Nathalie Mougenot

Heterozygous LmnadelK32 mice develop dilated cardiomyopathy through a combined pathomechanism of haploinsufficiency and peptide toxicity

Dilated cardiomyopathy (DCM) associates left ventricular (LV) dilatation and systolic dysfunction and is a major cause of heart failure and cardiac transplantation. LMNA gene encodes lamins A/C, proteins of the nuclear envelope. LMNA mutations cause DCM with conduction and/or rhythm defects. The pathomechanisms linking mutations to DCM remain to be elucidated. We investigated the phenotype and associated pathomechanisms of heterozygous Lmna(ΔK32/+) (Het) knock-in mice, which carry a human mutation. Het mice developed a cardiac-specific phenotype. Two phases, with two different pathomechanisms, could be observed that lead to the development of cardiac dysfunction, DCM and death between 35 and 70 weeks of age. In young Het hearts, there was a clear reduction in lamin A/C level, mainly due to the degradation of toxic ΔK32-lamin. As a side effect, lamin A/C haploinsufficiency probably triggers the cardiac remodelling. In older hearts, when DCM has developed, the lamin A/C level was normalized and associated with increased toxic ΔK32-lamin expression. Crossing our mice with the Ub(G76V)-GFP ubiquitin-proteasome system (UPS) reporter mice revealed a heart-specific UPS impairment in Het. While UPS impairment itself has a clear deleterious effect on engineered heart tissues force of contraction, it also leads to the nuclear aggregation of viral-mediated expression of ΔK32-lamin. In conclusion, Het mice are the first knock-in Lmna model with cardiac-specific phenotype at the heterozygous state. Altogether, our data provide evidence that Het cardiomyocytes have to deal with major dilemma: mutant lamin A/C degradation or normalization of lamin level to fight the deleterious effect of lamin haploinsufficiency, both leading to DCM.

Overexpression of cAMP-response element modulator causes abnormal growth and development of the atrial myocardium resulting in a substrate for sustained atrial fibrillation in mice

BACKGROUND AND METHODS Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques. RESULTS Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis. CONCLUSIONS Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice.

Resident PW1+ Progenitor Cells Participate in Vascular Remodeling During Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1(+) progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. OBJECTIVE To determine the role of pulmonary PW1(+) progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. METHODS AND RESULTS We investigated their contribution during chronic hypoxia-induced vascular remodeling in Pw1(nLacZ+/-) mouse expressing β-galactosidase in PW1(+) cells and in differentiated cells derived from PW1(+) cells. PW1(+) progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1(+) cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1(+) cells and the proportion of β-gal(+) vascular SMC were increased, indicating a recruitment of PW1(+) cells and their differentiation into vascular SMC during early chronic hypoxia-induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1(+) cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal(+) SMC were not derived from circulating bone marrow-derived PW1(+) progenitor cells, confirming a resident origin of the recruited PW1(+) cells. The number of pulmonary PW1(+) cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. CONCLUSIONS These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension-associated vascular remodeling.

Myoblasts and Embryonic Stem Cells Differentially Engraft in a Mouse Model of Genetic Dilated Cardiomyopathy

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with β-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

0386 : Direct thrombin inhibitors prevent atrial myocardial hypertrophy and atrial fibrillation susceptibility associated with heart failure in the rat

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia, which is characterized by a high risk of stroke due to increased thrombogenesis and thrombin formation in poorly contractile atria. In addition to its role in the coagulation cascade, thrombin has pleiotropic effects on cardiovascular system through the activation of protease activated receptor 1 (PAR 1). Here we examined whether thrombin contributes to the atrial myocardial remodeling that occurs during heart failure (HF) and whether direct thrombin inhibitors can prevent this remodeling process and AF progression. The study was conducted in a well established rat model of atrial dilation and susceptibility to AF associated with ischemic HF secondary to myocardial infarction (MI). The endogenous thrombin potential increased in plasma after MI, and remained elevated in HF rats. Treating MI rats for one or two months with direct thrombin inhibitors reduced the area and diameter of left atria. After two months, there was also a reduction of the duration of burst pacing induced AF and a downregulation of the expression of hypertrophic markers such as brain natriuretic peptide and β myosin heavy chain. The transcription factor NFATc3, known to be activated in cardiac hypertrophy, and the plasminogen activator inhibitor 1, which has both thrombotic and fibrogenic activities were also downregulated. Treating rats with a PAR 1 antagonist reproduced the effect of thrombin inhibitors. To study the role of thrombin on atrial myocardium in absence of other cardiac stimuli, atrial explant cultures were performed. In cultured atria, thrombin upregulated hypertrophic markers and plasminogen activator inhibitor 1 through PAR 1 and the Rho/Rho kinase pathway. These results indicate that thrombin is a potent hypertrophic factor for atrial myocardium; they point to chronic inhibition of thrombin/PAR1 pathways as a potential therapeutic option to prevent atrial remodeling and AF substrate formation. The author hereby declares no conflict of interest

0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV). We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes. Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV). Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice.

I039 Role of PRG-1, a neuronal phospholipid phosphatase, in the function of vascular smooth muscle cells

Proliferation and migration of vascular smooth muscle cells (VSMCs) leading to neointima formation are a hallmark of development and progression of some cardiovascular diseases. LPA (lysophosphatidic acid) which has been shown to induces a dedifferenciation of VSCM, resulting in cell proliferation and migration in vitro, and vascular remodelling in vivo. The aim of our project is to understand the role of PRG-1 (Plasticity Related Gene-1), or LPPR4 (Lipid Phosphate Phosphatase Related protein 4), in the regulation of VSMCs proliferation and migration induced by LPA. This enzyme acts as en ecto-enzyme dephosphorylating LPA, thereby inhibiting its activity, and was found in previous studies to be down-regulated in the media of aging and hypertensive rats. We showed that PRG-1 is expressed in the rat and human aortic media and rat VSMCs by PCR, immunofluorescence and western blot. This expression was highly decreased in cultured VSMCs as compared with what was observed in the aortic media, and restored by cells starvation, suggesting that PRG-1 expression is dependent on the differentiated phenotype of these cells. We studied the response to LPA of VSMCs infected with an adenovirus containing the human sequence of PRG1 (PRG1-VSMCs) or with an adenovirus expressing LacZ (LacZ-VSMCs). In vitro, LPA-induced migration of PRG1-VSMCs (measured by scrapewound assays) and LPA-induced proliferation (measured by BrdU incorporation) were inhibited (by 60 % and 40 % respectively) as compared with LacZ-VSMCs. In vivo, experiments of left carotid injury by ballooning followed by gene transfer with adenovirus injection, showed a 70 % reduction of neointima formation when carotids were infected with PRG-1 adenovirus as compared with infection with LacZ adenovirus. Current investigations indicate that PRG-1 seems to block Erk1/2 signalling pathway activated by LPA. Our results demonstrate that PRG-1 is expressed in the VSMCs of the media ; and this expression is modulated in conditions of vascular remodelling and primary culture. PRG-1 overexpression inhibits VSMCs migration and proliferation in vitro and neotintima formation in vivo. Taken together, these findings suggest that, in the media, PRG-1 plays a protective role in pathophysiological conditions.