Neural Crest is Involved in Development of Abnormal Myocardial Function

Around 85% of embryos homozygous for thesplotch(Sp2H) allele (Sp2H/Sp2H), aPax3 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 heartsin situat 13.5 dpc. The hearts ofSp2H/Sp2Hembryos with PTA clearly showed poor function when compared with normal litter mates. Contractile force was examined in detergent-skinned ventricular muscle strips fromSp2H/Sp2Hembryos at ages 12.5 and 13.5 dpc. There was no significant difference in the maximum force or in myosin content betweenSp2H/Sp2Hand control groups, indicating no significant dysfunction of the contractile apparatus in hearts fromSp2H/Sp2Hembryos. Ca2+transients were examined in enzymatically-dissociated ventricular myocytes and were significantly reduced in defective hearts, indicating that reduced cardiac function inSp2H/Sp2Hembryos 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 ≈3.2-fold inSp2H/Sp2Hhearts 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 deathin uterofrom cardiac failure. Interestingly,Sp2H/Sp2Hhearts without PTA had normal EC coupling. These results indicated that impaired EC coupling was secondary to thePax3 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.