The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine:glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT knockouts and wild-type littermates as well as in AGAT knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole-cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation was significantly lower in AGAT knockout compared to wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared to wild-type: LTCC conductance, the exchange constant of calcium transfer between subspace and cytosol, and SERCA activity. Since tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of post-translational regulatory mechanisms or structural reorganization. The AGAT knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.

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 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 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.

Creatine kinase (CK) functions as an energy buffer in muscles. Its substrate, creatine, is generated by L-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Creatine deficiency has more severe consequences for AGAT than GAMT KO mice. In the present study, to characterize their muscle phenotype further, we recorded the weight of tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GAS), plantaris (PLA) and soleus (SOL) from creatine-deficient AGAT and GAMT, KO and WT mice. In GAS, PLA and SOL representing glycolytic, intermediate and oxidative muscle, respectively, we recorded the activities of pyruvate kinase (PK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (CO). In AGAT KO compared to WT mice, muscle atrophy and differences in marker enzyme activities were more pronounced in glycolytic than oxidative muscle. In GAMT KO compared to WT, the atrophy was modest, differences in PK and LDH activities were minor, and CS and CO activities were slightly higher in all muscles. SOL from males had higher CS and CO activities compared to females. Our results add detail to the characterization of AGAT and GAMT KO skeletal muscle phenotypes and illustrate the importance of taking into account differences between muscles, and differences between sexes.

Biological measurements frequently involve measuring parameters as a function of time, space, or frequency. Later, during the analysis phase of the study, the researcher splits the recorded data trace into smaller sections, analyzes each section separately by finding a mean or fitting against a specified function, and uses the analysis results in the study. Here, we present the software that allows to analyze these data traces in a manner that ensures repeatability of the analysis and simplifies the application of FAIR (findability, accessibility, interoperability, and reusability) principles in such studies. At the same time, it simplifies the routine data analysis pipeline and gives access to a fast overview of the analysis results. For that, the software supports reading the raw data, processing the data as specified in the protocol, and storing all intermediate results in the laboratory database. The software can be extended by study- or hardware-specific modules to provide the required data import and analysis facilities. To simplify the development of the data entry web interfaces, that can be used to enter data describing the experiments, we released a web framework with an example implementation of such a site. The software is covered by open-source license and is available through several online channels.