A mouse model of inherited choline kinase ß-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia.

The CHKB gene encodes choline kinase ß, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.

Characterization of primary adult mouse cardiac fibroblast cultures.

The role of cardiac fibroblasts (CFs) in disease states has been a focus of cardiovascular research over the past decade. Here, we briefly describe methods for isolation and characterization of CFs from adult mouse ventricles. Primary cultures were stained using antibodies for several marker proteins such as α-smooth muscle actin (αSMA), vimentin, and discoidin domain receptor 2 (DDR2) to confirm the identity of CFs or cardiac myofibroblasts (CMFs). Most cells in primary cultures consisted of CFs, with very low frequencies of endothelial cells, cardiomyocytes, and smooth muscle cells. We compared marker expression between cultures that were not passaged (P0) or passaged for few times (P1-3). When compared with P1-3 cultures, P0 cultures consistently displayed a lower percentage of cells positive for αSMA and DDR2, whereas vimentin expression was significantly higher in P0 cultures compared with P1-3 cultures. P0 cells were also smaller in area than P1-3 cells. Further, P1-3 mouse CFs were found to express both ß1 and ß2 adrenergic receptors (ARs) and ß1ARs were more readily detected on the cell surface compared with ß2ARs. In summary, mouse CF cultures underwent phenotype conversion into CMFs after passaging, consistent with what is seen with CF cultures from other species.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential.

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

Effects of ß-adrenergic receptor drugs on embryonic ventricular cell proliferation and differentiation and their impact on donor cell transplantation.

ß-Adrenergic receptors (ß-ARs) and catecholamines are present in rodents as early as embryonic day (E)10.5. However, it is not known whether ß-AR signaling plays any role in the proliferation and differentiation of ventricular cells in the embryonic heart. Here, we characterized expression profiles of ß-AR subtypes and established dose-response curves for the nonselective ß-AR agonist isoproterenol (ISO) in the developing mouse ventricular cells. Furthermore, we investigated the effects of ISO on cell cycle activity and differentiation of cultured E11.5 ventricular cells. ISO treatment significantly reduced tritiated thymidine incorporation and cell proliferation rates in both cardiac progenitor cell and cardiomyocyte populations. The ISO-mediated effects on DNA synthesis could be abolished by cotreatment of E11.5 cultures with either metoprolol (a ß1-AR antagonist) or ICI-118,551 (a ß2-AR antagonist). In contrast, ISO-mediated effects on cell proliferation could be abolished only by metoprolol. Furthermore, ISO treatment significantly increased the percentage of differentiated cardiomyocytes compared with that in control cultures. Additional experiments revealed that ß-AR stimulation leads to downregulation of Erk and Akt phosphorylation followed by significant decreases in cyclin D1 and cyclin-dependent kinase 4 levels in E11.5 ventricular cells. Consistent with in vitro results, we found that chronic stimulation of recipient mice with ISO after intracardiac cell transplantation significantly decreased graft size, whereas metoprolol protected grafts from the inhibitory effects of systemic catecholamines. Collectively, these results underscore the effects of ß-AR signaling in cardiac development as well as graft expansion after cell transplantation.NEW & NOTEWORTHY ß-Adrenergic receptor (ß-AR) stimulation can decrease the proliferation of embryonic ventricular cells in vitro and reduce the graft size after intracardiac cell transplantation. In contrast, ß1-AR antagonists can abrogate the antiproliferative effects mediated by ß-AR stimulation and increase graft size. These results highlight potential interactions between adrenergic drugs and cell transplantation.

Divergent cell cycle kinetics of midgestation ventricular cells entail a higher engraftment efficiency after cell transplantation.

Cardiac progenitor cells (CPCs) in the primary and secondary heart fields contribute to the formation of all major cell types in the mammalian heart. While some CPCs remain undifferentiated in midgestation and postnatal hearts, very little is known about their proliferation and differentiation potential. In this study, using an Nkx2.5 cell lineage-restricted reporter mouse model, we provide evidence that Nkx2.5(+) CPCs and cardiomyocytes can be readily distinguished from nonmyocyte population using a combination of Nkx2.5 and sarcomeric myosin staining of dispersed ventricular cell preparations. Assessment of cell number and G1/S transit rates during ventricular development indicates that the proliferative capacity of Nkx2.5(+) cell lineage gradually decreases despite a progressive increase in Nkx2.5(+) cell number. Notably, midgestation ventricles (E11.5) contain a larger number of CPCs (∼2-fold) compared with E14.5 ventricles, and the embryonic CPCs retain cardiomyogenic differentiation potential. The proliferation rates are consistently higher in embryonic CPCs compared with myocyte population in both E11.5 and E14.5 ventricles. Results from two independent cell transplantation models revealed that E11.5 ventricular cells with a higher percentage of proliferating CPCs can form larger grafts compared with E14.5 ventricular cells. Furthermore, transplantation of embryonic ventricular cells did not cause any undesirable side effects such as arrhythmias. These data underscore the benefits of donor cell developmental staging in myocardial repair.

Atrial natriuretic peptide inhibits cell cycle activity of embryonic cardiac progenitor cells via its NPRA receptor signaling axis.

The biological effects of atrial natriuretic peptide (ANP) are mediated by natriuretic peptide receptors (NPRs), which can either activate guanylyl cyclase (NPRA and NPRB) or inhibit adenylyl cyclase (NPRC) to modulate intracellular cGMP or cAMP, respectively. During cardiac development, ANP serves as an early maker of differentiating atrial and ventricular chamber myocardium. As development proceeds, expression of ANP persists in the atria but declines in the ventricles. Currently, it is not known whether ANP is secreted or the ANP-NPR signaling system plays any active role in the developing ventricles. Thus the primary aims of this study were to 1) examine biological activity of ANP signaling systems in embryonic ventricular myocardium, and 2) determine whether ANP signaling modulates proliferation/differentiation of undifferentiated cardiac progenitor cells (CPCs) and/or cardiomyocytes. Here, we provide evidence that ANP synthesized in embryonic day (E)11.5 ventricular myocytes is actively secreted and processed to its biologically active form. Notably, NPRA and NPRC were detected in E11.5 ventricles and exogenous ANP stimulated production of cGMP in ventricular cell cultures. Furthermore, we showed that exogenous ANP significantly decreased cell number and DNA synthesis of CPCs but not cardiomyocytes and this effect could be reversed by pretreatment with the NPRA receptor-specific inhibitor A71915. ANP treatment also led to a robust increase in nuclear p27 levels in CPCs compared with cardiomyocytes. Collectively, these data provide evidence that in the developing mammalian ventricles ANP plays a local paracrine role in regulating the balance between CPC proliferation and differentiation via NPRA/cGMP-mediated signaling pathways.

Mechanisms of renal hyporesponsiveness to BNP in heart failure.

The B-type natriuretic peptide (BNP), a member of the family of vasoactive peptides, is a potent natriuretic, diuretic, and vasodilatory peptide that contributes to blood pressure and volume homeostasis. These attributes make BNP an ideal drug that could aid in diuresing a fluid-overloaded patient who had poor or worsening renal function. Despite the potential benefits of BNP, accumulating evidence suggests that simply increasing the amount of circulating BNP does not necessarily increase natriuresis in patients with heart failure (HF). Moreover, despite high BNP levels, natriuresis falls when HF progresses from a compensated to a decompensated state, suggesting the emergence of renal resistance to BNP. Although likely multifactorial, several mechanisms have been proposed to explain renal hyporesponsiveness in HF, including, but not limited to, decreased renal BNP availability, down-regulation of natriuretic peptide receptors, and altered BNP intracellular signal transduction pathways. Thus, a better understanding of renal hyporesponsiveness in HF is required to devise strategies to develop novel agents and technologies that directly restore renal BNP efficiency. It is hoped that development of these new therapeutic approaches will serve to limit sodium retention in patients with HF, which may ultimately delay the progression to overt HF.

A novel ß-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts.

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective ß-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

Signaling mechanisms regulating fibroblast activation, phenoconversion and fibrosis in the heart.

Cardiac fibroblasts (CFs) maintain the cardiac extracellular matrix (ECM) through myocardial remodelling. The remodelling process can become dysregulated during various forms of heart disease which leads to an overall accumulation of ECM. This results in cardiac fibrosis which increases the risk of heart failure in many patients. During heart disease, quiescent CFs undergo phenoconversion to an activated cell type called cardiac myofibroblasts (CMFs). Factors influencing phenoconversion include transforming growth factor ß (TGF-ß) which via SMADs (small mothers against decapentaplegic) activates the myofibroblast marker gene αSMA (α smooth muscle actin). Signaling molecules as diverse as NAD(P)H oxidase 4 (Nox4) and Wnt have been found to interact with TGF-ß signalling via SMADs. Pathways, including FAK/TAK/JNK and PI3K/Akt/rac have also been implicated in activating phenoconversion of fibroblasts. Another major contributor is mechanical stress exerted on CFs by ECM changes, which involves activation of ERK and subsequent αSMA expression. Other factors, such as the mast cell protease tryptase and the seeding density also affect the phenoconversion of fibroblast cultures in vitro. Further, reversal of myofibroblast phenotype has been reported by a negative regulator of TGF-ß, Ski, as well as the hormone relaxin and the second messenger cAMP. Targeting the signaling molecules involved in promoting phenoconversion of CFs to CMFs presents a possible method of controlling cardiac fibrosis. Here, we provide a brief review of signaling mechanisms responsible for phenoconversion and identify critical targets for the treatment of cardiac fibrosis.

Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart.

It has been shown that atrial natriuretic peptide (ANP) and its high affinity receptor (NPRA) are involved in the formation of ventricular conduction system (VCS). Inherited genetic variants in fatty acid oxidation (FAO) genes are known to cause conduction abnormalities in newborn children. Although the effect of ANP on energy metabolism in noncardiac cell types is well documented, the role of lipid metabolism in VCS cell differentiation via ANP/NPRA signaling is not known. In this study, histological sections and primary cultures obtained from E11.5 mouse ventricles were analyzed to determine the role of metabolic adaptations in VCS cell fate determination and maturation. Exogenous treatment of E11.5 ventricular cells with ANP revealed a significant increase in lipid droplet accumulation, FAO and higher expression of VCS marker Cx40. Using specific inhibitors, we further identified PPARγ and FAO as critical downstream regulators of ANP-mediated regulation of metabolism and VCS formation.

Role of D-type cyclins in heart development and disease.

A defining feature of embryonic cardiomyocytes is their relatively high rates of proliferation. A gradual reduction in proliferative capacity throughout development culminates in permanent cell cycle exit by the vast majority of cardiomyocytes around the perinatal period. Accordingly, the adult heart has severely limited capacity for regeneration in response to injury or disease. The D-type cyclins (cyclin D1, D2, and D3) along with their catalytically active partners, the cyclin dependent kinases, are positive cell cycle regulators that play important roles in regulating proliferation of cardiomyocytes during normal heart development. While expression of D-type cyclins is generally low in the adult heart, expression levels are augmented in association with cardiac hypertrophy, but are uncoupled from myocyte cell division. Accordingly, re-activation of D-type cyclin expression in the adult heart has been implicated in pathophysiological processes via mechanisms distinct from those that drive proliferation during cardiac development. Growth factors and other exogenous agents regulate D-type cyclin production and activity in embryonic and adult cardiomyocytes. Understanding differences in the precise intracellular mediators downstream from these signalling molecules in embryonic versus adult cardiomyocytes could prove valuable for designing strategies to reactivate the cell cycle in cardiomyocytes in the setting of cardiovascular disease in the adult heart.

Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research.

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.

Regulation of Transplanted Cell Homing by FGF1 and PDGFB after Doxorubicin Myocardial Injury.

There is no effective treatment for the total recovery of myocardial injury caused by an anticancer drug, doxorubicin (Dox). In this study, using a Dox-induced cardiac injury model, we compared the cardioprotective effects of ventricular cells harvested from 11.5-day old embryonic mice (E11.5) with those from E14.5 embryos. Our results indicate that tail-vein-infused E11.5 ventricular cells are more efficient at homing into the injured adult myocardium, and are more angiogenic, than E14.5 ventricular cells. In addition, E11.5 cells were shown to mitigate the cardiomyopathic effects of Dox. In vitro, E11.5 ventricular cells were more migratory than E14.5 cells, and RT-qPCR analysis revealed that they express significantly higher levels of cytokine receptors Fgfr1, Fgfr2, Pdgfra, Pdgfrb and Kit. Remarkably, mRNA levels for Fgf1, Fgf2, Pdgfa and Pdgfb were also found to be elevated in the Dox-injured adult heart, as were the FGF1 and PDGFB protein levels. Addition of exogenous FGF1 or PDGFB was able to enhance E11.5 ventricular cell migration in vitro, and, whereas their neutralizing antibodies decreased cell migration. These results indicate that therapies raising the levels of FGF1 and PDGFB receptors in donor cells and or corresponding ligands in an injured heart could improve the efficacy of cell-based interventions for myocardial repair.

A Cardiac Mitochondrial FGFR1 Mediates the Antithetical Effects of FGF2 Isoforms on Permeability Transition.

Mitochondria, abundant organelles in high energy demand cells such as cardiomyocytes, can determine cell death or survival by regulating the opening of mitochondrial permeability transition pore, mPTP. We addressed the hypothesis that the growth factor FGF2, known to reside in intracellular locations, can directly influence mitochondrial susceptibility to mPTP opening. Rat cardiac subsarcolemmal (SSM) or interfibrillar (IFM) mitochondrial suspensions exposed directly to rat 18 kDa low molecular weight (Lo-) FGF2 isoform displayed increased resistance to calcium overload-induced mPTP, measured spectrophotometrically as "swelling", or as cytochrome c release from mitochondria. Inhibition of mitochondrial protein kinase C epsilon abrogated direct Lo-FGF2 mito-protection. Exposure to the rat 23 kDa high molecular weight (Hi) FGF2 isoform promoted cytochrome c release from SSM and IFM under nonstressed conditions. The effect of Hi-FGF2 was prevented by mPTP inhibitors, pre-exposure to Lo-FGF2, and okadaic acid, a serine/threonine phosphatase inhibitor. Western blotting and immunoelectron microscopy pointed to the presence of immunoreactive FGFR1 in cardiac mitochondria in situ. The direct mito-protective effect of Lo-FGF2, as well as the deleterious effect of Hi-FGF2, were prevented by FGFR1 inhibitors and FGFR1 neutralizing antibodies. We propose that intracellular FGF2 isoforms can modulate mPTP opening by interacting with mito-FGFR1 and relaying isoform-specific intramitochondrial signal transduction.

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post-chamber formation.

There is scant information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Here we simultaneously tracked three lineage-specific markers (Nkx2.5, MLC2v, and ANF) and confirmed that CPCs with an Nkx2.5+MLC2v-ANF- phenotype are present in the embryonic (E) day 11.5 mouse ventricular myocardium. We demonstrated that these CPCs could give rise to working cardiomyocytes and conduction system cells. Using a two-photon imaging analysis, we found that the majority of CPCs are not capable of developing Ca2+ transients in response to beta-adrenergic receptor stimulation. In contrast, Nkx2.5+ cells expressing MLC2v but not ANF are capable of developing functional Ca2+ transients. We showed that Ca2+ transients could be invoked in Nkx2.5+MLC2v+ANF+ cells only upon inhibition of Gi, muscarinic receptors, or nitric oxide synthase (NOS) signaling pathways. Our data suggest that these inhibitory pathways may delay functional specification in a subset of developing ventricular cells.

Assessment of embryonic myocardial cell differentiation using a dual fluorescent reporter system.

Recent studies have identified the existence of undifferentiated myocardial cells during early embryonic as well as post-natal stages of heart development. While primitive cells present in the precardiac mesoderm can differentiate into multiple cell types of the cardiovascular system, the developmental potential of undifferentiated cells identified in the ventricular myocardium after chamber formation is not well characterized. A deeper understanding of mechanisms regulating myocardial cell differentiation will provide further insights into the normal and pathological aspects of heart development. Here, we showed that Nkx2.5 positive and sarcomeric myosin negative cells were predominantly localized in the right ventricular myocardium of CD1 mice at E11.5 stage. We confirmed that myocardial regions negative for saromeric myosin were also devoid of atrial natriuretic factor (ANF). These observations are consistent with our previous study, which showed that ANF expression is restricted to moderately differentiated and mature myocardial cells in E11.5 myocardium of C3H/FeJ mice. Further, we found that the receptor c-Kit, a marker for early embryonic myocardial progenitor cells, is not expressed in the undifferentiated cells of the E11.5 myocardium. To monitor the differentiation potential of Nkx2.5(+)/ANF(-) cells in vitro, we developed a novel double fluorescent reporter system. Subsequently, we confirmed that the majority of Nkx2.5(+)/ANF(-) cells expressed mature myocyte markers such as sarcomeric myosin, MLC2V and alpha-cardiac actin after 48 hrs in culture, albeit at lower levels compared to Nkx2.5(+)/ANF(+) or Nkx2.5(-)/ANF(+) cell populations. Our results suggest that fluorescent reporters under the control of lineage-specific promoters can be used to study myocardial cell differentiation in response to various exogenous or pharmacological agents.

Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium.

MHC-TGFcys33ser transgenic mice have elevated levels of active transforming growth factor (TGF)-beta1 in the myocardium. Previous studies have shown that these animals develop atrial, but not ventricular, fibrosis. Here we show that atrial fibrosis was accompanied with cardiomyocyte apoptosis. Although similar levels of cardiomyocyte apoptosis were present in the right and left atria of MHC-TGFcys33ser hearts, the extent of fibrosis was more pronounced in the right atrium. Thus, additional factors influence the degree of atrial fibrosis in this model. Tritiated thymidine incorporation studies revealed cardiomyocyte cell cycle activity in left atrial cardiomyocytes, but not in right atrial cardiomyocytes. These observations suggested that cardiomyocyte cell cycle activation ameliorated the severity of atrial fibrosis. To directly test this hypothesis, MHC-TGFcys33ser mice were crossed with MHC-cycD2 mice (which have constitutive cardiomyocyte cell cycle activity in the right atrium). Mice inheriting both transgenes exhibited right atrial cardiomyocyte cell cycle activity and a concomitant reduction in the severity of right atrial fibrosis, despite the presence of a similar level of cardiomyocyte apoptosis as was observed in mice inheriting the MHC-TGFcys33ser transgene alone. These data support the notion that cardiomyocyte cell cycle induction can antagonize fibrosis in the myocardium.

Quantification of cardiac fibrosis by colour-subtractive computer-assisted image analysis.

1. Quantification of fibrosis is a key parameter in the assessment of the severity of cardiovascular disease and efficacy of future candidate therapies. Computer-assisted methods are frequently used to assess cardiac fibrosis in several experimental models. A brief survey indicated that there is a clear dearth of literature outlining detailed methodologies for computer-based assessment of cardiac fibrosis. The purpose of the present study was to provide a reliable method for a systematic assessment of cardiac fibrosis. 2. We induced cardiac fibrosis by isoproterenol (ISO) infusion in adult CD1 male mice and quantified fibrosis using a recently developed colour-subtractive computer-assisted image analysis (CS-CAIA) technique. Here, we provided a detailed description of our methodology to facilitate its wider use by other researchers. 3. We showed that the severity of ISO-induced cardiac fibrosis was similar in the apex, mid-ventricular ring and base of the adult CD1 mouse heart. In contrast with other species, such as rats and dogs, we found that uniform expression of beta(1)-adrenoceptors between different regions in CD1 mouse hearts correlated well with uniform induction of cardiac fibrosis. 4. A previous study found a negative correlation between levels of myocardial fibrosis and the degree of cardiac hypertrophy in ISO-treated Wistar rats. In contrast, we found a similar degree of cardiac fibrosis in our ISO-treated CD1 mice. 5. Our results suggest that CD1 mice are an ideal model system to study catecholamine-induced cardiac remodelling, as well as to screen candidate antifibrotic agents for future therapies.

Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system.

Patients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation.