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Biomedical subjects

Joseph Y Cheung

Publications and source records attributed to Joseph Y Cheung.

28 records · Page 2Linked to original sources
PubMed2003

Effects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes.

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca(2+)](i) transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger (NCX1), Na(+)-K(+)-ATPase, and calsequestrin levels. SR Ca(2+) uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca(2+)](o), rASPLM myocytes had significantly (P < 0.003) lower contraction and [Ca(2+)](i) transient amplitudes than control GFP myocytes. At 5 mM [Ca(2+)](o), both contraction and [Ca(2+)](i) transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca(2+)](i) transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca(2+)](i) transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

Adenoviridae↗
PubMed2003

Influence of age and run training on cardiac Na+/Ca2+ exchange.

Effects of age and training on myocardial Na+/Ca2+ exchange were examined in young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT; 8- to 11-wk treadmill run training) male Fischer Brown Norway rats. Whole heart performance and isolated cardiocyte Na+/Ca2+ exchange characteristics were measured. At the whole heart level, a small but significant slowing of late isovolumic left ventricular (LV) relaxation, which may be indicative of altered Na+/Ca2+ exchange activity, was seen in hearts from AS rats. This subtle impairment in relaxation was not observed in hearts from AT rats. At the single-cardiocyte level, late action potential duration was prolonged, resting membrane potential was more positive, and overshoot potential was greater in cardiocytes from AS rats than from YS rats (P < 0.05). Training did not influence any of these age-related action potential characteristics. In electrically paced cardiocytes, neither shortening nor intracellular Ca2+ concentration ([Ca2+]i) dynamics was influenced by age or training. Similarly, neither age nor training influenced the rate of [Ca2+]i clearance via forward (Nain+ /Caout2+) Na+/Ca2+ exchange after caffeine-induced Ca2+ release from the sarcoplasmic reticulum or cardiac Na+/Ca2+ exchanger protein (NCX1) expression. However, when whole cell patch-clamp techniques combined with fluorescence microscopy were used to evaluate the ability of Na+/Ca2+ exchange to alter cytosolic [Ca2+] ([Ca2+]c) under conditions where membrane potential (Vm) and internal and external [Na+] and [Ca2+] could be controlled, we observed age-associated increases in forward Na+/Ca2+ exchange-mediated [Ca2+]c clearance (P < 0.05) that were not influenced by training. The age-related increase in forward Na+/Ca2+ exchange activity provides a hypothetical explanation for the late action potential prolongation observed in this study.

Action Potentials↗
PubMed2003

A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death.

TRPM2 is a Ca(2+)-permeable channel that is activated by oxidative stress and confers susceptibility to cell death. Here, an isoform of TRPM2 was identified in normal human bone marrow that consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Because of alternative splicing, a stop codon (TAG) is located at the splice junction between exons 16 and 17, resulting in deletion of the four C-terminal transmembrane domains, the putative calcium-permeable pore region, and the entire C terminus. This splice variant was found in other hematopoietic cells including human burst forming unit-erythroid-derived erythroblasts and TF-1 erythroleukemia cells. Endogenous expression of both the short form of TRPM2 (TRPM2-S) and the full length (TRPM2-L) was determined by reverse transcriptase-PCR, and localization of endogenous TRPM2 to the plasma membrane was demonstrated by confocal microscopy. Heterologous expression of TRPM2-S in HEK 293T cells demonstrated similar membrane localization as TRPM2-L, and coexpression of TRPM2-S did not alter the subcellular localization of TRPM2-L. The direct interaction of TRPM2-S with TRPM2-L was demonstrated with immunoprecipitation. H(2)O(2) induced calcium influx through TRPM2-L expressed in 293T cells. Coexpression of TRPM2-S suppressed H(2)O(2)-induced calcium influx through TRPM2-L. Furthermore, expression of TRPM2-S inhibited susceptibility to cell death and onset of apoptosis induced by H(2)O(2) in cells expressing TRPM2-L. These data demonstrate that TRPM2-S is an important physiologic isoform of TRPM2 and modulates channel activity and induction of cell death by oxidative stress through TRPM2-L.

Apoptosis↗
PubMed2002

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.

Action Potentials↗
PubMed2002

Rescue of contractile abnormalities by Na+/Ca2+ exchanger overexpression in postinfarction rat myocytes.

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca(2+)](i) levels at all extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca(2+)](o), subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca(2+)](o), supranormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca(2+) efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.

Adenoviridae↗
PubMed2002

Erythropoietin modulates calcium influx through TRPC2.

Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca](i)) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca](i) was examined. RT-PCR of TRPC 1-6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca](i). Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca](i) changes were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca](i) above base line (372 +/- 71%), which was significantly greater (p < or = 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca](i) required Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.

Animals↗
PubMed2002

Effects of Na(+)/Ca(2+) exchanger downregulation on contractility and [Ca(2+)](i) transients in adult rat myocytes.

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase abundance, SR Ca(2+) uptake, resting membrane potential, action potential amplitude and duration, L-type Ca(2+) current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca(2+) concentration ([Ca(2+)](o)) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents than control myocytes. At 0.6 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca(2+)](i) transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca(2+) influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.

Action Potentials↗
PubMed2002

Overexpression of phospholemman alters contractility and [Ca(2+)](i) transients in adult rat myocytes.

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990-1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h (P < 0.001) but no changes in Na(+)/Ca(2+) exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% (P < 0.005) and [Ca(2+)](i) transient amplitudes were 18% (P < 0.05) lower than control myocytes. At 0.6 mM [Ca(2+)](o), however, contraction and [Ca(2+)](i) transient amplitudes were significantly (P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca(2+)](o), the differences in contraction and [Ca(2+)](i) transient amplitudes were narrowed. This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca(2+) fluxes during excitation-contraction.

Adenoviridae Infections↗
PubMed2002

Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.

The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes.

Animals↗