| 摘要: | 背景 過氧體增殖劑活化受體 (PPARs-peroxisome proliferator-activated receptors)-γ 細胞核轉錄因子，對於心臟能量的表達，則可調控心臟脂質的生合成及醣類代謝。由於心肌中能量代謝的混亂已經被視為造成糖尿病心肌病變的一個重要觀點，因此對於評估 PPAR-γ 調控糖尿病的影響是很重要的。雖然 PPAR-γ 體具有抗炎作用，目前尚不清楚是否炎性細胞因子或 PPARs 配體可以調控 PPAR-γ 來調控心臟的功能。糖尿病及高血壓經常伴隨發生，因而導致心臟結構或功能異常，促使加速發病率及死亡率。雖然過去研究顯示 PPAR-γ 在糖尿病或高血壓的心臟疾病生理學中扮演重要角色，但目前尚不清楚糖尿病是否可以調控高血壓對 PPAR-γ 的影響。除此之外，對於 PPAR-γ 引起的配位體造成心肌功能障礙的機轉則尚未完全釐清。糖尿病及高血壓會影響心臟鈣離子的調節，而這對決定心臟功能的好壞扮演了不可缺的角色。然而，在心肌細胞中，PPAR-γ 配體對鈣離子調節的影響仍不清楚，需進一步的研究。
目的 首先，我們研究在心臟不同的區域中，糖尿病對 PPAR-γ 異構體表現的影響，並且探討在糖尿病心肌病變中，促炎性細胞因子及氧化壓力是否可以調控PPAR-γ。第二，我們探索在 HL-1 心肌細胞中，腫瘤壞死因子-α (tumor necrosis factor (TNF)-α) 和 PPARs 配體是否會直接影響 PPAR-γ 的表現。第三、我們評估糖尿病是否可以調控高血壓對心肌 PPAR-γ 異構體表現的影響。最後，我們要研究高血壓、糖尿病及 PPAR-γ 促效劑對鈣離子調控以及心室心肌的電生理特性的影響。
材料方法 研究1，雄性大鼠分為 Wistar 為控制組、糖尿病 (diabetic Wistar) 組和抗壞血酸治療糖尿病 (ascorbate-treated diabetic Wistar) 組。研究3，雄性大鼠分為自發性高血壓 (SHR)、糖尿病性高血壓(diabetic SHR)、糖尿病性高血壓給予 rosiglitazone (5mg/kg, rosiglitazone-treated diabetic SHR)、及 Wistar Kyoto (WKY) 為控制組。研究4，雄性大鼠分為 WKY、糖尿病WKY (diabetic WKY)、糖尿病WKY 給予 rosiglitazone (rosiglitazone-treated diabetic WKY)、SHR、diabetic SHR、rosiglitazone-treated diabetic SHR。研究2，HL-1 心肌細胞培養 24小時中，給予及無給予 TNF-α (1、10、25 和 50ng/ml）或 PPAR-γ 配位體- rosiglitazone, pioglitazone (0.1μM、1μM 和 10μM)。這些細胞也分別單獨給予 SN -50 (NF-κβ 抑制劑，50μg/ml)，抗壞血酸 (100μM) 和 輔酶 Q10 (10μM) 或同時給予 TNF-α。分析 PPAR-γ 異構體，採用逆轉錄酶和即時聚合酶連鎖反應定量 mRNA 和西方墨點法分析 (Western blot) 蛋白質分析 PPAR-γ 配位體、TNF-α、白細胞介 (IL-6) 和通過光度測量心臟 NAD(P)H 氧化酶 (oxidase) 的活性進行量化。研究4，利用 indo-1 螢光比例技術和全細胞鉗定來研究細胞內鈣離子(鈣離子流)、動作電位、離子流。 Western blot 評估肌漿網ATP酶 (SERCA2a)、鈉鈣交換體 (NCX) 和 ryanodine 受體 (RYR) 蛋白質的表現。
結果 研究1顯示控制組心臟組織PPAR-γ的mRNA和蛋白質表現量較低。在diabetic Wistar 心房和心室 PPAR-γ 蛋白質和 mRNA 在所有心臟部位表現量增加，而 ascorbate 會減少其表現量。此外，糖尿病會增加心房與心室 TNF-α 和 IL-6 蛋白質表現量及 NAD(P)H 氧化酶活性。Ascorbate 能減少心房增加 TNF-α 和 IL-6 蛋白質表現量及 NAD(P)H oxidase 活性，但心室部分只有減少增加的 NAD(P)H oxidase 活性。研究2 中，發現給予 TNF-α (50ng/ml) 24小時，會增加 PPAR-γ，但同時給予 SN-50 並沒有改變這些結果。然而，同時給予ascorbate能抑制 TNF-α 對 PPAR-γ 的影響，輔酶 Q10 能減輕 TNF-α 對 PPAR-γ 的影響。給予 24小時 rosiglitazone(10μM)、pioglitazone(10μM) 和 fenofibrate(10μM) 會增加 PPAR-γ mRNA的表現量。研究3顯示與控制組心臟相比，SHR 心臟在 PPAR-γ 的 mRNA 和蛋白質表現量會增加。SHR會誘發心臟 PPAR-γ 異構體 mRNA 和蛋白質的變化，在 diabetic SHR 中則會增強此變化，而給予 rosiglitazone 治療 diabetic SHR 能減少其反應。在SHR心臟的 TNF-α，IL-6 蛋白質表現量和 NAD(P)H oxidase 的活性是增加，而 diabetic SHR 則進一步加重其表現情形。在diabetic SHR 中，使用 rosilgitazone 治療可降低其 TNF-α、IL-6 蛋白質表現量和 NAD(P)H oxidase 活性。研究4，與 non-diabetic WKY 和 SHR 相比，diabetic WKY 和 diabetic SHR 有較小的肌漿網內鈣離子含量，以及有延長其衰退時間的短暫鈣離子流，並降低 SERCA2a、NCX 和 RYR 蛋白質表現量和減小的L-型鈣離子流。 Rosiglitazone 治療能減輕 diabetic rat 的鈣離子失調。糖尿病和高血壓都可延長動作電位，使用 rosiglitazone會增強其延長，並誘發觸發性動作電位。
結論 在 diabetic Wistar 心房和心室 PPAR-γ 異構體有不同的表現量，並且糖尿病藉由發炎性激素和氧化壓力調節 PPAR-γ 的表現。此外， HL–1 心肌細胞中發炎性激素TNF-α可以直接調節 PPAR-γ，這可能是氧化壓力引起的，而不是經由 NF-κβ 途徑。糖尿病與 PPAR-γ 配體可調控高血壓對心臟 PPAR-γ 異構體表現的影響。最後，我們也發現，糖尿病和高血壓調控鈣離子恆定。PPAR-γ 配體- rosiglitazone 顯著改變了鈣離子的調控和電生理特性，並在糖尿病伴隨高血壓時可能會引起心律失調的發生。
Background Peroxisome-proliferator-activated receptor-γ (PPAR-γ) is nuclear transcription factor expressed in the heart and modulates lipogenesis and glucose metabolism. Since energy derangement has emerged as an important aspect contributing to diabetic cardiomyopathy, it is therefore important to evaluate the diabetic effects on PPAR-γ regulation. Although PPAR-γ ligands have anti-inflammatory effects, it is not clear whether inflammatory cytokines or PPAR ligands regulate PPAR-γ to modulate cardiac functions. Diabetes and hypertension frequently co-exist and lead to cardiac structural or functional abnormalities, which accelerate the progression to morbidity and mortality. Although previous studies have shown that PPAR-γ has a pivotal role in the cardiac pathophysiology of diabetes or hypertension, it is not clear whether diabetes can modulate the cardiac effect of hypertension on PPAR-γ. Moreover, the mechanism of myocardial dysfunction-mediated by PPAR-γ ligands was not fully elucidated. Diabetes and hypertension have significant effects on cardiac calcium (Ca2+) regulation, which plays an essential role in determining cardiac function. However, the effect of PPAR-γ ligands on Ca2+ regulation in the cardiomyocytes is unclear and needs further investigation.
Objectives: First, we investigated the effects of diabetes on PPAR-γ expression in different cardiac regions and explored whether proinflammatory cytokines or oxidative stress can modulate PPAR-γ in diabetic hearts. Second, we evaluated the direct effects of TNF-α and PPAR ligands on the expression of PPAR-γ in HL-1 cardiomyocytes. Third, we evaluated whether diabetes can modulate the effect of hypertension on myocardial expressions of PPAR-γ. Lastly, we explored the effects of hypertension, diabetes, and PPAR-γ agonist-rosiglitazone on the regulation of Ca2+ and the electrophysiological characteristics of isolated ventricular myocytes.
Materials and Methods In study 1, male Wistar rats were separated into control, diabetes, and ascorbate-treated diabetic groups. While in Study 3, male rats were group into spontaneously hypertensive rat (SHR), diabetic SHR, diabetic SHR treated with rosiglitazone (5 mg/kg), and Wistar-Kyoto (WKY) as control. In Study 4, male rats were divided into WKY, diabetic WKY, diabetic WKY treated with rosiglitazone, SHR, diabetic SHR, and diabetic SHR treated with rosiglitazone. In Study 2, HL-1 cardiomyocytes were incubated with and without tumor necrosis factor (TNF)-α (1, 10, 25, and 50 ng/ml) or PPARs ligands-rosiglitazone, pioglitazone, fenofibrate (0.1 μM, 1 μM, and 10 μM) for 24 hours. The cells also received SN-50 (NF-κβ inhibitor, 50 μg/ml), ascorbic acid (100 μM) and coenzyme Q10 (10 μM) alone or combined with TNF-α. Real-time PCR and Western blot analysis were performed on PPAR-γ, TNF-α, and interleukin (IL)-6, and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity was quantified through photometric measurements. In Study 4, indo-1 fluorometric ratio technique and whole-cell patch clamp were used to investigate intracellular Ca2+ (Ca2+i), action potentials, and ionic currents in the ventricular myocytes from each group. Western blot was used to evaluate protein expressions of sarcoplasmic reticulum ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and ryanodine receptor (RyR).
Results Study 1 showed that in control hearts, PPAR-γ was the least expressed in mRNA and protein levels. Diabetes increased protein and mRNA levels of PPAR-γ in both atria and ventricles, and was attenuated after ascorbate treatment in all cardiac regions. Moreover, diabetes increased the TNF-α and IL-6 protein levels, and NAD(P)H oxidase activities in atria and ventricles. Ascorbate attenuated the increase of TNF-α, IL-6 protein levels, and NAD(P)H oxidase activity in the atria, but only attenuated the increase of NAD(P)H oxidase activities in the ventricles. In Study 2, incubation of TNF-α (50 ng/ml) for 24 hours increased PPAR-γ mRNA and protein levels, but this effect did not change after co-administration of SN-50. However, co-administration of ascorbic acid prevented TNF-α’s effect on PPAR-γ, and coenzyme Q10 partially attenuated TNF-α’s effect on PPAR-γ. In addition, administration of rosiglitazone (10 μM) and pioglitazone (10 μM) for 24 hours increased PPAR-γ mRNA levels. Study 3 showed that as compared to control hearts, SHR hearts had increased PPAR-γ mRNA and protein levels and was enhanced in diabetic SHR, which was attenuated in diabetic SHR treated with rosiglitazone. Cardiac TNF-α, IL-6 protein, and NAD(P)H oxidase activities were increased in SHR and were further aggravated in diabetic SHR. Rosilgitazone treatment decreased TNF-alpha, IL-6 protein, and NAD(P)H oxidase activities in diabetic SHR hearts. Study 4 showed that diabetic WKY and diabetic SHR had smaller sarcoplasmic reticulum Ca2+ contents, and Ca2+i transients with a prolonged decay portion, down-regulated SERCA2a, NCX, and RyR protein expressions and smaller L-type Ca2+ currents than non-diabetic WKY and SHR, respectively. The Ca2+ dysregulations in diabetes were attenuated in rats treated with rosiglitazone. Diabetes and hypertension both prolonged the action potential duration which were enhanced by the use of rosiglitazone, and induced the genesis of triggered activity.
Conclusions PPAR-γ is differentially expressed in the atria and ventricles of diabetic rats and diabetes can modulate PPAR-γ expression through inflammatory cytokines and oxidative stress. Furthermore, TNF-α can directly regulate PPAR-γ in HL-1 cardiomyocytes, which may be caused by the oxidative stress rather than NF-κβ pathway. Diabetes and PPAR-γ ligand modulated the hypertensive effects on cardiac PPAR-γ expression. Finally, we also found that diabetes and hypertension modulated Ca2+ handling. PPAR-γ ligand- rosiglitazone significantly changed the Ca2+ regulation and electrophysiological characteristics and may contain an arrhythmogenic potential in diabetes with hypertension. |
