Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

Complete failure of insulin-transmitted signaling, but not obesity-induced insulin resistance, impairs respiratory chain function in muscle.

The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.

Central FoxO3a and FoxO6 expression is down-regulated in obesity induced diabetes but not in aging.

BACKGROUND: Recent data suggest that insulin-like growth factor (IGF)-1 resistance in neurons prolongs longevity. In C. elegans this effect is mediated via DAF-16 the ortholog of the mammalian FoxO transcription factors. 3 different FoxO transcription factors (FoxOs) are expressed in rodent CNS: FoxO1, FoxO3a and FoxO6. METHODS: To define whether the different FoxOs are region-, sex- and age-specifically expressed, we analyzed FoxO mRNA levels in different brain regions from 6, 16, 60 and 100 weeks old mice using realtime-PCR. In addition, we fed mice a high fat diet (HFD) to experimentally induce obesity and diabetes and analyzed FoxO mRNA in the different brain regions. RESULTS: Interestingly, FoxO1 was predominantly expressed in the hippocampus whereas FoxO3a was quantitatively the most abundant FoxO in the neocortex. During aging, FoxO1 expression peaked in all brain regions at 16 weeks and FoxO6 showed its highest expression at 60 weeks in the parietal and occipital cortex. In 6 weeks old mice FoxO6 expression was higher in male compared to female mice in the hippocampus and all cortical regions. Surprisingly, in HFD animals FoxO3a was significantly less expressed in the cerebellum and all cortical regions compared to control animals. Even more dramatic, FoxO6 expression dropped about 80% in all brain regions in response to HFD. CONCLUSION: Thus, FoxOs in the CNS showed a highly distinct expression, which in addition was age- and sex-dependent. In contrast to FoxO1, FoxO3a and FoxO6 were specifically diminished in the CNS of HFD animals possibly contributing to the reduced lifespan observed in these animals.

Regulation and co-ordination of nuclear gene expression during mitochondrial biogenesis.

Biogenesis of mitochondria is happening constantly due to the physiological and developmental situation of a cell. As mitochondrial biogenesis is a complex process producing about 20 % of cellular protein, the expression of the 1000 genes involved is expected to be coordinated and regulated tightly. The variety of physiological stimuli and differentiation states lead to the idea of a complex network connecting many different regulatory pathways. By analysing nuclear encoded mitochondrial genes some of the factors involved in the regulation and coordination of mitochondrial gene expression were identified. These factors include general transcription factors such as Sp1 or YY1, as well as transcription factors specific for mitochondrial genes like the nuclear respiratory factors NRF1 and 2. An important control function linked to the physiological situation of a cell is triggered by hormones such as steroid and thyroid hormones. Even cell type-specific regulatory proteins like the myogenin transcription factor family have a strong influence on some mitochondrial genes in the specific cellular background. The regulatory function of most of these proteins can be modulated and enhanced by the coactivators PGC-1a and b and PRC. Although regulatory pathways have been characterized in more detail in recent years, no regulation mechanism has been shown to work on all analysed mitochondrial genes, and the general concept of mitochondrial regulation still remains unclear.

Mechanism of mammalian mitochondrial DNA replication: import of mitochondrial transcription factor A into isolated mitochondria stimulates 7S DNA synthesis.

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA-DNA transition. Time course and pulse-chase experiments showed that the half-life of this DNA is approximately 45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

TGF-beta(1) downregulates PTHrP in coronary endothelial cells.

Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system including coronary endothelial cells. Factors involved in the regulation of cardiac PTHrP expression have not been examined before. This study investigates the influence of transforming growth factor (TGF)-beta(1)on ventricular PTHrP expression. Coronary endothelial cells were isolated from ventricles of adult rats and PTHrP protein expression in these cultures was analysed by immunoblotting. TGF-beta(1)caused a concentration-dependent reduction in PTHrP protein within 24 h. In transgenic mice over-expressing TGF-beta(1)ventricular PTHrP protein expression and release was reduced compared to non-transgenic littermates. Similar concerns hold for PTHrP mRNA content (RT-PCR). Since ventricular TGF-beta(1)expression increases under pathophysiological conditions like arterial hypertension, ventricular PTHrP expression was further determined in aging spontaneously hypertensive (SHR-SP) and normotensive rats. TGF- beta(1)expression was increased in SHR-SP and ventricular PTHrP mRNA expression was downregulated at the age of 10 months. PTHrP expression did not recover in elder SHR-SP in which TGF-beta(1)expression was normalized again. Finally, we investigated ventricular PTHrP expression in rats after banding of the ascending aorta which generates a pressure induced hypertrophy without an induction of TGF-beta(1)expression. In ventricles from these animals, PTHrP expression was transiently increased and normalized at day 3. In conclusion, PTHrP expression was reduced under all conditions in which coronary endothelial cells were exposed to TGF-beta(1). PTHrP expression does not correlate with cardiac hypertrophy. Since coronary endothelial cells represent the majority of PTHrP producing cells in the ventricle its downregulation by TGF- beta(1)seems to be relevant for the paracrine effects of PTHrP.

Biogenesis of giant mitochondria during insect flight muscle development in the locust, Locusta migratoria (L.). Transcription, translation and copy number of mitochondrial DNA.

The biogenesis of giant mitochondria in flight muscle of Locusta migratoria (L.) was analyzed at the molecular level. During the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight, individual mitochondria have been shown to enlarge 30-fold and the fractional mitochondrial volume of muscle cells increases fourfold [Brosemer, R.W., Vogell, W. and Bücher, Th. (1963) Biochem. Z. 338, 854-910]. Within the same period, the activity of cytochrome c oxidase, containing subunits encoded on mitochondrial DNA, increased twofold. However, no significant change in mitochondrial DNA copy number, and even a threefold decrease in mitochondrial transcripts, was observed. Mitochondrial translation rate, measured in isolated organelles, was twofold higher in larval muscle, which can be explained only partly by the higher content of mitochondrial RNAs. Thus, rather unusually, in this system of mitochondrial differentiation, the mitochondrial biosynthetic capacity correlates with the rate of organelle biogenesis rather than the steady-state concentration of a marker enzyme. The copy number of mitochondrial DNA does not seem to play a major role in determining either mitochondrial transcript levels or functional mass.

Proliferation of mitochondria in chronically stimulated rabbit skeletal muscle--transcription of mitochondrial genes and copy number of mitochondrial DNA.

Mitochondrial proliferation was studied in chronically stimulated rabbit skeletal muscle over a period of 50 days. After this time, subunits of COX had increased about fourfold. Corresponding mRNAs, encoded on mitochondrial DNA as well as on nuclear genes, were unchanged when related to total tissue RNA, however, they were elevated two- to fivefold when the massive increase of ribosomes per unit mass of muscle was taken into account. The same was true for the mRNA encoding mitochondrial transcription factor A. Surprisingly, tissue levels of mtTFA protein were reduced about twofold, together with mitochondrial DNA. In conclusion, mitochondria are able to maintain high rates of mitochondrial transcription even in the presence of reduced mtTFA protein and mtDNA levels. Therefore, stimulated mtTFA gene expression accompanies stimulated mitochondrial transcription, as in other models, but it is not sufficient for an increase of mtDNA copy number and other, yet unknown, factors have to be postulated.

A variant form of the nuclear triiodothyronine receptor c-ErbAalpha1 plays a direct role in regulation of mitochondrial RNA synthesis.

In earlier research, we identified a 43-kDa c-ErbAalpha1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats.

In isolated cardiac myocytes, the direct effects of angiotensin II on cellular growth and gene expression were shown to be mediated by endothelin via the endothelin subtype A (ETA) receptor. To determine whether this pathway is also involved in the cardiovascular adaptations to a chronic activation of the renin-angiotensin system in vivo, the effects of a selective ETA receptor antagonist (LU 127043) were investigated in adult rats with renal artery stenosis. Four groups of rats (n=107) were studied over a period of 10 days after surgery: (1) sham-operated animals with saline administration, (2) rats subjected to left renal artery clipping with saline administration, (3) sham-operated rats with LU 127043 administration, and (4) rats subjected to left renal artery clipping with LU 127043 administration. LU 127043 (50 mg/kg) or saline was given by gavage twice daily starting 1 day before the operation. In clipped rats with saline administration, plasma renin activity, the ratio of left ventricular weight to body weight, and mRNAs for beta-myosin heavy chain and atrial natriuretic peptide were significantly elevated as early as 2 days after surgery. Blood pressure started to rise on the third postoperative day and attained a steady state hypertensive level by day 6. Blockade of ETA receptors had no effects on plasma renin activity or the time course of hypertension in clipped animals but completely prevented left ventricular hypertrophy and the re-expression of the beta-myosin heavy chain and atrial natriuretic peptide genes on day 2. While the expressions of the beta-myosin heavy chain and atrial natriuretic peptide genes were not different from saline-treated, clipped animals after day 4, the development of left ventricular hypertrophy remained markedly blunted (-50%) during ETA receptor blockade until day 10. These results show that a continuous blockade of ETA receptors significantly attenuates the development of left ventricular hypertrophy and, more transiently, fetal gene expression in the early phase of renovascular hypertension. Since neither blood pressure nor the increase in plasma renin activity was significantly altered by ETA receptor blockade, the inhibitory influences of the ETA receptor antagonist on left ventricular hypertrophy and gene expression were mediated most likely through a direct blockade of myocardial ETA receptors.

Stimulation of mitochondrial gene expression and proliferation of mitochondria following impairment of cellular energy transfer by inhibition of the phosphocreatine circuit in rat hearts.

Mitochondria proliferate when cellular energy demand increases. However, the pathways leading to enhanced expression of mitochondrial genes are largely unknown. We tested the hypothesis that an altered flux through energy metabolism is the key regulatory event by decreasing mitochondrial energy supply to rat heart cells by creatine depletion. Electron microscopy showed that the density of mitochondria increased by 75% in such hearts (p < 0.01). Levels of representative mRNAs encoded on mitochondrial DNA (mtDNA) or on nuclear chromosomes were elevated 1.5 to 2-fold (p < 0.05), while the mtDNA content was unchanged. The mRNA for the nuclear encoded mitochondrial transcription factor A (mtTFA) was increased after GPA feeding (p < 0.05). Thus, we have shown that an impairment of mitochondrial energy supply causes stimulation of gene expression resulting in mitochondrial proliferation, probably as a compensatory mechanism. The observed activation of the mtTFA gene corroborates the important function of this protein in nuclear-mitochondrial communication.

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats.

Left ventricular hypertrophy (LVH) is often associated with an impaired maximal coronary blood flow and increases the vulnerability of the heart tissue to ischaemia. In this study, the correlation between coronary blood flow and expression of the vascular endothelial growth factor (VEGF) mRNA was investigated. Using both haemodynamic measurements and analysis of mRNA, we have demonstrated that during development of LVH, in spontaneously hypertensive rats (SHR), an impaired maximal coronary flow at 12 weeks of age is associated with low levels of VEGF mRNA. However, in older SHR (32 weeks) with stabilised hypertrophy and a normal maximal coronary flow response, VEGF mRNA levels are increased 3-fold. These results suggest that the mechanism for the impaired flow, observed in some types of cardiac hypertrophy, might involve an inadequate growth of the coronary vessels due to insufficient activation of the VEGF gene.

Cytochrome oxidase activity and mitochondrial gene expression in skeletal muscle of patients with chronic obstructive pulmonary disease.

Several recent studies have suggested that skeletal muscle bioenergetics are abnormal in patients with chronic obstructive pulmonary disease (COPD). This study investigates the activity of cytochrome oxidase (COX), the terminal enzyme in the mitochondrial electron transport chain, and the expression of two mitochondrial DNA genes related to COX (mRNA of subunit I of COX [COX-I] and the RNA component of the 12S ribosomal subunit [12S rRNA]), in quadriceps femoris muscle biopsies obtained from COPD patients with various degrees of arterial hypoxemia, and from healthy sedentary control subjects of similar age. The activity of COX was measured spectrophotometrically in fresh tissue at 37 degrees C with excess substrate. RNA transcripts were measured using reverse transcription and polymerase chain reaction. The measurements of mRNA COX-I and 12S rRNA were normalized to the mRNA of actin, which is a housekeeping gene not influenced by hypoxia. We found that, compared with control subjects, COPD patients with chronic respiratory failure (PaO2 < 60 mm Hg) showed increased COX activity (p < 0.05). Further, the activity of COX was inversely related to arterial PO2 value (Rho -0.59, p < 0.01). The COX-I mRNA content was not different between patients and control subjects but patients with chronic respiratory failure had higher levels of 12S rRNA (p < 0.05), which were again inversely related to PaO2 (Rho -0.49, p < 0.05). These results indicate that the activity of COX is increased in skeletal muscle of patients with COPD and chronic respiratory failure, and they suggest that this is likely regulated at the translational level by increasing the number of mitochondrial ribosomes.

Regulation of mitochondrial transcription by mitochondrial transcription factor A.

In order to test the hypothesis that mitochondrial transcription factor A (mtTFA) regulates mitochondrial transcription in vivo, mtTFA was overexpressed in HeLa cells and imported into isolated rat liver mitochondria. Five hours after transfection with an eukaryotic expression vector, mitochondrial transcripts for cytochrome-c-oxidase subunit I and 12 S rRNA were increased over controls. In the presence of rat liver mitochondria, the 29 kDa mtTFA, generated by in vitro translation, was processed to a 24 kDa protein which was protected from protease digestion. This demonstrates that mtTFA was imported into the matrix. Incorporation of 32P-UTP into mitochondrial transcripts was stimulated following import of mTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription.

Quantification of transcript-to-template ratios as a measure of gene expression using RT-PCR.

A new protocol was established for the quantitative analysis of gene expression in small muscle biopsies. Reverse transcription-PCR was performed with a preparation of total nucleic acids (DNA+RNA), amplifying the sequences of interest (targets; mitochondrial transcripts: 12S rRNA, cytochrome-c-oxidase [COX I] mRNA) together with an endogenous, non-transcribed reference sequence (template: D-loop region of mtDNA). Synthesis of PCR products at consecutive cycles within the exponential phase was quantified by measuring incorporation of radioactivity. Product accumulation was determined by regression analysis of these data. Gene expression could then be quantified as a ratio of target transcripts to reference DNA. The results revealed a ratio of 12S rRNA:mtDNA and COX I mRNA:mtDNA of 14 and 2, respectively, or a ratio of 12 S rRNA:COX I mRNA of 7 in human left ventricle and are in good agreement with previously published values for rat liver and muscle. In addition to the investigation of mitochondrial gene expression in the steady state and during mitochondrial proliferation, this newly developed method will easily be applicable to expression analysis of any nuclear gene using an intron sequence as endogenous reference.

Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament.

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments (TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by TnI-(96-148). The regulatory complex composed of TnI-(96-148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnT.C while those regulatory complexes composed of TnT.C plus either TnI-(104-115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-S1 rate was mimicked by the regulatory complex composed of TnI-(1-116) plus TnT.C and was not seen with complexes composed with either TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC in the presence of calcium, its function is modulated by both the C-terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.

Dissociation of left ventricular hypertrophy, beta-myosin heavy chain gene expression, and myosin isoform switch in rats after ascending aortic stenosis.

BACKGROUND: Reexpression of the fetal beta-myosin heavy chain (beta-MHC) gene was reported to be a marker for phenotypic reprogramming and cardiac hypertrophy in rats. Recent in vitro studies strongly suggested a role of angiotensin II for phenotypic reprogramming. In the present investigation, beta-MHC gene expression was studied in an experimental model of pressure-over-load hypertrophy that is not associated with a concurrent activation of the circulating renin-angiotensin system. METHODS AND RESULTS: Hypertrophy was induced in rats by ascending aortic banding (n = 40). After 7 days, myosin contained 31% (P < .05) of the beta-MHC isoform in banded but < 5% in sham-operated animals. However, no specific elevation of beta-MHC mRNA levels was found in banded animals. In contrast, hearts of rats with abdominal aortic banding displayed a marked increase in beta-MHC mRNA levels (3-fold to 5-fold, P < .05). Both the left ventricular weight and left ventricular peak systolic pressure were significantly elevated compared with sham-operated animals (abdominal aortic banding, +13% and 164 +/- 7 mm Hg; ascending aortic banding, +27% and 191 +/- 9 mm Hg). Plasma renin activity was elevated in rats with abdominal aortic banding (2.5-fold, P < .05) but not in rats with ascending aortic banding. CONCLUSIONS: The results of the present work do not support the concept that increased beta-MHC gene expression is a general "stable late marker" of myocardial hypertrophy in rats. Our results suggest that the stimulation of the renin-angiotensin system is crucial for the activation of the beta-MHC gene.

Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation.

After cold exposure, cytochrome c oxidase (COX) activity increased about 2.5-fold within 2 weeks in the brown adipose tissue (BAT) of Djungarian hamsters. The mRNAs for COX subunits I and III and the 12 S rRNA, encoded on mitochondrial DNA (mtDNA), as well as mRNAs for COX subunits IV, Va and mitochondrial transcription factor A, encoded in the nucleus, were unchanged when expressed per unit of total tissue RNA. However, since total tissue RNA doubled per BAT depot, while total DNA remained unchanged, the actual levels of these transcripts were increased within BAT cells. In contrast, the abundance of mRNA for uncoupling protein was increased 10-fold, indicating specific activation of this gene. In addition, the maximal rate of protein synthesis analysed in a faithful in organello system was increased 2.5-fold in mitochondria isolated from BAT after 7 days of cold exposure. We conclude from these data that the biogenesis of thermogenic mitochondria in BAT following cold adaptation is achieved by increasing the overall capacity for synthesis of mitochondrial proteins in both compartments, by increasing their mRNAs as well as the ribosomes needed for their translation. In addition, the translational rate for COX subunits as well as all other proteins encoded on mtDNA is increased. Thus the pool of subunits encoded on mtDNA required for assembly of respiratory chain complexes is provided. By comparison with other models of increased mitochondrial biogenesis, we propose that thyroid hormone (generated within BAT cells by 5'-deiodinase, and induced upon sympathetic stimulation), which is a well known regulator of the biogenesis of mitochondria in many tissues, is also the major effector of these adaptive changes in BAT.

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle.

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 "mu"M), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 "mu"M and 150 "mu"M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.