(Received for publication, November 20, 1995; and in revised form, February 23, 1996)
From the
Dystrophin serves a variety of roles at the cell membrane through its associations, and defects in the dystrophin gene can give rise to muscular dystrophy and genetic cardiomyopathy. We investigated localization of cardiac dystrophin to determine potential intracellular sites of association. Subcellular fractionation revealed that while the majority of dystrophin was associated with the sarcolemma, about 35% of the 427-kDa form of dystrophin was present in the myofibrils. The dystrophin homolog utrophin was detectable only in the sarcolemmal membrane and was absent from the myofibrils as were other sarcolemmal glycoproteins such as adhalin and the sodium-calcium exchanger. Extraction of myofibrils with KCl and detergents could not solubilize dystrophin. Dystrophin could only be dissociated from the myofibrillar protein complex in 5 M urea followed by sucrose density gradient centrifugation where it co-fractionated with one of two distinctly sedimenting peaks of actin. Immunoelectron microscopy of intracellular regions of cardiac muscle revealed a selective labeling of Z-discs by dystrophin antibodies. In the genetically determined cardiomyopathic hamster, strain CHF 147, the time course of development of cardiac insufficiency correlated with an overall 75% loss of myofibrillar dystrophin. These findings collectively show that a significant pool of the 427-kDa form of cardiac dystrophin was specifically associated with the contractile apparatus at the Z-discs, and its loss correlated with progression to cardiac insufficiency in genetic cardiomyopathy. The loss of distinct cellular pools of dystrophin may contribute to the tissue-specific pathophysiology in muscular dystrophy.
The dystrophin gene encodes a 427-kDa protein in muscle and
brain tissues. Absence of this full-length form of dystrophin causes
severe, progressive muscle weakness in patients with Duchenne's
or Becker's muscular
dystrophies(1, 2, 3) . These patients may
also experience dilated cardiomyopathy, which in some cases may be much
more severe than the myopathy(37, 38, 39) .
The precise physiological role of dystrophin is not fully known
although, due to its subsarcolemmal localization, it has been proposed
to serve a variety of functions such as providing stability to the
membrane and signal
transduction(4, 9, 10, 14) .
Dystrophin forms a tight association with a glycoprotein complex
embedded in the plasma membrane(5, 6, 7) .
One of these glycoproteins, -dystroglycan, has been shown to bind
to the G-domains of laminin (8) and agrin to mediate
clustering of acetylcholine receptors(9, 10) . The
association of a pool of subsarcolemmal dystrophin with laminin in the
extracellular matrix via the membrane glycoproteins may be vital for
linking the cytoskeleton to the extracellular matrix, and the
disruption of this complex leads to increased membrane
fragility(4, 9, 10) . Alterations in
components of the dystrophin-glycoprotein-laminin complex, such as the
laminin-
2 gene product, and a 50-kDa dystrophin-associated
glycoprotein, adhalin, are associated with muscle membrane instability
and muscular dystrophy(11, 12, 13) . A
deficiency in adhalin and disruption of the membrane
dystrophin-glycoprotein complex has also been reported in the
cardiomyopathic hamster that experiences both hereditary cardiomyopathy
and myopathy(14) .
In addition to its role in membrane
stability and receptor clustering, recent studies imply that dystrophin
serves a unique role in signal transduction by localizing nitric-oxide
synthase to the sarcolemma (15) . In order to elucidate the
cellular roles of dystrophin, it is paramount to precisely define its
associations. Although it is well documented that dystrophin localizes
at the plasma membrane in a variety of
cells(16, 17, 18, 19) , studies
indicate that there may be tissue- and cell-specific differences in
dystrophin localization. For example, recent studies using confocal
imaging (20, 21, 22, 23) and
immunogold labeling (23) have shown that in addition to the
sarcolemma, a substantial pool of dystrophin also localizes at the
transverse tubules in cardiac muscle. Furthermore, cardiac dystrophin
was found to be distributed in three distinct subcellular pools, a
cytoplasmic pool, a membrane-bound pool not associated with
WGA()-binding glycoproteins, and a membrane-bound pool
associated with WGA-binding glycoproteins(20, 22) . In
view of the notion that dystrophin may serve diverse roles through its
associations, here we demonstrate a selective pool of dystrophin
molecules that associate with the myofibrils and localize to the
Z-discs in cardiac muscle. Furthermore, the specific loss of
myofibrillar dystrophin during progression to cardiac insufficiency in
the cardiomyopathic hamster CHF 147 suggests that dystrophin may serve
a unique role at the level of the sarcomere in addition to its recently
described membrane functions. The distinct pools of dystrophin and
their selective loss may be of significance in explaining the
tissue-specific pathophysiological responses in muscular
dystrophy(24) .
Subcellular fractions from fresh rabbit hearts were isolated as
described under ``Experimental Procedures.'' SDS-PAGE
analysis of the protein composition of the different fractions obtained
during myofibril isolation is shown in Fig. 1A. The
cardiac homogenate (lane H) was centrifuged to obtain a
supernatant fraction (lane S1) and a pellet fraction (lane
P1). The pellet was washed extensively with buffer II to obtain
supernatant (lane S2) and pellet (lane P2). Fraction
P2 was further extracted with Triton X-100 to obtain supernatant (lane S3) and pellet (lane P3). The final product
represented isolated myofibrils (MF). The MF fraction
consisted predominantly of myosin (200 kDa) and actin (42 kDa). Other
cytoskeletal proteins, such as -actinin (105 kDa) and desmin (55
kDa) were also present in this fraction.
Figure 1:
Presence of dystrophin in cardiac
myofibrils. A, Coomassie Blue staining of SDS gels
representing cardiac fractions obtained during isolation of myofibrils.
Each lane was loaded with 60 µg of proteins, and the proteins were
separated on a 6.25% SDS-polyacrylamide gel. H, homogenate; S1, supernatant fraction of the homogenate (buffer I wash); P1, pellet of the homogenate; S2, supernatant of the
buffer II wash; P2, pellet of the buffer II wash; S3,
supernatant of the Triton X-100 (buffer III) wash; P3, pellet
of the Triton X-100 wash; MF, the isolated myofibrils washed
with the suspension buffer. Standard protein markers (bands from the top to the bottom represent 211, 117, 81, 49, and 31
kDa, respectively). B, immunoblots of cardiac fractions
obtained from A. Panel a, anti--actinin antibody
(1:20,000) and anti-desmin ab (1:1,000); Panel b,
anti-Na
-Ca
exchanger antibody
(1:1000); Panel c, anti-dystrophin antibody (1:1000). The
mobility of myosin (My),
-actinin (
-A),
actin (Ac), desmin (Des),
Na
-Ca
exchanger (Na-Ca) and
dystrophin (Dys) is indicated.
The protein composition of
the myofibril preparation was further examined by immunoblotting with
various antibodies directed against known myofibrillar proteins and
dystrophin. Gels with identical sample loading to that shown in Fig. 1A were electroblotted onto nitrocellulose and
immunostained with the various antibodies (Fig. 1B).
Anti-desmin and anti--actinin staining indicated that these
proteins co-fractionated in a similar manner. The pellets of the washes
and the isolated myofibrils stained strongly for desmin and
-actinin (Fig. 1B, panel a, lanes P1, P2, P3, and MF), whereas the supernatant fractions had no
detectable desmin signal and stained only faintly for
-actinin (Fig. 1B, lanes S1, S2, and S3). Staining with
anti-dystrophin antibodies was readily detectable in the cardiac
homogenate, and it became stronger in the myofibrillar pellets obtained
after extraction with buffer I and buffer II (Fig. 1B, panel
c, lanes P1 and P2). In contrast to this, there was
little dystrophin staining found in the corresponding supernatant
fractions (Fig. 1B, lanes S1 and S2).
Furthermore, the nonionic detergent, Triton X-100, failed to extract a
significant amount of dystrophin from the pellet fraction (Fig. 1B, lane S3). After the Triton X-100
extractions the isolated myofibrils still contained a significant
amount of dystrophin staining (Fig. 1B, lane
MF).
To identify any cross-contamination of the myofibril
fractions with the sarcolemmal membrane, we investigated the presence
of the Na-Ca
exchanger which is a
transmembrane glycoprotein. Immunostaining with cardiac
Na
-Ca
exchanger antibody revealed
that some of this plasma membrane marker remained in the myofibril
pellet during the first two washes of the isolation procedure (Fig. 1B, panel b, lanes P1 and P2).
However, this residual membrane glycoprotein was completely removed
from the pellet fraction by extraction with Triton X-100 (Fig. 1B, lanes S3 and P3). This
immunostaining pattern for the various polypeptides was consistent in
all 18 different myofibril preparations examined.
Figure 2:
Presence of dystrophin in cardiac
sarcolemmal and myofibrillar fractions. A, Coomassie Blue
staining of the myofibrillar and sarcolemmal fractions obtained from
the same rabbit heart. Each lane was loaded with 5 µg of proteins,
and samples were separated on a 6.25% SDS-polyacrylamide gel. H, homogenate; S1, supernatant fraction of the
homogenate (buffer I) wash; P1, pellet of the homogenate; MF, the isolated myofibrils; C, crude cardiac
sarcolemma; SL, purified cardiac sarcolemma. Standard protein
markers (bands from the top to the bottom represent
211, 117, 81, 49, and 31 kDa, respectively). B, immunoblots of
cardiac myofibrillar and sarcolemmal fractions. Each lane was loaded
with 60 µg of protein, and samples were separated on a 6.25%
SDS-polyacrylamide gel and transferred to nitrocellulose for Western
blotting. Panel a, anti--actinin antibody (1:5000); Panel b, anti-desmin antibody (1:1000); Panel c,
anti-Na
-Ca
exchanger antibody
(1:1,000); anti-dystrophin antibody (1:1000). Dys, dystrophin; NaCa, Na
-Ca
exchanger; Des, desmin;
-A,
-actinin; My,
myosin. The data are typical of four independent
experiments.
As observed
with the previous fractionation procedure, desmin and -actinin
staining was intense in the myofibrillar fraction (Fig. 2B, lane MF) when compared with the
homogenate or sarcolemmal fractions. Immunostaining for the
Na
-Ca
exchanger revealed a 120-kDa
band in the crude sarcolemmal fraction (Fig. 2B, lane C) that was significantly stronger in the purified
sarcolemmal fraction (Fig. 2B, lane SL). A
70-kDa band also present with the 120-kDa band in the SL fraction was
most likely a degradation product of the
Na
-Ca
exchanger(32) .
Na
-Ca
exchanger staining was
undetectable in the myofibrillar fraction (Fig. 2B, lane MF). These findings indicate that the modified protocol
worked well and gave a clear separation of the sarcolemmal and
myofibrillar fractions.
Immunoblotting of the various fractions
obtained using the modified protocols revealed the presence of the
427-kDa form of dystrophin in the purified sarcolemma (Fig. 2B, lane SL) and in the myofibrillar
fraction (Fig. 2B, lane MF). Although the
intensity of dystrophin staining was slightly lower in the myofibrillar
fraction than in the purified sarcolemma, we reasoned that
quantitatively it may represent a substantial fraction of the total
dystrophin content of cardiac muscle. Western blots of the fractions
shown in Fig. 2B were therefore digitally scanned to
quantitate the signal intensity of the immunostaining per lane, and
this was normalized to the microgram of protein loaded per lane. These
values were then multiplied by the total amount of protein present in
each fraction to determine the relative amount of dystrophin,
Na-Ca
exchanger, desmin, and
-actinin present in the homogenate and the various fractions
prepared from it. The percent of dystrophin present in the membrane and
myofibrillar fractions was compared with the various markers. The
results show that approximately 35% ± 5 of the total dystrophin
present in the homogenate was recovered in association with the
myofibrillar fraction, whereas the recovery of desmin and
-actinin
in this fraction was calculated to be 88% ± 17 and 80% ±
18, respectively (n = 4). The total amount of
homogenate dystrophin that was recovered in the sarcolemmal fraction
was approximately 55%, compared with a 90% recovery of the
Na
-Ca
exchanger.
Dystrophin is known to localize at the sarcolemma in association with membrane glycoproteins. We examined the glycoprotein composition of the MF and SL fractions in a WGA overlay reaction (Fig. 3a). WGA staining of glycoproteins of various molecular mass was detected in the homogenate (lane H) and crude membranes (lane C), and this intensified in the purified sarcolemmal membranes (lane SL). There was weak WGA staining detected in the pellet (lane P1) of the homogenate of glycoproteins of 160, 220, and 300 kDa, and these were seen to enrich in the myofibrils (lane MF) purified from this pellet. These glycoproteins did not appear in the soluble fraction (lane S) or the crude (lane C) and purified membranes (lane SL). Immunoblotting with an antibody against adhalin, a 50-kDa dystrophin-associated sarcolemmal glycoprotein (Fig. 3b), revealed that while this glycoprotein was detectable in the homogenate (lane H) and enriched in the sarcolemmal membrane (lane SL), it was absent from the myofibrils (lane MF).
Figure 3: Distribution of glycoproteins in cardiac fractions. Panel a, WGA overlay and panel b, Western blotting with adhalin (Ad) antibodies of cardiac myofibrils (MF), sarcolemma (SL), crude membranes (C), and supernatant (S1) and pellet (P1) isolated from the same heart homogenate (H). Arrowheads indicate glycoproteins of 160, 220, and 300 kDa enriched in MF. The data are typical of two independent experiments.
Figure 4:
Effect of salt extraction on myofibrillar
dystrophin. Protein patterns (panel a) and immunoblots (panel b) of the fractions obtained from the isolation of
myofibrils with or without a KCl wash. Isolated myofibrils (MF) without KCl extraction; supernatant (S) and
pellet (P) obtained after KCl extraction of MF. Dys,
dystrophin. Dystrophin antibody was applied at a dilution of 1:500. The
data are typical of three independent experiments. My, myosin;
-A,
-actinin; Ac,
actin.
The dissociation of dystrophin
from myofibrils was further examined by solubilizing the myofibrillar
fraction in 5 M urea, followed by separating the proteins in a
5-30% continuous sucrose density gradient. Protein assay
indicated that more than 85% of total myofibrillar protein remained
soluble in the urea buffer after centrifugation at 100,000 g for 1 h. Western blot analysis confirmed that the dystrophin
was in the soluble fraction (data not shown). The protein patterns of
the various fractions obtained from the sucrose gradient fractionation
are shown in Fig. 5a. Myosin,
-actinin, and actin
were the major components in the sucrose gradient fractions. A single
myosin peak was clearly separated from a single
-actinin peak.
Actin appeared in two distinct peaks in the gradient. The first actin
peak (Fig. 5a, Fractions 14-19) was located near
the top of the sucrose gradient. This represented most of the actin
present in the myofibrillar fraction. A second smaller peak was
localized near the bottom of the sucrose gradient (Fig. 5a, Fractions 6-8). This peak did not
precisely correspond to the myosin peak, although it substantially
overlapped it. The lower actin peak did, however, precisely correspond
to a single dystrophin peak in the gradient (Fig. 5, a and b, Fractions 6-8).
Figure 5:
Sedimentation of myofibrillar dystrophin
in a sucrose density gradient. Isolated myofibrils were solubilized in
urea and separated in a 5-30% sucrose density gradient. Numbers
at the bottom of the figure indicate the fractions collected
from the bottom (lane 1) to the top (lane 23) of the
sucrose gradient. Coomassie Blue staining (a) and
immunoblotting (b) of proteins in the various fractions was
examined. Dystrophin antibody was applied at a dilution of 1:500. My, myosin; -A,
-actinin; Ac,
actin; Dys, dystrophin. The data are a typical of four
independent experiments.
Figure 6:
Dystrophin in skeletal and cardiac muscle.
Coomassie Blue staining of SDS gels (a) and immunoblotting (b) of cardiac (c) and skeletal (s) muscle
homogenate (H), myofibrils (MF), and sarcolemma (SL). Dystrophin antibody was applied at a dilution of 1:500. Dys, dystrophin; My, myosin; -A,
-actinin; Ac, actin. The data are typical of three
independent experiments.
Figure 7: Distribution of utrophin and dystrophin in the heart. All fractions were from the same rabbit heart. Each lane was loaded with 60 µg of proteins, and samples were separated on a 6.25% SDS-polyacrylamide gel. H, homogenate; S3, supernatant fraction of the Triton X-100 wash; MF, isolated myofibrils; C, crude cardiac sarcolemma; SL, purified cardiac sarcolemma. Dystrophin (Dys) antibody was applied at a dilution of 1:500. Monoclonal antibody against utrophin (Utr) was applied at a dilution of 1:500. The data are typical of three independent experiments.
Figure 8:
Localization of dystrophin at the
sarcomere monitored with immunogold 5-µm labeling on ultrathin
cryosections. A typical of 50 micrographs generated from four different
hearts is shown. Gold label (black dots) is seen along the Z
line with some labeling present at the sarcolemma. 58,750
.
Figure 9: Immunoblot analysis of dystrophin expression in normal and cardiomyopathic hamster heart. Subcellular fractions were isolated from cardiomyopathic CHF 147 strain (M) and normal hamster hearts (C) and separated in SDS-PAGE and analyzed in Western blots with affinity purified dystrophin antibodies. Lane S, sarcolemma; lane H, heart homogenate; lanes MF, myofibrils. Identical amounts of protein (40 µg) per lane were loaded between the myopathic and control groups. The results shown are typical of two to three independent experiments from groups of animals at day 180.
Figure 10: Evidence for myofibrillar dystrophin loss in the development of cardiomyopathy. A, myofibrils were prepared from hearts of cardiomyopathic hamsters (M) and normal control (C) hearts at ages 30, 60, 120, and 180 days and analyzed in Western blots with affinity purified dystrophin antibodies. B, Western blots were quantified by densitometry, and dystrophin content was plotted as a function of age in the normal (C dys) and cardiomyopathic (M dys) hamster. The desmin content in normal (C des) and cardiomyopathic (M des) hamsters on the same blot was also quantified by Western blotting and densitometry at the various ages shown. The results are typical of two to three independent experiments performed on two to three different groups of animals.
The present results demonstrate that a distinct pool of dystrophin molecules associated with the myofibrillar fraction and localized to the Z-discs in cardiac muscle. Whereas a majority of the dystrophin is localized at the surface membrane in association with glycoproteins, our results show that dystrophin at the Z-discs exists in the absence of any of these glycoproteins. Myofibrillar localization of dystrophin was exclusive to cardiac muscle, and its loss correlated with the development of cardiac insufficiency in the genetically determined cardiomyopathic hamster.
Dystrophin was tightly
associated with the cardiac myofibrillar fraction that was enriched in
cytoskeletal proteins such as -actinin and desmin. While
dystrophin associated with the sarcolemmal membrane was soluble in
detergents such as digitonin, Nonidet P-40, Triton X-100, and
deoxycholate as previously
reported(22, 40, 41, 42) , these
treatments failed to extract dystrophin from cardiac myofibrils. High
concentrations of KCl solubilized approximately 30% of the protein
content of isolated cardiac myofibrils, but dystrophin was not present
in the soluble fraction. Dissociation of dystrophin from other proteins
in the myofibrillar fraction could only be achieved by solubilization
of myofibrils in buffer containing 5 M urea followed by
separation of dissolved proteins on a sucrose density gradient. On the
sucrose gradient, the peak of myofibrillar dystrophin followed a
pattern different from the myosin and
-actinin peaks, but it
coincided with one of two distinct actin peaks, suggesting that
myofibrillar dystrophin may be associated with actin. Dystrophin is
known to bind actin via its N-terminal
domain(43, 44, 45) . Various isoforms of
actin are expressed by mature striated muscle. In addition to
muscle-specific actin present in the thin filaments, actin is also
present in the Z-disc and in the cytoskeletal scaffold surrounding
myofibrillar bundles(46) . Further work is therefore required
to determine the significance of the existence of two distinct pools of
actin in the myofibrillar fraction and the selective association of
dystrophin with one of these pools. Given our present observation of
immunogold labeling of Z-discs with dystrophin antibody in cardiac
muscle, the pool of actin cofractionating with dystrophin on the
sucrose gradient may represent Z-disc actin.
The tight association of cardiac dystrophin with the myofibrillar fraction suggests that dystrophin may play a unique role in the structure or function of the cardiac myofibril and that this pool of dystrophin may associate with proteins that are distinct from those of the sarcolemmal membrane dystroglycan complex. Accordingly, we found that adhalin, the 50-kDa dystrophin-associated glycoprotein, was present only in the sarcolemmal and not the myofibrillar fractions. The pool of myofibrillar dystrophin cannot therefore be associated with any of the glycoproteins found in the dystroglycan complex as is the case for a sarcolemmal pool of dystrophin. The model for the structure of the dystrophin-containing membrane cytoskeletal complex suggests that dystrophin is anchored in this complex via actin at its N-terminal domain and to one or more components of the membrane glycoprotein complex via its C-terminal domain. It will therefore be of interest to determine whether actin alone anchors dystrophin so tightly to the cardiac myofibrils or whether an additional dystrophin-binding protein is present in the Z-discs. In this regard dystrophin has been shown to localize nitric-oxide synthase to the sarcolemmal membrane and proposed to serve a role in nitric oxide signaling(15) . Whether dystrophin at the Z-disc can anchor such signaling molecules at the sarcomere is now under investigation. The origin and the nature of glycoproteins of 160, 220, and 300 kDa in the myofibrillar fraction are intriguing. These glycoproteins could not be extracted with detergents and high salt. Whether dystrophin can associate with any of these glycoproteins to anchor them at the Z-disc remains to be investigated. The dystrophin-related protein, utrophin, was exclusively distributed in the sarcolemmal membrane fraction of cardiac muscle and was absent from the myofibrillar fraction. This is consistent with the immunohistochemical localization of utrophin in heart sarcolemma(47) . The fact that utrophin was absent from the cardiac myofibrillar fraction argues against artificial redistribution of dystrophin during the subcellular fractionation procedure. The expression of substantial amounts of utrophin in normal adult cardiac muscle is another noteworthy difference between cardiac and skeletal muscle(29) .
Our finding that about 35% of the total cardiac
muscle content of the 427-kDa form of dystrophin is associated with
myofibrils points to a unique role for dystrophin in the
structure/function of the cardiac myofibril. It is evident that
dystrophin is a component not only of the membrane cytoskeleton but
also of the intracellular cytoskeletal network that organizes
myofibrils at the level of the Z-disc. It is of interest that the most
frequent cardiac abnormality observed in dystrophin deficiency is
dilated cardiomyopathy(37, 38, 39) , a
condition associated with cytoskeletal disorganization as well as
irregular sarcomeric and Z-disc structures(48) . The
cardiomyopathic hamster that exhibits autosomal recessive
cardiomyopathy and experiences muscular dystrophy has been widely used
as a model system. While the precise genetic defects remain to be
defined, several biochemical abnormalities associated with
Ca overload have been noted(49) . The CHF 147
strain of hamsters experience dilated cardiomyopathy and have been
shown to exhibit age-dependent morphological alterations in the
sarcomeric and Z-disc arrangements that correlate with the decrease in
contractile function and progression to cardiac insufficiency (33, 34, 35, 36) . Our results
indicating the loss of myofibrillar dystrophin during the time course
of these cardiac derangements suggest that dystrophin at the Z-disc may
play an essential role in maintaining structural integrity, and its
loss may contribute to the more severe cardiomyopathy compared with the
milder muscular dystrophy experienced by these animals. Furthermore,
the time-dependent increase in myofibrillar dystrophin paralleled the
enhanced contractile performance documented for the normal hamster
heart(36) , implying that dystrophin serves an important role
in the contractile apparatus. It should be noted that the expression of
desmin was essentially unaltered during the progression of cardiac
insufficiency in the CHF 147 hamster heart, and this is consistent with
results obtained in human-dilated cardiomyopathy(48) . The CHF
147 hamster is related to the BIO 14.6 cardiomyopathic strain that has
been reported to completely lack adhalin expression in both cardiac and
skeletal muscle with either no change or only a slight decrease in
membrane dystrophin levels(14, 50) . The lack of
adhalin has been suggested to result in disruption of the membrane
dystrophin-glycoprotein complex in the cardiomyopathic hamster BIO 14.6
strain(14, 50) . We also noted the absence of adhalin
in the CHF 147 hamster heart as early as day 30, prior to the onset of
any symptoms (data not shown), while the membrane pool of dystrophin
was reduced by only 28% at much later stages (i.e. day 180) of
the disease. The expression of another membrane cytoskeletal protein,
spectrin, was also unaltered in the cardiomyopathic hamster
heart(50, 51) . While the absence of adhalin may
contribute to cardiomyopathy and myopathy in these animals, our studies
show that it is the loss of dystrophin from the cardiac myofibrils that
correlates with progression to cardiac insufficiency. We suggest that
alterations in unique cellular pools of dystrophin in cardiac muscle
may underlie some of the differential pathophysiological responses of
cardiac and skeletal muscle observed in muscular dystrophy.