S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
Effect of metabolic substrates on intracellular Ca2+ homeostasis and contractility in cardiac myocytes of healthy and hypertensive rats
대사 물질에 의한 정상과 고혈압 백서 심장 세포의 칼슘 및 수축성 조절에 대한 연구
|dc.description||학위논문 (박사)-- 서울대학교 대학원 의과대학 의과학과, 2017. 8. 장은화.||-|
|dc.description.abstract||Metabolism is an important precondition of contractile function in cardiac myocyte. Until recently, glucose has been used as the source for myocyte ATP in majority of physiological experiments. In fact, fatty acids are the predominant metabolic substrates in healthy heart and such metabolism is altered in the myocardium under pathological stress. Accordingly, I aimed to test the effects of metabolic substrates supplementation (including glucose, fatty acids and other metabolic substrates, termed NF) on intracellular Ca2+ handling, myofilament Ca2+ sensitivity and contraction in left ventricular (LV) myocytes from normal and angiotensin II (Ang II)-induced hypertensive rats (HTN).
My results showed that NF significantly increased myocyte contraction and facilitated relaxation from normal heart. Similar to NF, supplementation of 3 fatty acids (linoleic acid, oleic acid and palmitic acid) also increased sarcomere shortening. However, inhibition of beta-oxidation process with carnitine palmitotransferase-1 (CPT-1) inhibitor, ranolazine, did not affect myocyte contraction increment in NF, indicating that glucose and fatty acid-dependent metabolism complement with each other in supporting cardiac contraction. In addition, NF increased the amplitudes of diastolic and systolic Ca2+ transients ([Ca2+]i), abbreviated time constant of [Ca2+]i decay (tau), but prolonged the peak duration of [Ca2+]i. Ca2+ content in the sarcoplasmic reticulum (SR) was increased by NF. Whole-cell patch-clamp experiments revealed that NF increased Ca2+ influx via L-type Ca2+ channels (LTCC, ICa-integral) and prolonged the action potential duration (APD). Furthermore, NF induced myofilament Ca2+ desensitization, which is responsible for NF-induced changes in [Ca2+]i handling. NF reduced intracellular pH ([pH]i). Buffering [pH]i with HCO3-/CO2 attenuated Δ [pH]i and reversed myofilament Ca2+ desensitization and Ca2+ transient in NF in normal heart. Collectively, NF stimulates myocyte contraction by increasing Ca2+ influx through LTCCs and Ca2+ release from the SR. Myofilament Ca2+ desensitization (via reducing [pH]i) contributes, at least in part, to NF-induced greater [Ca2+]i. In LV myocytes from HTN rats, where myofilament Ca2+ sensitivity was reduced and [Ca2+]i was greater, NF increased myocyte contraction and [Ca2+]i handling, reduced myofilament Ca2+ sensitivity further, despite that Ca2+ influx via LTCC was reduced.
-adrenergic stimulation with isoprenaline (ISO) significantly increased myocyte contraction in NF in both groups. In addition, ISO induced delayed-aftercontraction (DAC) and spontaneous Ca2+ transients (sCa) in NF and the frequency of DAC was ~3 fold higher in HTN. Biochemical experiments indicated that tyrosine phosphorylation of insulin receptor (IR), IR substrate-1 and endothelial NOS (eNOS) phosphorylation (eNOS-Ser1177) were blunted by NF in normal heart. In HTN, the insulin and eNOS-dependent signaling was reduced before NF application. Accordingly, insulin did not affect NF-enhancement of sarcomere shortening in basal or with ISO in both groups. Furthermore, NOS inhibition with L-NAME (1mM, 30 min ~ 1hr) failed to affect myocyte responses to NF in either group. These results suggest that insulin and eNOS-dependent signaling were impaired in LV myocyte with metabolic substrates supplementation.
Notably, NOS inhibition with L-NAME increased DAC and sCa by ISO in normal heart but reduced these parameters in HTN. Such effects were mimicked by nNOS inhibitor, SMTC, suggesting the role of nNOS. -1AR blockers nebivolol and cordycepin, two NO-potentiating chemicals, significantly decreased diastolic and peak amplitudes of Ca2+ transients with NF+ISO in both groups and abolished ISO-induced SCs in NF. KB-R7943, a Na+-Ca2+ exchanger inhibitor, abolished DAC in LV myocytes from sham and HTN.
Taken together, supplementation of myocardial favourable metabolic substrates affects Ca2+ handling and excitation-contraction coupling. Notably, myofilament Ca2+desensitization contributes to the regulation of intracellular [Ca2+]i homeostasis, an unrecognized regulation of myocyte [Ca2+]i and contraction. Impaired insulin/NO signaling is involved in arrhthmogenesis in NF under beta-adrenergic stimulation. In particular, nNOS exerts contrasting effects on DAC with NF in normal and HTN rat hearts.
Materials and Methods 10
Part I Assessment of myofilament Ca2+ sensitivity underlying cardiac excitation-contraction coupling. 22
Part II Cardiac inotropy, lusitropy, and Ca2+ handling with major metabolic substrates in rat heart. 26
Part III Metabolic substrates affect the LV myocyte contraction and [Ca2+]i by regulating myofilament Ca2+ sensitivity in Ang II-induced hypertensive rat heart. 40
Part IV 54
1. Metabolic substrate regulation of cardiac myocyte contraction and the roles of eNOS and nNOS in LV myocytes from sham rat heart. 54
2. Metabolic substrate regulation of cardiac myocyte contraction and the roles of eNOS and nNOS in LV myocytes from hypertensive rat heart. 59
Part I Assessment of myofilament Ca2+ sensitivity underlying cardiac excitation-contraction coupling. 80
Part II Cardiac inotropy, lusitropy, and Ca2+ handling with major metabolic substrate in rat heart. 83
Part III Effects of metabolic substrate on LV myocyte contraction in Ang II-induced hypertensive rat heart 87
Part IV Roles of eNOS and nNOS in metabolic substrate regulation of cardiac excitation contraction coupling in LV myocytes from sham and hypertensive rat hearts. 88
Abstract (in Korean) 106
|dc.subject||intracellular Ca2+ transient||-|
|dc.subject||myofilament Ca2+ sensitivity||-|
|dc.title||Effect of metabolic substrates on intracellular Ca2+ homeostasis and contractility in cardiac myocytes of healthy and hypertensive rats||-|
|dc.title.alternative||대사 물질에 의한 정상과 고혈압 백서 심장 세포의 칼슘 및 수축성 조절에 대한 연구||-|
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- College of Medicine/School of Medicine (의과대학/대학원)Dept. of Biomedical Sciences (대학원 의과학과)Theses (Ph.D. / Sc.D._의과학과)