Organization of the genetic locus for chicken myosin light chain kinase is complex: Multiple proteins are encoded and exhibit differential expression and localization

We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins. J. Cell. Biochem. 70:402–413, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of the kinase-related protein gene found at human chromosome 3q21 in a multi-gene cluster: organization, expression, alternative splicing, and polymorphic marker.

We report the amino acid sequence, genomic organization, tissue expression, and alternative splice patterns for the human kinase related protein (KRP) gene, as well as the discovery of a new CA repeat sequence polymorphic marker in an upstream intron of the myosin light chain kinase (MLCK) gene. The KRP/MLCK genetic locus is a prototype for a recently discovered paradigm in which an independently regulated gene for a non-enzymic protein is embedded within a larger gene for a signal transduction enzyme, and both classes of proteins are involved in the regulation of the same cellular structure. The MLCK/KRP gene cluster has been found only in higher vertebrates and is localized to human chromosome 3q21. The determination of the human KRP amino acid sequence through cDNA sequence analysis and its comparison to the exon/intron organization of the human KRP gene revealed an alternative splice pattern at the start of KRP exon 2, resulting in the insertion of a single glutamic acid in the middle of the protein. Examination of tissue distribution using Northern blot analysis revealed that the human expression pattern is more similar to the well-characterized chicken KRP gene expression pattern than to rodent or rabbit. Unexpected differences of the human gene from other species is the apparent expression of the human gene products in adult cardiac muscle, an observation that was pursued further by the production of a site-directed antiserum and immunohistochemistry analysis. The results reported here provide insight into the conserved and variable features of this late evolving genetic paradigm, raise new questions about the molecular aspects of cardiac muscle regulation, and provide tools needed for future clinical studies. The comparative analysis of the MLCK/KRP locus, combined with the recent discovery of a similar genomic relationship among other signal transduction proteins, suggest a diverse distribution of this theme among signal transduction systems in higher vertebrate genomes and indicate the utility of comparative genomics in revealing late evolving genetic paradigms.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

Cardiogel: A biosynthetic extracellular matrix for cardiomyocyte culture

Summary Tissue-cultured neonatal cardiomyocytes can be successfully maintained in culture on a variety of extracellular matrix components such as laminin, fibronectin, and interstitial collagens (Types I and III).In vivo, however, cardiomyocytes (as well as many other cells) exist in a highly complex extracellular matrix environment composed of, in addition to the above three components, other proteins, proteoglycans, and growth factors. We have developed a procedure for culturing cardiomyocytes on a naturally occurring complete extracellular matrix, Cardiogel. This substrate, synthesized by cardiac fibroblasts, contains laminin, fibronectin, Types I and III collagen, and proteoglycans. When compared to cardiomyocytes grown on laminin alone or fibronectin alone, Cardiogel-supported cardiomyocytes adhere more rapidly after plating, exhibit spontaneous contractility earlier, undergo cytoskeletal and myofibrillar differentiation earlier, and grow larger than their counterparts. We suggest that their superior growth characteristics reflect the synergistic effect of numerous extracellular matrix components’ signals in Cardiogel transduced by the cardiomyocyte cytoskeletal elements.     

Extracellular matrix dependence of organization of the cardiomyocyte contractile apparatus

In the process of cardiomyocyte cultivation, there occurs rearrangement of their contractile apparatus with transformation of typical myofibrils into structures of nonmuscle type and with subsequent restoration of the initial organization. The causes and mechanisms of the rearrangements described are unknown. In this study, it was shown that, at cultivation of cardiomyocytes on the individual proteins of the extracellular matrix (ECM), as well as on the matrix produced either by cardiac fibroblasts or by similar cardiomyocytes, the period in which the contractile apparatus is in a converted state is shortened, whereas soluble factors of the conditioned cultural medium do not produce any obvious effect on the above-indicated rearrangements. Using the method of extracellular matrix isolation adapted for cardiomyocytes, we have confirmed that, in the process of cultivation, cardiomyocytes themselves produce ECM that differs from ECM of cardiac fibroblasts by composition and varies with culturing time.     

Regulation of Cell Motility by Mitogen-activated Protein Kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Histological and ultrastructural abnormalities in murine desmoglein 2-mutant hearts

Mice carrying a deletion of the adhesive extracellular domain of the desmosomal cadherin desmoglein 2 develop an arrhythmogenic right ventricular cardiomyopathylike phenotype with ventricular dilation, fibrosis and arrhythmia. To unravel the sequence of myocardial alterations and to identify potential pathomechanisms, histological analyses were performed on mutant hearts from the juvenile to the adult state, i.e., between 2 and 13 weeks. At an age of 2 weeks 30% of mutants presented lesions,which were visible as white plaques on the heart surface or in the septum. From 4 weeks onwards, all mutants displayed a cardiac phenotype. Dying cardiomyocytes with calcification were found in lesions of all ages. But lesions of young mutant animals contained high amounts of CD45+ immune cells and little collagen fibers, whereas lesions of the older animals were collagen-rich and harbored only a small but still significantly increased number of CD45+ cells. Electron microscopy further showed that distinct desmosomes cannot be distinguished in intercalated discs of mutant hearts. Widening of the intercellular cleft and even complete dissociation of intercalated discs were often observed close to lesions. Disturbed sarcomer structure, altered Z-discs, multiple autophagic vacuoles and swollen mitochondria were other prominent pathological features. Taken together, the following scenario is suggested: mutant desmoglein 2 cannot fully support the increased mechanical requirements placed on intercalated disc adhesion during postnatal heart development, resulting in compromised adhesion and cell stress. This induces cardiomyocyte death, aseptic inflammation and fibrotic replacement. The acute stage of scar formation is followed by permanent impairment of the cardiac function.     

Increased expression of integrin-linked kinase improves cardiac function and decreases mortality in dilated cardiomyopathy model of rats

Aims

Integrin-linked kinase (ILK) is a multifunctional kinase linking the extracellular matrix to intracellular signaling pathways, whose activation in the heart gives rise to a number of functional consequences. The aim of this study is to demonstrate the therapeutic and survival benefit of cardiac ILK overexpression in a rat model of dilated cardiomyopathy.

Methods and Results

The dilated cardiomyopathy model was generated in rats by intraperitoneal administration of six equal doses of doxorubicin over a 2 week period. Five weeks after the first injection, echocardiographic analysis demonstrated impaired cardiac function and, at that point, recombinant adenoviral vector harboring ILK cDNA or vehicle was injected into the myocardium, and the rats re-studied 4 weeks later. Compared with vehicle injection, ILK treatment ameliorated inflammatory cell infiltration and cardiomyocyte degeneration, as well as left ventricular dilation and dysfunction. ILK treatment was also associated with a reduction in apoptosis and an increase in proliferation of cardiomyocytes, as well as decreased oxidative stress and autophagic vacuole accumulation. Importantly, mortality was lower in rats following ILK treatment than in those following vehicle injection. In cultured neonatal rat cardiomyocytes, we also found that ILK overexpression protected against doxorubicin-induced apoptosis, giving rise to an increase in their proliferation.

Conclusions

These data demonstrate for the first time that ILK gene therapy improves cardiac function and survival in a model of dilated cardiomyopathy, and this may be mediated through suppression of inflammation, prevention of ventricular remodeling, inhibition of cardiomyocyte apoptosis and autophagy, and stimulation of cardiomyocyte proliferation.     

Neurohormonal regulation of calcium in the cell

Neurohormone C (NC) is a glycopeptide isolated from bovine hypothalamus, which inhibits Ca-calmodulin (CaM)-dependent cAMP and cGMP phosphodiesterase (PDE) and is a regulator of Ca in the cell. Distribution of [⁴⁵Ca]CaCl₂ in the mitochondria and reticulum (SR) of heart and brain mitochondria and changes of Ca-binding proteins in these organelles under NC influence have been studied in the myocardium before and after isoproterenol-induced necrosis. Intraperitoneal administration of 80–100 mU of PDE inhibitory activity of NC to rats did not cause any noticeable changes in the protein content of intracellular organelles, but altered the affinity of certain proteins to⁴⁵Ca²⁺. This property of NC was especially noticable after isoproterenol necrosis. Necrotic injury of the myocardium induced Ca²⁺ storage in the mitochondria and SR of brain, and decreased the Ca²⁺ concentration in myocardial mitochondria. NC injection to the animals with necrosis was followed by Ca²⁺ release from all the studied organelles.     

The features of mitochondria of cardiomyocytes from rats with chronic heart failure

Electron-microscopy study of rat myocardium 2 weeks after a heart attack revealed significant alterations in the ultrastructure of cardiomyocytes than for the control. The location of myofibrils was less regular than for normal cells. The population of interfibrillar mitochondria decreased. Mitochondrial cristae were located less densely and formed cellated structures. Swollen mitochondria were observed in the periinfarction and intact areas, indicating the development of ischemia in the myocardium as a whole. Six months after the occlusion of coronary vessels alterations in the location of myofibrils and mitochondria were mainly observed in the peri-infarction area. Mitochondria also formed cellated structures. A 30% decrease in the density of the arrangement of the inner membranes of mitochondria on an area unit was found in the periinfarction zone. The ratio between the relative volumes of mitochondria and myofibrils in the cardiomyocytes of the peri-infarction area was increased by 20%. The area of mitochondria in the intact zone of the left ventricle was 30% greater than for the control. A study of isolated living cardiomyocytes revealed that the mitochondrial- membrane potential in the rats subjected to myocardial infarction half a year ago previously was significantly lower than for the mitochondrial-membrane potential in the control rats. Thus, cardiomyocytes that were similar to healthy cardiomyocytes in their morphology exhibited lower total mitochondrial-membrane potential, indicating their decreased energy state.     

Generation of cardiomyocytes by atrioventricular node cells in long-term cultures

Turnover of cardiac pacemaker cells may occur during the lifetime of the body, and we recently raised the hypothesis that specialized cardiac cells have in common the potential to generate cardiomyocytes from fibroblasts. To examine this hypothesis, we analyzed the ability of atrioventricular node cells (AVNCs) to generate functional cardiomyocytes in long-term culture. AVNCs were isolated from adult guinea pig hearts and cultured for up to three weeks. Under phase-contrast microscopic observation over time, it was found that within a week, a number of fibroblasts gathered around the AVNCs and formed cell clusters, and thereafter the cell clusters started to beat spontaneously. The nascent cell clusters expanded their area gradually by three weeks in culture and expressed specific cardiac genes and proteins. Maturation of newly formed cardiomyocytes seems to be slow in cultures of AVNCs compared with those of sinoatrial node cells. Stimulation of muscarinic receptors with acetylcholine induced a beating rate decrease which was blocked by atropine, and activation of adenylate cyclase activity with forskolin increased the beat rate, while stimulation of beta adrenoceptors by isoproterenol had no effect. These results indicate that AVNCs form a cluster of cells with properties of functional cardiomyocytes and provide evidence to support the hypothesis.     

Extracellular matrix remodeling is associated with the survival of cardiomyocytes in the subendocardial region of the ischemic myocardium

A significant amount of cardiomyocytes in subendocardial region survive from ischemic insults. In order to understand the mechanism by which these cardiomyocytes survive, the present study was undertaken to examine changes in these surviving cardiomyocytes and their extracellular matrix. Male C57BL/6 mice aged 8–12 weeks old were subjected to a permanent left anterior descending coronary artery ligation to induce ischemic injury. The hearts were collected at 1, 4, 7, or 28 days after the surgery and examined by histology. At day 1 after left anterior descending ligation, there was a significant loss of cardiomyocytes through apoptosis, but a proportion of cardiomyocytes were surviving in the subendocardial region. The surviving cardiomyocytes were gradually changed from rod-shaped to round-shaped, and appeared disconnected. Connexin 43, an important gap junction protein, was significantly decreased, and collagen I and III deposition was significantly increased in the extracellular matrix. Furthermore, lysyl oxidase, a copper-dependent amine oxidase catalyzing the cross-linking of collagens, was significantly increased in the extracellular matrix, paralleled with the surviving cardiomyocytes. Inhibition of lysyl oxidase activity reduced the number of surviving cardiomyocytes. Thus, the extracellular matrix remodeling is correlated with the deformation of cardiomyocytes, and the electrical disconnection between the surviving cardiomyocytes due to connexin 43 depletion and the increase in lysyl oxidase would help these deformed cardiomyocytes survive under ischemic conditions.     

Differences in phorbol-dependent phosphorylation of regulatory proteins and contraction of phasic and tonic smooth muscle

Smooth muscles are divided into slowly contracting tonic and relatively fast phasic muscles. In both cases Ca2+ is a key mediator of the contractile response. However, the appearance of a tonic component during sphincter or arterial muscle contraction and its absence in contracting visceral smooth muscle is characteristic of their difference. We have found that in chicken tissues phorbol 12,13-dibutyrate (PDBu) induces a sustained contraction in carotid arterial muscle, but provokes no contraction in phasic gizzard smooth muscle. Next we were aimed to find differences in PDBu-induced phosphorylation of the key proteins involved in regulation of smooth muscle contraction, i.e. caldesmon, myosin light chain kinase (MLCK), and the myosin light chain kinase-related protein (KRP, also known as telokin). Two correlative differences were observed. 1. PDBu stimulated phosphorylation of MLCK in tonic smooth muscle and had no effect on the level of MLCK phosphorylation in phasic muscle. Phosphopeptide mapping suggests the involvement of mitogen-activated protein (MAP) kinases in phosphorylation of MLCK in situ. 2. PDBu induced phosphorylation of MAP-kinase sites in caldesmon in both types of smooth muscle, but this phosphorylation had no significant effect on caldesmon functional activity in vitro. For the first time we have shown that in gizzard PDBu also stimulates a yet unknown transitory caldesmon-kinase different from protein kinase, C, Ca2+/calmodulin-dependent kinase II and casein kinase CK2. 3. No significant difference was found in the kinetics of PDBu-dependent phosphorylation of KRP in tonic and phasic smooth muscles. KRP was also demonstrated to be a major phosphoprotein in smooth muscle phosphorylated in vivo at several sites located within its N-terminal sequence. Protein kinases able to phosphorylate these sites were identified in vitro. Among them, MAP-kinase was suggested to phosphorylate a serine residue homologous to that phosphorylated in MLCK. 4. p42erk2 and p38 MAP-kinases were found in phasic and tonic smooth muscles. Both were responsive to PDBu in cultured chicken aortic smooth muscle cells, and their role in phosphorylation of MLCK and low molecular weight isoform of caldesmon was evaluated.     

Changes in β-adrenoceptors in heart failure due to myocardial infarction are attenuated by blockade of renin–angiotensin system

Earlier studies have revealed an improvement of cardiac function in animals with congestive heart failure (CHF) due to myocardial infarction (MI) by treatment with angiotensin converting enzyme (ACE) inhibitors. Since heart failure is also associated with attenuated responses to catecholamines, we examined the effects of imidapril, an ACE inhibitor, on the -adrenoceptor (-AR) signal transduction in the failing heart. Heart failure in rats was induced by occluding the coronary artery, and 3 weeks later the animals were treated with 1 mg/(kg·day) (orally) imidapril for 4 weeks. The animals were assessed for their left ventricular function and inotropic responses to isoproterenol. Cardiomyocytes and crude membranes were isolated from the non-ischemic viable left ventricle and examined for the intracellular concentration of Ca²⁺ [Ca²⁺]_i and -ARs as well as adenylyl cyclase (AC) activity, respectively. Animals with heart failure exhibited depressions in ventricular function and positive inotropic response to isoproterenol as well as isoproterenol-induced increase in [Ca²⁺]_i in cardiomyocytes; these changes were attenuated by imidapril treatment. Both ₁-AR receptor density and isoproterenol-stimulated AC activity were decreased in the failing heart and these alterations were prevented by imidapril treatment. Alterations in cardiac function, positive inotropic effect of isoproterenol, ₁-AR density and isoproterenol-stimulated AC activity in the failing heart were also attenuated by treatment with another ACE inhibitor, enalapril and an angiotensin II receptor antagonist, losartan. The results indicate that imidapril not only attenuates cardiac dysfunction but also prevents changes in -AR signal transduction in CHF due to MI. These beneficial effects are similar to those of enalapril or losartan and thus appear to be due to blockade of the renin–angiotensin system. (Mol Cell Biochem 263: 11–20, 2004)     

Exercise Protects against Chronic β-Adrenergic Remodeling of the Heart by Activation of Endothelial Nitric Oxide Synthase

Extensive data have shown that exercise training can provide cardio-protection against pathological cardiac hypertrophy. However, how long the heart can retain cardio-protective phenotype after the cessation of exercise is currently unknown. In this study, we investigated the time course of the loss of cardio-protection after cessation of exercise and the signaling molecules that are responsible for the possible sustained protection. Mice were made to run on a treadmill six times a week for 4 weeks and then rested for a period of 0, 1, 2 and 4 weeks followed by isoproterenol injection for 8 days. Morphological, echocardiographic and hemodynamic changes were measured, gene reactivation was determined by real-time PCR, and the expression and phosphorylation status of several cardio-protective signaling molecules were analyzed by Western-blot. HW/BW, HW/TL and LW/BW decreased significantly in exercise training (ER) mice. The less necrosis and lower fetal gene reactivation induced by isoproterenol injection were also found in ER mice. The echocardiographic and hemodynamic changes induced by β-adrenergic overload were also attenuated in ER mice. The protective effects can be sustained for at least 2 weeks after the cessation of the training. Western-blot analysis showed that the alterations in the phosphorylation status of endothelial nitric oxide synthase (eNOS) (increase in serine 1177 and decrease in threonine 495) continued for 2 weeks after the cessation of the training whereas increases of the phosphorylation of Akt and mTOR disappeared. Further study showed that L-NG-Nitroarginine methyl ester (L-NAME) treatment abolished the cardio-protective effects of ER. Our findings demonstrate that stimulation of eNOS in mice through exercise training provides acute and sustained cardioprotection against cardiac hypertrophy.     

β-AR blockers suppresses ER stress in cardiac hypertrophy and heart failure

BACKGROUND: Long-term β-adrenergic receptor (β-AR) blockade reduces mortality in patients with heart failure. Chronic sympathetic hyperactivity in heart failure causes sustained β-AR activation, and this can deplete Ca(2+) in endoplasmic reticulum (ER) leading to ER stress and subsequent apoptosis. We tested the effect of β-AR blockers on ER stress pathway in experimental model of heart failure. METHODS AND DISCUSSIONS: ER chaperones were markedly increased in failing hearts of patients with end-stage heart failure. In Sprague-Dawley rats, cardiac hypertrophy and heart failure was induced by abdominal aortic constriction or isoproterenol subcutaneous injection. Oral β-AR blockers treatment was performed in therapy groups. Cardiac remodeling and left ventricular function were analyzed in rats failing hearts. After 4 or 8 weeks of banding, rats developed cardiac hypertrophy and failure. Cardiac expression of ER chaperones was significantly increased. Similar to the findings above, sustained isoproterenol infusion for 2 weeks induced cardiac hypertrophy and failure with increased ER chaperones and apoptosis in hearts. β-AR blockers treatment markedly attenuated these pathological changes and reduced ER stress and apoptosis in failing hearts. On the other hand, β-AR agonist isoproterenol induced ER stress and apoptosis in cultured cardiomyocytes. β-AR blockers largely prevented ER stress and protected myocytes against apoptosis. And β-AR blockade significantly suppressed the overactivation of CaMKII in isoproterenol-stimulated cardiomyocytes and failing hearts in rats. CONCLUSIONS: Our results demonstrated that ER stress occurred in failing hearts and this could be reversed by β-AR blockade. Alleviation of ER stress may be an important mechanism underlying the therapeutic effect of β-AR blockers on heart failure.     

Hearts in adult offspring of copper-deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies

The long-term effects of low dietary copper (Cu) intake during pregnancy and lactation on cardiac mitochondria in first-generation adult rats was examined. Rat dams were fed diets containing either low (1 mg/kg Cu) or adequate (6 mg/kg Cu) levels of dietary Cu beginning 3 weeks before conception and ending 3 weeks after birth. Cytochrome c oxidase (CCO) activity was 51% lower in isolated cardiac mitochondria from 21-day-old offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. CCO activities in the cardiac mitochondria of 63- and 290-day-old offspring were 22% lower and 14% lower, respectively, in the offspring of Cu-deficient dams after they had been repleted with adequate dietary Cu from the time they were 21 days old. Electron micrographs showed that the size of residual bodies and the cellular volume they occupied in cardiomyocytes rose significantly between 63 and 290 days in the Cu-repleted offspring of Cu-deficient dams, but not in the offspring of Cu-adequate dams. The rate of hydrogen peroxide generation by cardiac mitochondria also was 24% higher in the 290-day-old repleted offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. The increase in hydrogen peroxide production by cardiac mitochondria and in the relative volume and size of dense deposits in cardiomyocytes is consistent with increased oxidative stress and damage resulting from prolonged reduction of CCO activity in the offspring of Cu-deficient dams.     

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (K_m = 200-400 M) is much lower than in isolated mitochondria (K_m = 10-20 M). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.