Full PapersRyanodine Receptor Expression in Embryonic Avian Cardiac Muscle
Abstract
The cardiac ryanodine receptor serves aa a sarcoplasmic reticulum calcium release channel and contributes to the rise in cytosolic calcium necessary for contraction of the heart. We have investigated the presence and oligomeric form of the ryanodine receptor during embryonic development of the avian heart. A single isoform of the ryanodine receptor was identified both in adult and in embryonic cardiac tissue from Days 4 to 20 of development, using anti-receptor monoclonal antibodies in conjunction with [3H]ryanodine binding. The results of sucrose density gradient sedimentation analysis and [3H]ryanodine binding indicated that the cardiac ryanodine receptor is present in a tetrameric form in both the adult and embryos at Day 6 of development. The observation of specific [3H]ryanodine binding in hearts from Days 4 and 5 of embryonic development also indicates the presence of a tetrameric receptor protein. Although the heart begins to beat at approximately 33-38 hr (Day 1.5) of embryonic development, we were unable to detect the cardiac ryanodine receptor, using biochemical or immunological techniques, prior to Embryonic Day 4. Ryanodine was found to alter the chronotropic state of intact hearts as early as Hamburger and Hamilton stages 15-19 (Embryonic Day 3) suggesting that receptor protein may be present in a limited subset of cells involved in pacemaker activity in the very early embryo. After Embryonic Day 4, ryanodine also exerted a negative inotropic effect on embryonic hearts. Our results suggest that a single isoform of the ryanodine receptor is present from Day 4 to Day 20 of embryonic development in avian cardiac muscle and that the ryanodine receptor assumes a tetrameric structure capable of forming a functional calcium release channel that participates in excitation-contraction coupling as early as Embryonic Day 4. In addition, a ryanodine receptor-related function may serve as a determinant of chronotropic effects in the very early embryonic heart.
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Calcium Buffering and Excitation-Contraction Coupling in Developing Avian Myocardium
2004, Biophysical JournalCitation Excerpt :Moreover, a comprehensive study of the morphology of the formation and maturation of SR junctional complexes is available only for the chick embryo (Protasi et al., 1996). CICR in chick develops early and is present well before hatching, probably by embryonic day (ED) 4–5, as ryanodine receptors are first detected at about this time (Dutro et al., 1993). The chick heart begins beating at ∼ED1.5 (Sperelakis, 1982).
This report provides a detailed analysis of developmental changes in cytoplasmic free calcium (Ca2+) buffering and excitation-contraction coupling in embryonic chick ventricular myocytes. The peak magnitude of field-stimulated Ca2+ transients declined by 41% between embryonic day (ED) 5 and 15, with most of the decline occurring between ED5 and 11. This was due primarily to a decrease in Ca2+ currents. Sarcoplasmic reticulum (SR) Ca2+ content increased 14-fold from ED5 to 15. Ca2+ transients in voltage-clamped myocytes after blockade of SR function permitted computation of the fast Ca buffer power of the cytosol as expressed as generalized values of Bmax and KD. Bmax rose with development whereas KD did not change significantly. The computed SR Ca2+ contribution to the Ca2+ transient and gain factor for Ca2+-induced Ca2+ release increased markedly between ED5 and 11 and slightly thereafter. These results paralleled the maturation of SR and peripheral couplings reported by others and demonstrated a strong relationship between structure and function in development of excitation-contraction coupling. Modeling of buffer power from estimates of the major cytosolic Ca binding moieties yielded a Bmax and KD in reasonable agreement with experiment. From ED5 to 15, troponin C was the major Ca2+ binding moiety, followed by SR and calmodulin.
T-type Ca<sup>2+</sup> current contribution to Ca<sup>2+</sup>-induced Ca<sup>2+</sup> release in developing myocardium
2003, Journal of Molecular and Cellular CardiologyIn normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electricallystimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.
Developmental changes of Ca<sup>2+</sup> handling in mouse ventricular cells from early embryo to adulthood
2002, Life SciencesTransplant of immature cardiomyocytes is recently attracting a great deal of interest as a new experimental strategy for the treatment of failing hearts. Full understanding of normal cardiomyogenesis is essential to make this regenerative therapy feasible. We analyzed the molecular and functional changes of Ca2+ handling proteins during development of the mouse heart from early embryo at 9.5 days postcoitum (dpc) through adulthood. From the early to the late (18 dpc) embryonic stage, mRNAs estimated by the real time PCR for ryanodine receptor (type 2, RyR2), sarcoplasmic reticulum (SR) Ca2+ pump (type 2, SERCA2) and phospholamban (PLB) increased by 3–15 fold in the values normalized to GAPDH mRNA, although Na+/Ca2+ exchanger (type 1, NCX1) mRNA was unchanged. After birth, there was a further increase in the mRNAs for RyR2, SERCA2 and PLB by 18–33 fold, but a 50% decrease in NCX1 mRNA. The protein levels of RyR2, SERCA2, PLB and NCX1, which were normalized to total protein, showed qualitatively parallel developmental changes. L-type Ca2+ channel currents (ICa-L) were increased during the development (1.3-fold at 18 dpc, 2.2-fold at adult stage, vs. 9.5 dpc). At 9.5 dpc, the Ca2+ transient was, unlike adulthood, unaffected by the SR blockers, ryanodine (5 μM) and thapsigargin (2 μM), and also by a blocker of the Ca2+ entry via Na+/Ca2+ exchanger, KB-R 7943 (1 μM). The Ca2+ transient was abolished after application of nisoldipine (5 μM). These results indicate that activator Ca2+ for contraction in the early embryonic stage depends almost entirely on ICa-L.
Cell biology of cardiac development
2001, International Review of CytologyBuilding a vertebrate heart is a complex task and involves several tissues, including the myocardium, endocardium, neural crest, and epicardium. Interactions between these tissues result in the changes in function and morphology (and also in the extracellular matrix, which serves as a substrate for morphological change) that are requisite for development of the heart. Some of the signaling pathways that mediate these changes have now been identified and several investigators are now filling in the missing pieces in these pathways in hopes of ultimately understanding the molecular mechanisms that govern healthy heart development. In addition, transcription factors that regulate various aspects of heart development have been identified. Transcription factors of the GATA and Nkx2 families are of particular importance for early specification of the heart field and for regulating expression of genes that encode proteins of the contractile apparatus. This chapter highlights some of the most significant discoveries made in the rapidly expanding field of heart development.
Differential distribution and subcellular localization of ryanodine receptor isoforms in the chicken cerebellum during development
1997, Brain ResearchThe distribution of ryanodine receptor (RyR) isoforms was examined using isoform-specific monoclonal antibodies in the developing chicken brain, from E18 through adulthood, using light and electron microscopic immunocytochemistry. Monoclonal antibody 110F is specific for the α-skeletal muscle form of RyR, while monoclonal antibody 110E recognizes both the β-skeletal muscle and cardiac isoforms, but does not distinguish between the two. Significant differences in the distribution of the α- and β/cardiac forms were observed. Labeling for the α-form was restricted to cerebellar Purkinje neurons while the β/cardiac form was observed in neurons throughout the brain. A major finding was the presence of labeling for the β/cardiac in presynaptic terminals of the parallel fibers in the molecular layer and the mossy fiber terminals in the granular layer glomeruli in late development and during adulthood. Labeling for the β/cardiac, but not the α-form, underwent a major redistribution in the cerebellum during the course of development. At 1 day of age, β/cardiac labeling was present mainly in Purkinje neurons. From 1 day to 4 weeks, immunolabeling for the β/cardiac form gradually disappeared from Purkinje neurons, but increased in granule cells. Within the molecular layer, the labeling pattern changed from being primarily within Purkinje dendrites to a more diffuse pattern. Electron microscopic examination of the cerebellar molecular layer of 2-week-old chicks revealed that β/cardiac-labeling was mainly present in the axons and presynaptic processes of the parallel fibers. No developmental changes were observed in other brain regions. This study represents the first demonstration of ryanodine receptor immunoreactivity in presynaptic boutons and suggests that the ryanodine receptor may modulate neurotransmitter release through local regulation of intracellular calcium in the parallel fiber synapse.
Development of mechanisms regulating intracellular Ca<sup>2+</sup> concentration in cardiac muscle cells of early chick embryos
1997, Developmental BiologyThe development of mechanisms for the regulation of intracellular-free calcium ion concentration ([Ca2+]i) was investigated in precardiac mesodermal cells (PMC) and cardiac muscle cells (CMC) from early chick embryos by microfluorometry using a Ca2+-sensitive fluorescent probe, fura-2, and transmission electron microscopy. Microfluorometry indicated that two types of regulatory mechanisms, involving the dihydropyridine receptor (DHPR) and the ryanodine receptor (RYR), are present in CMC when the heartbeat begins at the 8–9 somite stages. Nifedipine completely suppressed the beating of hearts isolated from embryos on Days 1.5 and 2. Ryanodine had no effect on the beating of hearts isolated from embryos on Day 1.5, though it completely suppressed beating in hearts from Embryonic Day 2. Microfluorometry revealed that a change occured in the Ca2+-regulating mechanisms of CMC on Day 2. Transmission electron microscopy showed the appearance in CMC, also on Day 2, of peripheral couplings with feet structures, and SR adjacent to the Z-line of myofibrils. These findings suggest that the calcium-induced calcium-release (CICR) mechanism appears in the CMC of the chick on the second day of embryonic development.