TBX5 transcription factor regulates cell proliferation during cardiogenesis.

Mutations in human TBX5, a member of the T-box transcription factor gene family, cause congenital cardiac septation defects and isomerism in autosomal dominant Holt-Oram syndrome. To determine the cellular function of TBX5 in cardiogenesis, we overexpressed wild-type and mutant human TBX5 isoforms in vitro and in vivo. TBX5 inhibited cell proliferation of D17 canine osteosarcoma cells and MEQC quail cardiomyocyte-like cells in vitro. Mutagenesis of the 5' end of the T-box but not the 3' end of the T-box abolished this effect. Overexpression of TBX5 in embryonic chick hearts showed that TBX5 inhibits myocardial growth and trabeculation. TBX5 effects in vivo were abolished by Gly80Arg missense mutation of the 5' end of the T-box. PCNA analysis in transgenic chick hearts revealed that TBX5 overexpression does suppress embryonic cardiomyocyte proliferation in vivo. Inhibitory effects of TBX5 on cardiomyocyte proliferation include a noncell autonomous process in vitro and in vivo. TBX5 inhibited proliferation of both nontransgenic cells cocultured with transgenic cells in vitro and nontransgenic cardiomyocytes in transgenic chick hearts with mosaic expression of TBX5 in vivo. Immunohistochemical studies of human embryonic tissues, including hearts, also demonstrated that TBX5 expression is inversely related to cellular proliferation. We propose that TBX5 can act as a cellular arrest signal during vertebrate cardiogenesis and thereby participate in modulation of cardiac growth and development.

Identification and localization of TBX5 transcription factor during human cardiac morphogenesis.

Mutations in the TBX5 transcription factor gene cause human cardiac malformation in Holt-Oram syndrome. To identify and localize TBX5 during cardiac morphogenesis, we performed immunohistochemical studies of TBX5 protein cardiac expression during human embryogenesis. Specific antibody to human TBX5 was generated in rabbits with a TBX5 synthetic peptide and affinity purification of antiserum. Anti-TBX5 was used in immunohistochemical analyses of human cardiac tissue. In embryonic and adult heart, TBX5 is expressed throughout the epicardium and in cardiomyocyte nuclei in myocardium of all four cardiac chambers. Endocardial expression of TBX5 is only present in left ventricle. Asymmetric left-sided transmyocardial gradients of TBX5 protein expression were observed in embryonic but not adult hearts. Human cardiac expression of TBX5 protein correlates with the cardiac manifestations of Holt-Oram syndrome. TBX5 transmyocardial protein gradients may contribute to normal patterning of the human heart during embryogenesis.

Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex.

Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

Atrial form and function: lessons from human molecular genetics.

Molecular genetic analyses of human hereditary disorders that affect cardiac atrial structure and function have recently identified several genes that regulate atrial morphogenesis. Mutations of the TBX5, NKX2.5, EVC, and PRKAR1 alpha genes all result in abnormalities of human atrial growth and development, and mutations in at least one gene results in familial atrial fibrillation and is as yet unidentified. Ongoing studies to find interactions between these transcription factors and intracellular signaling molecules and other as yet unknown genes are establishing critical pathways in human cardiogenesis. Human investigation and experimental animal models of heart development synergize to elucidate etiologies of common congenital heart disease.

Excessive microtubules are not responsible for depressed force per cross-bridge in cardiac neural-crest-ablated embryonic chick hearts.

Ablation of the cardiac neural crest (CNCA) in embryonic chicks results in a high incidence of persistent truncus arteriosus, a congenital heart defect associated with decreased myocardial contractility. Using left ventricular trabeculae from chicks at embryonic day (ED) 15, we have previously shown that the twitch force of intact preparations is significantly reduced whereas the maximal calcium-activated force of skinned preparations is not significantly different in CNCA and sham-operated animals. We also previously found that the ventricular content of myosin, as well as of actin and tropomyosin, was nearly doubled in ED 15 hearts after CNCA. Since the number of cross-bridges is proportional to the myosin concentration, these data suggest that the force exerted per cross-bridge is decreased in CNCA hearts. We investigated the possibility that the decrease in force per cross-bridge is caused by inhibition of the contractile apparatus by excessive microtubules. To the contrary, we found that the total beta-tubulin content and the fraction of beta-tubulin polymerized in microtubules measured by Western blotting was the same in ventricular muscle strips from CNCA and sham-operated embryos. Furthermore, exposure to microtubule-destabilizing agents did not improve the force-producing capability of the contractile apparatus in CNCA embryos. We conclude that depression of force per cross-bridge in hearts from CNCA embryos is not due to an excess of microtubules.

Neural crest is involved in development of abnormal myocardial function.

Around 85% of embryos homozygous for the splotch (Sp2H) allele (Sp2H/Sp2H), a Pax3 mutation, develop persistent truncus arteriosus (PTA), a defect related to the cardiac neural crest. These embryos die by 14.5 days post coitum. In an investigation of the cause of lethality in these embryos, we used digital video imaging microscopy to examine beating embryonic hearts in situ at 13.5 dpc. The hearts of Sp2H/Sp2H embryos with PTA clearly showed poor function when compared with normal litter mates. Contractile force was examined in detergent-skinned ventricular muscle strips from Sp2H/Sp2H embryos at ages 12.5 and 13.5 dpc. There was no significant difference in the maximum force or in myosin content between Sp2H/Sp2H and control groups, indicating no significant dysfunction of the contractile apparatus in hearts from Sp2H/Sp2H embryos. Ca2+ transients were examined in enzymatically-dissociated ventricular myocytes and were significantly reduced in defective hearts, indicating that reduced cardiac function in Sp2H/Sp2H embryos with PTA was due to impaired excitation-contraction (EC) coupling. Ca2+ currents were examined using the perforated patch clamp technique. The magnitude of the Ca2+ current was found to be reduced by approximately 3.2-fold in Sp2H/Sp2H hearts with PTA compared to normal. Since the sarcoplasmic reticulum is sparse or absent in the embryonic heart, the impaired EC coupling was due to the reduction in Ca2+ current. These observations suggest that neural crest abnormalities result in a defect in EC coupling, causing depressed myocardial function and death in utero from cardiac failure. Interestingly, Sp2H/Sp2H hearts without PTA had normal EC coupling. These results indicated that impaired EC coupling was secondary to the Pax3 mutation. The findings in this report indicate an important role for the neural crest in the development of normal myocardial function, and represent the first demonstration of impaired excitation-contraction coupling in a genetically-defined embryonic mammalian model of a cardiac structural defect.

Effect of cardiac neural crest ablation on contractile force and calcium uptake and release in chick heart.

Cardiac neural crest ablation (CNCA) in the chick embryo at stages 8-10 results in reduced contractility of the heart that can be observed as early as stage 14. We found that intact trabeculae from embryonic day (E) 15 experimental animals after CNCA display an approximately 50% decrease in twitch force relative to sham-operated E15 control animals. In control and CNCA trabeculae skinned in Triton X-100 and bathed in our standard solutions, neither maximum Ca(2+)-activated force nor Ca2+ sensitivity of the contractile apparatus was significantly different. CNCA resulted in a marked reduction in the magnitude of the Ca2+ transient in trabeculae, estimated using fura 2 acetoxymethyl ester. CNCA had no effect on the half-time of Ca2+ loading by the sarcoplasmic reticulum (SR) of saponin skinned trabeculae at fixed Ca2+. However, it slightly reduced the Ca2+ sensitivity of Ca2+ uptake by the SR. Its most dramatic effect was to essentially abolish Ca(2+)-induced Ca2+ release from the SR. These effects on Ca2+ metabolism explain, in part, the decrease in the intracellular Ca2+ transient and myocardial contractility observed with CNCA.