The creatine kinase energy transport system in the failing mouse heart

Characteristic alterations of the creatine kinase (CK) system occur in heart failure and may contribute to contractile dysfunction. We examined two mouse models of chronic cardiac stress, transverse aortic constriction (TAC) and coronary artery ligation (CAL), and examined the relationship of CK system changes with hypertrophy and heart failure development. C57Bl/6 mice were subjected to TAC or sham surgery and sacrificed after 2-10 weeks according to echocardiographic criteria of myocardial hypertrophy and function to create four groups representing progressive dysfunction from normal, through compensated hypertrophy, to heart failure. Only mice with congestive heart failure had LV total creatine concentration and total CK activity significantly lower than sham values (11% and 30% lower, respectively). However for all aortic banded mice, a linear relationship was observed between ejection fraction and estimated maximal CK reaction velocity. Mice with heart failure also had corresponding decreases in the activities of the Mito-, MM-, and MB-CK isoenzymes, while the BB isoform remained unchanged. To determine whether these changes were model specific, mice were subjected to CAL or sham operation and followed for 7 weeks. Quantitative changes in total creatine, total CK activity, Mito-CK and MM-CK activities were similar for CAL and TAC mice. We conclude that alterations in the creatine kinase system occur during heart failure in mice qualitatively similar to those occurring in larger animals and humans, suggesting that mice are a suitable model for studying the role of such changes in the pathogenesis of heart failure.     

Mechanisms of creatine depletion in chronically failing rat heart

The failing myocardium is characterised by energetic imbalance, reflected by reduced phosphocreatine and creatine content. These changes may contribute to cardiac dysfunction, yet mechanisms of creatine and phosphocreatine depletion are poorly understood. Creatine is taken up by the heart via the creatine transporter. We investigated the mechanisms leading to myocardial creatine depletion in heart failure. Therefore rats were subjected to chronic left coronary artery ligation (MI; n = 36) or to sham operation (sham; n = 25). After 8 weeks, hearts were perfused with 14C-creatine buffer to determine creatine uptake rates via the creatine transporter. Total creatine content was determined by HPLC. Creatine transport in sham hearts followed Michaelis-Menten kinetics with a V(max) of 5.9 +/- 0.5 nmol/min per gww. Heart failure led to a significant 30% decrease in intracellular creatine content and to a significant 26% reduction in creatine uptake (V(max) in MI 4.3 +/- 0.4 nmol/min per gww; P < 0.001 vs. sham). We conclude that depletion of creatine/phosphocreatine content in the failing heart is due to reduced sarcolemmal creatine uptake. The creatine transporter may be a potential therapeutic target to prevent energetic imbalance in heart failure.     

An adenylate kinase is involved in K<Subscript>ATP</Subscript> channel regulation of mouse pancreatic beta cells

Aims/hypothesis In a previous study, we demonstrated that a creatine kinase (CK) modulates K_ATP channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K_ATP channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. Methods Single channel activity was measured with the patch–clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. Results The ATP sensitivity of K_ATP channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K_ATP channel regulation. The observation that the stimulatory effect of AMP on K_ATP channels was prevented by the AK inhibitor P ¹,P ⁵-di(adenosine-5′)pentaphosphate (Ap₅A; 20 μmol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5′-(β,γ-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K_ATP channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. Conclusions/interpretation We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K_ATP channels. Thus, these enzymes determine the open probability of K_ATP channels and eventually the actual rate of insulin secretion.     

Mitochondrial tolerance to stress impaired in failing heart

Mitochondrial integrity is critical in the maintenance of bioenergetic homeostasis of the myocardium, with oxidative or metabolic challenge to mitochondria precipitating cell injury. In heart failure, where cardiac cells are exposed to elevated stress, mitochondrial vulnerability could contribute to the disease state. However, the mitochondrial response to stress is yet to be established in heart failure. Here, mitochondrial function and structure was evaluated prior and following stress using a transgenic (TG) model of heart failure, generated by cardiac overexpression of the cytokine TNFalpha. Compared to the wild type, mitochondria from TG failing hearts demonstrated impaired oxidative phosphorylation, mitochondrial DNA damage, reduced mitochondrial creatine kinase activity, abnormal calcium handling, and altered ultrastructure. Under anoxia/reoxygenation or calcium stress, mitochondria from failing hearts suffered exacerbated energetic failure with pronounced cytochrome c release. Thus, mitochondria from TNFalpha-TG failing hearts demonstrate structural and functional abnormalities, with reduced tolerance to stress manifested by impaired bioenergetics and increased susceptibility to injury. This abnormal vulnerability to stress underscores the impact of mitochondrial dysfunction in the pathobiology of heart failure.     

Inhibition of creatine kinase activity by lysine in rat cerebral cortex

Accumulation of lysine (Lys) in tissues and biochemical fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and also by other inherited neurometabolic disorders. In the present study, we investigated the in vitro effect of Lys on various parameters of energy metabolism in cerebral cortex of 30-day-old Wistar rats. We verified that total (tCK) and cytosolic creatine kinase activities were significantly inhibited by Lys, in contrast to the mitochondrial isoform which was not affected by this amino acid. Furthermore, the inhibitory effect of Lys on tCK activity was totally prevented by reduced glutathione, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, Lys did not affect ¹⁴CO₂ production from [U-¹⁴C] glucose (aerobic glycolytic pathway) and [1-¹⁴C] acetic acid (citric acid cycle activity) neither the various activities of the electron transfer chain and synaptic Na⁺K⁺-ATPase at concentrations as high as 5.0 mM. Considering the importance of creatine kinase (CK) activity for brain energy metabolism homeostasis and especially ATP transfer and buffering, our results suggest that inhibition of this enzyme by Lys may contribute to the neurological signs presented by symptomatic patients affected by FH and other neurodegenerative disorders in which Lys accumulates.     

磷酸肌酸钠对糖尿病合并心力衰竭老年患者的疗效观察

目的 观察磷酸肌酸钠对糖尿病合并心力衰竭老年患者的临床治疗效果.方法 选取2009年2月至6月在四川省人民医院住院治疗的糖尿病合并心力衰竭(纽约心脏病学会心功能分级Ⅲ～Ⅳ级)老年患者60例,采用抽签法随机分为对照组(n=30)和治疗组(n=30).在维持血糖、血压、血脂达标的情况下,对照组给予常规抗心力衰竭治疗,包括一般治疗与药物治疗(利尿剂、血管紧张素转换酶抑制剂、血管紧张素Ⅱ受体阻滞剂、β-受体阻滞剂、正性肌力药、醛固酮受体拮抗剂等),治疗组在此基础上静脉滴注磷酸肌酸钠2.0 g,每日1次,共14 d.治疗前后留取空腹血液标本,检测血糖、胰岛素、C肽、糖化血红蛋白、血浆脑钠肽、腺苷酸激活蛋白激酶,借助心脏超声检查评价患者心功能.应用t检验进行数据分析.结果 治疗后,2组患者心功能指标6 min步行试验、左心室射血分数、左心室缩短分数、E峰与A峰比值均显著改善,治疗组6 min步行试验[分别为(106±21)、(94±17)m]和E峰与A峰比值(分别为0.29 ±0.14、0.19±0.10)较对照组改善更好,差异有统计学意义(t值分别为2.776、3.099,均P＜0.05).治疗后,2组患者血浆脑钠肽、腺苷酸激活蛋白激酶、胰岛素抵抗及胰岛素敏感指数均显著改善,治疗组较对照组腺苷酸激活蛋白激酶[分别为(12±4)、(10±5)ng/L]和胰岛素抵抗(分别为1.7±0.9、0.9±0.4)改善更好,差异有统计学意义(t值分别为2.586、4.258,均P＜0.05).结论 磷酸肌酸钠可显著增强糖尿病合并心力衰竭老年患者心肌能量代谢,纠正胰岛素抵抗,调整心肌舒张功能,进而改善患者的临床症状及预后.     

心力衰竭中能量代谢紊乱及治疗的进展

心力衰竭是大多数心血管疾病的终末阶段,发病率和死亡率较高,严重危害人类的健康。心衰的发病机制复杂,尽管不同原发病导致心衰的机制存在差异,但心肌代谢异常、能量利用障碍是共同作用途径。目前改善心衰患者异常的心肌能量代谢已成为治疗心衰的有效手段,受到临床医生广泛关注。因此,本文就心衰与心肌能量代谢之间的关系及心肌能量代谢药物的应用进行综述,增进医务工作者对心衰及其治疗的理解。     

Creatine kinase in human erythrocytes: A newly detected genetic anomaly

Summary In a family of Italian origin, we found four members with a considerable activity of creatine kinase inside their erythrocytes. All other clinical and hematological findings were normal. The enzyme anomaly seems to be inherited in the autosomal mode.The creatine kinase (CK) activity in freshly drawn blood was about 12 U/g Hb. The activity was higher in young red cells than in older ones. Studies with specific antibodies against human CK isoenzymes revealed the CK activity in Offprint requests to: Prof. Dr. H. Arnold (address see above) the probands' red cells to be due to about 90 % to the BB-isoenzyme normally found in brain and nerve tissue.The presence of CK in the erythrocytes does not seem to have any consequences for the energy metabolism of these cells. Creatine concentration was slightly elevated, but creatine phosphate could not be detected.     

衰竭心肌能量代谢重构及β3肾上腺素能受体、过氧化物酶体增殖物激活受体α表达变化的研究

目的 通过检测衰竭与健康心肌组织代谢底物含量、相关酶活性及β3肾上腺素能受体、过氧化物酶体增殖物激活受体α（PPARα）基因和蛋白表达的变化,探讨衰竭心肌代谢重构的表现及可能机制。方法 选取6例意外伤亡健康者心肌组织和20例心外科瓣膜置换术的心力衰竭患者,检测心肌游离脂肪酸（FFA）、乳酸（LD）含量和ATP酶活力。应用逆转录聚合酶链反应、Western blot和免疫组化法分别检测心肌组织β3受体和PPARα mRNA表达以及蛋白和定位。结果 衰竭心肌FFA和LD含量明显增加（P〈0．01和P〈0．05）。Na^＋K^＋-ATP酶和Ca^2＋Mg^2＋-ATP酶活性则显著降低（P〈0．05和P〈0．01）。衰竭心肌β3肾上腺素能受体mRNA和蛋白表达明显高于正常心肌,而PPARα表达则显著低于正常心肌,但并未发生定位变化。结论心力衰竭时存在代谢重构,表现为心肌代谢底物发生转变,代谢相关酶活性下降等,β3肾上腺素能受体和PPARα在衰竭心肌分别上调和下调,可能参与心肌组织代谢重构。     

ATP flux through creatine kinase in the normal, stressed, and failing human heart

The heart consumes more energy per gram than any other organ, and the creatine kinase (CK) reaction serves as its prime energy reserve. Because chemical energy is required to fuel systolic and diastolic function, the question of whether the failing heart is "energy starved" has been debated for decades. Despite the central role of the CK reaction in cardiac energy metabolism, direct measures of CK flux in the beating human heart were not previously possible. Using an image-guided molecular assessment of endogenous ATP turnover, we directly measured ATP flux through CK in normal, stressed, and failing human hearts. We show that cardiac CK flux in healthy humans is faster than that estimated through oxidative phosphorylation and that CK flux does not increase during a doubling of the heart rate-blood pressure product by dobutamine. Furthermore, cardiac ATP flux through CK is reduced by 50% in mild-to-moderate human heart failure (1.6 +/- 0.6 vs. 3.2 +/- 0.9 micromol/g of wet weight per sec, P <0.0005). We conclude that magnetic resonance strategies can now directly assess human myocardial CK energy flux. The deficit in ATP supplied by CK in the failing heart is cardiac-specific and potentially of sufficient magnitude, even in the absence of a significant reduction in ATP stores, to contribute to the pathophysiology of human heart failure. These findings support the pursuit of new therapies that reduce energy demand and/or augment energy transfer in heart failure and indicate that cardiac magnetic resonance can be used to assess their effectiveness.     

ATP-sensitive K+ channel channel/enzyme multimer: metabolic gating in the heart

Cardiac ATP-sensitive K(+) (K(ATP)) channels, gated by cellular metabolism, are formed by association of the inwardly rectifying potassium channel Kir6.2, the potassium conducting subunit, and SUR2A, the ATP-binding cassette protein that serves as the regulatory subunit. Kir6.2 is the principal site of ATP-induced channel inhibition, while SUR2A regulates K(+) flux through adenine nucleotide binding and catalysis. The ATPase-driven conformations within the regulatory SUR2A subunit of the K(ATP) channel complex have determinate linkage with the states of the channel's pore. The probability and life-time of ATPase-induced SUR2A intermediates, rather than competitive nucleotide binding alone, defines nucleotide-dependent K(ATP) channel gating. Cooperative interaction, instead of independent contribution of individual nucleotide binding domains within the SUR2A subunit, serves a decisive role in defining K(ATP) channel behavior. Integration of K(ATP) channels with the cellular energetic network renders these channel/enzyme heteromultimers high-fidelity metabolic sensors. This vital function is facilitated through phosphotransfer enzyme-mediated transmission of controllable energetic signals. By virtue of coupling with cellular energetic networks and the ability to decode metabolic signals, K(ATP) channels set membrane excitability to match demand for homeostatic maintenance. This new paradigm in the operation of an ion channel multimer is essential in providing the basis for K(ATP) channel function in the cardiac cell, and for understanding genetic defects associated with life-threatening diseases that result from the inability of the channel complex to optimally fulfill its physiological role.     

Growth hormone and heart failure: Oxidative stress and energetic metabolism in rats

Several evidences point for beneficial effects of growth hormone (GH) in heart failure (HF). Taking into account that HF is related with changes in myocardial oxidative stress and in energy generation from metabolic pathways, it is important to clarify whether GH increase or decrease myocardial oxidative stress and what is its effect on energetic metabolism in HF condition. Thus, this study investigated the effects of two different doses of GH on energetic metabolism and oxidative stress in myocardium of rats with HF. Male Wistar rats (n=25) were submitted to aortic stenosis (AS). The HF was evidenced by tachypnea and echocardiographic criteria around 28 weeks of AS. The rats were then randomly divided into three groups: (HF) with HF, treated with saline (0.9% NaCl); (HF-GH1), treated with 1 mk/kg/day recombinant human growth hormone (rhGH), and (HF-GH2) treated with 2 mg/kg/day rhGH. GH was injected, subcutaneously, daily for 2 weeks. A control group (sham; n=12), with the same age of the others rats was evaluated to confirm data for AS. HF had lower IGF-I (insulin-like growth factor-I) than sham-operated rats, and both GH treatments normalized IGF-I level. HF-GH1 animals had lower lipid hydroperoxide (LH), LH/total antioxidant substances (TAS) and glutathione-reductase than HF. Glutathione peroxidase (GSH-Px), hydroxyacyl coenzyme-A dehydrogenase, lactate dehydrogenase(LDH) were higher in HF-GH1 than in HF. HF-GH2 compared with HF, had increased LH/TAS ratio, as well as decreased oxidized glutathione and LDH activity. Comparing the two GH doses, GSH-Px, superoxide dismutase and LDH were lower in HF-GH2 than in HF-GH1. In conclusion, GH effects were dose-dependent and both tested doses did not aggravate the heart dysfunction. The higher GH dose, 2 mg/kg exerted detrimental effects related to energy metabolism and oxidative stress. The lower dose, 1mg/kg GH exerted beneficial effects enhancing antioxidant defences, reducing oxidative stress and improving energy generation in myocardium of rats with heart failure.     

The role of arginine vasopressin and its receptors in the normal and failing rat heart

OBJECTIVE: To evaluate the role of arginine vasopressin (AVP) in the normal and post-myocardial infarction (post-MI) hearts, and to investigate whether chronic AVP receptor antagonism can attenuate post-infarct ventricular remodeling. BACKGROUND: A number of neurohormones, like norepinephrine and angiotensin II, have detrimental effects in heart failure (HF) and inhibiting them is beneficial. AVP shares some important properties with these hormones and is activated in HF. However, its role in the syndrome of HF, especially the effect of AVP inhibition, is largely undefined. METHODS AND RESULTS: Effects of AVP-V1a and AVP-V2 receptor stimulation on normal rat cardiomyocyte contractile function, intracellular calcium [Ca2+]i, inositol 3 phosphate (IP3) generation and cell survival were studied. In post-MI rats, AVP receptor function was assessed in cardiomyocytes as well as isolated working hearts. AVP receptor total number (ligand binding) and mRNA levels (RT-PCR) were measured in myocytes. Finally, the effects of chronic AVP-V1a blockade were assessed in post-MI rats using echo and morphometry to measure ventricular remodeling. Normal cardiomyocytes showed a dose-dependent increase in myocyte contractile function, [Ca2+]i, and IP3 generation in response to AVP-V1a receptor stimulation. AVP-V2 agonists had no effect. Cells from MI hearts showed reduced inotropic response to AVP-V1a stimulation. Myocyte AVP-V1a receptor number and receptor mRNA were decreased. Prolonged exposure to AVP reduced cellular viability. However, chronic AVP-V1a blockade did not attenuate structural remodeling or improve function in post-MI hearts. CONCLUSIONS: Isolated adult rat cardiomyocytes bear AVP-V1a receptors that mediate inotropic effects through the IP3 pathway. These receptors are functionally and numerically downregulated in the post-MI remodeled hearts. Despite evidence that AVP is cytotoxic to cells, chronic AVP-V1a receptor blockade does not attenuate post-MI ventricular remodeling in this model. Thus, AVP may not play a major role in the structural progression of HF.     

曲美他嗪对心力衰竭大鼠心功能自由基代谢心肌纤维化及心肌超微结构的影响

目的:与倍他乐克进行对照,观察曲美他嗪(TMZ)对心肌梗死后心力衰竭大鼠的血流动力学参数、自由基代谢、心肌纤维化、血浆心钠素(ANP)和病理形态学方面(包括光镜和电镜)的影响,分析其产生作用的可能机制。方法:取雄性SD大鼠制成心肌梗死后心力衰竭模型,随机分为3组:心力衰竭对照组(S组);心力衰竭服用倍他乐克10mg·kg-1·d-1组(B组);心力衰竭服用TMZ10mg·kg-1·d-1组(T组)。每组8只,用药共6周。另设8只大鼠为假手术组(C组)。实验结束时进行血流动力学测定,血浆ANP、血清透明质酸(HA)、层黏蛋白(LN)、血浆和心肌组织匀浆中过氧化物歧化酶(SOD)、丙二醛(MDA)等指标,以及病理形态学的检测等。结果:用药6周后,与C组比较,S组、T组和B组左室收缩压(LVSP)下降,左室舒张末期压力(LVEDP)升高,±dp/dt降低;T组、B组与S组比较,LVSP上升,LVEDP下降,±dp/dt上升,T组和B组之间差异无统计学意义。S组、B组和T组的血浆SOD与C组相比,明显降低;S组的血浆MDA值与C组相比增高,T组血浆MDA值与其他3组比较降低。和C组相比,S、B和T组心肌组织的SOD均降低;B、T组的心肌SOD值较S组增高;S组的心肌MDA值较C组高。S、B和T组的血浆ANP值均显著高于C组,B组和T组明显低于S组。B、T、S组的血清HA值均比C组高,B、T组HA值与S组相比降低。光镜和电镜结果显示,T组和B组的心肌损害程度,较S组明显减轻。结论:TMZ可以改善心力衰竭大鼠的左室压力指标,改善自由基代谢,降低血浆ANP、HA值,改善心肌的病理和超微结构。     

β-肾上腺素受体激酶与心力衰竭治疗的研究进展

全球大约有2 300万人患有心力衰竭,对β-肾上腺素受体的反应性降低是心力衰竭的特征并由此导致心脏功能异常。通过转基因的办法抑制β-肾上腺素能受体激酶1(βARK1)进而改善衰竭心脏的收缩功能和对β-肾上腺素能神经兴奋的反应性,使得βARK1的抑制成为重要的治疗心力衰竭的手段。本文就βARK1研究的最新进展作一综述。     

Cardiac phenotype of mitochondrial creatine kinase knockout mice is modified on a pure C57BL/6 genetic background

Discrepant results for the phenotype of mitochondrial creatine kinase knockout mice (Mt-CK^−/−) could be due to mixed genetic background and use of non-littermate controls. We therefore backcrossed with C57BL/6J for > 8 generations, followed by extensive in vivo cardiac phenotyping. Echocardiography and in vivo LV haemodynamics were performed in independent cohorts at 20-40 weeks and 1 year. No significant differences were observed for ECG, LV volumes, pressures, and systolic or diastolic function compared to littermate controls. Furthermore, responses to dobutamine were not different, indicating preserved contractile reserve. Contrary to published reports using Mt-CK^−/− on a mixed background, we observed normal LV weights even in year old mice, and gene expression of common hypertrophic markers were not elevated. However, previously undetected adaptations were observed: an increase in activity of the cytosolic MM-CK isoenzyme (+ 20% vs WT, P = 0.0009), and of citrate synthase (+ 18% vs WT, P = 0.0007), a marker for mitochondrial volume. In a 3-week voluntary wheel running protocol, Mt-CK^−/− ran significantly less per day (P = 0.009) and attained lower maximum speed compared to controls (P = 0.0003), suggesting impaired skeletal muscle function. MM-CK isoenzyme activity was significantly elevated in soleus but not gastrocnemius muscle of KO mice, and citrate synthase activities were normal in both, suggesting compensatory mechanisms are incomplete in skeletal muscle. Conclusions: in contrast to previous reports using a mixed genetic background, Mt-CK^−/− on a C57BL/6 background do not develop LV hypertrophy or dysfunction even up to 1 year, and this may be explained by a compensatory increase in MM-CK activity and mitochondrial volume.     

Altered hetero- and homeometric autoregulation in the terminally failing human heart

OBJECTIVE AND METHODS: To further investigate length-dependent force generation in human heart, nonfailing (donor hearts, NF) and terminally failing (heart transplants, dilated cardiomyopathy, DCM) left ventricular myocardium was studied under various preload (4-40 mN/mm2) or length conditions. In addition, morphological studies (van Giesson Trichrome staining, electron microscopy) were performed. RESULTS: In NF, a biphasic increase in force of contraction (FOC) was observed after elevating the preload (4-40 mN/mm2): there was an immediate fast increase (FOCf,), followed by a slow increase over several minutes (FOCs), which was paralleled by an increase in the systolic fura-2 transient. In DCM, FOCf, FOCs and the systolic fura-2 transient were blunted and diastolic tension was increased at increasing muscle length. Only in NF, a stretched induced increase in diastolic fura-2 ratio was observed. In DCM, no obvious interstitial fibrosis and no difference in basement membrane structure and attachment were observed. CONCLUSIONS: Since FOCf has been attributed to the Frank-Starling mechanism, whereas FOCs represents a length-dependent increase in the intracellular Ca2+-transient, the impaired length-dependent force generation in failing myocardium results from a dysregulation of both myofibrillar Ca2+-sensitivity as well as the intracellular Ca2+-homeostasis. Interstitial fibrosis may have only minor impact on force generation in human end-stage heart failure.