Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2) = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84).A change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination