Secondary Sphere Formation Enhances the Functionality of Cardiac Progenitor Cells

Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant → primary cardiosphere (CS) formation → sphere-derived cells (SDCs) in adherent culture condition → secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy.     

Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice

Cardiac progenitor cells are a potential source of cell therapy for heart failure. Although recent studies have shown that transplantation of cardiac stem/progenitor cells improves function of infarcted hearts, the precise mechanisms of the improvement in function remain poorly understood. The present study demonstrates that transplantation of sheets of clonally expanded stem cell antigen 1–positive (Sca-1–positive) cells (CPCs) ameliorates cardiac dysfunction after myocardial infarction in mice. CPC efficiently differentiated into cardiomyocytes and secreted various cytokines, including soluble VCAM-1 (sVCAM-1). Secreted sVCAM-1 induced migration of endothelial cells and CPCs and prevented cardiomyocyte death from oxidative stress through activation of Akt, ERK, and p38 MAPK. Treatment with antibodies specific for very late antigen-4 (VLA-4), a receptor of sVCAM-1, abolished the effects of CPC-derived conditioned medium on cardiomyocytes and CPCs in vitro and inhibited angiogenesis, CPC migration, and survival in vivo, which led to attenuation of improved cardiac function following transplantation of CPC sheets. These results suggest that CPC transplantation improves cardiac function after myocardial infarction through cardiomyocyte differentiation and paracrine mechanisms mediated via the sVCAM-1/VLA-4 signaling pathway.     

静脉移植骨髓间质干细胞改善大鼠梗死心脏功能

目的:探讨经外周静脉途径移植骨髓间质干细胞治疗急性心肌梗死的有效性和可能的机制。方法:采用左前降支冠状动脉结扎术制备大鼠心肌梗死模型,24h后经尾静脉注入同种异体骨髓间质干细胞或磷酸缓冲液;分别于移植后3d和1个月取材,应用ELISA和免疫组化技术分析细胞移植对血管内皮生长因子(VEGF)表达的影响,通过免疫组化染色观察移植细胞的分化情况并计数血管;超声心动图连续检测大鼠心功能在术后各时间点的变化。结果:细胞移植术后3d,移植组VEGF蛋白的表达量明显高于对照组(P<0.01),免疫组化显示移植组心脏的梗死区及其周边区域的细胞内VEGF阳性着色明显强于对照组,1个月时明显减弱。移植术后1个月,在部分移植细胞中检测到心肌细胞或血管内皮细胞特异性蛋白的表达,血管计数显示移植组血管数量较同期对照组增加1.4倍(P<0.01);同时移植组心功能较对照组明显改善(P<0.01)。结论:通过促进梗死周边缺血心肌的血管新生和移植细胞分化,骨髓间质干细胞经外周静脉移植可作为急性心肌梗死治疗的另一条有效路径。     

Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF

Using a rat model of ischemia/reperfusion injury, we demonstrate here that HGF is cardioprotective due to its antiapoptotic effect on cardiomyocytes. Following transient myocardial ischemia and reperfusion, c-Met/HGF receptor expression rapidly increased in the ischemic myocardium, an event accompanied by a dramatic increase in plasma HGF levels in the infarcted rats. When endogenous HGF was neutralized with a specific antibody, the number of myocyte cell deaths increased markedly, the infarct area expanded, and the mortality increased to 50%, as compared with a control group in which there was no mortality. Plasma from the myocardial infarcted rats had cardioprotective effects on primary cultured cardiomyocytes, but these effects were significantly diminished by neutralizing HGF. In contrast, recombinant HGF administration reduced the size of infarct area and improved cardiac function by suppressing apoptosis in cardiomyocytes. HGF rapidly augmented Bcl-xL expression in injured cardiomyocytes both in vitro and in vivo. As apoptosis of cardiomyocytes is one of the major contributors to the pathogenesis in subjects with ischemia/reperfusion injury, prevention of apoptosis may prove to be a reasonable therapeutic strategy. Supplements of HGF, an endogenous cardioprotective factor, may be found clinically suitable in treating subjects with myocardial infarction.     

Differential myocardial infarct repair with muscle stem cells compared to myoblasts.

Myoblast transplantation for cardiac repair has generated beneficial results in both animals and humans; however, poor viability and poor engraftment of myoblasts after implantation in vivo limit their regeneration capacity. We and others have identified and isolated a subpopulation of skeletal muscle-derived stem cells (MDSCs) that regenerate skeletal muscle more effectively than myoblasts. Here we report that in comparison with a myoblast population, MDSCs implanted into infarcted hearts displayed greater and more persistent engraftment, induced more neoangiogenesis through graft expression of vascular endothelial growth factor, prevented cardiac remodeling, and elicited significant improvements in cardiac function. MDSCs also exhibited a greater ability to resist oxidative stress-induced apoptosis compared to myoblasts, which may partially explain the improved engraftment of MDSCs. These findings indicate that MDSCs constitute an alternative to other myogenic cells for use in cardiac repair applications.     

Basophils balance healing after myocardial infarction via IL-4/IL-13

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6C^lo macrophages toward increased numbers of inflammatory Ly6C^hi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6C^hi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.     

A novel protective effect of erythropoietin in the infarcted heart

Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.     

Sonic hedgehog is a critical mediator of erythropoietin-induced cardiac protection in mice

Erythropoietin reportedly has beneficial effects on the heart after myocardial infarction, but the underlying mechanisms of these effects are unknown. We here demonstrate that sonic hedgehog is a critical mediator of erythropoietin-induced cardioprotection in mice. Treatment of mice with erythropoietin inhibited left ventricular remodeling and improved cardiac function after myocardial infarction, independent of erythropoiesis and the mobilization of bone marrow–derived cells. Erythropoietin prevented cardiomyocyte apoptosis and increased the number of capillaries and mature vessels in infarcted hearts by upregulating the expression of angiogenic cytokines such as VEGF and angiopoietin-1 in cardiomyocytes. Erythropoietin also increased the expression of sonic hedgehog in cardiomyocytes, and inhibition of sonic hedgehog signaling suppressed the erythropoietin-induced increase in angiogenic cytokine expression. Furthermore, the beneficial effects of erythropoietin on infarcted hearts were abolished by cardiomyocyte-specific deletion of sonic hedgehog. These results suggest that erythropoietin protects the heart after myocardial infarction by inducing angiogenesis through sonic hedgehog signaling.     

Cellular mechanisms underlying cardiac engraftment of stem cells

Introduction: Over the past decade, it has become clear that long-term engraftment of any ex vivo expanded cell product transplanted into injured myocardium is modest and all therapeutic regeneration is mediated by stimulation of endogenous repair rather than differentiation of transplanted cells into working myocardium. Given that increasing the retention of transplanted cells boosts myocardial function, focus on the fundamental mechanisms limiting retention and survival of transplanted cells may enable strategies to help to restore normal cardiac function.

Areas covered: This review outlines the challenges confronting cardiac engraftment of ex vivo expanded cells and explores means of enhancing cell-mediated repair of injured myocardium.

Expert opinion: Stem cell therapy has already come a long way in terms of regenerating damaged hearts though the poor retention of transplanted cells limits the full potential of truly cardiotrophic cell products. Multifaceted strategies directed towards fundamental mechanisms limiting the long-term survival of transplanted cells will be needed to enhance transplanted cell retention and cell-mediated repair of damaged myocardium for cardiac cell therapy to reach its full potential.     

The potential of embryonic stem cells to treat heart disease

Cell therapy is currently receiving growing interest as a new means of repairing infarcted myocardium. Despite the encouraging experimental results yielded by autologous skeletal myoblasts and bone marrow-derived hematopoietic and mesenchymal stem cells, there is increasing evidence that the plasticity of these adult cells is more limited than initially thought and that, consequently, their conversion into cardiomyocytes is unlikely or, at best, quantitatively very limited. As the engrafted cells should electromechanically interact with host cardiac cells to form a functional syncytium, attention is now increasingly focused on cells that feature a true cardiomyogenic differentiation potential, enabling them to connect with the neighboring cardiomyocytes. In this setting, embryonic stem cells are particularly attractive, since they can be precommitted towards a cardiac lineage and complete their full maturation in vivo, possibly under the influence of host tissue-associated paracrine signaling pathways. Although a potential clinical use of embryonic stem cells is still fraught with difficulty (amplification, purification and immunogenicity), available experimental data suggest a consistent efficacy in repairing infarcted myocardium, which has stimulated efforts to address these issues.     

Carvedilol Enhances Mesenchymal Stem Cell Therapy for Myocardial Infarction via Inhibition of Caspase-3 Expression

Adult stem cells have shown great promise toward repairing infarcted heart and restoring cardiac function. Mesenchymal stem cells (MSCs), because of their inherent multipotent nature and their ability to secrete a multitude of growth factors and cytokines, have been used for cardiac repair with encouraging results. Preclinical studies showed that MSCs injected into infarcted hearts improve cardiac function and attenuate fibrosis. Although stem cell transplantation is a promising therapeutic option to repair the infarcted heart, it is faced with a number of challenges, including the survival of the transplanted cells in the ischemic region, due to excessive oxidative stress present in the ischemic region. The objective of this study was to determine the effect of Carvedilol (Carv), a nonselective β-blocker with antioxidant properties, on the survival and engraftment of MSCs in the infarcted heart. MSCs were subjected to a simulated host-tissue environment, similar to the one present in the infarcted myocardium, by culturing them in the presence of hydrogen peroxide (H(2)O(2)) to induce oxidative stress. MSCs were treated with 2.5 μM Carv for 1 h in serum-free medium, followed by treatment with H(2)O(2) for 2 h. The treated cells exhibited significant protection against H(2)O(2)-induced cell death versus untreated controls as determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. Likewise, transplantation of MSCs after permanent left coronary artery ligation and treatment of animals after myocardial infarction (MI) with Carv (5 mg/kg b.wt.) led to significant improvement in cardiac function, decreased fibrosis, and caspase-3 expression compared with the MI or MSC-alone groups.     

Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)-derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6-10-fold because of induction of proliferation on purification. Long-term engraftment (4-5 months) was observed when co-transplanting selected ES cell-derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell-derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.     

Isolation and expansion of resident cardiac progenitor cells

After myocardial infarction, loss of viable cardiomyocytes severely impairs cardiac function. Recently, stem cell transplantation has been put forward as a promising approach to repair the damaged heart. Although several clinical trials have already been performed, the dominant beneficial effects are probably due to neoangiogenesis and arteriogenesis. However, replacement of cardiomyocytes is vital to improve cardiac function in the long term. Stem cells and progenitor cells, with the capacity to differentiate into cardiomyocytes, have been described in both embryonic and adult tissues. Upon stimulation, cardiac progenitor cells proliferate and differentiate into cardiomyocytes, vascular smooth muscle cells, and endothelial cells. Currently however, high proliferation rates and differentiation of cardiac progenitor cells beyond the fetal stage have not yet been achieved. Full differentiation into adult cardiomyocytes in vitro and in vivo might be important for efficient integration with the host environment and therefore more research is needed to study factors that influence proliferation and differentiation. Here we will discuss the isolation of cardiac progenitor cells, their potential to differentiate into various cell types needed for cardiac repair, the possible mechanisms behind these events, and how these cells may be implemented in future clinical settings.     

The response of neonatal rat ventricular myocytes to lipopolysaccharide-induced stress

Sepsis induced by exposure to lipopolysaccharide (LPS) can be life-threatening and lead to multiple-organ dysfunction. Sepsis-associated cardiac dysfunction is a primary cause of mortality. The response of isolated cardiac myocytes to LPS exposure is poorly understood. Cultured neonatal rat ventricular cardiomyocytes were used to evaluate the response to LPS exposure. Other authors have reported that LPS exposure at doses sufficient to induce tumor necrosis factor alpha (TNF-alpha) production and apoptosis in adult cardiomyocytes do not induce apoptosis in neonatal cardiomyocytes. We therefore hypothesized that neonatal cardiomyocytes have innate protective mechanisms that protect from septic damage. Cultured neonatal rat ventricular cardiomyocytes were stimulated by exposure to LPS for varying lengths of time. NFkappaB signaling pathways, TNF-alpha production, and Akt activation were monitored. We also assessed the induction of apoptosis in these cells by monitoring caspase-3 activity. LPS rapidly stimulates nuclear translocation of NFkappaB and Akt activation. TNF-alpha production is also stimulated. However, high doses of LPS are unable to induce apoptosis in these cells, and protection is not a function of Akt activation. LPS treatment also stimulated the levels of cyclooxygenase-2 and the production of downstream metabolites, specifically PGE2 and 15deoxyDelta12-14PGJ2 (15dPGJ2). Specific inhibition of cyclooxygenase-2 activity induced apoptosis in the presence of LPS, whereas direct exposure to 15dPGJ2 at pharmacological levels induced apoptosis. Neonatal rat ventricular cardiomyocytes have innate protective mechanisms that prevent apoptotic cell death after LPS exposure. Metabolic products of arachidonic acid metabolized by the cyclooxygenase pathway can be potentially apoptotic or antiapoptotic. The balance of these products within these cells may define the cellular response to LPS exposure.     

Cardiac remodeling by fibrous tissue after infarction in rats

After transmural myocardial infarction (MI), extensive myocardial remodeling by fibrous tissue appears in both infarcted and noninfarcted myocardium, which contributes to ventricular diastolic dysfunction. In the present study we sought to assess the time course of collagen remodeling in the infarcted rat hearts by detecting spatial and time-dependent cellular events related to collagen synthesis and degradation 2 to 28 days after left coronary artery ligation. In infarcted hearts, and compared with findings in sham-operated and unoperated rat hearts, we found the following: (1) macrophages infiltrated into sites of MI and visceral pericardium on day 2 and gradually disappeared after day 14; (2) myofibroblasts (MyoFb) first appeared at these sites of repair on day 3 and remained abundant thereafter at all time points examined; (3) transforming growth factor-beta1 (TGF-beta1) mRNA was enhanced in infarcted and noninfarcted myocardium on day 2 and remained throughout 28 days; (4) type I and III collagen mRNAs began to increase at and remote to MI on day 3 and remained elevated thereafter; (5) matrix metalloproteinase-1 mRNA was significantly increased at and remote to MI on day 3, declined to the control level on day 7, and remained low thereafter; (6) tissue inhibitor of matrix metalloproteinase (TIMP)-I, -II, and -III mRNAs were markedly elevated at sites of repair on day 3 and sustained throughout 28 days; (7) fibrillar collagen accumulation that was evident at and remote to MI on day 7 continued to accumulate thereafter at each site over 4 weeks. When compared with findings in unoperated rat heart, pericardial fibrosis was evident in both infarcted and noninfarcted heart, and the temporal response of collagen generation/ degradation in pericardium was similar to that in infarcted myocardium. Thus collagen synthesis is activated in both infarcted and noninfarcted rat myocardium after transmural anterior infarction and is persistent throughout the 28-day period of study, whereas early collagen degradation is short lived and inactivated in the fibrogenic phase. Activated TGF-beta1 mRNA expression is accompanied by the appearance of MyoFb and the expression of fibrillar collagens and TIMPs, suggesting that this fibrogenic cytokine may contribute to collagen remodeling in the rat heart after MI.     

Epicardial Placement of Mesenchymal Stromal Cell-sheets for the Treatment of Ischemic Cardiomyopathy; In Vivo Proof-of-concept Study

Transplantation of bone marrow mesenchymal stromal cells (MSCs) is an emerging treatment for heart failure. We have reported that epicardial placement of MSC-sheets generated using temperature-responsive dishes markedly increases donor MSC survival and augments therapeutic effects in an acute myocardial infarction (MI) model, compared to intramyocardial (IM) injection. This study aims to expand this knowledge for the treatment of ischemic cardiomyopathy, which is likely to be more difficult to treat due to mature fibrosis and chronically stressed myocardium. Four weeks after MI, rats underwent either epicardial MSC-sheet placement, IM MSC injection, or sham treatment. At day 28 after treatment, the cell-sheet group showed augmented cardiac function improvement, which was associated with over 11-fold increased donor cell survival at both days 3 and 28 compared to IM injection. Moreover, the cell-sheet group showed improved myocardial repair, in conjunction with amplified upregulation of a group of reparative factors. Furthermore, by comparing with our own previous data, this study highlighted similar dynamics and behavior of epicardially placed MSCs in acute and chronic stages after MI, while the acute-phase myocardium may be more responsive to the stimuli from donor MSCs. These proof-of-concept data encourage further development of the MSC-sheet therapy for ischemic cardiomyopathy toward clinical application.     

Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice

MMPs are implicated in LV remodeling after acute myocardial infarction (MI). To analyze the role of MMP-2, we generated MI by ligating the left coronary artery of MMP-2-KO and WT mice, the latter of which were administered orally an MMP-2-selective inhibitor or vehicle (TISAM). The survival rate was significantly higher in MMP-2-KO and TISAM-treated mice than in control WT mice. The main cause of mortality in control WT mice was cardiac rupture, which was not observed in MMP-2-KO or TISAM-treated mice. Control WT mice, but not MMP-2-KO or TISAM-treated mice, showed activation of the zymogen of MMP-2, strong gelatinolytic activity, and degradation of ECM components, including laminin and fibronectin, in the infarcted myocardium. Although infarcted cardiomyocytes in control WT mice were rapidly removed by macrophages, the removal was suppressed in MMP-2-KO and TISAM-treated mice. Macrophage migration was induced by the infarcted myocardial tissue from control WT mice and was inhibited by treatment of macrophages with laminin or fibronectin peptides prior to migration assay. These data suggest that inhibition of MMP-2 activity improves the survival rate after acute MI by preventing cardiac rupture and delays post-MI remodeling through a reduction in macrophage infiltration.     

Toll-like receptor 2 activation by bacterial peptidoglycan-associated lipoprotein activates cardiomyocyte inflammation and contractile dysfunction

OBJECTIVE: Although cardiac dysfunction plays an important role in the pathogenesis of sepsis, the mechanisms that underlie cardiac dysfunction in sepsis remain poorly understood. Bacterial peptidoglycan-associated lipoprotein (PAL), an outer-membrane protein of Gram-negative bacteria, was recently found to be released into the bloodstream in sepsis and to cause inflammation and death in mice. The present studies assessed the effects of PAL on cardiomyocyte function and its signal transduction in cardiomyocytes. DESIGN: Randomized prospective animal study. SETTING: Research laboratory. SUBJECTS: Male C57BL/6 mice, B6;129S-Tnfrsf1a(tm1Imx) Tnfrsf1b(tm1Imx)/J knockout mice, Toll-like receptor 2 (TLR2) knockout mice, and myeloid differentiation factor 88 (MyD88) knockout mice. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Immunohistochemical staining and immunoblot analysis indicated that intravenously injected PAL bound to myocardium. Injection of PAL decreased cardiac function in vivo. Challenge with PAL altered cell shortening and Ca2+ transients in isolated mouse cardiomyocytes but not in cardiomyocytes isolated from TLR2 -/- and MyD88 -/- mice. Cytokine profiling arrays demonstrated that tumor necrosis factor-alpha (TNFalpha), granulocyte colony-stimulating factor, and interferon-gamma-production were elevated in PAL-treated cardiomyocytes. Increased TNFalpha production was abolished in MyD88 -/- cardiomyocytes but restored by adenovirally mediated expression of MyD88. PAL did not affect cell shortening and Ca2+ cycling in cardiomyocytes obtained from mice deficient for TNFalpha receptor (TNFR) 1 and TNFR2 (TNFR1/2 -/-). CONCLUSION: Our data reveal that PAL uses the TLR2/MyD88 signaling cascade to induce cardiomyocyte dysfunction and inflammatory responses and that TNFalpha is a major mediator of PAL-induced dysfunction in cardiomyocytes. These studies suggest that circulating PAL and other TLR2 agonists may contribute to cardiac dysfunction in sepsis.     

RXRalpha overexpression in cardiomyocytes causes dilated cardiomyopathy but fails to rescue myocardial hypoplasia in RXRalpha-null fetuses

Retinoid X receptor alpha-null (RXRalpha-null) mutants exhibit hypoplasia of their ventricular myocardium and die at the fetal stage. In the present study, we wished to determine whether transgenic re-expression of RXRalpha in mutant cardiac myocytes could rescue these defects. Two transgenic mouse lines specifically overexpressing an RXRalpha protein in cardiomyocytes were generated, using the cardiac alpha-myosin heavy chain (alpha-MHC) promoter. Breeding the high copy number transgenic line onto an RXRalpha-null genetic background did not prevent the myocardial hypoplasia and fetal lethality associated with the RXRalpha(-/-) genotype, even though the transgene was expressed in the ventricles as early as 10. 5 days post-coitum. These data suggest that the RXRalpha function involved in myocardial growth may correspond to a non-cell-autonomous requirement forsignal orchestrating the growth and differentiation of myocytes. Interestingly, the adult transgenic mice developed a dilated cardiomyopathy, associated with myofibrillar abnormalities and specific deficiencies in respiratory chain complexes I and II, thus providing an additional model for this genetically complex disease.