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Beta-adrenergic stimulation disassembles microtubules in neonatal rat cultured cardiomyocytes through intracellular
1First Department of Medicine, Osaka University School of Medicine, Suita, Japan.
Abstract:
Catecholamine cardiotoxicity is attributable in part to Ca2+ overload. To test whether the cytoskeletal structures of microtubules in cardiomyocytes are reversibly injured by catecholamine through excessive Ca2+ influx, morphological changes in the microtubules of neonatal rat myocytes were studied by immunohistochemical technique during exposure to norepinephrine (NE). In intact myocytes, microtubules appeared as a filamentous network throughout the cytoplasm and around the nucleus. NE exposure (10 mumol/L) for > 30 minutes elicited microtubular disassembly in a duration-dependent fashion without any irreversible change in sarcomere structure, and this abnormality recovered within 24 hours after cessation of stimulation. Microtubular disruption scores obtained by semiquantitative assessment were significantly increased in a dose-dependent manner (10.8 +/- 4.0 in the control condition, 23.4 +/- 4.7 at 60 minutes with 10 mumol/L NE), whereas they were significantly attenuated by pretreatment with propranolol (100 mumol/L; score, 11.8 +/- 3.3) but not with phentolamine (100 mumol/L; score, 26.4 +/- 4.8). Isoproterenol (1 mumol/L) and denopamine (10 mumol/L) mimicked the effects of NE, but phenylephrine did not, indicating that NE-induced microtubular disassembly is mediated by beta 1-adrenergic receptor stimulation. This beta-adrenergic receptor-mediated insult was significantly attenuated by a decrease in Ca2+ concentration in the medium from 2 to 0.5 mmol/L and by pretreatment with diltiazem (1 mumol/L). In contrast, microtubular disassembly was induced by an increase in Ca2+ concentration in the medium and an administration of the Ca2+ ionophore A23187, even without beta-adrenergic receptor stimulation. Involvement of intracellular hypoxia and activation of Ca(2+)-calmodulin-dependent kinase or Ca(2+)-dependent neutral protease were excluded from possible mechanisms; however, inhibition of tubulin polymerization by excessive Ca2+ influx during beta-adrenergic receptor stimulation may be primarily involved. We conclude that microtubular structures that support cellular integrity are reversibly injured by beta-adrenergic receptor stimulation through excessive Ca2+ influx.
Insights
Catecholamine exposure causes reversible injury to cardiomyocyte microtubules via excessive calcium influx, primarily through beta-adrenergic receptors. This disruption of microtubule structure impacts cellular integrity but recovers over time.
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Pharmacology
Background:
- Catecholamine cardiotoxicity is partly due to calcium (Ca2+) overload.
- The impact of catecholamines on cardiomyocyte cytoskeletal structures, specifically microtubules, requires further investigation.
Purpose of the Study:
- To investigate whether catecholamines, through excessive Ca2+ influx, cause reversible injury to microtubule cytoskeletal structures in cardiomyocytes.
- To elucidate the specific adrenergic receptor subtypes involved in catecholamine-induced microtubule damage.
Main Methods:
- Neonatal rat myocytes were exposed to norepinephrine (NE) and other adrenergic agonists.
- Immunohistochemical techniques were used to assess morphological changes in microtubules.
- Semiquantitative scoring assessed microtubule disruption, and effects of various drugs and Ca2+ concentrations were evaluated.
Main Results:
- Norepinephrine exposure induced dose- and duration-dependent microtubule disassembly, which was reversible within 24 hours.
- The effect was mediated by beta-1 adrenergic receptor stimulation, as indicated by isoproterenol and denopamine effects, and blocked by propranolol but not phentolamine.
- Reduced extracellular Ca2+ and diltiazem pretreatment attenuated NE-induced microtubule damage, while increased Ca2+ or A23187 induced disassembly independently.
Conclusions:
- Beta-adrenergic receptor stimulation leads to reversible microtubule injury in cardiomyocytes through excessive Ca2+ influx.
- This mechanism involves the inhibition of tubulin polymerization by elevated intracellular Ca2+.
- Microtubule damage affects cellular integrity but is a reversible consequence of catecholamine exposure.