Related Experiment Videos
Ryanodine and dihydropyridine binding patterns and ryanodine receptor mRNA levels in myopathic hamster heart
W G Lachnit1, M Phillips, K J Gayman
1Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis 95616.
Insights
Cardiomyopathy in hamsters shows altered calcium channel densities. Partially purified membranes reveal increased voltage-dependent Ca2+ channels (VDCC) and Ca2+ release channels (CICR), suggesting changes in SR Ca2+ transport.
Area of Science:
- Cardiovascular Research
- Molecular Cardiology
- Biochemistry
Background:
- Cardiomyopathy involves altered cardiac function and calcium handling.
- Voltage-dependent Ca2+ channels (VDCC) and Ca2+ induced Ca2+ release channels (CICR) are crucial for excitation-contraction coupling.
Purpose of the Study:
- To investigate the densities and functional properties of VDCC and CICR in cardiomyopathic hamster hearts.
- To determine if observed changes are due to transcriptional upregulation or altered protein fractionation.
Main Methods:
- Utilized radioligand binding assays with [3H]PN-200 for VDCC and [3H]ryanodine for CICR.
- Compared receptor densities and mRNA levels in normal versus cardiomyopathic hamster hearts.
- Assessed channel sensitivity to activators (Ca2+, doxorubicin) and inhibitors.
Main Results:
- Partially purified membranes from cardiomyopathic hearts showed a twofold increase in VDCC and CICR binding sites.
- Crude membranes showed no difference in VDCC but decreased CICR binding sites and mRNA.
- Myopathic heart membranes exhibited increased sensitivity to doxorubicin and Ca2+ activation and inhibition of Ca2+ uptake.
Conclusions:
- Increased VDCC and CICR binding in partially purified fractions likely results from altered T-tubule and SR fractionation, not transcriptional changes.
- Functional alterations in CICR channels may impair SR Ca2+ transport, contributing to cardiomyopathy progression.
- Deficiency in dystrophin-associated glycoprotein may underlie these observed changes.
Abstract:
We have determined the densities of sarcolemmal voltage-dependent Ca2+ channels (VDCC) and Ca(2+)-induced Ca2+ release channels (CICR) of sarcoplasmic reticulum (SR) in the cardiomyopathic hamster heart using [3H]PN-200 and [3H]ryanodine, respectively. Partially purified cardiac membrane preparations from myopathic animals exhibit a twofold higher capacity to bind both [3H]PN-200 and [3H]ryanodine. Crude particulate membrane fractions from normal and cardiomyopathic animals reveal no significant difference in receptor densities for [3H]PN-200, whereas densities for [3H]ryanodine binding sites and mRNA levels are significantly (P < 0.05) diminished in cardiomyopathic animals. Inhibition of [3H]ryanodine binding by either Ca2+ or Mg2+ (in mM) as well as temperature dependence for receptor activation for [3H]ryanodine (Q10) is not significantly different, whereas membranes isolated from cardiomyopathic hearts are 1.4-fold and threefold more sensitive to activation by doxorubicin and Ca2+ (in microM), respectively. Vesicles isolated from myopathic hearts are more sensitive to inhibition of Ca2+ uptake by doxorubicin. The higher densities of binding sites for [3H]PN-200 and [3H]ryanodine observed in partially purified membrane fractions from cardiomyopathic hearts are more likely the result of altered patterns with which T-tubule and CICR channels fractionate in preparations from cardiomyopathic hamster heart rather than transcriptional upregulation and may be a consequence of the deficiency in a dystrophin-associated glycoprotein recently identified. Downregulation and functional changes in CICR channels may alter SR Ca2+ transport and contribute to the progression of cardiomyopathy in the hamster.