Requirement of protein kinase D1 for pathological cardiac remodeling.
ABSTRACT The adult heart responds to biomechanical stress and neurohormonal signaling by hypertrophic growth, accompanied by fibrosis, diminished pump function, and activation of a fetal gene program. Class II histone deacetylases (HDACs) suppress stress-dependent remodeling of the heart via their association with the MEF2 transcription factor, an activator of heart disease. Protein kinase D (PKD) is a stress-responsive kinase that phosphorylates class II HDACs, resulting in their dissociation from MEF2 with consequent activation of MEF2 target genes. To test whether PKD1 is required for pathological cardiac remodeling in vivo, we generated mice with a conditional PKD1-null allele. Mice with cardiac-specific deletion of PKD1 were viable and showed diminished hypertrophy, fibrosis, and fetal gene activation as well as improved cardiac function in response to pressure overload or chronic adrenergic and angiotensin II signaling. We conclude that PKD1 functions as a key transducer of stress stimuli involved in pathological cardiac remodeling in vivo.
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ABSTRACT: An increase in the mass of left ventricular muscle is intimately associated with most chronic diseases of the heart.1- 6 Classically, left ventricular hypertrophy, which represents an extreme increase in left ventricular mass, has been thought to represent a reaction to pressure or volume overload.7- 8 In the short run, increases in left ventricular mass may be beneficial by allowing the heart to compensate for increased wall stress and potential hemodynamic compromise; in the long run, left ventricular hypertrophy is harmful.8JAMA The Journal of the American Medical Association 12/2004; 292(19):2396-8. · 30.03 Impact Factor
Requirement of protein kinase D1 for pathological
Jens Fielitz*, Mi-Sung Kim*, John M. Shelton†, Xiaoxia Qi*, Joseph A. Hill†, James A. Richardson*‡,
Rhonda Bassel-Duby*, and Eric N. Olson*§
Departments of *Molecular Biology,†Internal Medicine, and‡Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, TX 75390-9148
Contributed by Eric N. Olson, December 27, 2007 (sent for review December 14, 2007)
The adult heart responds to biomechanical stress and neurohor-
monal signaling by hypertrophic growth, accompanied by fibrosis,
diminished pump function, and activation of a fetal gene program.
Class II histone deacetylases (HDACs) suppress stress-dependent
remodeling of the heart via their association with the MEF2
transcription factor, an activator of heart disease. Protein kinase D
(PKD) is a stress-responsive kinase that phosphorylates class II
HDACs, resulting in their dissociation from MEF2 with consequent
activation of MEF2 target genes. To test whether PKD1 is required
for pathological cardiac remodeling in vivo, we generated mice
with a conditional PKD1-null allele. Mice with cardiac-specific
fibrosis, and fetal gene activation as well as improved cardiac
function in response to pressure overload or chronic adrenergic
and angiotensin II signaling. We conclude that PKD1 functions as
a key transducer of stress stimuli involved in pathological cardiac
remodeling in vivo.
cardiac hypertrophy ? histone deacetylase ? stress-responsive kinase
stresses, such as increased cardiac afterload due to hypertension,
aortic valve stenosis, or acute myocardial infarction. Cardiomy-
ocytes respond to such stress stimuli by increasing cell size,
reorganizing sarcomeres to enhance contractility and activating
a fetal cardiac gene program (1–3). Although these responses
may initially normalize wall stress, prolonged hypertrophy in-
creases the risk for chamber dilation, heart failure and sudden
death (4, 5). A complex set of signaling pathways and down-
stream transcription factors underlie these responses of the heart
to acute and chronic injury (6).
Class II histone deacetylases (HDACs) function as negative
regulators of pathological cardiac remodeling through associa-
tion with the myocyte enhancer factor-2 (MEF2) transcription
factor and other prohypertrophic transcriptional regulators (7–
9). Mice lacking either HDAC5 or HDAC9 are sensitized to
cardiac stress (7, 9), whereas mice lacking MEF2D display an
impaired response to stress signals that normally lead to hyper-
trophy, fibrosis, and fetal gene activation (10). Manipulation of
the HDAC-MEF2 axis may therefore impact the prognosis and
outcome of heart disease.
The actions of class II HDACs are controlled by signal-
dependent phosphorylation (11–14). When unphosphorylated,
class II HDACs localize to the nucleus where they associate with
MEF2 and silence MEF2 target genes. Extracellular stimuli
transmitted through G protein-coupled receptors activate pro-
tein kinase D (PKD) and Ca2?/calmodulin-dependent kinases
(CaMKs), which phosphorylate class II HDACs, triggering their
nuclear export, relieving MEF2 repression, and promoting
pathological cardiac remodeling (13–15).
Cardiac PKD is activated in response to hypertension, pres-
sure overload, and chronic neurohormonal signaling (15–19).
Knockdown of PKD1 expression with siRNA blunts agonist-
dependent hypertrophy, whereas in vivo cardiac-specific expres-
he adult heart undergoes left ventricular hypertrophy and
myocardial remodeling when subjected to pathological
sion of constitutively active PKD1 causes a brief phase of cardiac
hypertrophy, followed by chamber dilation and impaired systolic
function and death (16).
To further define the functions of PKD1 in the heart, we
generated mice with a cardiac-specific mutation of the Prkcm
gene, which encodes PKD1. Mice lacking cardiac PKD1 display
an impaired response to stress signals that normally lead to
cardiac hypertrophy, fibrosis and fetal gene activation. These
findings demonstrate that PKD1 activity plays a key role in
mediating stress-dependent remodeling and reprogramming of
gene expression in the adult heart.
Cardiac-Specific Deletion of PKD1. Because PKD1 is expressed in
numerous tissues (20), we used the Cre-loxP recombination
system to generate a conditional PKD1 (Prkcm) allele that could
be deleted specifically in cardiomyocytes. LoxP sites were in-
serted into the Prkcm locus to flank exons 12 through 14, which
encode part of the catalytic domain of PKD1, including the ATP
binding motif that is essential for kinase function (Fig. 1A).
Expression of Cre recombinase results in deletion of the region
between the loxP sites, eliminating the function of PKD1 as a
kinase. To determine the outcome of a complete loss of function
of PKD1, we deleted the gene using a CAG-Cre transgene, which
expresses Cre recombinase in the embryo at the zygote stage
(21). The homozygous PKD1 mutant allele caused embryonic
lethality with incomplete penetrance, so we generated a car-
diomyocyte-specific deletion of PKD1 using ?-MHC-Cre trans-
genic mice (22) that express Cre recombinase specifically in
Mice with cardiac-specific deletion of PKD1 (referred to as
PKD1 cKO) were indistinguishable from their WT littermates.
Deletion of the genomic region between the loxP sites was
confirmed by PCR of genomic DNA (Fig. 1B). RT-PCR of RNA
from adult heart showed that exon 11 was spliced to exon 15 in
the mutant allele, resulting in a frame-shift within the coding
region of the PKD1 mRNA (Fig. 1C). Western blot analysis of
indicating that the truncated mRNA was unstable or the result-
ing protein product was rapidly degraded (Fig. 1D). Quantifi-
cation of PKD1 mRNA by real-time RT-PCR revealed a 5-fold
reduction of PKD1 mRNA in PKD1 cKO hearts (Fig. 1E). The
observed residual expression of PKD1 mRNA most likely re-
flects PKD1 expression in fibroblasts, endothelial, smooth mus-
cle, and immune cells within the heart. Quantification of Prkcm2
Author contributions: J.F., R.B.-D., and E.N.O. designed research; J.F., M.-S.K., J.M.S., X.Q.,
J.A.H., and J.A.R. performed research; J.F., J.M.S., X.Q., and J.A.R. contributed new re-
agents/analytic tools; J.F., J.A.R., R.B.-D., and E.N.O. analyzed data; and J.F., R.B.-D., and
E.N.O. wrote the paper.
The authors declare no conflict of interest.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
February 26, 2008 ?
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(encoding PKD2) and Prkcn (encoding PKD3) mRNA expres-
sion in PKD1 cKO hearts showed no compensatory up-
regulation of these genes (Fig. 1E).
PKD1 cKO Mice Are Resistant to Hypertrophy and Fibrosis in Response
to Pressure Overload.TheheartsofWTandPKD1cKOmicewere
comparable in size in the absence of stress (Fig. 2 A and B).
Twenty-one days after thoracic aortic constriction (TAC), WT
mice showed a 47% increase in heart weight/tibia length (HW/
TL) with pronounced thickening of the left ventricular free wall
and interventricular septum (Fig. 2 A and B). In contrast, PKD1
cKO mice showed only a 23% increase in HW/TL with a minimal
increase in wall thickness. Pressure overload hypertrophy in WT
mice is accompanied by extensive fibrosis of the ventricular wall,
as detected by Masson’s trichrome staining (Fig. 2A). There was
a dramatic reduction in Masson’s trichrome staining of cKO
hearts compared with WT littermates (Fig. 2A).
Cardiac Function in PKD1 cKO Mice. At baseline, no significant
differences were seen in the left ventricular end-diastolic diam-
eter (LVIDd), left ventricular end-systolic diameter (LVIDs),
heart rate, or fractional shortening (FS) between WT and PKD1
cKO mice, as measured by echocardiography (Fig. 2C and Table
in LVIDs accompanied by a pronounced reduction in cardiac
contractility, as indicated by decreased FS. In contrast, PKD1
cKO animals were remarkably resistant to left ventricular dila-
tion and its concomitant decrease in contractility (Fig. 2C and
Table 1). Additionally, WT animals experienced a reduction
in heart rate, indicative of cardiac demise, which was not
observed in PKD1 cKO mice. These data demonstrate that
PKD1 is required for a maximal cardiac remodeling response
and functional deterioration of the heart in response to pressure
Fetal Gene Activation Is Blunted in PKD1 cKO Hearts in Response to
TAC. PKD1 was also essential for maximal fetal gene activation
in response to TAC. Up-regulation of the hypertrophic gene
markers, atrial natriuretic factor (ANF), brain natriuretic peptide
PKD1 locus and targeting strategy. LoxP sites were inserted in the introns
kinase domain including the ATP-binding motif. The neomycin resistance
achieved by breeding PKD1loxP/loxPmice to either CAG-Cre or ?-MHC-Cre
and circled. (B) PCR genotyping to distinguish PKD1 alleles. PCR products
corresponding to WT (151 bp), PKD1loxP(loxP) (255 bp), and PKD1 KO (359 bp)
are shown. The positions of the primers that produce these PCR products are
labeled b and c for WT and PKD1loxPand a and d for PKD1 KO and are circled
in A. (C) RT-PCR to detect WT and mutant PKD1 transcripts. The PKD1 mutant
allele lacks exons 12, 13, and 14. GAPDH was detected as a loading control.
Locations of primers used for RT-PCR are shown on the left. (D) Western blot
analysis of PKD1 in cardiac extracts from WT and PKD1 mutant mice. GAPDH
protein was used as a loading control. (E) Expression of PKD1 transcripts
detected by quantitative PCR. Total RNA isolated from ventricles of 8-week-
old male mice was used for cDNA synthesis and subsequent quantitative PCR
(n ? 6 for each genotype). P ? 0.01. Error bars indicate ?SEM.
Generation of mice with a conditional PKD1 mutation. (A) Mouse
WT and PKD1 mutant mice subjected to either a sham operation (WT and
PKD1 cKO, n ? 6) or TAC (Top; WT, n ? 12; PKD1 cKO, n ? 11). Histological
sections stained with H&E (Middle) or Masson’s trichrome to detect fibrosis
(Bottom). (Scale bars: Top and Middle, 2 mm; Bottom, 40 ?m.) (B) Heart
weight/tibia length (HW/TL) ratios (?SEM) of WT and PKD1 cKO mice were
determined 21 days after TAC. (C) PKD1 cKO mice display less left ventricular
dilation during systole (LVIDs) and a less pronounced decrease in fractional
shortening (FS) in response to TAC than WT mice.
www.pnas.org?cgi?doi?10.1073?pnas.0712265105 Fielitz et al.
(BNP) and myosin, heavy polypeptide 7, cardiac muscle, beta
of procollagen, type I, alpha 2 (Col1a2), which is up-regulated
during cardiac fibrosis, was also compromised in PKD1 cKO
mice. Baseline expression of fetal cardiac genes was unaltered in
PKD1 cKO mice, suggesting that deletion of PKD1 does not
itself impose a stress on the heart.
PKD1 cKO Mice Are Resistant to Angiotensin II-Dependent Hypertro-
phy. PKD1 is activated by angiotensin II (AngII) in vitro (23). We
therefore examined whether PKD1 was necessary for cardiac
hypertrophy in response to chronic AngII stimulation by adminis-
tering AngII to mice over 14 days using osmotic minipumps (3.0
mg/kg/day). Compared with WT mice, which showed a 16%
in HW/TL (Fig. 4 A and B). There was also a reduction in fibrosis
as seen by Masson’s trichrome staining of cKO hearts compared
Col1?2 expression was also compromised in PKD1 cKO mice (Fig.
effects of AngII on the heart. However, because the dose of AngII
used in these experiments has been shown to induce hypertension
(24, 25), we cannot distinguish whether the loss of PKD1 in the
heart prevents left ventricular hypertrophy by specifically antago-
nizing AngII signaling in cardiomyocytes or by preventing hyper-
trophy in response to hypertension.
PKD1 cKO Mice Are Resistant to Isoproterenol-Dependent Hypertro-
phy. In contrast to Ang II, isoproterenol (ISO), a ?-adrenergic
agonist, does not activate PKD1 in vitro (16). To examine
whether PKD1 is necessary for cardiac hypertrophy in response
to ?-adrenergic stimulation, ISO was administered to mice over
7 days by using osmotic minipumps (8.7 mg/kg/day). Unexpect-
edly, we observed that the hearts of PKD1 cKO mice showed
only a 21% increase in HW/TL compared with the 37% increase
in WT mice (Fig. 5 A and B) and diminished expression of
stress-response genes (Fig. 5C). Thus, contrary to in vitro find-
ings, these results suggest that PKD1 is necessary for cardiac
hypertrophy in response to chronic ?-adrenergic stimulation in
vivo. It is possible that chronic long-term administration of ISO
indirectly activates other pathways such as the ?-adrenergic
The results of this study demonstrate that PKD1, the major
PKD isoform in the heart (19), is a critical component of the
signaling pathways through which pressure overload, AngII,
and adrenergic signaling drive pathological cardiac remodel-
ing. PKD is a potent kinase for class II HDACs (15, 16), which
function as signal-responsive repressors of cardiac hypertro-
phy, at least in part through their repressive influence on
MEF2 (7–9). These findings provide the first evidence that
deletion of a class II HDAC kinase in vivo diminishes stress-
induced hypertrophy. The blunted hypertrophic response of
Table 1. Echocardiographic analysis of PKD1 mutant mice after TAC
AnimalLVIDd, mmLVIDs, mm FS, %HR, min?1
3.10 ? 0.1
3.92 ? 0.15
3.48 ? 0.1
3.31 ? 0.28
1.15 ? 0.11
1.92 ? 0.29
1.24 ? 0.09
1.26 ? 0.26
63.2 ? 2.5
51.7 ? 5
64.1 ? 2.3
63.1 ? 4.6
660 ? 27
635 ? 35
660 ? 17
720 ? 15
0.73 ? 0.05
1.05 ? 0.1
0.83 ? 0.03
0.99 ? 0.05
end-systolic diameter; FS, fractional shortening; HR, heart rate; PWd, posterior wall thickness in diastole.
Transcripts for markers of hypertrophy in hearts from WT and PKD cKO mice
were detected by quantitative PCR 21 days after TAC (n ? 3–9 per group).
Values indicate relative expression level to a WT sham-operated group
(?SEM). ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; ?-MHC,
?-myosin heavy chain; Col1a2, procollagen, type I, ?2.
Diminished fetal gene activation in PKD1 cKO mice after TAC.
sion. (A) Hearts of WT and PKD1 cKO mice treated with either saline vehicle
(WT and PKD1 cKO, n ? 6) or AngII (3.0 mg/kg per day) for 14 days (Top; WT,
n ? 9; PKD1 cKO, n ? 8), histological sections stained with H&E (Middle), or
Masson’s trichrome to detect fibrosis (Bottom). (Scale bars: Top and Middle, 2
mm; Bottom, 40 ?m.) (B) Heart weight/tibia length (HW/TL) ratios (?SEM) are
shown as bar graphs (n ? 7–9). (C) Transcripts for markers of hypertrophy in
hearts from WT and PKD cKO mice treated with either saline vehicle or AngII.
Values indicate relative expression level to a WT sham-operated group
(?SEM). ANF, atrial natriuretic factor; ?-MHC, ?-myosin heavy chain; Col1?2,
procollagen, type I, ?2.
Diminished hypertrophic response of PKD1 cKO mice to AngII infu-
Fielitz et al.
February 26, 2008 ?
vol. 105 ?
no. 8 ?
PKD1 cKO mice is similar to that of mice lacking MEF2 (10),
further substantiating the hypertrophic signaling pathway from
PKD1 to MEF2 in vivo.
Signal-Dependent Control of PKD. The canonical pathway for
activation of PKD involves PKC-mediated phosphorylation of
from repression by the amino-terminal pleckstrin homology
(PH) domain (26). Ca2?-independent, nonconventional PKCs,
the dominant regulators of PKD activity, have been linked to
cardiomyocyte hypertrophy and death (17, 18, 27, 28).
In cultured cardiomyocytes, PKD1 is activated by a subset of
stress stimuli, such as AngII and phenylephrine but not by
isoproterenol (16, 23). Surprisingly, however, both AngII- and
isoproterenol-induced cardiac hypertrophy was blunted in mice
harboring the PKD1 deletion. The apparent requirement of
PKD1 for cardiac hypertrophy in response to isoproterenol in
vivo might reflect secondary signals leading to PKD1 activation,
which do no exist after short-term exposure of cultured cardi-
omyocytes to isoproterenol.
Potential Redundancy of PKD Isoforms. PKD1 is one of three PKD
family members that share homology in two amino-terminal
cysteine-rich domains that mediate binding to diacylglycerol, an
internal PH domain and carboxyl-terminal catalytic domains
(29). Each PKD isoform is capable of phosphorylating the class
II HDACs 4, 5, 7, and 9 on the serines that mediate nuclear
export via 14–3-3, suggesting the potential for redundant control
of class II HDACs by PKD family members. Indeed, siRNA
knockdown of PKD1 expression in cultured cardiomyocytes
blunts but does not eliminate HDAC5 nuclear export (15). In
addition, disruption of both PKD1 and PKD3 is necessary to
block HDAC5 phosphorylation in response to antigen receptor
signaling in chicken B lymphocytes (30). The blunted hypertro-
phic response of PKD1 cKO mice indicates that PKD2 and
PKD3 cannot fully compensate for the loss of PKD1. The
residual hypertrophy and fetal gene activation in PKD1 cKO
as other stress-responsive kinases such as CaMK. In this regard,
deletion of calcineurin and its target transcription factor NFAT
also blunts, but does not abolish hypertrophy (31, 32) further
suggesting redundancy in hypertrophic signaling pathways.
Additional Cardiac Functions of PKD. PKD has also been implicated
in the phosphorylation of cardiac troponin I (33, 34) and
numerous other substrates (19). Interestingly, PKD phosphory-
lates the same sites in troponin I as PKA, resulting in reduced
myofilament Ca2?sensitivity (34), whereas other PKA sites in
phospholamban and cardiac myosin-binding protein C are not
targeted by PKD. However, for the majority of PKA targets that
play a role in excitation–contraction coupling, such as the
ryanodine receptor and the L-type calcium channel, it is un-
known whether PKD is capable of phosphorylating their PKA
sites and thereby regulating their activity.
Circumstantial evidence also suggests a role for PKD in the
control of cardiac fibrosis. The profibrotic mineralocorticoid,
aldosterone, can activate PKD (35), and PKD signaling was
recently shown to stimulate aldosterone production in adrenal
cells through up-regulation of aldosterone synthase (36). These
findings suggest that PKD contributes to a positive-feedback
loop that promotes cardiac fibrosis.
The activation of PKD by diverse stimuli that lead to
pathological cardiac remodeling, its activation in failing human
hearts, ability to drive hypertrophy and heart failure in trans-
genic mice (17, 18), and its apparent requirement for a
maximal hypertrophic remodeling response in mice, as shown
in the present study, point to PKD as a promising therapeutic
target for cardiac hypertrophy and heart failure. PKD has also
been implicated in myocardial responses to ischemia, angio-
genesis, and platelet activation (19), pointing to the potential
of PKD inhibitors as therapeutics for diverse cardiovascular
Materials and Methods
Generation of PKD1 Knockout Mice and Animal Experiments. Details of mouse
information (SI) Materials and Methods.
Surgical Manipulations and Echocardiography. Methods for TAC and implan-
tation of AngII and Iso pumps are described in SI Materials and Methods.
Histology. Methods for histology are described in SI Materials and Methods.
RNA analysis. Methods for RNA analysis are described in SI Materials and
between groups were analyzed by Mann–Whitney U test or two-sided Stu-
dent’s t test. Statistics were calculated with Excel and SPSS software. A P value
?0.05 was considered to be statistically significant.
(A) Hearts of WT and PKD1 cKO mice chronically infused with either saline
vehicle (WT and PKD1 cKO; n ? 6) or isoproterenol (8.7 mg/kg per day) for 7
days (Top; n ? 11; PKD1 cKO, n ? 16), histological sections stained with H&E
(Middle) or Masson’s trichrome to detect fibrosis (Bottom). (Scale bars: Top
and Middle, 2 mm; Bottom, 40 ?m.) (B) Heart weight/tibia length (HW/TL)
hypertrophy in hearts from WT and PKD cKO mice infused with either saline
vehicle or isoproterenol. Values indicate relative expression level to a WT
heavy chain; Col1?2, procollagen, type I, ?2.
Diminished hypertrophic response of PKD1 cKO mice to ISO infusion.
www.pnas.org?cgi?doi?10.1073?pnas.0712265105 Fielitz et al.
ACKNOWLEDGMENTS.We thank M. Avkiran and T. McKinsey for comments
on the manuscript, M. D. Schneider (Baylor College of Medicine, Houston,
TX) for the ?-MHC-Cre mouse line, and K. Song for helpful discussions.
E.N.O. was supported by grants from the National Institutes of Health, the
Donald W. Reynolds Center for Clinical Cardiovascular Research, and
the Robert A. Welch Foundation. J.F. was supported by a fellowship from
the Muscular Dystrophy Association and the Pfizer Fellowship of the
German Society of Cardiology.
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