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Abstract

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being mitochondrial creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their solei for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode. The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA solei had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered cell energetics and metabolism in the formation of these crystalline structures are discussed.

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

“…We have taken advantage of this fact to equilibrate liposomes with the ampholytic¯uorochrome DiOC 6 (3) (573 Da), which freely distributes between the external and internal compartment, followed by cyto¯uorometric determination of DiOC 6 (3) retention. We interpret a reduction in DiOC 6 (3) retention (which correlates with an increase in malate, ATP, or calcein e ux) (Beutner et al, 1996;Marzo et al, 1998;O'Gorman et al, 1997) to re¯ect an increase in membrane permeability. As shown in Figure 5a, t-BHP and diamide cause DiOC 6 (3) e ux from control liposomes containing PTPC, yet fail to act on liposomes generated in the absence of PTPC (not shown).…”

“…PTPC containing the ANT, cyclophilin D, porin, and hexokinase were enriched from rat brain and incorporated into liposomal membranes. In response to PTPC opening agents such as atractyloside or Ca 2+ , small molecules (51500 Da) such as malate (106 Da), ATP (509 Da), or calcein (622 Da) are readily released from such PTPC liposomes, suggesting a general increase in membrane permeability (Beutner et al, 1996;Marzo et al, 1998;O'Gorman et al, 1997). We have taken advantage of this fact to equilibrate liposomes with the ampholytic¯uorochrome DiOC 6 (3) (573 Da), which freely distributes between the external and internal compartment, followed by cyto¯uorometric determination of DiOC 6 (3) retention.…”

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

“…These microcompartments control metabolite channeling between CK and ANT (Fig. 8), thus participating in the CK/phosphocreatine energy circuit (2) and possibly regulating mitochondrial permeability transition, one of the pathways leading to apoptosis (14,15,57). Under pathological conditions, these microcompartments can be disrupted, thus potentially leading to energy deficits and increased apoptosis (58 -60).…”

“…ADP is subsequently reimported into the matrix space via ANT, an obligatory ATP/ADP antiporter, and phosphocreatine is released into the cytosol via mitochondrial porin (or voltagedependent anion channel, VDAC) in the outer mitochondrial membrane. This intimate exchange of substrates and products, the so-called functional coupling or metabolite channeling (10,11), fulfills important functions that may vary among different tissues, species, and developmental states (12,13): (i) phosphocreatine becomes the high energy intermediate that is exported from mitochondria into the cytosol (3); (ii) locally generated ADP stimulates oxidative phosphorylation (11); and (iii) ADP channeled through the MtCK/ANT interaction inhibits the Ca 2ϩ -induced opening of the mitochondrial permeability transition pore (14,15), a well known trigger of apoptosis (16,17). Thus, overexpression of uMtCK in many malignant cancers with especially poor prognosis (18,19) may be related to high energy turnover and failure to eliminate cancer cells via apoptosis.…”

Novel Mitochondrial Creatine Transport Activity

“…for energetic purposes, or whether Cr, a highly abundant zwitterionic guanidino compound in the cytosol, could fulfill some protective role as an osmolyte to guarantee mitochondrial integrity under conditions of cellular stress. Interestingly enough, Cr has been shown to exert marked protection against Ca 2ϩ -induced mitochondrial permeability transition pore opening (39), an early event of cellular apoptosis. The importance of assessing the pathway(s) and cellular location of Cr transport is further highlighted by a description of the first patients with an X-linked genetic disease due to defects of the CRT gene (SLC6A8) (40).…”