Arezu Jahani-Asl

Marc Germain

Ruth S Slack
email: rslack@uottawa.ca

Mitochondria: Joining Forces to Thwart Cell Death

Mitochondria are highly dynamic organelles that undergo constant cycles of fusion and fission. An additional level of regulation of mitochondrial function, which is particularly important in neurons, is their active transport along microtubules. Recent evidence suggests that the mitochondrial fusion/fission machinery, as well as the molecular motors responsible for their movement, constitute powerful regulatory control points that directly impact metabolism and regulation of cell death. This is true for not only apoptosis, but also for excitotoxicity where calcium overload is a major component of the cell death process. In this review, we will describe the molecular mechanisms regulating fusion and fission and how this impinges on cell survival in the context of acute neuronal injury.

A C C E P T E D M A N U S C R I P T Introduction

As the powerhouse of the cell, mitochondria play a crucial role in energy metabolism. In addition, recent research has highlighted other aspects of mitochondrial function that play key roles in cell survival and death [1,2]. Mitochondria are highly dynamic organelles that undergo constant cycles of fusion and fission. This dynamic behaviour of mitochondria is required for proper mitochondrial function and distribution and plays an important role in cell death. Mitochondrial dynamics also impinge on a second important mitochondrial function, calcium buffering, which regulates signal transduction pathways, metabolism and cell death. An additional level of regulation, which is particularly important in neurons, is the active transport of mitochondria along microtubules. These processes regulate not only normal function and distribution of mitochondria but also several types of cell death such as apoptosis and excitotoxicity. Below, we describe the molecular mechanism regulating fusion and fission and their impact on cell survival in the context of acute neuronal injury.

Mechanisms that regulate mitochondrial fusion and fission

In healthy cells, mitochondrial fusion and fission are ongoing processes that are essential for development and survival (reviewed in [1,2]). For example, fission is required during cell division to properly distribute mitochondria to daughter cells [3,4] while fusion allows rapid mixing of mitochondrial content in order to buffer localized substrate deficits or complement damaged mitochondrial DNA [5,6]. In neurons, mitochondrial fission is essential for mitochondrial transport to their potential docking sites in axons and dendrites and is tightly associated with development of spines and synapses [7,8]. In addition, mitochondrial dynamics play a role in the proper recruitment of mitochondria to strategic locations such as areas of high metabolic demand. This is especially important in neurons because of their peculiar morphological characteristics. At the molecular level, three key GTPases mediate mitochondrial fusion and fission events in mammalian cells (Figure 1). These include mitofusins (MFN1/2), Optic Atrophy 1 (OPA1) and Dynamin Related Protein 1 (DRP1) which are briefly described below (for a recent review, see [9].

Mitofusins MFN1 and MFN2 are integral outer mitochondrial membrane (OMM) proteins that regulate OMM fusion. Although MFN1 and MFN2 have overlapping functions, they are also believed to have distinct roles [10][11][12]. MFN1 is mainly involved in the docking of two juxtaposed mitochondria while MFN2 could potentially act as a regulatory GTPase [11][12][13][14]. Specific roles for MFN2 include modulation of metabolism by affecting the electron transport chain [15,16] and tethering of endoplasmic reticulum to mitochondria [17]. Of note, GTP hydrolysis by MFN2 is not required for fusion as a hydrolysis deficient mutant of MFN2 (MFN2 RasG12V ) can still induce mitochondrial fusion [13,14]. In addition, point mutations in MFN2 are associated with Charcot Marie Tooth Neuropathy type II, a peripheral nerve neuropathy causing progressive muscle weakness and muscular atrophy [18].

OPA1 OPA1, an intermembrane space protein associated with the inner mitochondrial membrane (IMM), regulates IMM fusion as well as cristae structure [19][20][21]. There are eight OPA1 splice variants as well as several proteases that can cleave its transmembrane domain, suggesting a complex mode of regulation. [22][23][24]. Recent studies have also

A C C E P T E D M A N U S C R I P T

demonstrated that both its full-length (L-OPA1) and cleaved (S-OPA1) forms are required for IMM fusion [23,24]. The functional importance of OPA1 was highlighted by the discovery that mutations in OPA1 lead to a severe neuropathy, autosomal dominant optic atrophy (ADOA), characterized by a progressive degeneration of optic nerve, moderate to severe loss of visual acuity, and abnormalities of colour vision [25,26].

DRP1 DRP1 is a key regulator of mitochondrial fission that is recruited to fission sites along mitochondria and is actively involved in the scission process. Recruitment of DRP1 to mitochondria is regulated by several post-translational modifications such as phosphorylation and SUMOylation [27]. DRP1 plays an essential role in several aspects of mitochondrial function. For example, in neurons, DRP1-mediated fission allows distribution of mitochondria along dendrites and is essential for synapse development [8,28,29].

Mitochondrial dynamics and cell death

As outlined above, mitochondria play a vital role in cellular homeostasis, while regulation of their dynamics is central to proper mitochondrial function. Paradoxically, mitochondria also play a key role in the regulation of cell death, and mitochondrial dynamics constitute an integral part of the pathways leading to the demise of the cell. In the central nervous system, two major types of cell death occur following acute injury, apoptosis and excitotoxicity, both of which are sensitive to the modulation of the fission/fusion machinery.

Classical Apoptotic Cell death

Classical apoptotic cell death is a tightly regulated process controlled by members of the BCL-2 family of proteins. Induction of apoptosis by intracellular signals such as DNA damage or ER stress leads to the activation of the pro-apoptotic BCL-2 homologues BAX and BAK, which promote cytochrome c release from mitochondria and subsequent activation of caspases, the apoptotic proteases. Apoptosis plays an important role in injury-induced neuronal death. Indeed, biochemical hallmarks of apoptosis have been observed in cells following both focal and global ischemia [30,31] while animals overexpressing BCL-2 are more resistant to stroke [32,33]. One important apoptotic trigger following neuronal injury is the reactive oxygen species (ROS) that are generated during hypoxia/reperfusion. ROS cause DNA damage, leading to p53-dependent activation of apoptosis [34][35][36] Multiple studies have substantiated a link between mitochondrial dynamics and the classical apoptotic pathway. For example, in neuronal cells, mitochondria undergo excessive fission in response to ROS, DNA damage, and NO, which can be prevented by the expression of MFN2 or a dominant-negative form of DRP1 (K38A) [14,37]. Dominant-negative DRP1 also rescued cell survival under these conditions, indicating that Drp1-mediated mitochondrial fission is an early event contributing to the collapse of mitochondria and cell death. Similarly, loss of MFN1, MFN2 or OPA1 leads to mitochondrial fragmentation and increased sensitivity to cell death stimuli [20,38,39], while overexpression of these proteins leads to better survival in several cell types including neurons [14,37]. Importantly, in the absence of injury, loss of MFN2 results in

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT
neuronal loss in vitro [14], and degeneration of purkinje neurons in vivo [40], highlighting the importance of mitochondrial fusion proteins in neuronal survival. At the molecular level, two potentially overlapping mechanisms have been proposed to explain the importance of fission during apoptosis. First, apoptotic DRP1-dependent mitochondrial fragmentation is associated with opening of the cristae structure and mobilisation of cytochrome c, which can then be rapidly released upon OMM permeabilisation [41,42]. Consequently, interfering with mitochondrial fission using a dominant-negative DRP1 prevents the reorganization of the cristae, dampening cytochrome c release and caspase activation [41]. Second, several components of the fission/fusion machinery have been associated with the regulation of BAX/BAK activation. For example, DRP1 and MFN2 have been found in puncti with BAX and BAK [43]. In addition, overexpression of MFN2 or the MFN2 RasG12V mutant inhibits BAX activation and protects against cytochrome c release in cell lines and primary neurons [13,14]. MFN2 has also been suggested to interact with the anti-apoptotic BCL-2 homologue BCL-X L and its c elegans homologue CED9 [44], although the significance of this interaction for cell survival has not been addressed. Other proteins involved in mitochondrial fusion/fission also interact with BAX. For example, Endophilin B1 (Bif-1, SH3GLB1), a protein of the endophilin family that plays a role in membrane dynamics, has been shown to regulate both mitochondrial dynamics and activation of BAX [45,46]. Interestingly, the cross-talk between mitochondrial dynamics and BCL-2 homologues seems to go both ways. Indeed, BCL-X L was shown to enhance mitochondrial dynamics and increase mitochondrial biomass, leading to increased synapse formation in cultured primary neuron [44,47,48]. Similarly, deficiency of both BAX and BAK in the BAX/BAK double knockout (DKO) cells caused fragmentation of the mitochondrial network. This defect was mapped to alterations in the regulation of MFN2 [49], as BAX promoted the recruitment of MFN2 into foci associated with fission/fusion sites. Of note, a constitutively active, GTP-bound form of Mfn2 (MFN2 RasG12V ) reinstated fusion in MFN2 knockout but not DKO cells where it also failed to redistribute to fission/fusion foci [49]. Similar clusters containing BAX/BAK, MFN2 and DRP1 have previously been described in apoptotic cells and associated with the cytochrome c release activity of BAX. It is therefore possible that the enhanced anti-apoptotic activity of MFN2 RasG12V relates to its inability to be recruited to the apoptotic BAX foci, which could interfere with the activation of BAX within these foci. However, this remains to be tested, as other models of BAX activation suggest that BAX-induced cytochrome c release is driven by much smaller BAX oligomers that directly permeabilize the OMM [50][51][52].

Excitotoxic cell death

Apoptosis is an important mode of cell death in most mammalian tissue during development and in the context of cellular damage. However, a second mode of cell death, excitotoxicity, is a major cause of neuronal loss following acute brain injury. Excitotoxicity is caused by an excessive synaptic release of the neurotransmitter glutamate, causing the overactivation of glutamate receptors. Several downstream pathways then lead to a BAX/BAK-independent form of cell death with characteristics of both apoptosis and necrosis (reviewed in [53,54]). For example, activation of NO synthase by PSD95 downstream of the NMDA receptor leads to the generation of highly reactive peroxynitrites, causing cellular damage and cell death. Excessive calcium influx

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

following NMDAR overactivation also activates several potentially deleterious signalling cascades. A major effect of calcium overload is the alteration of mitochondrial function. Mitochondria act as a calcium buffer following calcium release from the endoplasmic reticulum or through cell surface receptors [55], but also respond to calcium influx by increasing their metabolism [56]. However, if mitochondrial calcium increases over a certain value, a channel freely permeable to water and ions, the permeability transition pore (PTP), is open [57]. Opening of the PTP causes swelling and rupture of the OMM in a process that is completely distinct from BAX/BAK-dependent OMM permeabilisation. This leads to the disruption of energy production and release of cytotoxic factors, such as Apoptosis-Inducing Factor (AIF) and EndoG, from mitochondria. These factors then converge on the nucleus, causing DNA fragmentation and cell death. In addition, calcium-dependent activation of µ-calpain promotes the release of AIF from mitochondria [58,59]. Calcium overload-induced mitochondria dysfunction also leads to the formation of ROS, causing further cellular damage.

As with apoptosis, components of the fission/fusion machinery influence excitotoxic cell death. For example, NO-induced toxicity in primary cortical neurones involves mitochondrial fragmentation and is prevented by overexpression of MFN1 or dominantnegative DRP-1 [37]. Similarly, overexpression of MFN2 or OPA1 protected neurons from NMDA-induced cell death (Jahani-Asl et al, submitted). However, neither downregulation of DRP1 or overexpression of MFN1 rescued NMDAR overactivationinduced toxicity [37], suggesting that mitochondrial fusion alone might not be sufficient and that other pathways, such as calcium handling by mitochondria, might be involved. Links between calcium and mitochondrial structure and function are well documented. For example, sustained calcium release from the ER cause DRP1-dependent mitochondrial fragmentation [60], while Miro, a mitochondrial protein containing a EFhand calcium binding domain, regulates mitochondrial transport in a calcium dependent manner. Interestingly, MFN2 has recently been shown to tether mitochondria and ER, affecting ER calcium content [17]. The close association between ER and mitochondria is indeed thought to be critical for calcium handling. A possible link between mitochondrial calcium and OPA1 activity was also recently highlighted by the observation that LETM1, a mitochondrial EF-hand-containing protein, affects OPA1 processing and mitochondrial structure [61]. Calcium-induced modulation of OPA1 activity could potentially help preserve mitochondrial structure at higher calcium load by maintaining cristae structure.

In addition to the influence of the core fission/fusion machinery, mitochondrial movements play an important role in calcium handling. Mitochondria are transported in an anterograde fashion along microtubules by the motor protein Kinesin-1, which binds to mitochondria through an adaptor complex formed of Miro and Milton [62,63]. In the presence of high calcium levels, Kinesin-1 is dissociated from microtubules through the action of calcium-bound Miro, concentrating mitochondria at sites of high calcium concentration. This serves two purposes: it allows increased buffering of calcium by mitochondria and also increases local ATP production to help pump calcium out of the cell. The significance of this was highlighted by the observation that a Miro mutant that did not respond to calcium increased NMDA toxicity. Overexpression of Miro can also increase mitochondrial length, which could further help buffering calcium [63]. However, as mitochondrial fission is required for proper distribution of mitochondria to synaptic terminals, the balance between fission, fusion and mitochondrial movements rather than

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

activation of one or the other is likely to be the critical readout. This is exemplified by the mitochondrial phenotype in Alzheimer's disease neurons, where both fission and fusion proteins are affected, resulting in a reduced axonal distribution of mitochondria [64] . In that context, expression of DRP1 improved synaptic function following Amyloid-ß treatment by promoting the redistribution of mitochondria to synapses.

Concluding Remarks

In summary, a strong link has been uncovered in recent years between cell death and mitochondrial dynamics. Several questions remain however, especially with regards to excitotoxicity where the interplay between fission/fusion and mitochondrial movements is likely to play a critical role. For example, it still remains to be determined whether fusion per se is protective, or if other cytoprotective roles of fusion proteins (such as signalling functions) are the dominant factor. The balance between fission and fusion also needs to be taken in account, as fission is required to distribute mitochondria to their proper location within the cell. Given these caveats, modulation of these pathways will likely have far-reaching consequences beyond the regulation of calcium signalling described here, but that warrant further research. ) are located on the outer mitochondrial membranes (OMM) and regulate fusion of the OMM. Mfns interact on apposing mitochondria to mediate their tethering (Arrow 1). This results in OMM fusion (Arrow 2). OPA1, an intermembrane space GTPase which is found in association with the inner mitochondrial membrane (IMM), mediates fusion of IMM (Arrow 3). (B) The key regulator of mitochondrial fission is Dynamin Related Protein 1 (DRP1). DRP1 is a cytosolic GTPase which is recruited to the mitochondria at the site of scission (Arrow 1). DRP1 forms a ring like structure on the mitochondria surface. The energy from the GTP hydrolysis drives constriction of the membranes (Arrow 2).

ACCEPTED MANUSCRIPT

A C C E P T E D M A N U S C R I P T

Figure 1 .

 1 Figure 1. Regulation of mitochondrial fission/fusion(A) Mitofusins (Mfn1 and Mfn2) are located on the outer mitochondrial membranes (OMM) and regulate fusion of the OMM. Mfns interact on apposing mitochondria to mediate their tethering (Arrow 1). This results in OMM fusion (Arrow 2). OPA1, an intermembrane space GTPase which is found in association with the inner mitochondrial membrane (IMM), mediates fusion of IMM (Arrow 3). (B) The key regulator of mitochondrial fission is Dynamin Related Protein 1 (DRP1). DRP1 is a cytosolic GTPase which is recruited to the mitochondria at the site of scission (Arrow 1). DRP1 forms a ring like structure on the mitochondria surface. The energy from the GTP hydrolysis drives constriction of the membranes (Arrow 2).

Acknowledgement

We would like to thank Drs. Heidi McBride and Melissa Kelly for critical reading of this manuscript. We are also grateful to the Life Side (especially Delphie Dugal-Tessier and Matthew Andrusiak) for providing the title. This work is supported by a grant from HSFO to RSS. AJA is holds a CIHR studentship. MG holds a HSFO research fellowship.


PhD Nicola J A Scott
email: nicola.scott@otago.ac.nz

Leigh J Ellmers

John G Lainchbury

Nobuyo Maeda

Oliver Smithies

A Mark Richards

Vicky A Cameron

Npr-1 signaling in cardiac development 1 INFLUENCE OF NATRIURETIC PEPTIDE RECEPTOR-1 ON SURVIVAL AND CARDIAC HYPERTROPHY DURING DEVELOPMENT

Keywords: Akt1, atrial natriuretic peptide, calcineurin A, cardiac hypertrophy, Connexin 43, gene expression, heart development, Npr1, transcription factors

The heart adapts to an increased workload through the activation of a hypertrophic response within the cardiac ventricles. This response is characterized by both an increase in the size of the individual cardiomyocytes and an induction of a panel of genes normally expressed in the embryonic and neonatal ventricle, such as atrial natriuretic peptide (ANP). ANP and brain natriuretic peptide (BNP) exert their biological actions through activation of the natriuretic peptide receptor-1 (Npr1). The current study examined mice lacking Npr1 (Npr1 -/-) activity and investigated the effects of the absence of Npr1 signaling during cardiac development on embryo viability, cardiac structure and gene and protein expression. Npr1 -/-embryos were collected at embryonic day (ED) 12.5, 15.5 and neonatal day 1 (ND 1). Npr1 -/- embryos occurred at the expected Mendelian frequency at ED 12.5, but knockout numbers were significantly decreased at ED 15.5 and ND 1. There was no indication of cardiac structural abnormalities in surviving embryos. However, Npr1 -/-embryos exhibited cardiac enlargement (without fibrosis) from ED 15.5 as well as significantly increased ANP mRNA and protein expression compared to wild-type (WT) mice, but no concomitant increase in expression of the hypertrophy-related transcription factors, Mef2A, Mef2C, GATA-4, GATA-6 or serum response factor (SRF). However, there was a significant decrease in Connexin-43 (Cx43) gene and protein expression at mid-gestation in Npr1 -/-embryos. Our findings suggest that the mechanism by which natriuretic peptide signaling influences cardiac development in Npr1 -/-mice is distinct from that seen during the development of pathological cardiac hypertrophy and fibrosis. The decreased viability of Npr1 -/-embryos may result from a combination of cardiomegaly and dysregulated Cx43 protein affecting cardiac contractility.

INTRODUCTION

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) regulate blood pressure by vasodilation, by increasing vascular permeability resulting in a net movement of fluid out of the vascular compartment and by modulating the rate of diuresis and natriuresis through the kidneys [START_REF] Holmes | Renal, endocrine and haemodynamic effects of human brain natriuretic peptide in normal men[END_REF][START_REF] Espiner | Natriuretic hormones[END_REF]. During development the natriuretic peptide system is functional by mid-gestation, and is capable of responding to volume stimuli, regulating blood pressure and salt and water balance in the developing embryo [START_REF] Lang | The effect of intravenous saline loading on plasma levels of brain natriuretic peptide in man[END_REF][START_REF] Jaekle | Hemodynamic and hormonal responses to atrial distension in the ovine fetus[END_REF]. Circulating levels of ANP have been shown to be higher in the fetus than in adults, and fetal ventricles express higher levels of both ANP and BNP than adult ventricles [START_REF] Cameron | The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta[END_REF][START_REF] Kingdom | Atrial natriuretic peptide in the fetus[END_REF]. Both ANP and BNP are expressed within the myocardium from ED 8.0 and are two of the first markers of heart formation [START_REF] Christoffels | Chamber formation and morphogenesis in the developing mammalian heart[END_REF][START_REF] Zeller | Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis[END_REF].

After birth, ANP is down regulated in the ventricles, and in the adult expression in the atria is 1000-fold higher than in the ventricles. Re-activated ventricular ANP expression occurs in response to cardiac stress and serves as the most highly conserved marker of hypertrophy [START_REF] Day | Ventricular atriopeptin: unmasking of mRNA and peptide synthesis by hypertrophy or dexamethasone[END_REF]. In association with the reappearance of ANP expression in the adult ventricle, cardiac contractile protein genes, normally only active during cardiac development, are also re-expressed leading to the identification of the embryonic gene program characteristic of pathological ventricular hypertrophy [START_REF] Lattion | Myocardial recruitment during ANF mRNA increase with volume overload in the rat[END_REF][START_REF] Takayanagi | Synthesis and presence of atrial natriuretic factor in rat ventricle[END_REF].

Transgenic mouse models lacking the natriuretic peptide receptor (NPR)-A gene (Npr1 -/-) have been widely used to illustrate the essential role of natriuretic peptide signaling in suppressing the development of cardiac hypertrophy and fibrosis on cardiac function and survival [START_REF] Oliver | Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A[END_REF][START_REF] Oliver | Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner[END_REF][START_REF] Knowles | Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice[END_REF][START_REF] Kuhn | Progressive cardiac hypertrophy and dysfunction in atrial natriuretic peptide receptor (GC-A) deficient mice[END_REF][START_REF] Ellmers | Ventricular expression of natriuretic peptides in Npr1(-/-) mice with cardiac hypertrophy and fibrosis[END_REF]. These studies have focussed on the effect of disrupted natriuretic peptide signalling in adult animals only. Further investigations utilizing these and other animal models have characterized the molecular mechanisms by which natriuretic peptide signaling mediates the hypertrophic response in a variety of pathological states [START_REF] Ellmers | Ventricular expression of natriuretic peptides in Npr1(-/-) mice with cardiac hypertrophy and fibrosis[END_REF][START_REF] Lopez | The guanylyl cyclase-deficient mouse defines differential pathways of natriuretic peptide signaling[END_REF][START_REF] Molkentin | 2nd, Cytoplasmic signaling pathways that regulate cardiac hypertrophy[END_REF][START_REF] Vellaichamy | Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A[END_REF].

Furthermore, fetal models of cardiovascular pathophysiology suggest the natriuretic peptide system in the fetal heart and circulation may regulate both physiological fluid balance and cellular development within cardiomyocytes [START_REF] Walther | Dual natriuretic peptide response to volume load in the fetal circulation[END_REF]. Both immune and non-immune fetal hydrops are associated with higher than normal fetal concentrations of circulating ANP [START_REF] Nimrod | Atrial natriuretic peptide production in association with nonimmune fetal hydrops[END_REF], while fetal cardiac distress is associated with increased BNP expression reminiscent of heart failure in the adult [START_REF] Itoh | Brain natriuretic peptide levels in the umbilical venous plasma are elevated in fetal distress[END_REF]. Despite this, few studies have investigated natriuretic peptide signaling during the process of heart development itself.

A large number of studies have identified the critical role of natriuretic peptide signaling in the development of pathological hypertrophy in adults. In this study we tested the hypothesis that ablation of Npr1 signaling during cardiac development would result in the early establishment of a hypertrophic phenotype. The aim of this study was to determine if gene and protein expression during cardiac development in Npr1 -/-mice was characteristic of pathological or physiological cardiac hypertrophy.

A C C E P T E D

M A N U S C R I P T

ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 6

MATERIALS AND METHODS

Generation and genotyping of mice.

Npr1 -/-mice and WT controls were generated as previously described [START_REF] Oliver | Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A[END_REF].

Experiments were performed according to the protocols approved by the Animal Ethics Committee of the University of Otago.

Male and female Npr1 -/-and WT embryos from timed matings between Npr1 +/- dams and Npr1 -/-sires and WT dams and sires were collected at ED 12.5 and 15.5 and neonatal pups collected at ND 1. Npr1 -/-dams were not used for breeding in this study due to reduced conception rates, poor maternal behaviour and increased rates of infanticide in the few successful pregnancies observed. Npr1 -/-embryos and ND 1 pups from these matings were not used in the current study. Embryos were collected after sacrificing the pregnant dams by cervical dislocation following Halothane BP™ (Nicolas Piramal I Ltd, London) anesthesia. Embryos were rapidly dissected out of the uterine horns and separated from the placenta in ice-cold, RNase-free, phosphate buffered saline solution. Embryos were digitally photographed against a grid and ruler to assess body dimensions. The embryonic left hind limb was removed for DNA extraction and the embryos immersion-fixed in 10% neutral buffered formalin. ND 1 pups were sacrificed by rapid decapitation, tails removed for DNA extraction and whole bodies immersion-fixed as above. Genomic DNA was extracted via a standard phenol-chloroform extraction protocol from embryonic left hind limbs or tail-tips of ND 1 mice and genotyped as previously described [START_REF] Oliver | Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A[END_REF][START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF].

Systolic and diastolic blood pressures in conscious dams were measured by a non-invasive, computerized tail-cuff system (ADInstruments, Dunedin, New Zealand) as previously described [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF]. The mice were familiarized to the procedure by being placed in the restrainer and the tail cuff system for a period of 7 days, after which baseline mean arterial pressure (MAP) measurements were made for each animal (mean of at least ten recordings), and repeated at 10 and 15 days post coitum (dpc) or until sacrifice.

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 7

Gender determination of collected embryos.

To determine the gender of embryos a PCR-based assay was established utilizing a variant of the Structural Maintenance of Chromosomes (SMC) gene located on the X-chromosome (Accession number AC083816), which has a Ychromosome homolog containing a 30 bp intronic deletion. The region of the gene surrounding the deletion site was amplified, using the following primers (forward-5'CCGCTGCCAAATTCTTTGG and reverse-5'CTCAAAAGCCAAAAGCTTCA).

Amplification of the X-chromosomal SMC variant produced a 330 bp fragment and the Y-chromosomal variant, produced a 300 bp fragment visualized by gel electrophoresis.

Histological characterization of Npr1

ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 8

thickness in each chamber were determined on transverse sections under light microscopy with an eyepiece graticule (Olympus Model WH10X3) using a modified

Cavalieri method [START_REF] Gundersen | The efficiency of systematic sampling in stereology and its prediction[END_REF]. ED 12.5 and 15.5 hearts were viewed at a total magnification of 100×, while ND 1 hearts were viewed at a total magnification of 40×. Additionally, the number of nuclei per photographic field was recorded within representative regions of the free wall of the mid left ventricle (LV) at 400× magnification. In all cases the mean of 5 independent sections per specimen was used for statistical analysis.

RNA isolation and quantitative real-time PCR analysis.

Total RNA was isolated from whole ED 12.5 and 15.5 embryos and excized hearts (atria and ventricles combined) of ND 1 as described previously (n=6 per gender and genotype at each gestational age) [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF]. Briefly tissues were homogenized in a Retsch MM301 tissue mill at 30 Hz for 10 minutes in 800µl prechilled TRIzol™ (Invitrogen, Carlsbad, CA). Chloroform (160µl) was added and samples were centrifuged at 12,000g for 15 minutes at room temperature. The RNAcontaining supernatant was purified by RNeasy Midi and Mini columns according to manufacturer's instructions (Qiagen, Hilden, Germany).

Quantitative real-time PCR (RT-PCR) analysis was performed on cDNA generated from 2.5µg of total RNA as previously described [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF]. Oligonucleotide primer sequences and PCR annealing temperatures are given in Table 1. PCR reactions were performed in a total volume of 30µl containing 1µl cDNA, 0.4mM primers, 1× PCR buffer, 0.2mM dNTPs, 1.5mM MgCl2, 10nM Sybr Green 1 (Roche, IN) and 1U TAQ-Ti DNA polymerase (Fisher Biotec, Australia). The PCR reaction conditions were optimized for each gene of interest, and PCR products were confirmed by sequencing on an ABI 3100-Avant Genetic Analyzer (Foster City, CA).

Levels of mRNA expression were evaluated by quantitative RT-PCR in either a

Rotor-Gene RG-3000 real-time PCR machine (Corbett Research, Australia) or a

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 9

Light Cycler 480 (Roche Diagnostics, IN) as previously described [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF]. For each assay a hotstart at 96°C was performed for 2 minutes followed by 30 cycles of denaturation at 94°C for 30s, annealing for 35s at the gene-specific annealing temperature (Table 1) and extension at 72°C for 30s, after which a melt curve was performed. Each sample was assayed in duplicate and gene expression levels were quantified against a standard curve and expressed as amount of message per µg of total RNA.

Immunohistochemistry.

Npr1 -/-and WT mice were collected at ED 12.5 and 15.5 and ND1 (n=6 per gender and genotype at each gestational age), fixed in 10% formalin overnight at 4°C followed by ethanol dehydration and paraffin embedding. Immunohistochemistry was performed in duplicate using indirect immunoperoxidase staining (Envision®+ Dual Link System-HRP, DakoCytomation, CA), on 3 µm serial sections on poly-L-lysine coated slides.

Primary antibodies used for immunohistochemistry included, GATA-4, Mef2, SRF from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) and Cx43 (Zymed laboratories, San Francisco, CA, USA). ANP antiserum was a generous gift from Associate Professor Tim Yandle (Endolab, University of Otago-Christchurch). ANP antiserum was raised in New Zealand white rabbits injected and boosted at monthly intervals with rat ANP (3-28) conjugated to bovine thyroglobulin followed by boosting with human ANP (1-28) conjugated to bovine thyroglobulin. The ANP antiserum exhibited high cross-reactivity to human, rat and mouse ANP (>95%), but <5% crossreactivity to other natriuretic peptides.

Briefly, immunohistochemistry involved deparaffinized sections being heattreated for 2 minutes in citrate buffer (pH 6.0), quenching of endogenous peroxidase activity with 3% hydrogen peroxidase in distilled water (15 mins at room temperature)

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 10

and non-specific binding of IgG suppressed with 1% normal goat serum in phosphate buffered saline (15 mins at room temperature). The sections were incubated with the primary antisera diluted to working concentrations of 1:300 for ANP, Cx43, Mef2 and GATA-4 and 1:500 for SRF at room temperature for 1 hour then visualized using liquid DAB+ (DakoCytomation, CA). Sections were counter-stained with alum hematoxylin. Negative controls were obtained by omission of the primary antibody.

Representative antibody staining within the left ventricular free wall and left atria was photographed for semi-quantitative densitometry of DAB+ staining using light microscopy under bright-field illumination (Olympus BX50 microscope) and Leica digital camera, DFC420. Intensity of DAB+ staining was determined using MetaMorph™ image analysis software (v6.2.6, Molecular Devices), as previously described [START_REF] Dragunow | Image-based highthroughput quantification of cellular fat accumulation[END_REF][START_REF] Graham | Induction of Krox-24 by endogenous cannabinoid type 1 receptors in Neuro2A cells is mediated by the MEK-ERK MAPK pathway and is suppressed by the phosphatidylinositol 3-kinase pathway[END_REF].

Statistical analysis.

All results are expressed as means ± SEM. The effects of genotype (Npr1 -/-vs WT), gender and gestational age were tested by three-way factorial ANOVA.

Subsequent comparisons between genotypes at specific ages were performed using independent t-tests. Bivariate relationships between expression levels for all genes and proteins were tested with Pearson's correlation. All statistical analyses were performed using SPSS version 11 (SPSS Inc., Chicago, Illinois). A p value of <0.05 was considered significant.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 11

RESULTS

Colony genotype distribution.

The genotype distributions at weaning (approximately four weeks after birth), identified that within this colony genotype distributions were non-Mendelian, with significantly fewer than expected Npr1 -/-mice surviving to four weeks of age (Figure 1A, 17% Npr1 -/-, 58% Npr1 +/-and 25% WT mice respectively, n=250, p=0.009).

Litter sizes were comparable between WT and Npr1 +/-dams collected at ED 12.5 (Npr1 +/-5.1±0.8 vs WT 6.3±1.1), at ED 15.5 (Npr1 +/-5.9±0.5 vs WT 6.2±0.7) and at ND 1 (Npr1 +/-5.3±0.5 vs WT 5.6±0.6, n=332 from 59 litters). The effect of genotype on gestational survival, genotype distribution during embryogenesis was studied in embryos collected from Npr1 +/-/Npr1 -/-timed matings (Figure 1B). At ED 12.5 the numbers of Npr1 -/-embryos and Npr1 +/-embryos were approximately equivalent, as expected from heterozygote-knockout matings (45% Npr1 +/-and 55%

Npr1 -/-). By ED 15.5 the proportion of Npr1 -/-embryos had declined to 39%. At ND 1 the number of Npr1 -/-mice was further reduced, with only 34% of the collected embryos identified as Npr1 -/-, instead of the expected 50% (n=130, p<0.05).

Maternal blood pressure during pregnancy.

Blood pressure was monitored throughout pregnancy in Npr1 -/-, Npr1 +/-and WT dams. At baseline, mean arterial pressure (MAP, Figure 1C) was significantly different between genotypes, with the average MAP of WT dams being 121 mmHg compared to either Npr1 +/-or Npr1 -/-(139 and 162 mmHg, respectively, p<0.05). The MAP in all three genotypes increased significantly with duration of pregnancy (p<0.001), but there were no differences in the extent to which blood pressure increased between genotypes. The observed initial difference in MAP due to genotype was maintained and remained significant throughout pregnancy (p<0.001).

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 12

3.3 Embryonic Npr1 -/-and WT heart structure.

There were no physical abnormalities evident between Npr1 -/-and WT embryos at any age (Figures 2A andB.) No cardiac anatomical abnormalities were apparent in H&E stained serial sections of the thoracic regions of Npr1 -/-compared with WT mice at any of the gestational ages studied (Figure 2B). Masson's Trichrome staining did not identify aberrant collagen deposition within the hearts of Npr1 -/-mice at any of the gestational ages studied (Figure 2C).

Both ED 12.5 and 15.5 Npr1 -/-mice tended to be lighter than WT embryos (Table 2), however, the difference was not significant. There was no overall difference in body weight at ND 1 between genotypes. As an alternative measure embryonic crown-rump lengths (in mm) were calculated from the digital photos taken at embryo collection (ED 12.5 Npr1 -/-9.33±0.39 vs WT 9.7±0.12, ED 15.5 Npr1 -/- 14.39±0.22 vs WT 14.42±0.22), with no significant difference in embryo length at either gestational age (p=0.078 and 0.937 respectively).

Heart dimensions were compared between Npr1 -/-and WT mice from ED 15.5 (at ED 12.5 heart dimensions were not quantifiable). An increase in heart size was apparent from ED 15.5 in Npr1 -/-mice compared to WT (p=0.003, Figure 3A), while male and female Npr1 -/-hearts at ND 1 were 22% and 29% larger respectively than WT littermates (p=0.0008). In addition, a significant increase in the ratio between heart size and chest size, an indication of cardiomegaly, was observed in ND 1

Npr1 -/-mice compared to WT (p<0.05, Figure 3B).

In Npr1 Furthermore while there was no significant difference in the number of cardiomyocyte nuclei within the left ventricular free wall at either ED 12.5 and ND 1, ED 15.5 Npr1 -/- mice had significantly fewer nuclei than WT mice (17 nuclei vs 21 nuclei, p=0.0019, Figure 3C), indicating cardiac enlargement resulted from hypertrophy rather than hyperplasia.

Natriuretic peptide expression during development.

As expected, quantitative real-time PCR demonstrated higher mRNA levels of both the cardiac natriuretic peptides, ANP and BNP, in Npr1 -/-mice compared to WT at all three gestational ages. In particular ANP message expression was significantly increased in Npr1 -/-mice at ED 12.5 (whole embryos) and ND 1 (isolated hearts, Figure 4A) when compared to WT. While not significant, BNP gene expression levels tended to be higher in Npr1 -/-mice compared to WT at all three gestational ages (Figure 4B).

Immunohistochemical analysis showed aggregations of ANP protein throughout the atria and within the trabecular region of the cardiac ventricles of Npr1 -/-and WT mice during gestation (Figure 4C). ANP protein staining was significantly greater in both the left atria (Figure 4D) and LV (Figure 4E) of Npr1 -/- compared to WT mice at all three gestational time points. As expected ANP protein expression within the LV was significantly greater in Npr1 -/-mice than WT mice (193.6±7 in Npr1 -/-vs 124.8±5 in WT, p<0.001) and was maximally expressed at ED 15.5. There was no significant gender influence on ANP protein expression in either the atria or ventricles (p=0.570 and 0.859 respectively). In WT mice, levels of ANP expression within the left atria increased significantly with gestational age (p<0.001), however, in Npr1 -/-mice left atrial ANP protein levels appeared to peak at ED 15.5.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 14

Transcription factor expression during development.

Gene and protein expression levels of transcription factors implicated in cardiac development; GATA-4, GATA-6, Mef2A, Mef2C and SRF, were investigated in Npr1 -/-mice compared to WT and are shown in Table 3. GATA-4 mRNA and Mef2 protein levels were significantly reduced in Npr1 -/-mice at ND1, but neither gene or protein expression of the other transcription factors was altered.

Gestational age significantly influenced gene expression levels (p<0.001 for all genes except BNP where p=0.008). All transcription factor gene expression was significantly correlated to gestational age (Pearson's correlation coefficients of r=-0.422, 0.561, 0.665, 0.725 and 0.614, p<0.01 for Mef2A, Mef2C, GATA-4, GATA-6

and SRF respectively).

Renin-Angiotensin system gene expression during development.

Gene expression levels of Angiotensinogen, the Angiotensin II type I receptor (AT1R) and the angiotensin converting enzyme (ACE) were investigated. There was no significant difference in expression levels between genotypes at any stage of development (Table 4).

Hypertrophy-related gene expression during development.

Expression levels of markers of physiologically derived cardiac hypertrophy, Akt1, and pathological cardiac hypertrophy, Calcineurin A, were investigated (Table 4). While both genes were differentially expressed with gestational age, Akt1 expression was significantly reduced in Npr1 -/-mice only at ED12.5 compared to WT (p=0.024), while Calcineurin A expression was significantly increased in the hearts of ND 1 Npr1 -/-mice (p=0.031).

Connexin 43 gene and protein expression in Npr1 -/-mice during

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 15

development.

Cx43 gene and protein expression decreased significantly with gestational age irrespective of Npr1 genotype (Figure 5A). However at ND 1 mRNA levels were significantly higher in Npr1 -/-hearts compared to WT (p=0.044, Figure 5A).

Additionally, developmental age, gender and Npr-1 genotype in combination influenced Cx43 gene expression (p=0.019).

Cx43 protein is detected in the adult myocardium along the myocyte margins, with Cx43 positive protein regions forming aggregations of gap junctions. During cardiac development Cx43 protein was expressed within both the myocardium of the cardiac atria and ventricles in both Npr1 -/-and WT mice (Figure 5B), but excluded from the regions of the cardiac cushions and cardiac valves (Figure 5B). Maximum Cx43 protein expression was evident at ED 12.5 in both Npr1 -/-and WT mice. Cx43 hemi-channel aggregations at the myocardial cell membrane were also detected in the myocardium from ED 12.5; however they were more diffuse in localization compared to the high density of aggregations evident in the hearts of ND 1 and adult mice.

Cx43 protein expression was significantly different between genotypes dependent on gestational age (p=0.014 Figure 5C), with levels of Cx 43 protein significantly reduced in the ventricles of Npr1 -/-mice at ED 15.5 (p=0.029).

DISCUSSION

In the current study, Npr-1 gene ablation was shown to reduce gestational survival and contribute to left ventricular hypertrophy (LVH) in the developing hearts of Npr1 -/-mice. Despite the presence of cardiomyocyte hypertrophy, there was no significant change in gene expression of hypertrophy-related transcription factors.

There was, however, a decrease in Cx43 protein expression at mid-gestation. The absence of any apparent gross developmental abnormalities in the whole embryos or apparent defects of cardiac structure, suggests that the decreased viability of Npr1 -/- embryos may result from a combination of cardiomegaly, increased calcineurin A expression and decreased Cx43 protein affecting cardiac contractility. This is the first study to examine cardiac structure, gene expression and protein expression changes in Npr1 -/-mice during embryonic development.

Npr-1 gene ablation is characterized by incremental increases in blood pressure relative to the number of disrupted alleles [START_REF] Oliver | Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner[END_REF], as well as the development of cardiac hypertrophy and fibrosis in post-natal life [START_REF] Oliver | Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A[END_REF]. The findings from this study suggest that Npr-1 activity is important in embryonic survival as significantly fewer than expected Npr1 -/-embryos survived to birth and subsequently to weaning.

Reduced post-natal survival has been previously reported in an alternative strain of Npr-1 null mice, where the total number of null mice at three weeks of age was only 70% of that expected [START_REF] Lopez | Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide[END_REF]. It was reported that a large proportion of the gestational deaths were attributable to fetal hydrops. Within our colony, the proportion of Npr1 -/-mice surviving at four weeks of age was comparable at 68%.

Furthermore we identified that the decline in the number of Npr1 -/-mice occurred across a continuum from ED 12.5 until birth, indicating that Npr1 -/-embryos were implanted at a normal rate but a degree of embryonic lethality was evident at mid/late gestation. Fetal hydrops was not observed in our colony, nor was there evidence of cardiac structural abnormalities among the surviving embryos at collection to account for loss of developmental viability. However, significant left ventricular hypertrophy was apparent from ED 15.5 onwards in Npr1 -/-mice.

Maternal hypertension has been shown to damage and prematurely age the placental vasculature [START_REF] Soma | Morphologic changes in the hypertensive placenta[END_REF] and is also associated with inter-uterine growth retardation, reduced birth weight and increased neonatal death [START_REF] Martyn | Growth in utero, adult blood pressure, and arterial compliance[END_REF][START_REF] Xiong | Impact of pregnancy-induced hypertension on fetal growth[END_REF]. In surrogate studies using spontaneously hypertensive rats Di Nicolantonio et al. have shown that the genetic identity of the fetus determines the growth rate of both the fetus and the placenta independent of maternal phenotype [START_REF] Di Nicolantonio | Fetal versus maternal determinants of the reduced fetal and placental growth in spontaneously hypertensive rats[END_REF]. In this study maternal blood pressure was monitored throughout pregnancy. While there was a significant genotype influence on blood pressure, the degree of blood pressure elevation as pregnancy progressed was not significantly different between Npr1 +/-, Npr1 -/-and WT dams. Nor were there significant differences in embryo body weight between genotypes at any time point investigated. There was no indication maternal hypertension contributed to the gestational death or resulted in reduced birth weight of Npr1 -/-mice during development.

Histological examination identified an increase in heart dimensions and subsequent heart size to chest size ratio due to thickening of the left heart structures, namely the LV free-wall and IVS, in Npr1 -/-mice from ED 15. is due to cell division [START_REF] Leu | Characterisation of postnatal growth of the murine heart[END_REF]. In the current study the decreased nuclei number suggests the increase in heart mass of Npr1 -/-mice during gestation resulted from enlargement of the myocardial cells (hypertrophy), rather than through increased cell numbers (hyperplasia) in the left ventricle.

Several studies using both experimental manipulation and genetic models have explored the gene expression changes associated with pathological cardiac hypertrophy [START_REF] Knowles | Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice[END_REF][START_REF] Kishimoto | A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy[END_REF]. These studies and our data strongly suggest that ANP-

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development 18

mediated Npr-1 signaling has a direct inhibitory effect on cardiac hypertrophy.

Horsthuis et al. have shown that distinct regulatory sequences and divergent regulatory pathways control fetal ANP expression and the reactivation of ventricular ANP expression during cardiac disease [START_REF] Horsthuis | Npr-1 signaling in cardiac development 23[END_REF]. Despite the increased levels of natriuretic peptide gene and protein expression, there was no detected increase in gene expression of the hypertrophy-related, cardiac transcription factors; Mef2A, Mef2C, GATA-4, GATA-6 and SRF or genes of the renin-angiotensin system. This contrasts with adult Npr1 -/-mice, in which we previously reported cardiac hypertrophy was correlated to increased expression of both the GATA-4 and Mef2C genes in aged animals [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF]. Interestingly our observation of unaltered RAS gene expression in embryos contrasts with a study by Li et al [START_REF] Li | Guanylyl cyclase-A inhibits angiotensin II type 1A receptor-mediated cardiac remodeling, an endogenous protective mechanism in the heart[END_REF] where cardiac hypertrophy and fibrosis in adult Npr1 -/-mice was ascribed to augmented cardiac AT1R signaling. This further illustrates differential role of natriuretic peptide signaling in the developing and mature cardiovascular systems. In the absence of up-regulation of traditional transcriptional markers of pathological cardiac hypertrophy we investigated the expression of Atk1, a down-stream effector of the phosphoinositol-3 kinase p110α subunit (PI3K-P110α), recently implicated as key component of physiological cardiac hypertrophy [START_REF] Mcmullen | Differences between pathological and physiological cardiac hypertrophy: novel therapeutic strategies to treat heart failure[END_REF][START_REF] Porrello | Heritable pathologic cardiac hypertrophy in adulthood is preceded by neonatal cardiac growth restriction[END_REF][START_REF] Rigor | Phosphoinositide 3kinase Akt signaling pathway interacts with protein kinase Cbeta2 in the regulation of physiologic developmental hypertrophy and heart function[END_REF]. Contrary to our expectations significant down-regulation of expression was observed at ED 12.5. Interestingly, Calcineurin A expression increased in Npr1 -/-mice from ED 15.5 (not significant) culminating in significantly elevated expression at ND 1 once cardiomyocyte hypertrophy is observed. These result complement our previous data where calcium/calmodulin signaling was implicated in the early establishment of cardiac hypertrophy in young Npr1 -/-mice [START_REF] Ellmers | Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis[END_REF].

Cx43, like the natriuretic peptides, is a marker of myocardial cell development during embryogenesis [START_REF] Christoffels | Chamber formation and morphogenesis in the developing mammalian heart[END_REF]. In the heart, Cx protein-derived gap junctions provide the pathway for intercellular current flow, enabling coordinated action potential This study has demonstrated that during embryonic development of Npr1 -/- mice, there is progressive LVH from ED 15.5, but no concomitant up-regulation of hypertrophy-associated, transcription factor genes or components of the reninangiotensin system. However increased ANP expression during embryonic development was associated with reduced expression of both Cx43 and SRF genes in Npr1 -/-mice. While the morphological cardiac enlargement in embryonic and adult Npr1 -/-mice is similar, the different molecular characteristics of LVH evident in Npr1 -/- mice from ED 15.5 compared to adult Npr1 -/-mice indicates that the role of natriuretic peptide signaling in cardiac development is distinct from that traditionally attributed to either pathological or physiological cardiac remodeling.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development

5 .

 5 Previously Kuhn et al., have shown Npr-1 null mice exhibit cardiomyocyte enlargement at both 4 and 12 months of age [15]. Leu et al. have shown that during the first four post-natal days the volume of cardiomyocytes remains constant despite a concomitant increase in cardiac mass, indicating that normal cardiac growth during this developmental phase

  in cardiacdevelopment 19 propagation and contraction. In adult ventricular muscle, cell-to-cell coupling is formed predominantly by Cx43 channels. Deranged expression and organization of Cx43 gap junctions in the ventricle have been demonstrated in a variety of cardiovascular pathologies, including hypertrophy[START_REF] Severs | Gap junction alterations in human cardiac disease[END_REF]. Cardiomyocyte-restricted deletion of Cx43 has been shown to result in increased gestational attrition of null mice from ED 13.5 through to post-natal day 6 in the absence of major cardiac structural malformations[START_REF] Eckardt | Cardiomyocyte-restricted deletion of connexin43 during mouse development[END_REF]. Dysregulation of Cx43 activity has been associated with impaired electrical action of the heart, namely an increased prevalence of tachyarrhythmias[START_REF] Beauchamp | Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice[END_REF][START_REF] Danik | Modulation of cardiac gap junction expression and arrhythmic susceptibility[END_REF], such impaired cardiac conduction might account for the observed gestation deaths in Npr1 -/-mice, particularly the sporadic rate of deaths occurring along a developmental continuum.

Figure legends: Figure 1 .

 1 Figure legends: Figure 1. Npr1 genotype distribution at weaning (A) and during gestation (B) and maternal blood pressure changes during gestation (C). Black, dark grey and light grey bars represent Npr1 -/-, Npr1 +/-and WT groups respectively. Significant differences between the genotypes are indicated by *p<0.05, **p<0.01 and †p<0.001. Maternal blood pressures were calculated from n=8 per group, except for Npr1 -/- where n=4.

Figure 2 .

 2 Figure 2. Representative digital photographs of male mice at EDs 12.5 and 15.5 (A), comparative composite H&E stained hearts of Npr1 -/-and WT mice at ED 12.5 in the transverse plane, ED 15.5 and ND 1 hearts in the coronal plane (B), and Masson's trichrome stained paraffin sections from the left ventricle (C). The top row images are comparative sections from a 6-month old Npr1 -/-mouse, on the left a section through the LV wall and on the right a positively stained cardiac valve. Row 2 displays composite photomerges of H&E stained sections from ED 12.5 Npr1 -/-and WT mice. Rows 3-5 are representative Masson's trichrome stained sections from ED 12.5, ED 15.5 and ND 1 mice respectively. Structures labeled: A=right atria; RV= right ventricle; LV=left ventricle; white arrow indicates the pericardium. Scale bar represents 100 µm.

Figure 3 .

 3 Figure 3. Calculated heart size at ED 15.5 and ND 1 (A) and calculated heart size to chest size ratio at ED 15.5 and ND 1 in Npr1 -/-mice compared to WT (B), left ventricular free-wall myocyte nuclei count (C) in Npr1 -/-mice compared to WT and paraffin embedded H&E stained sections from the left ventricular free-wall (D). Black and light grey bars represent Npr1 -/-and WT groups respectively. The y axis for both A and B are in arbitrary units determined from and eye piece graticule as described in the methods. Significant differences between the genotypes are indicated by *p<0.05 and **p<0.01. Scale bar represents 100 µm.

Figure 4 .

 4 Figure 4. Gestational expression of ANP mRNA (A), BNP mRNA (B), left ventricular ANP protein staining (C), and quantitation of staining for left atrial ANP protein (D) and left ventricular ANP protein (E) in Npr1 -/-mice compared to WT control mice. ^Quantitated gene levels are expressed as picograms of message per microgram of total RNA (pg/µg total RNA), except for ND 1 where ANP expression gene expression is expressed as nanograms of message per microgram of total RNA (ng/ µg total RNA). Scale bar represents 100 µm and red asterixis denote left ventricular lumen. Protein expression represents the accumulation of ANP protein measured by immunohistochemistry using MetaMorph™ image analysis software. Black and grey bars represent Npr1 -/-and WT groups respectively. Significant differences between the genotypes are indicated by *p<0.05 and †p<0.001.

Figure 5 .

 5 Figure 5. Gestational expression of Cx43 mRNA (A) and protein (B, C) in Npr1 -/-and WT mice. (B) Representative micrographs of Cx43 protein expression in whole hearts of ED 12.5 and left ventricles of ED 15.5 and ND 1 mice. (C) Quantitation of Cx43 protein expression in the left ventricle of Npr1 -/-and WT mice. Quantitated gene levels are expressed as picograms of message per microgram of total RNA (pg/µg total RNA). Protein expression represents the accumulation of Cx43 protein measured by immunohistochemistry using MetaMorph™ image analysis software. Black and grey bars represent Npr1 -/-and WT groups respectively. Significant differences between the genotypes are indicated by *p<0.05. Scale bar represents 100 µm.

  -/-and WT embryos.

	M A N U S C R I P T
	Before being sectioned, embryos and ND 1 pups were either transferred to 10% neutral-buffered formalin containing 10% sucrose as a cryoprotectant for 24 hours and then embedded in OCT medium (Miles; Elkhart, IN, n=6 per gender and genotype at each gestational age), or paraffin embedded (n=6 per gender and genotype at each gestational age). An equal number of embryos and neonates per group were embedded such that the heart could be sectioned in either the transverse or sagittal plane. The use of two anatomical planes ensured that cardiac morphology A C C E P T E D
	could be thoroughly examined, especially the locations of the great vessels and the
	structures of the interventricular and interatrial septa. Histological analysis was
	performed on 20-µm-thick serial cryostat sections and 3-µm-thick serial paraffin
	sections (Leica Microsystems), stained with either hematoxylin and eosin (H&E) or
	Masson's trichrome and examined under bright-field illumination (Olympus BX50).
	Composite photo images of the entire heart structure were compiled using
	Photoshop Elements 4.0 (Adobe Systems Inc). Heart size for each embryo, as well
	as individual chamber dimensions, ventricular septal length and width, and free wall

  -/-mice the increased heart size was due to significantly increased volume (7.97±0.13 vs 7.01±0.21 units, p=0.611) or thickness of right ventricular free wall (1.84±0.01 vs 1.73±0.01 units, p=0.179) between genotypes.

	ACCEPTED MANUSCRIPT Npr-1 signaling in cardiac development	13
	M A N U S C R I P T
	A C C E P T E D
	interventricular septal thickness (3.16±0.03 vs 2.36±0.01 graticule units, p=0.0002)
	and a trend towards increased left ventricular free wall thickness (2.22±0.07 vs

1.82±0.02 graticule units, p=0.070) compared to WT mice (2.22±0.07 vs 1.82±0.02 graticule units, p=0.070). There were no significant differences in left ventricular chamber

Npr-1 signaling in cardiac development

Acknowledgements. This work was supported by the National Heart Foundation of New Zealand (Grant number 1056). The authors would like to thank Mr Howard Potter (Molecular Pathology, Canterbury Health Labs) for work that involved sequencing PCR products, Ms Rachel Purcell (Children's Cancer Research Group, University of Otago-Christchurch) for help with Immunohistochemistry techniques, Professor Mike Dragunow (Department of Pharmacology and Clinical Pharmacology, University of Auckland) for MetaMorph data analysis and Dr Anna Pilbrow (Christchurch Cardioendocrine Research Group, University of Otago) for statistical advice.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Npr-1 signaling in cardiac development