Previous studies suggest native cardiac I Kr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular I Kr channels are heteromers containing two ␣ subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits.Long QT syndrome (LQTS) 1 is an inherited or acquired disease associated with episodic ventricular arrhythmias and sudden death. One form of inherited LQTS (LQTS-2) results from mutations in the human Ether-a-go-go-Related Gene 1 (hERG1or KCNH2) (1). hERG1 encodes a potassium channel with biophysical and pharmacological properties similar to those of cardiac I Kr , thus explaining the underlying cause of LQTS-2 as a defect in this repolarizing current (2, 3). In mammalian heart, two ERG 2 transcripts, 1a and 1b, encode proteins differing in their amino-terminal sequence (see Fig. 1A) and gating properties (4,5). Expressed in Xenopus oocytes, these subunits preferentially form heteromultimers (4). However, despite high levels of ERG1b transcript (4), first generation ERG1 antibodies against a common epitope identified only ERG1a protein in native tissue (6, 7), suggesting that ERG1b subunits do not contribute to cardiac I Kr channels. Here we provide the first direct evidence for ERG1b protein expression, localization, and co-assembly with ERG1a in cardiac ventricular myocytes. These findings indicate cardiac I Kr channels are minimally composed of ERG1a and -1b ␣ subunits. EXPERIMENTAL PROCEDURESCell Lines and Antibodies-Human embryonic kidney 293 (HEK-293) cell lines stably expressing wild-type hERG1a have been described previously (8, 9). Cell lines stably expressing hERG1a and -1b were prepared by transfection of HEK-293/hERG1a stable cells with hERG1b containing a Kozak consensus sequence (4) cloned into the BamHI/EcoRI sites of pcDNA3.1z (Invitrogen). Separate cell colonies were selected after plating cells at low density and grown in media containing 100 g/ml Zeocin, 500 g/ml neomycin for selection. All HEK-293 cells were cultured in Dulbecco's modified Eagle's medium at 37°C. The pan-ERG1 antibody, ERG1-KA, has been described previously (10). ERG1 isoform-specific antibodies were produced by Bethyl Laboratories (Montgomery, TX) in rabbits. Antisera were affinity-purified using the same peptides employed in immunization. The sequence for the ERG1b peptide is amino acids 12-25 (GALRPRAQKGRVRR), and the sequence for ERG1a is amino acids 140 -153 (SPAHDTNHRG-PPTS) (Neoclo...

Primary ovarian insufficiency (POI) is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR) as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours) than granulosa cells (measurable at 4 hours), as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10–12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult in the ovary must act almost immediately to prevent acute insult as significant damage was seen in stroma cells within the first two hours.

Doxorubicin (DXR) is a frontline chemotherapy agent implicated in unintended ovarian failure in female cancer survivors. The fertility preservation techniques currently available for cancer patients are often time and cost prohibitive and do not necessarily preserve endocrine function. There are no drug-based ovary protection therapies clinically available. This study provides the first investigation using dexrazoxane (Dexra) to limit DXR insult in ovarian tissue. In KK-15 granulosa cells, a 3-h DXR treatment increased double-strand (ds) DNA breaks 40%-50%, as quantified by the neutral comet assay, and dose-dependent cytotoxicity. Dexra exhibited low toxicity in KK-15 cells, inducing no DNA damage and less than 20% cell loss. Cotreating KK-15 cells with Dexra prevented acute DXR-induced dsDNA damage. Similarly, Dexra attenuated the DXR-induced 40%-65% increase in dsDNA breaks in primary murine granulosa cells and cells from in vitro cultured murine ovaries. DXR can cause DNA damage either through a topoisomerase II-mediated pathway, based on DXR intercalation into DNA, or through oxidative stress. Cotreating KK-15 cells with 2 μM Dexra was sufficient to prevent DXR-induced, but not H(2)O(2)-induced, DNA damage. These data indicated the protective effects are likely due to Dexra's inhibition of topoisomerase II catalytic activity. This putative protective agent attenuated downstream cellular responses to DXR, preventing H2AFX activation in KK-15 cells and increasing viability as demonstrated by increasing the DXR lethal dose in KK-15 cells 5- to 8-fold (LD(20)) and primary murine granulosa cells 1.5- to 2-fold (LD(50)). These data demonstrate Dexra protects ovarian cells from DXR insult and suggest that it is a promising tool to limit DXR ovarian toxicity in vivo.

Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment.

Advances in cancer treatment utilizing multiple chemotherapies have dramatically increased cancer survivorship. Female cancer survivors treated with doxorubicin (DXR) chemotherapy often suffer from an acute impairment of ovarian function, which can persist as long-term, permanent ovarian insufficiency. Dexrazoxane (Dexra) pretreatment reduces DXR-induced insult in the heart, and protects in vitro cultured murine and non-human primate ovaries, demonstrating a drug-based shield to prevent DXR insult. The present study tested the ability of Dexra pretreatment to mitigate acute DXR chemotherapy ovarian toxicity in mice through the first 24 hours post-treatment, and improve subsequent long-term fertility throughout the reproductive lifespan. Adolescent CD-1 mice were treated with Dexra 1 hour prior to DXR treatment in a 1:1 mg or 10:1 mg Dexra:DXR ratio. During the acute injury period (2–24 hours post-injection), Dexra pretreatment at a 1:1 mg ratio decreased the extent of double strand DNA breaks, diminished γH2FAX activation, and reduced subsequent follicular cellular demise caused by DXR. In fertility and fecundity studies, dams pretreated with either Dexra:DXR dose ratio exhibited litter sizes larger than DXR-treated dams, and mice treated with a 1:1 mg Dexra:DXR ratio delivered pups with birth weights greater than DXR-treated females. While DXR significantly increased the “infertility index” (quantifying the percentage of dams failing to achieve pregnancy) through 6 gestations following treatment, Dexra pretreatment significantly reduced the infertility index following DXR treatment, improving fecundity. Low dose Dexra not only protected the ovaries, but also bestowed a considerable survival advantage following exposure to DXR chemotherapy. Mouse survivorship increased from 25% post-DXR treatment to over 80% with Dexra pretreatment. These data demonstrate that Dexra provides acute ovarian protection from DXR toxicity, improving reproductive health in a mouse model, suggesting this clinically available drug may provide ovarian protection for cancer patients.