Avian Pathology

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Pathophysiology and pathological remodelling

associated with dilated cardiomyopathy in broiler
chickens predisposed to heart pump failure

A. A. Olkowski, C. Wojnarowicz & B. Laarveld

To cite this article: A. A. Olkowski, C. Wojnarowicz & B. Laarveld (2020) Pathophysiology and
pathological remodelling associated with dilated cardiomyopathy in broiler chickens predisposed to
heart pump failure, Avian Pathology, 49:5, 428-439, DOI: 10.1080/03079457.2020.1757620

To link to this article: https://doi.org/10.1080/03079457.2020.1757620

Published online: 22 May 2020.

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AVIAN PATHOLOGY
2020, VOL. 49, NO. 5, 428–439
https://doi.org/10.1080/03079457.2020.1757620

ORIGINAL ARTICLE

Pathophysiology and pathological remodelling associated with dilated

cardiomyopathy in broiler chickens predisposed to heart pump failure
A. A. Olkowskia, C. Wojnarowiczb and B. Laarvelda
a
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada; bPrairie Diagnostic Services, Veterinary
Pathology, University of Saskatchewan, Saskatoon, Canada

ABSTRACT ARTICLE HISTORY

Broiler chickens selected for rapid growth are highly susceptible to dilated cardiomyopathy Received 21 January 2020
(DCM). In order to elucidate the pathophysiology of DCM, the present study examines the Accepted 14 April 2020
fundamental features of pathological remodelling associated with DCM in broiler chickens
KEYWORDS
using light microscopy, transmission electron microscopy (TEM), and synchrotron Fourier Broiler; dilated
Transform Infrared (FTIR) micro-spectroscopy. The morphological features and FTIR spectra of cardiomyopathy; cardiac
the left ventricular myocardium were compared among broiler chickens affected by DCM protein; conformational
with clinical signs of heart pump failure, apparently normal fast-growing broiler chickens changes
showing signs of subclinical DCM (high risk of heart failure), slow-growing broiler chickens
(low risk of heart failure) and Leghorn chickens (resistant to heart failure, used here as
physiological reference). The findings indicate that DCM and heart pump failure in fast-
growing broiler chickens are a result of a complex metabolic syndrome involving multiple
catabolic pathways. Our data indicate that a good deal of DCM pathophysiology in chickens
selected for rapid growth is associated with conformational changes of cardiac proteins, and
pathological changes indicative of accumulation of misfolded and aggregated proteins in the
affected cardiomyocytes. From TEM image analysis it is evident that the affected
cardiomyocytes demonstrate significant difficulty in the disposal of damaged proteins and
maintenance of proteostasis, which leads to pathological remodelling of the heart and
contractile dysfunction. It appears that the underlying causes of accumulation of damaged
proteins are associated with dysregulated auto phagosome and proteasome systems, which,
in susceptible individuals, create a milieu conducive for the development of DCM and heart
failure.

RESEARCH HIGHLIGHTS

. The light and electron microscopy image analyses revealed degenerative changes and
protein aggregates in the cardiomyocytes of chickens affected by DCM.
. The analyses of FTIR spectra of the myocardium revealed that DCM and heart pump failure in
broiler chickens are associated with conformational changes of myocardial proteins.
. The morphological changes in cardiomyocytes and conformational changes in myocardial
proteins architecture are integral constituents of pathophysiology of DCM in fast-growing
broiler chickens.

Introduction
Meat-type chickens (broilers) genetically selected for
rapid growth are highly susceptible to heart failure,
and many succumb to heart pump failure and develop
ascites (for review see Olkowski, 2007). For several dec-
ades this condition has been recognized as one of the
leading causes of morbidity, mortality, and whole car-
cass condemnations in commercial broiler flocks
throughout the world (Olkowski et al., 1996; Maxwell
& Robertson, 1997; Jakob et al., 1998; Kumor & Olk-
owski, 2001; Kittelsen et al., 2015; Part et al. 2016; Mwi-
mali et al., 2018). Mortality and morbidity associated
with heart failure in commercial broilers are of great
economic significance. Maxwell and Robertson
(1997) estimated the global losses in the broiler
industry associated with ascites at around $1 billion
per annum.
Broilers showing clinical signs of heart pump failure
and ascites typically present gross pathological changes
including severe dilation of the heart chambers and
thinning of the ventricular wall consistent with dilated
cardiomyopathy (DCM) (Olkowski et al., 1998, 2001).
On necropsy examination, the affected hearts appear
flaccid, which on palpation is manifested as loss of
muscle tone and turgor typical of a normal heart (Olk-
owski et al., 1998, 2001, 2005a, b). However, it is impor-
tant to stress that similar changes are also observed on
post mortem examination in many apparently normal
fast growing broilers, which indicates a wide spread

CONTACT A. A. Olkowski andrew.olkowski@usask.ca

© 2020 Houghton Trust Ltd

subclinical form of DCM (Olkowski et al., 1998, 2001;
Olkowski, Abbott, et al., 2005; Olkowski, Duke, et al.,
2005; Wilson et al., 2016).
Considerable research efforts over the last three dec-
ades have been committed to unravel the aetiology of
heart failure and ascites in broiler chickens (Peacock
et al., 1989; Decuypere et al., 1994; Fedde et al., 1998;
Martinez-Lemus et al., 1998; Olkowski et al., 1998,
1999, 2001, 2003; Olkowski, Abbott, et al., 2005; Olk-
owski, Duke, et al., 2005; Buys et al., 1999, 2007; Has-
sanzadeh et al., 2001; Wideman, 2001; Luger et al.,
2002; Deng et al., 2006; Druyan et al., 2007; Nain
et al., 2008a, b; Hassanpour et al., 2019). While pro-
gress has been made in the understanding of some
metabolic and physiological variables associated with
ascites, many aspects of the pathogenesis of DCM
remain obscure. In particular, the pathophysiology of
heart contractile dysfunction in broilers observed on
echocardiographic examination (Martinez-Lemus
et al., 1998; Olkowski, Abbott, et al., 2005; Deng
et al., 2006) remains unclear.
Some aspects of poor performance of the heart in
broilers can be associated with thyroid disturbances
(Decuypere et al., 1994; Luger et al., 2002) and insuffi-
ciency in cardiac energy metabolism (Olkowski et al.,
2007; Nain et al., 2008a). It is also noteworthy that
the myocardium of broilers with signs of DCM and
heart pump failure shows biochemical evidence of
severe oxidative stress (Nain et al., 2008b), and this fac-
tor may have detrimental effects on biochemical and
mechanical function of the contractile apparatus. In
this regard, there is a growing body of evidence that
oxidative stress can induce post-translational modifi-
cations of proteins and trigger dysregulation of protein
folding, and clearance of misfolded proteins has been
observed to be associated with many diseases, includ-
ing heart failure (for recent review see Tomin et al.,
2019).
Interestingly, over the last decade, new research
findings pointed out that conformational changes in
cardiac proteins represent an integral element of the
pathogenesis of DCM. Gianni et al. (2010) provided
the first evidence that protein misfolding and aggrega-
tion is implicated in the aetiology and pathogenesis of
idiopathic DCM in humans. More recent studies pro-
vided further evidence that proteasome dysfunction
plays a central role in human heart failure (Willis &
Patterson, 2013; del Monte & Agnetti, 2014; McLendon
& Robbins, 2015; Gilda & Gomes, 2017; Tomin et al.,
2019).
With deference to recent advances in heart failure
research, and based on our previous observations (Olk-
owski et al., 2001; Nain et al., 2008b), we hypothesized
that the pathophysiology of heart pump failure in broi-
lers may involve discrete structural changes in cardiac
muscle architecture associated with misfolding and
aggregation of myocardial proteins. Accordingly, the
AVIAN PATHOLOGY 429
goal of the present study was to examine microscopic
features of cardiomyocytes and determine whether
conformational changes in myocardial proteins may
be involved in the natural history of DCM in broiler
chickens that eventually result in fulminant heart
pump failure.
Materials and methods
General
The experimental design, management of birds and
feeding regimes were as described previously (Olk-
owski, Abbott, et al., 2005; Olkowski, Duke, et al.,
2005). Briefly, the chickens used in this study rep-
resented a random sample obtained from basic flocks
consisting of 50 fast-growing broilers, 50 feed
restricted, slow-growing broilers, and 50 Leghorns.
The broilers allocated to the slow-growing group
were derived from the same population as the fast-
growing broilers, and subjected to a feed restriction
regime (70% ad libitum) from day 7 onwards. All
other groups of birds were fed ad libitum. Water was
provided to all groups ad libitum. In the present
study we examined structural changes in the cardio-
myocytes and conformational characteristics of myo-
cardial proteins in chicken groups categorized
according to risk of heart failure as follows: Leghorn
chickens (resistant to heart failure) used in this study
as physiological reference; slow-growing broiler chick-
ens where growth rate was controlled by dietary restric-
tion (low risk of heart failure); fast-growing, ad libitum
fed broiler chickens (high risk of heart failure), and
broilers showing DCM and clinical signs of heart
pump failure.
Experimental protocols were approved by the Uni-
versity of Saskatchewan Animal Care Committee and
procedures were performed in accordance with the
guidelines on the care and use of farm animals in
research, teaching and testing (Canadian Council on
Animal Care, 2009).
Clinical and post mortem evaluation
All chickens were subjected to daily clinical evaluation.
A standard experimental protocol included monitoring
for overt clinical signs of heart pump failure as assessed
by presence of signs such as hypoxaemia (blood gas
analyses), cyanosis (clinical evaluation), and ascites
(post mortem examination) as previously described
(Olkowski, Abbott, et al., 2005; Olkowski, Duke, et
al., 2005). Following the completion of the trial, all
chickens were euthanized by cervical dislocation and
subjected to detailed post mortem evaluation. Diagnosis
of DCM and heart pump failure was based on clinical
observations and post mortem signs of DCM. Diagnosis
of subclinical DCM was made when apparently normal
broilers showed signs of ventricular dilation on post
430 A. A. OLKOWSKI ET AL.

mortem examination. Gross evaluation of the hearts
and procedures of heart tissue procurement for light
microscopy, transmission electron microscopy (TEM)
and Fourier Transform Infrared (FTIR) evaluation
were performed immediately after the chickens were
euthanized.
Light microscopy
For light microscopic examination, hearts were fixed in
phosphate buffered 10% formalin solution, and blocks
of tissue sections taken midway from the apex were
embedded in paraffin. Longitudinal and transverse sec-
tions (5 μm in thickness) were processed for light
microscopy and stained with haematoxylin and eosin
(H&E) as previously described (Olkowski et al., 2003).
Transmission electron microscopy
For TEM evaluation, heart tissue samples (mid-sec-
tions of septum and left ventricular wall) from normal
chickens (n = 3), chickens showing signs of subclinical
DCM (n = 3), and chickens showing signs of DCM
with fulminant heart pump failure (n = 3) were pro-
cessed as described previously (Olkowski et al., 2001).
FTIR micro-spectroscopy
For FTIR study, left ventricular muscle sections were
obtained from randomly selected Leghorns (n = 3),
slow-growing broiler chickens (n = 3), apparently nor-
mal fast growing broilers affected by subclinical DCM
(n = 3), and chickens showing DCM with clinical
signs of heart pump failure and ascites (n = 5). All
heart samples were processed immediately after pro-
curement using the following steps: (1) cryo preser-
vation of heart tissue by submerging in optimal
cutting temperature (OCT) compound and flash freez-
ing in liquid nitrogen; (2) cutting of frozen tissue
blocks at 6 µm sections using a micro-cryotome at
−25°C; (3) placing of sections on low emittance micro-
scope slides (MirrIR slides, Kevley Technologies, Ches-
terfield, OH, USA) for FTIR imaging and micro-
spectroscopy; and (4) drying in vacuum chamber.
FTIR micro-spectroscopy, mapping, and data acqui-
sition were performed according to the generic flow
chart presented in Figure 1.
Results
Figure 2 shows representative examples of heart anat-
omy observed on post mortem evaluation in Leghorn
chickens (used here as physiological reference), slow-
growing broiler chickens (low risk of heart failure),
fast-growing broilers affected by subclinical DCM,
and broilers with advanced DCM and showing clinical
signs of heart pump failure.

Figure 1. Technical approach to FTIR micro-spectroscopy. The FTIR spectra were obtained from a randomly selected area (approxi-
mately 250 µm2) of the myocardium prepared as described in the text using the mid IR beam line as infrared (IR) source at the
Canadian Light Source (University of Saskatchewan, Saskatoon, SK, Canada) facility using a Hyperion 3000 IR microscope coupled
to a Tensor 27 interferometer (Bruker Optics, Billerica, MA). A 15x magnification Schwarzschild objective was used to focus IR light
on the sample and collect the reflected beam. A KBr-supported Ge multilayer beam-splitter and a 64 × 64 pixel Focal Plane MCT
detector (Santa Barbara Corp., Santa Barbara, CA, USA) were used to measure spectra in the mid-infrared spectral region. Interfer-
ograms were recorded by scanning the moving mirror at 2.2 kHz to an upper frequency limit of 3950 cm−1 and with a spectral
resolution of 4 cm−1. 4 × 4 pixel binning was performed during acquisition. Single channel traces were obtained using the fast
Fourier transform algorithm, without zero-filling, after applying a Blackman-Harris 3-Term apodization function. Data analysis
was performed using OPUS version 6.5 (Bruker Optics, Billerica, MA, USA).

Leghorn chickens (resistant to heart failure) and
slow-growing broiler chickens (low risk of heart fail-
ure) showed bullet-shaped hearts (Figure 2(a,b))
which is a typical heart shape for chickens. In contrast,
apparently normal fast-growing broiler chickens with
subclinical DCM (Figure 2(c)) and broilers with clini-
cal signs of heart pump failure and ascites (Figure 2
(d)) showed more spherical-shaped hearts with clear
evidence of chamber dilation. Notably, in clear contrast
to normal hearts, the dilated hearts from both clinical
and subclinical cases appeared flaccid and lacked the
normal tone of healthy heart muscle which is mani-
fested by a collapsing ventricular wall.
On light microscopic evaluation, the hearts from
chickens showing gross signs of DCM and clinical
signs of heart pump failure presented histopathological
evidence of profound degenerative changes (Figure 3).
It is noteworthy that the architecture of the myocar-
dial syncytium was profoundly disturbed which is evi-
denced by disordered and interrupted myofibrils.
Prominent sarcoplasmic eosinophilia with loss of stria-
tions indicated aggregation of damaged proteins in the
cardiomyocytes, whereas pyknosis and karyolysis indi-
cated pathological changes in the nucleus. Taken
together, these changes showed evidence of deteriorat-
ing and dying cardiomyocytes.
The TEM examination revealed profound degenera-
tive changes in the cardiomyocytes of chickens affected
Figure 2. Typical gross anatomy of chicken hearts obtained
from a Leghorn chicken (a), slow-growing broiler chicken (b),
fast-growing broiler chicken with subclinical DCM (c), and a
broiler chicken showing clinical signs of heart pump failure
(d). Cardiac structures are identified as follows; LV (left ventri-
cle), RV (right ventricle), LA (left atrium), and RA (right atrium).
AVIAN PATHOLOGY 431
Figure 3. Left ventricular myocardium from a broiler chicken
with advanced DCM. The affected heart tissue shows signs of
congestion and interstitial oedema (red arrows). Numerous car-
diomyocytes exhibit advanced degenerative changes mani-
fested by prominent sarcoplasmic eosinophilia with loss of
striations (stars), vacuolated sarcoplasm (black arrowheads),
nuclear pyknosis (white arrowheads), and karyolysis (black
arrows). Bar = 25 μm. Colour online.
by DCM. Figure 4 shows representative examples of
ultrastructural features in the cardiomyocytes from
normal chickens and chickens showing signs of DCM
and heart pump failure.
The ultrastructural changes described above in
Figure 4 were qualitatively similar in the cardiomyo-
cytes of all chickens showing post mortem signs of
DCM, but there were differences in magnitude of the
lesions between subclinical and clinical cases.
In chickens with subclinical DCM the lesions were
mostly focal, less pronounced, and confined to a smal-
ler area, whereas in chickens showing clinical signs of
heart pump failure the lesions were more prominent,
multifocal or locally extensive.
Spectral FTIR analyses
Detailed FTIR spectra analyses were performed using
the 2nd derivative. For the purpose of this study, infer-
ences regarding conformational structure of proteins
are based on contrast between the reference spectra
obtained from myocardium from Leghorn chicken
and myocardium from chickens affected by DCM
and clinical signs of heart failure (Figure 5)
It is noteworthy that a normal reference chicken
heart protein spectrum (Figure 5, top panel) showed
predominantly α-helix conformation, whereas the
chicken heart affected by DCM showed a protein spec-
trum indicative of changes consistent with random
coil, β-sheet, β-turns, and aggregate conformations
(Figure 5, bottom panel).
The FTIR spectral analysis provided information on
the spatial distribution of changes in protein confor-
mation in the cardiac muscle of respective risk groups
(Figure 6).
432 A. A. OLKOWSKI ET AL.
Figure 4. Transmission electron micrographs of the left ventri-
cular myocardium from a normal chicken (a) and a broiler
chicken with DCM and ascites (b). The normal chicken myocar-
dium shows morphology of intact sarcomere with clearly deli-
neated typical banding, and mitochondria with well-defined
matrix and cristae. In contrast, the DCM affected myocardium
shows distinct pathological changes in the contractile appar-
atus evidenced by rarified and interrupted myofibrils, and dis-
turbed Z disc banding of the sarcomere (b, white arrow heads).
There is evident degeneration of mitochondrial matrix and cris-
tae (b, white arrows). Noteworthy is the occurrence of vacuolar
auto phagosome (b, black arrow heads) and dense body pha-
gosome structures containing amorphous matter and small
electron dense protein aggregates (b, black arrows). Also note-
worthy are multifocal and locally extensive clusters of electron-
dense amorphous large protein aggregates (b, ovals) which are
not engulfed by autophagy structures. Noteworthy is virtual
absence of dense body auto phagosome structures and elec-
tron dense protein aggregates in normal myocardium. Bar =
1μm.
Notably, the cardiac muscle protein conformation
pattern in Leghorn chickens, a strain of slow-growing
chickens used in the present study as physiological
reference (Figure 6(a)) showed predominant α-helix
conformation (wave number 1648–1660) with only
very minor deflections in the regions of β-sheet and β
turns (wave number 1674–1695). In contrast, all
FTIR tracings obtained from broiler chickens, i.e. a
strain selected for rapid growth (Figure 6(b–d)) showed
various degrees of additional deflections indicative of
changes consistent with random coil, β-sheet, β-
turns, and aggregate conformations (as referenced in
Figure 5). It is noteworthy, however, that in the cat-
egory of broiler chickens, the conformational changes
were clearly discernible even in the slow-growing
group (low risk of heart failure), where growth rate
was controlled by dietary restriction (Figure 6(b)).
These conformational changes were more prominent
in apparently normal, fast-growing broilers with sub-
clinical DCM (Figure 6(c)), and were most pronounced
in hearts from birds showing DCM and clinical signs of
heart pump failure (Figure 6(d)).
Discussion
Commercial broiler chickens genetically selected for
rapid growth are inherently predisposed to heart failure
(for review see Olkowski, 2007). In clinical terms, heart
failure in commercial broilers occurs in an acute form,
where sudden death is associated with catastrophic
ventricular fibrillation (Olkowski & Classen, 1997),
and chronic form, with clinical signs of hypoxaemia,
cyanosis, and ascites typically associated with heart
pump failure (Olkowski et al., 1998, 2001; Olkowski,
Abbott, et al., 2005; Olkowski, Duke, et al., 2005).
The inherent susceptibility of fast-growing broiler
chickens to heart pump failure is well evidenced by
the high prevalence of DCM, both in clinical and sub-
clinical form, observed in commercial broiler popu-
lations on post mortem examination (Olkowski et al.,
1998; 2001; Olkowski, Abbott, et al., 2005; Olkowski,
Duke, et al., 2005; Wilson et al., 2016). Moreover, a
recent cross-sectional study by Gesek et al. (2016) pro-
vided further evidence that many otherwise normal
broiler chicks, in any particular population, present
histopathological changes in the myocardium, and
this indicates that many young fast-growing broiler
chicks are at high risk of developing fulminant DCM
and heart pump failure.
The pathogenesis of heart failure involves the
dynamic and diverse myocardial processes generally
characterised as the pathological remodelling (Fedak
et al., 2005a, b). By definition, cardiac remodelling
involves genome expression resulting in molecular,
cellular and interstitial changes, and manifested as
changes in size, shape and function of the heart
(Cohn et al., 2000). Previous studies based on echocar-
diographic examination provided evidence that DCM
in broiler chickens is associated with ventricular con-
tractile dysfunction (Olkowski, Abbott, et al., 2005;
Deng et al., 2006). The present study provides further
evidence that DCM in broiler chickens is associated
with significant pathological remodelling of the heart,
clearly discernible by change in size, shape, and muscle
tone of the heart. Pathological remodelling is also well
evidenced by microscopic changes, demonstrated on
both light and electron microscopy.
Previously we reported that DCM in broiler chick-
ens is associated with pathological changes in myocar-
dial extracellular matrix (ECM) and structural and
molecular changes in contractile apparatus (Olkowski
et al., 2001). With regard to the changes in ECM, sub-
sequent research has shown that ECM remodelling is a
central process underlying the maladaptive
 AVIAN PATHOLOGY 433

Figure 5. Representative patterns of FTIR spectra, 2nd derivative of amide I bands (1600–1700cm−1) obtained from heart sections
of a Leghorn chicken (top panel) and from heart sections of a broiler chicken showing clinical signs of heart pump failure and DCM
(lower panel). The grey bars below spectra panels indicate respective IR band deflections corresponding to wave numbers charac-
teristic of α-helix: 1648–1660; β-sheet: 1623–1641 and 1674–1695; β turns: 1674–1695; random coil 1642–1657; aggregates 1610–
1630. The reference values for amide I frequency ranges were adopted in principle from Barth & Zscherp (2002); Barth (2007) and
Ganim et al. (2008). We specifically were interested in examining the infrared spectra at the frequency between 1600 and
1700cm−1, because a strong signal in this region refers to the amide linkage between amino acid residues relevant to confor-
mational arrangement of protein molecules.

reorganization of cardiac size, shape, and function
during the progression of heart failure (Fedak et al.,
2005b). Building on our previous observation in
respect to structural and molecular changes in contrac-
tile apparatus (Olkowski et al., 2001), the present study
revealed several important findings that provide a new
perspective on the pathological remodelling of the con-
tractile apparatus.
Of particular interest is the observation from TEM
showing rarified myofibril structures along with inten-
sely disturbed architecture of Z-disc of the sarcomere
(Figure 4(b)), which is consistent with the changes in
the molecular profile of the two key sarcomere pro-
teins, titin and myosin, observed in the previous
study (Olkowski et al., 2001), and provides better
understanding of the pathological remodelling at ultra-
structural level, especially in view of advances in
research on the role of titin in sarcomere insufficiency
in DCM (Haghighi et al., 2015).
An important observation from the present study
that provides a new perspective on pathophysiology
of DCM in chickens selected for rapid growth is that,
in contrast to normal chickens, the cardiomyocytes of
chickens affected by DCM show an abundance of pro-
minent auto phagosome structures. This indicates that
DCM is associated with abnormally elevated cellular
demand for disposal of damaged, unfolded, or mis-
folded proteins. Autophagy is an evolutionary con-
served catabolic process that ensures continuous
removal of damaged cell organelles, damaged,
unfolded, or misfolded proteins, and protein aggregates
in order to maintain cellular homeostasis (Kroemer &
Levine, 2008). In cardiomyocytes, protein homeostasis
(proteostasis) is critically important for the proper
function of the heart pump (Henning & Brundel,
2017). The heart is a functional syncytium, thus the
physiological integrity of every individual cardiomyo-
cyte is critical for the proper function of the heart as
434 A. A. OLKOWSKI ET AL.

Figure 6. Representative randomly selected tracings of the amide I region of FTIR (2nd derivative) from the left ventricular myo-
cardium obtained from Leghorn chicken (a), slow-growing broiler chicken (b), broiler chicken showing subclinical signs of DCM (c),
and broiler chicken showing clinical signs of heart pump failure and marked DCM (d). The panels show stacks of randomly selected
tracings from the map of spectra obtained from respective risk groups in the amide I region (for technical reference see Figure 1).
The pattern of FTIR deflections was similar in all individuals within each group. The most prominent magnitude of spectral changes
noted in broilers vs Leghorns are shaded in grey.

a pump. It has been shown that the disruption of con-
tractile function can be caused by accumulation of
protein aggregates. In human DCM, progression of
heart pump failure is characterized by loss of proteos-
tasis which leads to the formation of aggresomes in car-
diomyocyte cytoplasm. Similar pathophysiology
appears to be associated with DCM and heart failure
in chickens selected for rapid growth. Taken together,
the findings from TEM are consistent with functional
deficiency of the heart in fast-growing broilers relative
to Leghorns, and help in understanding of the mechan-
isms associated with the heart’s contractile dysfunction
observed on echocardiographic examination in broilers
showing signs of DCM (Martinez-Lemus et al., 1998;
Olkowski et al., 2005a; Deng et al., 2006).
Notably, as evidenced in the present study, in
chicken DCM, the affected cardiomyocytes appear to
be loaded with dense body autophagy structures con-
taining small electron-dense protein aggregates, and
extensive clusters of electron-dense amorphous large
protein aggregates which are not engulfed by autop-
hagy structures. The occurrence of protein aggregates
outside of the auto phagosome structure suggests dys-
regulation of proteolytic function in the cardiomyo-
cytes. Taken together, this indicates that the affected
cardiomyocytes are stressed and struggling with the
disposal of damaged proteins. Dysregulation of the
autophagy system and impaired proteasome function
in broiler heart tissue may be among key factors under-
lining predisposition of broilers to heart failure associ-
ated with altered expression of autophagy related
genes. This reasoning is supported by the observation
that inhibition of proteasome induced heart dysfunc-
tion (Herrmann et al. 2013), and the recent study of
Gil-Cayuela et al. (2019) where the authors observed
changes in expression of autophagy related genes in
human cases of DCM.
Impaired proteasome is a significant factor in heart
failure pathogenesis (Wang et al., 2011). We would
argue that a robust proteasome system in chicken car-
diomyocytes may be critically important in maintain-
ing cardiac health, as fast-growing broilers are
continuously subjected to high levels of metabolic
and oxidative stress (Olkowski et al., 2007; Nain
et al., 2008a, b). In the present study, the findings
from TEM analyses indicate that the cardiomyocytes
of fast-growing broilers affected by DCM (both clinical
and subclinical) have difficulty in disposing protein
aggregates. In respect to this, it is reasonable to infer
that fast-growing broiler chickens may be at high risk
of proteasome system insufficiency, because of a mis-
match between high demand for disposal of damaged
proteins on one hand, and deficiency in expression of
the proteasome system on the other. Such reasoning

is well supported by the recent study of Zhang et al.
(2018) where direct comparison of cardiac gene
expression during early development between strains
of slow-growing and fast-growing broiler chickens
revealed lowered expression of genes encoding for the
ubiquitin-proteasome system in a fast-growing strain.
Interestingly, ingenuity pathway analysis in the
study of Zhang et al. (2018) suggested that, in fast-
growing broilers, lipid accumulation, oxidative stress
and endoplasmic reticulum stress may be related to
increased cardiac dysfunctions, increased cell apoptosis,
and inhibited cell cycle. Notably, endoplasmic reticu-
lum stress is associated with the accumulation of mis-
folded and unfolded proteins (Wang et al., 2017)
Recent research indicates that accumulation of mis-
folded proteins is associated with oxidative stress, and a
defective autophagy pathway is associated with
accumulation of proteinaceous aggregates in the form
of aggresomes in the cardiomyocytes (Caragnano
et al., 2019). A perturbed reduction–oxidation homeo-
stasis in the cell can affect protein conformation and
trigger a pathogenic cascade of protein aggregation
(Christians & Benjamin, 2012). In respect to this, it is
noteworthy that oxidative stress is a prominent feature
in the myocardium of fast-growing chickens (Nain
et al., 2008b). Broiler chickens in a commercial setting
are frequently subjected to various forms of environ-
mental stress and metabolic stress associated with
rapid growth. The study by Nain et al. (2008b)
described several biomarkers of oxidative stress in the
heart tissue of fast-growing broilers, and showed that
oxidative stress is a prominent integral component of
DCM pathophysiology. Hence the high level of protein
catabolism observed in the cardiac tissues of broilers
affected by DCM is not surprising, but the accumu-
lation of protein aggregates in broiler cardiomyocytes
observed in the present study indicates impaired clear-
ance of protein damaged by oxidation, as the oxidized
protein aggregates are resistant to proteolytic degra-
dation and obstruct function of the proteasome (Ayya-
devara et al., 2016). Oxidized proteins tend to
precipitate and, if they are not degraded, the conse-
quence is the formation of proteinaceous aggregates
which are cardio-toxic (Willis & Patterson, 2013; San-
dri & Robins, 2014; McLendon & Robbins, 2015). Since
the proteasome plays an important role in the degra-
dation of oxidized proteins (Jung et al. 2014), insuffi-
ciency of this system in the affected cardiomyocytes
will eventually disrupt normal cellular processes by a
wide range of mechanisms (Gilda & Gomes, 2017).
Association of proteinaceous aggregates with DCM
in fast-growing broilers is evidenced by light
microscopy and TEM image analyses, and further
confirmed by FTIR spectroscopy. FTIR spectroscopy
is a powerful technique, which is specifically useful
for studies on the conformational changes in proteins,
and has been widely used to investigate the process of
AVIAN PATHOLOGY 435
protein misfolding and aggregate formation in many
diseases (Miller et al., 2013). This technique was
recently used in the identification of markers for patho-
logical remodelling in tachycardia-induced dilated car-
diomyopathy (Benitez-Amaro et al., 2018) and studies
on protein aggregates in cardiomyocytes (Ami et al.,
2018).
In the present study, the FTIR spectra analyses
revealed conformational changes consistent with
damaged, unfolded or misfolded cardiac proteins,
and more specifically indicative of occurrence of
protein aggregates in the cardiomyocytes. However,
most likely, the conformational changes detected by
FTIR spectral analyses also include more discreet
changes in secondary structure of some cardiac pro-
teins that are not yet apparent on light microscopic
or even TEM examination, but may affect morphology
of the heart and function of the heart pump.
Changes in cardiac muscle protein conformation
indicative of structural disarray, such as random coil,
β turns and aggregate or rigid β-sheet conformation
observed in the present study, provide a plausible
explanation for the gross morphological and functional
changes in the heart of chickens at high risk of heart
pump failure. In particular, characteristic features
such as the loss of cardiac muscle turgor, resulting in
flaccidity of the ventricular wall, are consistent with
the loss of elastic recoil, which is an essential element
of normal heart muscle compliance attributable to α-
helix conformation of major contractile (myosin, tro-
ponin, tropomyosin) and structural (titin) proteins.
In order to perform mechanical work in harmony
with metabolic factors, the contractile proteins in the
cardiomyocyte must be properly configured (Lopes &
Elliot, 2014; Yar et al., 2014). Several recent studies
have emphasized the importance of the secondary
structure of cardiac proteins in the pathogenesis of
heart pump failure. It has been shown that mutation
of cardiac contractile and structural proteins affecting
helical structure are associated with cardiomyopathy
(Voelkel & Linke, 2011; Pinotsis et al., 2012; Preller
& Manstein, 2013; Swindle et al., 2014; Cheng & Reg-
nier, 2016; Pérez-Serra et al., 2016).
Conformational changes in secondary structure of
contractile and structural proteins may affect mor-
phology and function of the heart as the elastic recoil
of cardiac contractile proteins is an essential factor of
cardiac muscle compliance. Notably, the α-helical
conformation provides the elasticity of proteins,
whereas the β-sheet, random coil, disordered turns
and twists, and protein aggregate conformations
would decrease elasticity. Therefore, even small
changes in conformation of the cardiac proteins
towards random coil, β-sheet, β-turns, and aggregate
conformations, as observed in the present study,
may have a significant impact on morphology and
overall contractile performance of the heart. The
436 A. A. OLKOWSKI ET AL.
present FTIR findings help in explaining our previous
observations where heart pump function indices, such
as declining heart rate and lowered fractional shorten-
ing, are clearly discernible in chickens showing subcli-
nical changes of heart morphology, and are prominent
in clinically affected chickens (Olkowski et al., 2001;
Olkowski, Abbott, et al., 2005; Olkowski, Duke, et
al., 2005).
The findings from FTIR analysis provide supportive
evidence that conformational changes of cardiac pro-
teins are intimately linked to the predisposition of
chickens selected for rapid growth to heart failure, as
a similar pattern of conformational changes (differing
only in magnitude) was seen in all groups of broiler
chickens (susceptible to heart failure), but not in Leg-
horn chickens (resistant to heart failure). However, it
is noteworthy that, although the chicken genotype
selected for rapid growth is generally predisposed to
heart pump failure, the development of the clinical
condition is strictly dependent on rapid growth factors,
as the risk of heart failure can be readily abrogated
when the growth rate is controlled by dietary restric-
tion (Olkowski et al., 1998, 2001; Olkowski, Abbott,
et al., 2005; Olkowski, Duke, et al., 2005; Nain et al.,
2008b).
Predisposition of broiler chickens to heart failure
has been attributed to the genetic selection of broilers
for rapid growth and feed conversion efficiency
(Havenstein et al., 1994; Scheele, 1997). However, to
the best of our knowledge no specific genes associated
with heart failure in chickens have been identified,
although, admittedly, there is a dearth of studies on
genetic aspects of heart failure in broilers. While
some genetic mutation cannot be excluded at this
time, it is more likely that the predisposition of chick-
ens selected for rapid growth to heart failure is associ-
ated with polymorphism of the phenotype and
epigenetic modification. Based on our research, we
can argue that the pathophysiology of DCM and
heart failure is strictly associated with physiological
and metabolic mechanisms correlated with rapid
somatic body mass accrual, since controlling growth
by dietary restriction practically eliminates signs of
DCM and the risk of heart failure (Olkowski et al.,
1998, 2001, 2005a, b). Thus, if rapid growth is a prere-
quisite for the development of DCM, then it seems
doubtful that susceptibility of chickens selected for
rapid growth is solely rooted in the genome. On the
other hand, epigenetic modification can react to cues
from the environment, nutrition, and various meta-
bolic pathways. Therefore, induced epigenetic changes
can result in permanent maintenance of the acquired
phenotype, since epigenetic modification is a stable
way of regulating gene expression independent of
changes in the nucleotide sequence, as the regulation
of gene expression occurs at the transcriptional level
via DNA methylation and histone modifications.
Epigenetics may provide a new framework for the
search to unravel the underlying mechanisms predis-
posing chickens selected for rapid growth to heart fail-
ure. In this respect, considering the findings of
Gilsbach et al. (2014) who reported involvement of
demethylation in the titin gene in an experimental
model of heart failure, it is possible that the changes
in molecular weight profile of titin observed in chick-
ens showing signs of DCM and heart failure documen-
ted in our previous study (Olkowski et al., 2001), and
pathological remodelling of the sarcomere observed
in the present study, can be associated with epigenetic
modification of the cardiac titin gene, as well as other
genes. This reasoning is supported by a recent study
by Tabish et al. (2019) who provided further evidence
that development of DCM in a mouse model is associ-
ated with significant epigenetic changes in key signal-
ling pathways involved in pathological cardiac
remodelling and heart contractile dysfunction. Taken
together, our observations, and the findings of others,
suggest that epigenetic modifications may well play
an important role in gene expression, which can be
the underlying cause of the predisposition of chickens
selected for rapid growth to heart failure.
This concept is supported by findings from the
recent study of Zhang et al. (2018) who observed the
lower expression of genes encoding ATP synthase pro-
teins, based on transcriptome differences in the fast-
growing broiler chicken strain in comparison to the
slow-growing strain. Notably, the critically important
metabolic stress factors in the myocardium of fast-
growing chickens, and significant high risk factors in
the pathogenesis of DCM and heart failure, are related
to the insufficiency of high energy substrates and
energy metabolism (Olkowski et al., 2007; Nain et al.,
2008a), and the observations of Zhang et al. (2018)
provide a better understanding of the underlying
cause for insufficiency of a key high energy substrate
ATP. Given that the insufficiency of energy substrate
and metabolism in the hearts of chickens selected for
rapid growth can be readily abrogated by controlling
growth rate by dietary restriction (Olkowski et al.,
2007; Nain et al., 2008a), and in the context of data
provided by Zhang et al. (2018), we would argue that
the apparent downturn in ATP synthase gene
expression in the fast-growing strain of broiler chick-
ens is most likely associated with epigenetic
modification.
In summary, the findings from the present study
indicate that DCM and heart pump failure in fast-
growing broiler chickens is a result of a complex meta-
bolic syndrome involving multiple pathways. Our data
indicate that a good deal of DCM and heart pump fail-
ure pathophysiology, in chickens selected for rapid
growth, is associated with conformational changes of
cardiac proteins, most likely resulting from posttran-
slational modification, oxidative damage, unfolding

or misfolding, and protein aggregate accumulation. It is
evident that the affected cardiomyocytes demonstrate
significant difficulty in disposal of damaged proteins
and maintenance of proteostasis, which leads to patho-
logical remodelling of the heart and contractile dys-
function. It appears that the underlying causes of
accumulation of damaged proteins are associated
with dysregulated auto phagosome and proteasome
systems, which, in susceptible individuals, create a
milieu conducive for the development of DCM. Future
research to unravel the underlying factors predisposing
fast-growing chickens to heart failure should explore
epigenetic modification of gene expression and post-
translational modifications of cardiac proteins.
Acknowledgements
This research was funded by grants provided by the Poultry
Industry Council and NSERC. FTIR analyses were per-
formed at the Canadian Light Source, which is funded by
the Canada Foundation for Innovation, the Natural Sciences
and Engineering Research Council of Canada, the National
Research Council Canada, the Canadian Institutes of Health
Research, the Government of Saskatchewan, Western Econ-
omic Diversification Canada, and the University of
Saskatchewan.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the Natural Sciences and Engin-
eering Research Council of Canada (NSERC) [grant number
CRDPJ – 311700-04 ]; Poultry Industry Council [grant num-
ber 164].
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