Our study identifies for the first time a molecular mechanism involving RyR2 dysfunction due to depressed calstabin2 expression and intracellular Ca2+
leak in Boxer dog ARVC. In our study we report markedly decreased myocardial calstabin2 expression, resulting in calstabin2 depletion in the RyR2 complex, as well as calstabin2 depletion within homogenates of whole cardiac LV tissue. Calstabin2 depletion from RYR2 has been previously demonstrated in both human26
models of HF.27
A study in dogs with pacing-induced HF revealed significantly decreased calstabin2 protein in the RyR2 complex as compared to control.11
PKA hyperphosphorylation of RyR2, a defect that is variably associated with the heart failure phenotype, chronic catecholamine stimulation, and depletion of calstabin2 from RyR2,26,28
was not found in Boxer dogs with ARVC, implicating reduced expression and decreased calstabin2 protein as likely mechanism of calstabin2 deficiency in the RYR2 channel complex.
A previous study indicated that RyR2 message and protein was decreased in both the RV and LV in Boxer dogs with ARVC, with the greatest reductions occurring in the RV.25
In our study, by immunoprecipitating the RyR2 macromolecular complex, we were able to standardize the amount of RyR2 protein used for immunoblotting and directly compare calstabin2 in the RyR2 complex between study groups. We found calstabin2 depletion in the LV of Boxer dog ARVC hearts, and this finding along with the presence of fatty infiltration of the LV, suggests that further comparative studies using RV and interventricular tissue from affected dogs and controls should be perfomed.29
Previous studies in Boxer dogs were not able to link ARVC to either the RyR2 gene25
or five different desmosomal genes, including plakophilin-2, plakoglobin, desmoplakin, desmoglein-2, and desmocollin. 30
In our study, direct sequencing of DNA samples from 10 affected Boxers with ARVC did not identify a causative mutation in the estimated promoter, exonic, or splice site regions of calstabin2. The mechanisms underlying the decreased calstabin2 mRNA expression in Boxer dogs with ARVC requires further investigation and may be associated with decreased mRNA stability and/or abnormalities of calstabin2 transcription.
Our study has several limitations. One criticism may be the use of non-Boxer dogs as controls and our findings should be corroborated by additional studies using well-defined control groups of healthy Boxers. In this study, we chose to use non-Boxer controls to avoid the inadvertent inclusion of individuals affected by subclinical (concealed) cardiomyopathy as a result of the high prevalence of ARVC in Boxer dogs15
and felt that significant breed differences involving important cardiac proteins were unlikely. Likewise, we compared our Boxer dog DNA samples to two Labrador retriever dogs since the published canine genome was developed from an adult Boxer dog that may not have been evaluated for ARVC.31
Another limitation involves the examination of RYR2 function from only LV samples of Boxer dogs, and further studies utilizing RYR2 isolated from the interventricular septum and RV of both control and affected animals are warranted. Our tissue samples were those of “convenience”, that is, gathered from a tissue bank that had been previously collected by the investigators (MAO, KMM). Due to the desire to compare different forms of canine cardiomyopathy, and the unavailability of RV tissue samples from control and Doberman pinschers with DCM, we chose to utilize LV samples from all groups. A third important limitation involves uncertainty if the calstabin2 deficiency we report is a primary or secondary abnormality. Boxer dogs demonstrated decreased calstabin2 expression compared to another form of naturally-occurring DCM in Dobermans, however expression in dogs undergoing rapid ventricular pacing was similar to that found in Boxers. Interestingly, all forms of cardiomyopathy evaluated in this study demonstrated decreased calstabin2 expression as compared to control, highlighting the possibility that decreased expression could be a secondary change in response to the heart failure phenotype; however in another breed of dog (Great Dane), which is also commonly afflicted with cardiomyopathy, calstabin2 expression as measured by a second generation oligonucleotide microarray is upregulated (unpublished data) and these apparent differences warrant further study. Even if calstabin2 deficiency is a secondary abnormality, restoration of calstabin2-RYR2 stoichiometry may be an attractive therapeutic target to help reduce incidence of arrhythmias.30
Finally, Xiao et al. recently reported the absence of inducible ventricular arrhythmias in calstabin2-null mice even when stimulated with epinephrine or caffeine,10
and further studies that specifically investigate the effect of RYR2 dysfunction on production of arrhythmias in Boxer dogs are needed.
Our findings of calstabin2 depletion in the RyR2 complex in the hearts of Boxer dogs with arrhythmogenic cardiomyopathy indicate a specific molecular mechanism with direct pathophysiological implications for the canine disease phenotype. Our results, together with previous studies, indicate that Boxer dogs with calstabin2 deficiency may represent a potentially important abnormality in Boxer ARVC.