RXRα overexpression in cardiomyocytes causes dilated cardiomyopathy but fails to rescue myocardial hypoplasia in RXRα-null fetuses
Vemparala Subbarayan, … , Pierre Chambon, Philippe Kastner
Vemparala Subbarayan, … , Pierre Chambon, Philippe Kastner
Published February 1, 2000
Citation Information: J Clin Invest. 2000;105(3):387-394. https://doi.org/10.1172/JCI8150.
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Article

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

Abstract

Retinoid X receptor α–null (RXRα-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 RXRα in mutant cardiac myocytes could rescue these defects. Two transgenic mouse lines specifically overexpressing an RXRα protein in cardiomyocytes were generated, using the cardiac α-myosin heavy chain (α-MHC) promoter. Breeding the high copy number transgenic line onto an RXRα-null genetic background did not prevent the myocardial hypoplasia and fetal lethality associated with the RXRα–/– genotype, even though the transgene was expressed in the ventricles as early as 10.5 days post-coitum. These data suggest that the RXRα 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.

Authors

Vemparala Subbarayan, Manuel Mark, Nadia Messadeq, Pierre Rustin, Pierre Chambon, Philippe Kastner

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Figure 1

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Molecular characterization of MyHCα-RXRα transgenic mice. (a) Schematic ...
Molecular characterization of MyHCα-RXRα transgenic mice. (a) Schematic representation of the transgene construct (not at scale). Black boxes indicate exons 1–3 of the MyHCα gene, which correspond to 5′-untranslated sequences. P1 and P2 are the RT-PCR primer used in the experiment shown in Figure 2a. Probe A corresponds to a EcoRI-NdeI fragment from the MyHCα gene, and probe B corresponds to a EcoRI-KpnI fragment containing all RXRα coding sequences. Fragments detected by these probes after EcoRI or BamHI digestion are shown. B, BamHI; E, EcoRI; K, KpnI; N, NotI; Nd, NdeI; X, XhoI. (b) Southern blot analysis. A total of 10 μg of BamHI- or EcoRI-restricted genomic DNA from WT mice or mice from lines 41 or 54 were analyzed by Southern blotting with probes A and probe B, as indicated. Arrows point to the bands of the expected size, whereas stars point to additional bands having undergone some deletions or rearrangements. (c) RT-PCR analysis of tissue samples from line 41. Lanes 1–7 correspond to: (1) adult ventricle with the reverse transcriptase omitted in the RT step, (2) adult ventricle, (3) adult atrium, (4) lungs, (5) skeletal muscle, (6) liver, and (7) brain. The top panel corresponds to transgene transcript amplification with the transgene-specific primers depicted in a; the bottom panel corresponds to the control amplification of 36B4 gene transcripts (39). (d) Northern blot analysis of transgene expression in line 41. A total of 20 μg of total RNA from adult heart (lane 1), lungs (lane 2), spleen (lane 3), skeletal muscle (lane 4), kidney (lane 5), and liver (lane 6) were analyzed by Northern blotting with a cDNA probe corresponding to a 2-kb fragment containing the RXRα coding sequences. The bottom panel corresponds to the ethidium bromide staining of ribosomal 28S RNA of the same RNA sample separated on an identical gel. (e) Western blot analysis. A total of 50 μg of nuclear extracts prepared from WT liver (L) or heart (H) and from heart from transgenic mice from lines 41 or 54, as well as a control extract of bacterially produced RXRα (lane 5), was separated on a 8% SDS-polyacrylamide gel and revealed with polyclonal antibodies directed against either COOH-terminal RXRα epitopes (top panel) or an NH2-terminal RXRα epitope (bottom panel). Note that transgenic hearts express an RXRα polypeptide that is smaller than WT RXRα and not detected by the NH2-terminal antibody. Smaller fragments seen in lanes 3 and 5 likely correspond to proteolytic degradation products.