Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

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.

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication.

Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT-/- mouse hearts.

Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

Intraspinal MDL28170 microinjection improves functional and pathological outcome following spinal cord injury.

Although calpain (calcium-activated cysteine protease) inhibition represents a rational therapeutic target for spinal cord injury (SCI), few studies have reported improved functional outcomes with post-injury administration of calpain inhibitors. This reflects the weak potency and limited aqueous solubility of current calpain inhibitors. Previously, we demonstrated that intraspinal microinjection of the calpain inhibitor MDL28170 resulted in greater inhibition of calpain activity as compared to systemic administration of the same compound. In the present study, we evaluated the ability of intraspinal MDL28170 microinjection to spare spinal tissue and locomotor dysfunction following SCI. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon impactor at the 200-kdyn force setting. Open-field locomotion was evaluated until 6 weeks post-injury. Histological assessment of tissue sparing was performed at 6 weeks after SCI. The results demonstrate that MDL28170, administered with a single post-injury intraspinal microinjection (50 nmoles), significantly improves both locomotor function and pathological outcome measures following SCI.

Hyperhomocysteinemia in movement disorders: Current evidence and hypotheses.

Elevated plasma levels of homocysteine (Hcy) are a risk factor for systemic vascular diseases, stroke and vascular dementia. In recent years, increasing Hcy levels have been detected in neurological disorders that are not vascular in origin including Alzheimer's Disease and movement disorders (MD) such as idiopathic Parkinson's Disease (PD), Huntington's Disease (HD) and primary dystonia. Hyperhomocysteinemia (HHcy) in PD results from L-Dopa administration and its O-methylation dependent from catechol-O-methyltransferase and may be implicated in the development of motor complications and non-motor symptoms, such as dementia. In a recent study, HHcy has been evidenced in HD patients, compared to controls. Because mutated Huntington protein influences Hcy metabolism by modulating cystathionine-beta-synthase activity, Hcy could represent a biological marker of neurodegeneration and could explain the leading role of cardiovascular and cerebrovascular diseases as causes of death in HD. Finally, several cases of homocystinuria associated with dystonia, and some recent reports of elevated Hcy in patients with primary adult onset dystonia have been published. Increased Hcy plasma levels may have important implications in patients affected by these basal ganglia disturbances, by exerting neurotoxic effects, contributing to neurotransmitter imbalance in motor circuits, and increasing the risk for vascular insults and cognitive dysfunctions.

Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis.

The first inborn error of creatine metabolism (guanidinoacetate methyltransferase [GAMT] deficiency) has recently been recognized in an infant with progressive extrapyramidal movement disorder. The diagnosis was established by creatine deficiency in the brain as detected by in vivo magnetic resonance spectroscopy and by defective GAMT activity and two mutant GAMT alleles in a liver biopsy. Here, we describe characteristic guanidino-compound patterns in body fluids of this index patient with GAMT deficiency. Concentrations of guanidino compounds (creatine and guanidinoacetate) and creatinine were determined by cation-exchange chromatography and by color reaction with picric acid, respectively, in urine, plasma, and cerebrospinal fluid (CSF). Creatine concentrations were low in plasma, CSF, and urine while guanidinoacetate concentrations were markedly elevated. Daily urinary creatinine excretion was low, whereas creatinine concentrations in random urine samples were not always discriminative. Guanidino compound to creatinine ratios were not informative, as low creatinine concentrations resulted in high values for all determined compounds. During a 22-month period of oral treatment with creatine-monohydrate, plasma and urinary creatine concentrations increased to levels high above the normal range, and daily urinary creatinine excretion-proportional to total body creatine-became normalized. Guanidinoacetate concentrations remained elevated even during additional substitution of ornithine, which inhibits guanidinoacetate synthesis in vitro. The results indicate that GAMT deficiency can be recognized noninvasively by determination of guanidino compounds (creatine and guanidinoacetate) in body fluids. A deficiency of creatine, but not an accumulation of guanidinoacetate, can be corrected by treatment with oral creatine substitution.

Models of disuse: a comparison of hindlimb suspension and immobilization.

The effects of 1 and 2 wk of hindlimb suspension (HS) on rat skeletal muscle function were determined and the results compared with those obtained previously with hindlimb immobilization (HI). Both models of disuse (HS and HI) primarily affected slow-twitch muscle. Each decreased the isometric twitch duration in the slow-twitch soleus; however, the HS-mediated effect was entirely a result of a shortened contraction time (CT), whereas HI reduced one-half relaxation time (1/2 RT) as well as CT. Soleus muscle mass and peak tetanic tension (Po) declined with disuse. The HS effect on muscle mass and Po was variable, however, for all experiments HS produced atrophy equal to or greater than HI. A major difference existed in the effects of HS and HI on the maximal speed of soleus muscle shortening (Vmax). One and 2 wk of HS produced increases in Vmax to 4.45 +/- 0.34 and 6.83 +/- 0.74 fiber lengths/s, respectively, compared with control velocities of 3.05 +/- 0.08. By contrast over a similar time period, HI had no significant effect on soleus Vmax. The increase in Vmax at 14 days of HS was associated with, and perhaps caused by, the increased expression of a second faster migrating isozyme of myosin. The new native isozyme comigrated with fast myosin, but its light chain subunits contained only LC1s and LC2s. The mechanism responsible for the increase is unknown. One plausible explanation is that the apparent HS-mediated modification in muscle fiber type is dependent on the elimination of loadbearing or isometric contractions, a condition that does not exist during HI.

The clinical significance of serum alkaline phosphatase isoenzymes in locomotor diseases.

AP isoenzymes were estimated in 292 patients with locomotor diseases and in 124 healthy controls. The diagnostic usefulness of AP determination is increased by estimation of isoenzymes. Investigations were made to study the biological profile of organ specific AP activities: 1. Rheumatoid arthritis and Reiter's syndrome - the total AP and L-AP activities were increased. 2. Ankylosing spondylitis treated by physiotherapy - the total AP, B-AP and I-AP activities were increased. After drug therapy an increase occurred also in L-AP activity while I-AP activity showed no significant change. 3. Progressive OA of hip and knee showed increased levels of total AP and B-AP activities. 4. Degenerative diseases of the spine, chiefly cases of discopathy, showed significantly reduced levels of AP and B-AP activities. 5. In osteoporosis there was an increase in total AP, L-AP, B-AP and I-AP activities. 6. In the active generalised form of Paget's disease, increased levels were found of total AP, B-AP, I-AP and L-AP activities. 7. In neoplastic diseases the isoenzymes can help to reveal metastatic dissemination and thus aid preoperative evaluation. 8. In gout and hyperuricemic syndromes there was a relative increase of B-AP activity and non-significant fall of L-AP activity. Increased levels of L-AP occured in patients with gallbladder disease, after immunosuppressive therapy or after infectious hepatitis. A fall of L-AP levels was found after Corticotrophin and after intraarticular administration of Kenalog. Increased B-AP activities occurred after total hip replacement, in acute or chronic pyelonephritis and in active osteonecrosis and osteoporosis. Anabolic therapy caused a significant fale of B-AP activity to fall significantly. Reduced B-AP levels were also found after antibiotic therapy. Increased I-AP activity was found in cases of osteoporosis, and in secondary amyloidosis; reduced I-AP activity was seen in mucous colitis. The activity of I-AP is assumed to increase as a result of the changed intestinal calcium and phosphorus regulation occurring in association with the enhanced bone tissue metabolism. From this point of view an order of significance is given for the activity of bone pathology in the separate diagnostic groups of locomotor diseases.

Alterations in the accumulation patterns of polyamines in brains of myelin-deficient mice.

Quaking mutants and jimpy mutants of mice have known deficiencies of myelination of the central nervous system, as well as lesser involvement of the peripheral nervous system. Both mutants also have altered polyamine synthesis and accumulation, particularly in the hindbrain and spinal column. The ratio of spermidine/spermine, which generally is higher in tissues with high rates of biosynthetic activity, was significantly lower in the mutants as compared to their normal siblings. In quaking mutants, 5 months of age, the spermidine concentration of hindbrain and spinal column was 60% that of controls. In contrast, the decreased spermidine/spermine ratio in jimpy mutants resulted from a marked increase in the spermine concentration in both forebrain and hindbrain. Alterations in the spermidine/spermine ratio could lead to reductions in the biosynthetic potential of the brain during development.