Contribution Of Inositol Research Articles

Modeling inherited short-coupled polymorphic ventricular tachycardia using patient specific hiPSC-derived cardiomyocytes and CRISPR/Cas 9 technology

Polymorphic ventricular tachycardia (PMVT) can occur in patients with structurally normal hearts and in 8% of cases can lead to sudden cardiac death, typically exercise-induced. The role of the cardiac type 2 ryanodine receptor (RyR2) in pathogenesis of PMVT presenting at rest is unclear. We aimed here at modelling PMVT observed in a patient harboring the RyR2-H29D mutation by comparing the molecular and functional properties of RyR2-H29D hiPSC-derived cardiomyocytes (hiPSC-CMs) with their isogenic control counterparts with a particular focus on the RyR2 properties. We collected blood samples from the patient and generated several clones of RyR2-H29D hiPSC, in addition to generating an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. We used fluorescent confocal microscopy, patch-clamp and video-image-based analysis to investigate the molecular and functional consequences of the RyR2-H29D mutation. We first hypothesized that PMVT hiPSC-CMs expressing the RyR2-H29D mutation would exhibit abnormal Ca 2+ homeostasis. Thus, we measured and analyzed the intracellular Ca 2+ variation. We found that the RyR2-H29D hiPSC-CMs exhibit clone-independent aberrant properties including intracellular sarcoplasmic reticulum (SR) Ca 2+ leak through RyR2 under physiological pacing. The contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling exacerbate the abnormal intracellular Ca 2+ release in the RyR2-H29D hiPSC-CMs. Moreover, the RyR2-H29D hiPSC-CMs exhibit RyR2 post-translational remodeling, shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. These abnormalities are fully reversed with isogenic control. Our results suggest that RyR2-mediated Ca 2+ leak induces an impairment of Ca 2+ homeostasis and provide support to decipher the molecular mechanisms of short-coupled PMVT at rest.

Modeling inherited short-coupled polymorphic ventricular tachycardia using patient specific hiPSC-derived cardiomyocytes and CRISPR/Cas 9 technology

Polymorphic ventricular tachycardia (PMVT) can occur in patients with structurally normal hearts and in 8% of cases can lead to sudden cardiac death, typically exercise-induced. The role of the cardiac type 2 ryanodine receptor (RyR2) in pathogenesis of PMVT presenting at rest is unclear. We aimed here at modelling PMVT observed in a patient harboring the RyR2-H29D mutation by comparing the molecular and functional properties of RyR2-H29D hiPSC-derived cardiomyocytes (hiPSC-CMs) with their isogenic control counterparts with a particular focus on the RyR2 properties. We collected blood samples from the patient and generated several clones of RyR2-H29D hiPSC, in addition to generating an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. We used fluorescent confocal microscopy, patch-clamp and video-image-based analysis to investigate the molecular and functional consequences of the RyR2-H29D mutation. We first hypothesized that PMVT hiPSC-CMs expressing the RyR2-H29D mutation would exhibit abnormal Ca 2+ homeostasis. Thus, we measured and analyzed the intracellular Ca 2+ variation. We found that the RyR2-H29D hiPSC-CMs exhibit clone-independent aberrant properties including intracellular sarcoplasmic reticulum (SR) Ca 2+ leak through RyR2 under physiological pacing. The contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) exacerbate the abnormal intracellular Ca 2+ release in the RyR2-H29D hiPSC-CMs. Moreover, the RyR2-H29D hiPSC-CMs exhibit RyR2 post-translational remodeling, shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. These abnormalities are fully reversed with isogenic control. Our results suggest that RyR2-mediated Ca 2+ leak induces an impairment of Ca 2+ homeostasis and provide support to decipher the molecular mechanisms of short-coupled PMVT at rest.

‘Eventless’ InsP3‐dependent SR‐Ca2+ release affecting atrial Ca2+ sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca(2+) release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca(2+)-induced Ca(2+) release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca(2+) release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm)(-1) s(-1)). These individual photolytically triggered Ca(2+) release events were identified as Ca(2+) sparks, which originated from RyR openings. This was verified by Ca(2+) spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca(2+) release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca(2+) flux but eventless SR-Ca(2+) release through InsP3R were characterized using SR-Ca(2+) leak/SR-Ca(2+) load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca(2+) spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca(2+) flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Visualization of Ins(1,4,5)P3 dynamics in living cells: two distinct pathways for Ins(1,4,5)P3 generation following mechanical stimulation of HSY-EA1 cells

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] generation on the mechanical-stimulation-induced Ca(2+) response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P(3) ([IP(3)](i)), as indicated by the Ins(1,4,5)P(3) biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P(3) wave as [IP(3)](i) increased in the stimulated region. A small transient [IP(3)](i) increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P(3) responses and resultant Ca(2+) releases. The purinergic receptor blocker suramin completely blocked increases in [IP(3)](1) and the Ca(2+) release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P(3) in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP(3)](i) increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P(3)-induced Ca(2+) release was larger than that of Ca(2+) entry in the Ca(2+) response to mechanical stimulation in HSY-EA1 cells.

Multimodality of Ca2+ Signaling in Rat Atrial Myocytes

It has been suggested that the multiplicity of Ca(2+) signaling pathways in atrial myocytes may contribute to the variability of its function. This article reports on a novel Ca(2+) signaling cascade initiated by mechanical forces induced by "puffing" of solution onto the myocytes. Ca(i) transients were measured in fura-2 acetoxymethyl (AM) loaded cells using alternating 340- and 410-nm excitation waves at 1.2 kHz. Pressurized puffs of bathing solutions, applied by an electronically controlled micro-barrel system, activated slowly (approximately 300 ms) developing Ca(i) transients that lasted 1,693 +/- 68 ms at room temperature. Subsequent second and third puffs, applied at approximately 20 s intervals activated significantly smaller or no Ca(i) transients. Puff-triggered Ca(i) transients could be reactivated once again following caffeine (10 mM)-induced release of Ca(2+) from sarcoplasmic reticulum (SR). Puff-triggered Ca(i) transients were independent of [Ca(2+)](o), and activation of voltage-gated Ca(2+) or cationic stretch channels or influx of Ca(2+) on Na(+)/Ca(2+)exchanger, because puffing solution containing no Ca(2+), 10 microM diltiazem, 1 mM Cd(2+), 5 mM Ni(2+), or 100 microM Gd(3+) failed to suppress them. Puff-triggered Ca(i) transients were enhanced in paced compared to quiescent myocytes. Electrically activated Ca(i) transients triggered during the time course of puff-induced transients were unaltered, suggesting functionally separate Ca(2+) pools. Contribution of inositol 1,4,5-triphosphate (IP(3))-gated or mitochondrial Ca(2+) pools or modulation of SR stores by nitric oxide/nitric oxide synthase (NO/NOS) signaling were evaluated using 0.5 to 500 microM 2-aminoethoxydiphenyl borate (2-APB) and 0.1 to 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 mM Nomega-Nitro-L-arginine methyl ester (L-NAME) and 7-nitroindizole, respectively. Only FCCP appeared to significantly suppress the puff-triggered Ca(i) transients. It was concluded that neither Ca(2+) influx nor depolarization was required for activation of this signaling pathway. These studies suggest that pressurized puffs of solutions activate a mechanically sensitive receptor, which signals in turn the release of Ca(2+) from a limited Ca(2+) store of mitochondria. How mechanical forces are sensed and transmitted to mitochondria to induce Ca(2+) release and what role such a Ca(2+) signaling pathway plays in the physiology or pathophysiology of the heart remain to be worked out.

O-226 - Contribution of inositol 1,4,5-triphosphate to the Ca2+ release through Na+/Ca2+ exchanger

O-226 - Contribution of inositol 1,4,5-triphosphate to the Ca2+ release through Na+/Ca2+ exchanger