Cristián Ibarra

Insulin-like Growth Factor-1 Induces an Inositol 1,4,5-Trisphosphate-dependent Increase in Nuclear and Cytosolic Calcium in Cultured Rat Cardiac Myocytes*

In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Ca2+i levels apparent both in the nucleus and cytosol, releasing this ion from intracellular stores through an inositol 1,4,5-trisphosphate (IP3)-dependent signaling pathway. Intracellular IP3 levels increased after IGF-1 stimulation in both the presence and absence of extracellular Ca2+. A different spatial distribution of IP3 receptor isoforms in cardiomyocytes was found. Ryanodine did not prevent the IGF-1-induced increase of Ca2+i levels but inhibited the basal and spontaneous Ca2+i oscillations observed when cardiac myocytes were incubated in Ca2+-containing resting media. Spatial analysis of fluorescence images of IGF-1-stimulated cardiomyocytes incubated in Ca2+-containing resting media showed an early increase in Ca2+i, initially localized in the nucleus. Calcium imaging suggested that part of the Ca2+ released by stimulation with IGF-1 was initially contained in the perinuclear region. The IGF-1-induced increase on Ca2+i levels was prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM, thapsigargin, xestospongin C, 2-aminoethoxy diphenyl borate, U-73122, pertussis toxin, and βARKct (a peptide inhibitor of Gβγ signaling). Pertussis toxin also prevented the IGF-1-dependent IP3 mass increase. Genistein treatment largely decreased the IGF-1-induced changes in both Ca2+i and IP3. LY29402 (but not PD98059) also prevented the IGF-1-dependent Ca2+i increase. Both pertussis toxin and U73122 prevented the IGF-1-dependent induction of both ERKs and protein kinase B. We conclude that IGF-1 increases Ca2+i levels in cultured cardiac myocytes through a Gβγ subunit of a pertussis toxin-sensitive G protein-PI3K-phospholipase C signaling pathway that involves participation of IP3.

Local Control of Nuclear Calcium Signaling in Cardiac Myocytes by Perinuclear Microdomains of Sarcolemmal Insulin-Like Growth Factor 1 Receptors

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca2+ handling system is physically and functionally segregated from the cytosolic Ca2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca2+ signaling in response to an extracellular ligand.

Local Control of Nuclear Ca2+ Signalling in Cardiac Myocytes by Perinuclear Microdomains of Sarcolemmal IGF-1 Receptors

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca2+ handling system is physically and functionally segregated from the cytosolic Ca2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca2+ signaling in response to an extracellular ligand.

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca2+) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are intracellular Ca2+ release channels that mediate Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores. The three IP3R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP3R by the endogenous modulators IP3, Ca2+, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP3R subtype in shaping cytosolic Ca2+ oscillations.

Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi.

Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart

BackgroundCardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.ResultsAdult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.ConclusionThe new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.

An integrated mechanism of cardiomyocyte nuclear Ca2 + signaling

In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation.

Intracellular calcium release modulates polycystin-2 trafficking

BackgroundPolycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.MethodsWe investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.ResultsWe demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.ConclusionsThese novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.

Sublethal caspase activation promotes generation of cardiomyocytes from embryonic stem cells.

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit+/α-actininlow cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.

Role of Heterotrimeric G Protein and Calcium in Cardiomyocyte Hypertrophy Induced by IGF-1

In the heart, insulin‐like growth factor‐1 (IGF‐1) is a peptide with pro‐hypertrophic and anti‐apoptotic actions. The pro‐hypertrophic properties of IGF‐1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF‐1 also increases intracellular Ca2+ levels through a pertussis toxin (PTX)‐sensitive G protein. Here we investigate whether this Ca2+ signal is involved in IGF‐1‐induced cardiomyocyte hypertrophy. Our results show that the IGF‐1‐induced increase in Ca2+ level is abolished by the IGF‐1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca2+‐dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF‐1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF‐1‐dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF‐1‐induced cardiomyocyte hypertrophy, determined by cell size and β‐myosin heavy chain (β‐MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF‐1‐induced cardiac hypertrophy. We conclude that IGF‐1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ‐subunits of a PTX‐sensitive G protein/Ca2+/PKCα/ERK pathway without the participation of calcineurin. J. Cell. Biochem. 115: 712–720, 2014.