肌酸激酶(CK)被认为是心脏主要的磷酸转移系统,对于克服扩散限制和调节线粒体呼吸很重要。在缺乏l-精氨酸的肌酸缺陷小鼠中,它是底物受限的:甘氨酸脒基转移酶(AGAT)或胍乙酸N-甲基转移酶(GAMT)。我们的目的是确定表达,活动,己糖激酶(HK)和腺苷酸激酶(AK)的线粒体偶联,因为这些代表替代能量转移系统。在透化心肌细胞中,我们评估了HK产生了多少内源性ADP,AK,或CK刺激线粒体呼吸,有多少被引导到线粒体。在整个心脏匀浆中,胞质和线粒体部分,我们测量了AK的活动,CK,和香港。最后,我们评估了主要HK的表达,AK,和CK同工型。总的来说,HK刺激的呼吸,AK,CK分别为25、90和80%,分别,最大呼吸率,20、0和25%,分别,被引导到线粒体。活动,分布,和香港的表达,AK,在GAMT敲除(KO)小鼠中,CK没有改变。在AGATKO小鼠中,我们发现AK没有变化,但是我们在线粒体部分发现了更高的HK活性,HKI的更大表达,但是HK对呼吸的刺激较低。我们的发现表明,小鼠心脏较少依赖磷酸转移系统来促进ADP穿过线粒体膜的流动。在AGATKO小鼠中,这是纯肌酸缺乏的模型,HK的变化可能反映了代谢的变化以及影响线粒体调节和活性氧的产生。在肌酸缺乏的AGAT-/-和GAMT-/-小鼠中,心肌肌酸激酶系统是底物受限的。尚不清楚己糖激酶和腺苷酸激酶的亚细胞定位和线粒体ADP通道是否可以作为替代的磷酸转移系统进行补偿。我们的结果显示腺苷酸激酶没有变化,它是心脏肌酸激酶的主要替代品。然而,我们发现己糖激酶I在AGAT-/-心肌细胞中的表达和活性增加。这可能会影响线粒体调节和活性氧的产生。
Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.