Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca(2+)-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.     

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.     

miRNAs got rhythm

Despite significant advances in treatments, cardiovascular disease (CVD) remains the leading cause of human morbidity and mortality in developed countries. The development of novel and efficient treatment strategies requires an understanding of the basic molecular mechanisms underlying cardiac function. MicroRNAs (miRNAs) are a family of small nonprotein-coding RNAs that have emerged as important regulators in cardiac and vascular developmental and pathological processes, including cardiac arrhythmia, fibrosis, hypertrophy and ischemia, heart failure and vascular atherosclerosis. The miRNA acts as an adaptor for the miRNA-induced silencing complex (miRISC) to specifically recognize and regulate particular mRNAs. Mature miRNAs recognize their target mRNAs by base-pairing interactions between nucleotides 2 and 8 of the miRNA (the seed region) and complementary nucleotides in the 3'-untranslated region (3'-UTR) of mRNAs and miRISCs subsequently inhibit gene expression by targeting mRNAs for translational repression or cleavage. In this review we summarize the basic mechanisms of action of miRNAs as they are related to cardiac arrhythmia and address the potential for miRNAs to be therapeutically manipulated in the treatment of arrhythmias.     

Redox interaction of Mn-bicarbonate complexes with reaction centres of purple bacteria

It is found that dark reduction of photooxidized primary electron donor P870+ in reaction centres from purple anoxygenic bacteria (two non-sulphur Fe-oxidizing Rhodovulum iodosum and Rhodovulum robiginosum, Rhodobacter sphaeroides R-26 and sulphur alkaliphilic Thiorhodospira sibirica) is accelerated upon the addition of Mn2+ jointly with bicarbonate (30-75 mM). The effect is not observed if Mn2+ and HCO3(-) have been replaced by Mg2+ and HCO2(-), respectively. The dependence of the effect on bicarbonate concentration suggests that formation of Mn2+-bicarbonate complexes, Mn(HCO3)+ and/or Mn(HCO3)2, is required for re-reduction of P870+ with Mn2+. The results are considered as experimental evidence for a hypothesis on possible participation of Mn-bicarbonate complexes in the evolutionary origin of oxygenic photosynthesis in the Archean era.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.     

Thymidine phosphorylating enzymes in the gonads of marine invertebrates

The activities of enzymes involved in the consecutive phosphorylation of thymidine were revealed in the gonad extracts of marine invertebrates. Along with thymidine kinase activity, thymidilate kinase activity was revealed in all the studied species; however, the specific activities of nucleoside and nucleotide kinases varied in different species of mollusks, sea stars and sea urchins. Thymidine and thymidilate kinases were isolated from the gonads of the scallop Mizuhopecten yessoensis and some of their enzymat properties were studied. The thymidine kinase of M. yessoensis catalyzed the phosphorylation of thymidine and deoxycytidine at a lesser rate, but didn’s use purine ribo-and deoxyribonucleosides or pyrimidine ribonucleosides as phosphate acceptors. The thymidilate kinase carried out both TMP and dCMP phosphorylation. As well as ATP, the enzymes of M. yessoensis were also able to use dATP, dGTP, GTP, UTP and CTP as donors of phosphate groups. The thymidine kinase activity was inhibited by TMP, TTP and dCTP.     

Budding yeast Pch2, a widely conserved meiotic protein, is involved in the initiation of meiotic recombination

Budding yeast Pch2 protein is a widely conserved meiosis-specific protein whose role is implicated in the control of formation and displacement of meiotic crossover events. In contrast to previous studies where the function of Pch2 was implicated in the steps after meiotic double-strand breaks (DSBs) are formed, we present evidence that Pch2 is involved in meiotic DSB formation, the initiation step of meiotic recombination. The reduction of DSB formation caused by the pch2 mutation is most prominent in the sae2 mutant background, whereas the impact remains mild in the rad51 dmc1 double mutant background. The DSB reduction is further pronounced when pch2 is combined with a hypomorphic allele of SPO11. Interestingly, the level of DSB reduction is highly variable between chromosomes, with minimal impact on small chromosomes VI and III. We propose a model in which Pch2 ensures efficient formation of meiotic DSBs which is necessary for igniting the subsequent meiotic checkpoint responses that lead to proper differentiation of meiotic recombinants.     

Fundamental cell cycle kinases collaborate to ensure timely destruction of the synaptonemal complex during meiosis

The synaptonemal complex (SC) is a proteinaceous macromolecular assembly that forms during meiotic prophase I and mediates adhesion of paired homologous chromosomes along their entire lengths. Although prompt disassembly of the SC during exit from prophase I is a landmark event of meiosis, the underlying mechanism regulating SC destruction has remained elusive. Here, we show that DDK (Dbf4‐dependent Cdc7 kinase) is central to SC destruction. Upon exit from prophase I, Dbf4, the regulatory subunit of DDK, directly associates with and is phosphorylated by the Polo‐like kinase Cdc5. In parallel, upregulated CDK1 activity also targets Dbf4. An enhanced Dbf4‐Cdc5 interaction pronounced phosphorylation of Dbf4 and accelerated SC destruction, while reduced/abolished Dbf4 phosphorylation hampered destruction of SC proteins. SC destruction relieved meiotic inhibition of the ubiquitous recombinase Rad51, suggesting that the mitotic recombination machinery is reactivated following prophase I exit to repair any persisting meiotic DNA double‐strand breaks. Taken together, we propose that the concerted action of DDK, Polo‐like kinase, and CDK1 promotes efficient SC destruction at the end of prophase I to ensure faithful inheritance of the genome.