Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria.

In this study a Ca(2+) sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca(2+)-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca(2+) sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca(2+) transient. Cytoplasmic (Fura-2) and mitochondrial Ca(2+) (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca(2+) loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca(2+) sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K(d) of approximately 220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca(2+) transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca(2+) release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by approximately 65% and increased the time to 50% decay, whilst cytosolic Ca(2+) transients were unchanged. The mitochondrial Ca(2+) uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca(2+). The mitochondrial-targeted Ca(2+) probe indicates sustained and transient phases of mitochondrial Ca(2+) signal, which are dependent on cytoplasmic Ca(2+) levels and require a functional mCU.

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.