Loss of mitochondrial DNA in rabbit bladder smooth muscle following partial outlet obstruction results from lack of organellar DNA replication

When the rabbit bladder outlet is partially obstructed, the relative amount of mitochondrial (mt) DNA per cell in bladder smooth muscle falls rapidly. In order to assess whether this loss of organellar genome results from att enuation of mt DNA replication, we cloned portions of rabbit genes specifyi ng the single-strand binding (SSB) protein required for initiation of mt DN A replication, and the catalytic subunit of DNA polymerase, gamma (pol gamm a), the replication enzyme itself. We then designed primer-probe systems fo r real-time RT-PCR (TaqMan(TM)) analyses for each gene. These were used to assess mRNA in preparations from bladder smooth muscle and mucosa from rabb its subjected to surgical obstruction of the bladder outlet for up to 14 da ys. mRNA from the pol gamma gene remained essentially at control level in s mooth muscle and mucosa in all samples. In mucosa, mRNA from the SSB protei n gene remained virtually at control levels in all samples, as did mt genom e copy number. In smooth muscle, however, levels of this mRNA declined by > 95% within 3 days of obstruction and remained at that level through 14 days ; this attenuation of SSB protein mRNA paralleled the loss of mt DNA in the same smooth muscle samples. Thus, lack of mt SSB protein, and consequently attenuated mt DNA replication, is a primary factor in the loss of mt genom e copies in bladder smooth muscle after outlet obstruction in the rabbit mo del of benign bladder dysfunction.