untreated H9c2 cells and H9c2 cells exposed for 48 h to
20 lM pavetamine were harvested by trypsinization. Cells were
washed with PBS and resuspended in 1200 ll PBS. Thereafter,
500 ll of the cell suspensions were transferred to 1.5 ml microcen-
trifuge tubes and centrifuged for 1 min at 720g. The supernatants
were removed and the cell pellets resuspended in 500 ll of 5 lM
digitonin in 0.1 M citric acid (pH 4.5), and incubated for 20 min
at room temperature. The cells were then centrifuged at 5000g
for 1 min and the supernatants were collected. An amount of
500 ll of each supernatant was added to 500 ll of 3.75 mM p-
nitrophenyl-N-acetyl-b-D-glucosaminide in 0.1 M citric acid (pH
4.5) and incubated at 37 ?C for 2 h. The reaction was stopped by
the addition of 500 ll of 0.1 M carbonate buffer, pH 10.0. The
absorbance values were measured at a wavelength of 404 nm in
a UV-160A UV/visible spectrophotometer (Shimadzu, North Amer-
ica). The protein content was determined with a Bio-Rad protein
assay reagent (Bio-Rad Laboratories, California, USA). The results
were expressed as the mean ± standard error of the mean (SEM).
The Student’s t-test was used for statistical analysis of the data,
with p values of <0.05 considered to be significant.
2.7. Determination of acid phosphatase activity
The activity of acid phosphatase was determined according to
the method of Malagoli et al., 2006. Briefly, H9c2 cells cultured
on coverslips were treated with 20 lM pavetamine for 48 h in a
humidified 5% CO2incubator. For the acid phosphatase activity as-
say, both control and pavetamine-treated cells were incubated for
4 h at 37 ?C in a 0.1 N sodium acetate–acetic acid buffer (pH 5.0)
Fig. 1. H9c2 cells stained with ER Tracker for labeling of sarcoplasmic reticula (SR). (A) untreated control cells; (B–D) cells treated with 20 lM pavetamine for 24 h; (E–F) cells
treated with 20 lM pavetamine for 48 h; (G–H) cells treated with 3 lM thapsigargin for 24 h.
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containing 0.01% naphthol phosphate AS-BI, 2% N0-N0-dimethyl-
formamide 0.06% fast red violet LB and 0.5 mM MnCl2. Light
microscopy images were acquired using a microscope (Leica, Mod-
el CTR6000, Germany) fitted with a digital camera system (Leica,
DFC490, Germany).
3. Results
The SR of H9c2 cells treated with 20 lM pavetamine (Fig. 1B)
differed in appearance from that of the control cells (Fig. 1A). The
SR of treated cells were less compact than those of the control cells
and appeared more granular (Fig. 1B–F), or even collapsed (Fig. 1C).
The SR in Fig. 1D appeared more abundant than those of the con-
trol cells Fig. 1A). After exposure to pavetamine for 48 h, the SR
accumulated around the nuclei (Fig. 1E and F). In Fig. 1G and H,
cells treated with 3 lM thapsigargin demonstrated more numer-
ous and denser SR compared to the control cells (Fig. 1A).
Staining of H9c2 cells exposed to 20 lM pavetamine using
MitoTracker Green revealed abnormal mitochondria (Fig. 2B–D).
In the control cells (Fig. 2A), the mitochondria were evenly distrib-
uted around the nucleus, whereas after 24 h of pavetamine treat-
ment, the mitochondria relocated towards one pole of the cell
(Fig. 2B) and became more elongated (Fig. 2C). The mitochondria
of H9c2 cells exposed for 48 h to 20 lM pavetamine (Fig. 2D) dis-
integrated and the fluorescence faded rapidly.
Staining of the lysosomes with the LysoSensor probe revealed a
number of aberrations in cells treated with pavetamine (Fig. 3). An
increase in the number and size of lysosomes was noted in H9c2
cells exposed for 48 h to 20 lM pavetamine (Fig. 3B–D), in compar-
ison to untreated control cells (Fig. 3A). The lysosomes relocated to
the periphery of the cell (Fig. 3B–D), whilst those in Fig. 3C were
markedly swollen, with weak fluorescence on the outer edge. The
average activity of hexosaminidase in the treated cells was
635.5 ± 44.2 lM/h/mg protein compared to 243.8 ± 21.4 lM/h/mg
protein in the control cells (Fig. 4). Digitonin-permeabilized frac-
tions represented the enzyme activity in the cytosol (Theodossiou
et al., 2006). H9c2 cells treated with 20 lM pavetamine for 48 h
showed more intense staining with the substrate for acid phospha-
tase (an enzyme specific for lysosomes) (Fig. 5C and D) than the
untreated control cells (Fig. 5A). There was light pink staining of
the lysosomes around the nucleus in the control cells, whilst the
treated cells were characterised by deep purple staining in all areas
of the cytosol, indicating increased enzyme activity.
The effect of pavetamine on the cytoskeleton of H9c2 cells was
investigated using phalloidin-FITC (Fig. 6). In the control cells, both
Fig. 2. H9c2 cells stained with MitoTracker Green for labeling of mitochondria. (A) Untreated control cells; (B–C) cells treated with 20 lM pavetamine for 24 h; (D) cells
treated with 20 lM pavetamine for 48 h.
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thick and thin bundles were stained with phalloidin, with the
thicker filaments located at the periphery of the cells (Fig. 6A).
The filaments appeared throughout the cells as a mesh-like net-
work. The H9c2 cells treated with 20 lM pavetamine for 24 h
showed differences in cell shape and F-actin patterns, although
the intensity of the fluorescent staining was comparable to that
of the control cells (Fig. 6B and C). The F-actin was ruffled around
the nucleus (Fig. 6B), or lost its mesh-like appearance and became
parallel in orientation (Fig. 6C). Fluorescent staining was much less
intense or even absent, with only the nuclei being stained, in the
H9c2 cells treated for 48 h with pavetamine (Fig. 6D). Within
5 min of exposure of H9c2 cells to 12 lM CytoD, the F-actin net-
work became severely disrupted (Fig. 6E). Dissolution of stress fi-
bres and numerous small and highly fluorescent cytoplasmic
aggregates, or foci, appeared, with clumping in some areas of the
cells.
4. Discussion
This fluorescent subcellular investigation demonstrated that
pavetamine caused alterations to the SR, mitochondria, lysosomes
and F-actin of H9c2 cells, all of which have important functions in
the cell. These findings concur with a previous TEM study (Ellis
et al., 2010), in which pavetamine was shown to cause damage
to the mitochondria and SR of H9c2 cells after 24 h exposure and
the appearance of secondary lysosomes after 48 h exposure.
Pretorius et al. (1973) reported a depressed uptake of calcium
ions by isolated fragments of the SR from sheep dosed with pave-
tamine-producing P. pygmaeum plant material, suggesting that the
Fig. 3. H9c2 cells stained with Lysosensor probe, which stains both lysosomes and late endosomes. (A) untreated control cells stained with Lysosensor probe; (B–D) cells
treated with 20 lM pavetamine for 48 h and then stained with Lysosensor probe.
Control Pavetamine
0
100
200
300
400
500
600
700
Control
Pavetamine
µM/h/mg protein
Fig. 4. Lysosomal hexosaminidase enzyme activity of untreated control and
pavetamine-treated H9c2 cells after 48 h exposure. H9c2 cells treated with
pavetamine had statistically significant higher hexosaminidase activity than the
control cells as analyzed with the Student’s t-test (p < 0.05). The results presented
the average of three independent enzyme activity determinations.
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Fig. 5. Acid phosphatase enzyme activity of untreated control and pavetamine-treated H9c2 cells. (A) untreated control cells; (B–D) H9c2 cells treated with 20 lM
pavetamine after 48 h exposure. The experiments were repeated three times independently with similar results.
Fig. 6. H9c2 cells stained with phalloidin-FITC which binds to the F-actin cytoskeleton. (A) untreated control cells; (B–C) cells treated for 24 h with 20 lM pavetamine; (D)
cells treated for 48 h with 20 lM pavetamine; (E) cells treated with 12 lM cytoD (an inhibitor of actin polymerization) for 5 min.
C.E. Ellis et al./Toxicology in Vitro 24 (2010) 1258–1265
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intracellular Ca2+homeostasis is altered during gousiekte. They
concluded that reduced contractility of hearts affected by gous-
iekte can be directly correlated with the altered Ca2+homeostasis.
Disturbances in any of these functions will lead to SR stress, which
will alter the efficiency of protein synthesis and protein folding. In
this study the SR stained with the ER Tracker probe showed abnor-
malities in the cells treated with pavetamine, indicating ER stress.
Treatment with thapsigargin, a SR stressor, produced similar re-
sults. Thapsigargin blocks the SERCA pump and prevents the trans-
port of Ca2+back into the SR, thus causing depletion in the Ca2+
stores of the SR, with a resultant rise in cytosolic Ca2+levels (Prasad
and Inesi, 2009).
The MitoTracker Green probe diffuses passively across the sar-
colemma and accumulates in active mitochondria, irrespective of
the mitochondrial membrane potential (DWm). In this study, the
morphology of the mitochondria of pavetamine-treated cells was
adversely affected and the rapid fading of fluorescent staining indi-
cated that the mitochondria were inactive. The mitochondrion is
the power engine of the cell by virtue of generating ATP through
oxidative phosphorylation. Snyman et al., (1982) investigated the
energy production in hearts of sheep dosed with P. pygmaeum
and found a decreased level of ATP and creatine phosphate (CrP),
with increased lactate levels, reflecting a shift towards anaerobic
metabolism. They further demonstrated a reduced uptake of oxy-
gen in isolated mitochondria.
The LysoSensor probe is a dye that is acidotropic and accumu-
lates in acidic organelles like late endosomes and lysosomes as a
result of protonation (Lin et al., 2001). Polyamines accumulate in
polyamine-sequestering vesicles that co-localize with acidic vesi-
cles of the late endocytic compartment and the trans Golgi (Soulet
et al., 2004). Amine drugs such as procaine, nicotine and atropine
cause multiple large vacuoles to form (Morissette et al., 2008).
These vacuoles are formed by vacuolar (V)-ATPase as a result of
the cell’s response to concentrated cationic drugs. Lipophilic weak
bases, such as monoamines and diamines, are used to study vacu-
olar acidification (Millot et al., 1997). Lysosomotropism is a term
used to describe the accumulation of basic compounds inside
acidic organelles. Basic compounds reaching the acidic milieu of
lysosomesbecomeprotonated
resulting in their accumulation inside the lysosomes (Lemieux
et al., 2004). According to the results obtained in this study, pave-
tamine caused an increase in the amount and size of lysosomes. To
further investigate the lysosomes, two enzymatic assays were per-
formed. There was an increase in the activity of cytosolic hexosa-
minidase in cells treated with pavetamine, indicating that the
lysosomal membranes became more permeable. Acid phosphatase
activity in the treated cells was also increased in comparison to the
control cells. There are three proteolytic systems in cells: protea-
somes, calpains and the lysosomal hydrolases (Bartoli and Richard,
2005). If the lysosomal hydrolases escape from lysosomes, they can
have a devastating effect on cellular and extracellular matter (Be-
chet et al., 2005). The lysosomal endopeptidases, known as cathep-
sins, can hydrolyse myofibrillar proteins like troponin T, myosin
heavy chain, troponin I and tropomyosin (Bechet et al., 2005). Cal-
pain degrades the cytoskeleton and myofibril proteins such as tro-
ponin I, troponin T, desmin, fodrin, filamin, C-protein, nebulin,
gelsolin, vinculin and vimentin, leading to impairment of the ac-
tin–myosin interaction (Lim et al., 2004). The lysosomal damage
observed in the current study might explain the typical ultrastruc-
tural feature of gousiekte in hearts, namely degeneration of the
myofibrils (Prozesky et al., 2005). It is possible that the lysosomal
endopeptidases may play a part in the degradation of the
myofibrils.
Phalloidin, a toxin from the mushroom Amanita phalloides, is an
actin filament stabilizing compound (Kustermans et al., 2008).
Coupling of phalloidin to fluorescent dyes therefore renders it a
andmembrane-impermeable,
useful tool to study the cytoskeleton. Phalloidin stains only fila-
mentous actin with seven or more monomeric actin molecules
(Visegrady et al., 2005). In this study treatment of cells with pave-
tamine caused alterations in the F-actin cytoskeleton. The F-actin
became ruffled around the nuclei and lost its mesh-like appear-
ance. In some cells, F-actin was absent, with only the nuclei being
stained. CytoD caused severe disruption of the F-actin network.
The effect of pavetamine on F-actin was different to that of CytoD.
Changes to the F-actin after pavetamine treatment occurred much
later than the CytoD-induced alterations. Owing to the complexity
of the cytoskeleton, many other proteins, like the actin-binding, ac-
tin-severing and actin-capping proteins, may be involved in the
cytotoxicity caused by pavetamine. Two possible consequences of
disruption of the cytoskeleton by pavetamine may include altered
protein synthesis (by interfering with transcription) and changes
to the L-type calcium channels. Disruption of F-actin decreases cal-
cium current (Lader et al., 1999).
In conclusion, the cytotoxicity of pavetamine is exerted through
mitochondrial damage, SR stress, increased lysosomal activity and
damage to the F-actin network. All of these cell components play
crucial roles in cell homeostasis. Despite these serious alterations,
the affected animal survives for 4–8 weeks after ingestion of the
pavetamine-containing plants. In future studies, some of the car-
diac contractile proteins in rat neonatal cardiomyocytes will be im-
muno-labeled to identify which proteins are degraded during
gousiekte. The activity of non-lysosomal enzymes, the proteasome
and calpains will also be investigated to clarify their role in the
degradation of the myofibres seen in gousiekte.
Conflict of interest
The authors declare that there is no conflict of interest.
Ethical statement
The authors declare that this work has not been published else-
where and that no animal experiments were carried out in the
present study.
Acknowledgements
We would like to thank Mr. A. Hall (Laboratory for Microscopy
and Microanalysis, University of Pretoria) for the CLSM work. We
would like to thank Prof. Mary-Louise Penrith and Dr. K. Gilling-
water for editing this manuscript. This investigation was funded
by the North-West and Gauteng Provinces, as well as by the
Department of Paraclinical Sciences, University of Pretoria.
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