This is the first study to report a SIDS-associated mutation in GJA1
-encoded Cx43 that causes formation of a biophysically non-functional gap junction channel. We discovered two novel Cx43 mutations in two unrelated SIDS victims, with both mutations involving highly conserved residues and absent in over 1000 ethnic-matched controls. The first, E42K, localizes to the transmembrane region near the extracellular loops, which are important for proper formation of fully functioning connexons.19
S272P localizes to the cytoplasmic C-terminus, near a region of multiple serine phosphorylation sites.19
However, functional studies for S272P showed wildtype trafficking and gap junctional coupling, whereas E42K demonstrated a profound reduction in junctional conductance for cells expressing either the E42K-Cx43 mutation alone or together with WT, similar to the phenotype of other disease-associated connexin mutations.11, 20
In addition, immunostaining of the decedent’s tissue revealed patchy loss of Cx43, suggestive of somatic mosaicism similar to that seen in cases of atrial fibrillation induced by somatic connexin 40 and 43 mutations10, 11
and highly predictive of an arrhythmogenic substrate. We predict therefore that while the death of the E42K-positive case may be directly due to mutation-induced pathology, the death of the S272P-positive case remains unexplained, since our investigations demonstrated wildtype junctional physiology with this second case.
Functional gap junction channels are formed by two hemichannels (connexons) in adjacent cells, each consisting of a hexamer of one or more types of connexin protein subunits, and allow for intercellular communication via the passage of electrical impulses between myocytes and thus facilitate synchronous contraction of the myocardium.21, 22
Cx43 is the major gap junction protein in ventricular tissue.21
Global deletion of the GJA1
gene in mice results in death shortly after birth, primarily due to structural abnormalities in the cardiac outflow tract present well before birth.12, 13
In contrast, Cx43 conditional knockout mice (CKO) show no significant differences in their phenotype or behavior compared to their control littermates and yet die suddenly at 81.4 ± 3.3 days.23
As shown by immunohistochemistry and Western immunoblotting studies, by 45 days, Cx43 expression in the ventricles was reduced to 18% in the CKO animals compared to controls. High power views showed areas where cells formed rare plaques alternating with clusters of Cx43-positively stained cells. The QRS amplitude was progressively decreased in the CKO animals and paralleled the loss of Cx43 expression. Programmed electrical stimulation induced sustained polymorphic VT in 8 of 10 CKO animals at 45 days, while only non-sustained VT was induced in only 1/3 of the control mice. Yet, despite the increased susceptibility of CKO mice to lethal arrhythmias, hemodynamic parameters and ventricular contractility of CKO mice at all time points was no different to controls.23
Moreover, chimeric Cx43 mice, which were generated by introducing Cx43-deficient embryonic stem cells in wildtype recipient blastocysts, also demonstrated conduction delay and spontaneous ventricular tachycardia in the absence of structural changes.24
Gap junction expression in those hearts was highly abnormal with foci of Cx43-deficient myocytes interspersed throughout an otherwise well-coupled myocardial syncitium.25
Telemetry recordings in the chimeric animals showed increased frequency of premature ventricular contractions compared to control littermates. Moreover, multiple morphologies of ventricular ectopy were recorded suggesting the presence of multiple arrhythmogenic foci. It is true that the chimeric animals showed decreased ventricular systolic function (fractional shortening was 33.2 ± 2.08% in the chimeric animals compared to 41.0 ± 1.27% in controls). However, although the LV systolic pressures were decreased in the chimeras, there were no differences in the diastolic pressures between chimeric and control animals. Moreover, there was no evidence of ventricular dilatation or hypertrophy.25
Taken together, these results suggest that gap junction remodeling predisposes to arrhythmias but does not obligate any significant mechanical dysfunction. It is likely that the cardiac tissue in our decent mimics this latter instance, and such findings in murine models suggest why no cardiac abnormalities were detected at autopsy to render a non-SIDS diagnosis and why the infant was able to survive two months before death.
Loss of ventricular gap junction function underlies formation of an arrhythmogenic substrate in animal models.24, 26, 27
However, as the previously discussed studies support, it is likely that heterogenous Cx43 loss, not reduction of Cx43 per se
, creates an arrhythmogenic substrate whereby safe, albeit slow, conduction is dissipated and disrupted by sinks of well-coupled cells. Thus the spontaneous ventricular tachyarrhythmias and sudden death noted in cardiac-restricted knockout mice may actually stem from low residual mosaic expression of Cx43. The pro-arrhythmic nature of heterogeneous Cx43 expression is further supported by the observation that heterogeneous populations of neonatal murine ventricular myocytes have pro-arrhythmic impulse propagation.28
Moreover, Boulaksil et al. demonstrated increased heterogeneity of Cx43 in both congestive heart failure patients with documented ventricular tachycardias as well as in a heart failure mouse model which showed inducible polymorphic VT in the setting of heterogeneous Cx43 loss.29
In this case, whether the heterogeneity of Cx43 is related to the polymorphic morphology of the tachycardias remains unsolved. Lastly, a Cx43 somatic mutation was recently found to underlie lone atrial fibrillation in a patient.11
Immunohistochemistry of the patient’s atrial tissue showed a mosaic pattern of intracellular retention of Cx43 and a mosaic pattern of aberrant gap junction formation, very much like the images we obtained from our case. Expression of the mutant Cx43 form in oocyte pairs from the genus Xenopus showed a dramatic decrease in cell-cell coupling conductance.11
We hypothesize, given the immunostaining pattern of the decedent, that mutant E42K-Cx43, although trafficking properly in an in vitro
setting, does not properly interact with adjacent Cx43 to form fully functional gap junctions, forming an arrhythmogenic substrate. We know that other cardiac disorders trigger reduction in Cx43 expression,30, 31
and it is likely that such changes may not be detected at levels assessed by our plaque formation assay. Further, our immunostaining assay is optimized to detect high levels of Cx43 at gap junctions.32
A change in expression may reduce Cx43 to a level undetectable by our current assay. This may explain why our in vitro
data in N2A cells demonstrates proper trafficking whereas intact decedent heart tissue demonstrates a mosaic pattern.
Since this infant was not wearing a Holter monitor at the time of his tragic demise, it is impossible to verify our postulated exit rhythm of ventricular fibrillation. Nevertheless, E42K-Cx43 was identified in a sudden death cohort, in a gene with known association with cardiac disease, was located in a highly conserved region of the gene, and absent in over 1000 ethnic-matched alleles. In addition, the heterogeneous staining pattern of the infant’s cardiac tissue offers strong evidence that this mutation conferred a sudden death predisposing arrhythmogenic phenotype. Further, this deceased infant’s loss-of-function E42K-Cx43 mutation and likely somatic mosaicism essentially represents the human equivalent of the chimeric Cx43 mouse. The S272P-Cx43 mutation had a wildtype phenotype in our investigations and therefore, although rare and highly conserved, it cannot at this time be considered a sudden-death predisposing mutation.
In conclusion, this is the first study to implicate a mutation in a connexin protein in the pathogenesis of SIDS. E42K-Cx43 demonstrated a severe loss-of-function phenotype, and is located in a crucial conserved region of the protein. While such SIDS-associated Cx43 mutations are rare, this study provides evidence for the contribution of Cx43 dysregulation to sudden death, and contributes to the growing body of literature implicating cardiac arrhythmias as the cause for a subset of SIDS.