Extremely acidophilic microorganisms (optimum growth pH of 3) maintain a near neutral cytoplasmic pH via several homeostatic mechanisms, including an inside positive membrane potential created by potassium ions. Transcriptomic responses to pH stress in the thermoacidophilic archaeon, Sulfolobus acidocaldarius were investigated by growing cells without added sodium and/or potassium ions at both optimal and sub-optimal pH. Culturing the cells in the absence of added sodium or potassium ions resulted in a reduced growth rate compared to full-salt conditions as well as 43 and 75 significantly different RNA transcript ratios, respectively. Differentially expressed RNA transcripts during growth in the absence of added sodium ions included genes coding for permeases, a sodium/proline transporter and electron transport proteins. In contrast, culturing without added potassium ions resulted in higher RNA transcripts for similar genes as a lack of sodium ions plus genes related to spermidine that has a general role in response to stress and a decarboxylase that potentially consumes protons. The greatest RNA transcript response occurred when S. acidocaldarius cells were grown in the absence of potassium and/or sodium at a sub-optimal pH. These adaptations included those listed above plus osmoregulated glucans and mechanosensitive channels that have previously been shown to respond to osmotic stress. In addition, data analyses revealed two co-expressed IclR family transcriptional regulator genes with a previously unknown role in the S. acidocaldarius pH stress response. Our study provides additional evidence towards the importance of potassium in acidophile growth at acidic pH.
A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image-and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine-an open channel blocker of HCNs-for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755). Heart rate variability (HRV) reflects the inter-beat variation of the RR interval. HRV is controlled by the sinoatrial node, which receives input from the autonomic nervous system. Autonomic imbalance has been associated with work stress and other modifiable or non-modifiable risk factors 1 , and is reflected in lower HRV. Lower HRV has been associated with higher cardiac morbidity and mortality 2 , as well as with a higher risk of all-cause mortality 3. HRV can be quantified non-invasively using an ECG, making HRV a useful clinical marker for perturbations of the autonomic nervous system. However, the mechanisms influencing HRV remain poorly understood. Recently, we and others identified the first loci that are robustly associated with HRV, using a meta-analysis of genome-wide association studies (GWAS) with data from 53,174 participants 3. Five of the identified loci had
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