Contribution of TRPV4 to enhanced calcium cycling and cardiac arrhythmia following ischemia-reperfusion in aged mouse hearts

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI--COLUMBIA AT REQUEST OF AUTHOR.] Cardiomyocyte Ca2+ homeostasis is altered with aging and predisposes the Aged heart to Ca2+ intolerance and arrhythmia. Transient Receptor Potential Vanilloid 4 (TRPV4) is an osmotically-activated cation channel and channel expression is increased in cardiomyocytes of Aged mice. The central goal of this work was to determine the role of TRPV4 in calcium handling and arrhythmogenesis in response to hypoosmotic stress and following ischemia-reperfusion (I/R). Hypoosmotic stress induced an increase in calcium transient amplitude in cardiomyocytes isolated from Aged mice which was followed by an increased incidence of arrhythmic Ca2+ events and Ca2+ waves. This effect was prevented by TRPV4 inhibition and was absent in cardiomyocytes from Young mice. Cardiac contractile function, membrane potential, and cardiac ECG was monitored in Langendorff-perfused hearts during I/R. Aged hearts responded to I/R with an initial increase in contractile function, membrane depolarization, and incidence of ventricular arrhythmia during reperfusion. This effect was attenuated by TRPV4 inhibition and was absent in hearts of Young mice. Also, in hearts of Aged, TRPV4 inhibition decreased the percent of damaged tissue following I/R compared to untreated conditions. Finally, Langendorff-perfused hearts from Aged mice expressing a genetically encoded Ca2+ sensor (GCaMP6f) were subjected to I/R and demonstrated an increased Ca2+ transient amplitude and incidence of arrhythmic Ca2+ waves compared to Aged mice treated with TRPV4 inhibition. These findings suggest that TRPV4 may contribute to initial inotropy followed by pro-arrhythmic cardiomyocyte Ca2+ signaling, arrhythmogenesis, and cell death following I/R in the Aged heart.

Hypoosmotic stress induces R loop formation in nucleoli and ATR/ATM-dependent silencing of nucleolar transcription

The contribution of nucleoli to the cellular stress response has been discussed for over a decade. Stress-induced inhibition of RNA polymerase I-dependent transcription is hypothesized as a possible effector program in such a response. In this study, we report a new mechanism by which ribosomal DNA transcription can be inhibited in response to cellular stress. Specifically, we demonstrate that mild hypoosmotic stress induces stabilization of R loops in ribosomal genes and thus provokes the nucleoli-specific DNA damage response, which is governed by the ATM- and Rad3-related (ATR) kinase. Activation of ATR in nucleoli strongly depends on Treacle, which is needed for efficient recruitment/retention of TopBP1 in nucleoli. Subsequent ATR-mediated activation of ATM results in repression of nucleolar transcription.

Synergistic Effects of Temperature and Salinity on the Gene Expression and Physiology of Crassostrea virginica

The eastern oyster, Crassostrea virginica, forms reefs that provide critical services to the surrounding ecosystem. These reefs are at risk from climate change, in part because altered rainfall patterns may amplify local fluctuations in salinity, impacting oyster recruitment, survival, and growth. As in other marine organisms, warming water temperatures might interact with these changes in salinity to synergistically influence oyster physiology. In this study, we used comparative transcriptomics, measurements of physiology, and a field assessment to investigate what phenotypic changes C. virginica uses to cope with combined temperature and salinity stress in the Gulf of Mexico. Oysters from a historically low salinity site (Sister Lake, LA) were exposed to fully crossed temperature (20°C and 30°C) and salinity (25, 15, and 7 PSU) treatments. Using comparative transcriptomics on oyster gill tissue, we identified a greater number of genes that were differentially expressed (DE) in response to low salinity at warmer temperatures. Functional enrichment analysis showed low overlap between genes DE in response to thermal stress compared with hypoosmotic stress and identified enrichment for gene ontologies associated with cell adhesion, transmembrane transport, and microtubule-based process. Experiments also showed that oysters changed their physiology at elevated temperatures and lowered salinity, with significantly increased respiration rates between 20°C and 30°C. However, despite the higher energetic demands, oysters did not increase their feeding rate. To investigate transcriptional differences between populations in situ, we collected gill tissue from three locations and two time points across the Louisiana Gulf coast and used quantitative PCR to measure the expression levels of seven target genes. We found an upregulation of genes that function in osmolyte transport, oxidative stress mediation, apoptosis, and protein synthesis at our low salinity site and sampling time point. In summary, oysters altered their phenotype more in response to low salinity at higher temperatures as evidenced by a higher number of DE genes during laboratory exposure, increased respiration (higher energetic demands), and in situ differential expression by season and location. These synergistic effects of hypoosmotic stress and increased temperature suggest that climate change will exacerbate the negative effects of low salinity exposure on eastern oysters.

TRPV4 increases cardiomyocyte calcium cycling and contractility yet contributes to damage in the aged heart following hypoosmotic stress

Aims Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia–reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress. Methods and results TRPV4 protein expression increased in cardiomyocytes of Aged (24–27 months) mice compared with Young (3–6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 μmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4−/−) mice. In Aged, pharmacological inhibition of TRPV4 (1 μmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage. Conclusions Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.