Utilization of the genetically encoded calcium indicator Salsa6F in cardiac applications.

Calcium signaling is a critical process required for cellular mechanisms such as cardiomyocyte contraction. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

Regulation of calcium entry by cyclic GMP signaling in Toxoplasma gondii.

Ca2+ signaling impacts almost every aspect of cellular life. Ca2+ signals are generated through the opening of ion channels that permit the flow of Ca2+ down an electrochemical gradient. Cytosolic Ca2+ fluctuations can be generated through Ca2+ entry from the extracellular milieu or release from intracellular stores. In Toxoplasma gondii, Ca2+ ions play critical roles in several essential functions for the parasite, like invasion of host cells, motility, and egress. Plasma membrane Ca2+ entry in T. gondii was previously shown to be activated by cytosolic calcium and inhibited by the voltage-operated Ca2+ channel blocker nifedipine. However, Ca2+ entry in T. gondii did not show the classical characteristics of store regulation. In this work, we characterized the mechanism by which cytosolic Ca2+ regulates plasma membrane Ca2+ entry in extracellular T. gondii tachyzoites loaded with the Ca2+ indicator Fura-2. We compared the inhibition by nifedipine with the effect of the broad spectrum TRP channel inhibitor, anthranilic acid or ACA, and we find that both inhibitors act on different Ca2+ entry activities. We demonstrate, using pharmacological and genetic tools, that an intracellular signaling pathway engaging cyclic GMP, protein kinase G, Ca2+, and the phosphatidyl inositol phospholipase C affects Ca2+ entry and we present a model for crosstalk between cyclic GMP and cytosolic Ca2+ for the activation of T. gondii's lytic cycle traits.

Cardiac Localized Polycystin-2 plays a Functional Role in Natriuretic Peptide Production and its Absence Contributes to Hypertension.

Cardiovascular complications are the most common cause of mortality in patients with autosomal dominant polycystic kidney disease (ADPKD). Hypertension is seen in 70% of patients by the age of 30 prior to decline in kidney function. The natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are released by cardiomyocytes in response to membrane stretch, increasing urinary excretion of sodium and water. Mice heterozygous for Pkd2 have attenuated NP responses and we hypothesized that cardiomyocyte-localized polycystin proteins contribute to production of NPs. Cardiomyocyte-specific knock-out models of polycystin-2 (PC2), one of the causative genes of ADPKD, demonstrate diurnal hypertension. These mice have decreased ANP and BNP expression in the left ventricle. Analysis of the pathways involved in production, maturation, and activity of NPs identified decreased transcription of CgB, PCSK6, and NFAT genes in cPC2-KOs. Engineered heart tissue with human iPSCs driven into cardiomyocytes with CRISPR/Cas9 KO of PKD2 failed to produce ANP. These results suggest that PC2 in cardiomyocytes are involved in NP production and lack of cardiac PC2 predisposes to a hypertensive volume expanded phenotype, which may contribute to the development of hypertension in ADPKD.

Cardiovascular perspectives of the TRP channel polycystin 2.

Calcium release from the endoplasmic reticulum (ER) is predominantly driven by two key ion channel receptors, inositol 1, 4, 5-triphosphate receptor (InsP3R) in non-excitable cells and ryanodine receptor (RyR) in excitable and muscle-based cells. These calcium transients can be modified by other less-studied ion channels, including polycystin 2 (PC2), a member of the transient receptor potential (TRP) family. PC2 is found in various cell types and is evolutionarily conserved with paralogues ranging from single-cell organisms to yeasts and mammals. Interest in the mammalian form of PC2 stems from its disease relevance, as mutations in the PKD2 gene, which encodes PC2, result in autosomal dominant polycystic kidney disease (ADPKD). This disease is characterized by renal and liver cysts, and cardiovascular extrarenal manifestations. However, in contrast to the well-defined roles of many TRP channels, the role of PC2 remains unknown, as it has different subcellular locations, and the functional understanding of the channel in each location is still unclear. Recent structural and functional studies have shed light on this channel. Moreover, studies on cardiovascular tissues have demonstrated a diverse role of PC2 in these tissues compared to that in the kidney. We highlight recent advances in understanding the role of this channel in the cardiovascular system and discuss the functional relevance of PC2 in non-renal cells.

Utilization of the genetically encoded calcium indicator Salsa6F in cardiac applications.

Calcium signaling is a critical process required for cellular mechanisms such as cardiac contractility. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

Calcium signaling in polycystic kidney disease- cell death and survival.

Polycystic kidney disease is typified by cysts in the kidney and extra-renal manifestations including hypertension and heart failure. The main genetic underpinning this disease are loss-of function mutations to the two polycystin proteins, polycystin 1 and polycystin 2. Molecularly, the disease is characterized by changes in multiple signaling pathways including down regulation of calcium signaling, which, in part, is contributed by the calcium permeant properties of polycystin 2. These signaling pathways enable the cystic cells to survive and avoid cell death. This review focuses on the studies that have emerged in the past 5 years describing how the structural insights gained from PC-1 and PC-2 inform the calcium dependent molecular pathways of autophagy and the unfolded protein response that are regulated by the polycystin proteins and how it leads to cell survival and/or cell death.

Polycystin-2 (PC2) is a key determinant of in vitro myogenesis.

The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca2+) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations, and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca2+ signals and metabolic pathways.

Tagging of Weakly Expressed Toxoplasma gondii Calcium-Related Genes with High-Affinity Tags.

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca2+ concentration is tightly regulated at the physiological cytosolic concentration of 50-100 nm. The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca2+ channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high-performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy-terminus tagging plasmid containing the previously characterized "spaghetti monster-HA" (smHA) or "spaghetti monster-MYC" (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii.

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for the cellular and developmental changes that support T. gondii parasitism. The characterization of the molecular players and pathways directly activated by Ca2+ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.

A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.