Eduard I DEDKOV

Abstract: We compared the effects of heart rate reduction (HRR) by the hyperpolarization-activated pacemaker current (I(f)) channel inhibitor ivabradine (MI+Iva) and the beta(1)-blocker atenolol (MI+Aten) on ventricular remodeling and perfusion after myocardial infarction (MI) in middle-aged (12 mo) Sprague-Dawley rats. Mean HRR was virtually identical in the two treated groups (19%). Four weeks after coronary artery ligation, maximal myocardial perfusion fell in the MI group but was preserved in infarcted rats treated with either Iva or Aten. However, coronary reserve in the remodeled hearts was preserved only with Iva, since Aten treatment elevated baseline perfusion in response to a higher wall stress. The higher maximal perfusion noted in the two treated groups was not due to arteriogenesis or angiogenesis. Plasma levels of angiotensin (ANG) II and myocardial ANG type 1 (AT(1)) receptor and transforming growth factor (TGF)-beta1 were reduced during the first week of treatment by both Iva and Aten. Moreover, treatment also reduced arteriolar perivascular collagen density. Despite these similar effects of Iva and Aten on vascularity and ANG II, Iva, but not Aten, attenuated the decline in ejection fraction and lowered left ventricular (LV) end-diastolic volume (LVEDV)-to-LV mass ratio, determined by echocardiography. In conclusion, 1) Iva has advantages over Aten in postinfarction therapy that are not due to differential effects of the drugs on heart rate, and 2) age limits growth factor upregulation, angiogenesis, and arteriogenesis in the postinfarcted heart.

Abstract: Leptofibrils, or leptomeres, remain the least studied cytoskeletal structures in muscle cells, and their function and mechanism of assembly are still poorly understood. Our ultrastructural study of the surviving cardiac myocytes located in the perinecrotic border zone of the infarcted left ventricle in rats revealed intense formation of leptofibrils and leptofibrillar clusters during 4-15 days following experimental myocardial infarction. In the perinecrotic myocytes, leptofibrils developed predominantly in the subsarcolemmal areas, near disassembled intercalated discs and at the sites of intense myofibrillogenesis in the peripheral zones of the sarcoplasm. We found that the development of these structures occurred before or at the time of assembly of myofibrils. In our material, leptofibrils consisted of longitudinally oriented filamentous bundles inserted in electron dense Z-band-like material and periodically crossed by 3-8 bands of this material with the period of cross-striation of 120-210 nm. The presence of leptofibrils in growing cytoplasmic processes and ruffles developing in the border zone in the areas of lost intercellular contacts indicates their formation de novo during post-infarction period. We observed four major morphological types of localization of these structures: (1) direct contact of one end of leptofibrils with Z bands of nascent, mature or disassembling myofibrils; (2) direct contact with the sarcolemma: (a) multifocal attachment of leptofibrils to the sarcolemma through the lateral surfaces of their minute Z band-like structures; (b) attachment of one or both ends of leptofibrils to the sarcolemma without contacts or in contact with myofibrils; (3) attachment of leptofibrils to subsarcolemmal accumulations of electron dense Z-band material in newly formed fasciae adherentes of the remodeled intercalated disks; (4) clustering and contacts of leptofibrils with one another predominantly at the level of their Z bands. Interestingly, most leptofibrils of all four types were topographically associated with the system of T-tubules, the sarcoplasmic reticulum and subsarcolemmal vesicles. Serial sections through the areas containing leptofibrils indicate their spindle-like or nearly cylindrical shape. Thus, we found that leptofibrils assemble in terminally differentiated cardiac myocytes following destabilization of their differentiated state and partial dedifferentiation induced by myocardial infarction. The results of this study demonstrate that formation of leptofibrils, earlier described mainly in the developing and malignant muscle, is temporally associated with adaptive structural remodelling and the activation of myofibrillogenesis in functionally overloaded cardiac myocytes of adult animals. Our findings suggest that re-expression of some structural characteristics of the embryonic muscle appear to represent one of the mechanisms that underlie adaptive plasticity of the myocardium following injury and under conditions of hyperfunction.

Abstract: We tested the hypothesis that chronically reducing the heart rate in infarcted middle-aged rats using ivabradine (IVA) would induce arteriolar growth and attenuate perivascular collagen and, thereby, improve maximal perfusion and coronary reserve in the surviving myocardium. Myocardial infarction (MI) was induced in 12-mo-old male Sprague-Dawley rats, which were then treated with either IVA (10.5 mg.kg(-1).day(-1); MI + IVA) or placebo (MI) via intraperitoneal osmotic pumps for 4 wk. Four weeks of IVA treatment limited the increase in left ventricular end-diastolic pressure and the decrease in ejection fraction but did not affect the size of the infarct, the magnitude of myocyte hypertrophy, or the degree of arteriolar and capillary growth. However, treatment reduced interstitial and periarteriolar collagen in the surviving myocardium of MI + IVA rats. The reduced periarteriolar collagen content was associated with improvement in maximal myocardial perfusion and coronary reserve. Although the rates of proliferation of periarteriolar fibroblasts were similar in the MI and MI + IVA groups, the expression levels of the AT(1) receptor and transforming growth factor (TGF)-beta(1) in the myocardium, as well as the plasma level of the ANG II peptide, were lower in treated rats 14 days after MI. Therefore, our data reveal that improved maximal myocardial perfusion and coronary reserve in MI + IVA rats are most likely the result of reduced periarteriolar collagen rather than enhanced arteriolar growth.

Abstract: Syndecan-4 and its cytoplasmic binding partner, synectin, are known to play a role in FGF-2 signaling and vascular growth. To determine their roles in coronary artery/arteriolar formation and growth, we compared syndecan-4 and synectin null mice with their wild-type counterparts. Image analysis of arterioles visualized by smooth muscle alpha-actin immunostaining revealed that synectin (-/-) mice had lower arteriolar length and volume densities than wild-type mice. As shown by electron microscopic analysis, arterioles from the two did not differ in morphology, including their endothelial cell junctions, and the organization and distribution of smooth muscle. Using micro-computer tomography, we found that the size and branching patterns of coronary arteries (diameters > 50 microm) were similar for the two groups, a finding that indicates that the growth of arteries is not influenced by a loss of synectin. Syndecan-4 null male mice also had lower arteriolar length densities than their gender wild-type controls. However, female syndecan-4 null mice were characterized by higher arteriolar length and volume densities than their gender-matched wild-type controls. Thus, we conclude that both synectin and syndecan-4 play a role in arteriolar development, a finding that is consistent with previous evidence that FGF-2 plays a role in coronary arterial growth. Moreover, our data reveal that gender influences the arteriolar growth response to syndecan-4 but not to synectin.

Abstract: OBJECTIVE: Peripheral blood contains primitive (stem cell-like) and monocytic-like endothelial cell progenitors. Diabetes apparently converts these primitive progenitors, from a pro-angiogenic to anti-angiogenic phenotype. Monocytic progenitors seem to be less affected by diabetes, but potential pro-angiogenic activities of freshly isolated monocytic progenitors remain unexplored. We compared the ability of primitive and monocytic endothelial cell progenitors to stimulate vascular growth and healing in diabetes and investigated potential molecular mechanisms through which the cells mediate their in vivo effects. METHODS AND RESULTS: Human CD34+ primitive progenitors and CD14+ monocytic progenitors were injected locally into the ischemic limbs of diabetic mice. CD14+ cell therapy improved healing and vessel growth, although not as rapidly or effectively as CD34+ cell treatment. Western blot analysis revealed that cell therapy modulated expression of molecules in the VEGF, MCP-1, and angiopoietin pathways. CONCLUSIONS: Injection of freshly isolated circulating CD14+ cells improves healing and vascular growth indicating their potential for use in acute clinical settings. Importantly, CD14+ cells could provide a therapeutic option for people with diabetes, the function of whose CD34+ cells may be compromised. At least some progenitor-induced healing probably is mediated through increased sensitivity to VEGF and increases in MCP-1, and possibly modulation of angiopoietins.

Abstract: Previous studies have not addressed regional differences in adaptive arteriolar growth in the surviving left ventricular (LV) myocardium after infarction in appropriately aged animals, namely middle-aged or older. Accordingly, we examined the adaptive postinfarction growth of arterioles in two distinct regions, i.e., the LV free wall (LVFW) and septum, of middle-aged rats. We induced a myocardial infarction (MI) in 12-mo-old rats to analyze 1) protein expression in VEGF/Flt-1/Flk-1 and angiopoietin (Ang)-1/Ang-2/Tie-2 systems, 2) the arteriolar DNA synthesis, 3) the extent of the arteriolar bed, and 4) the alteration in minimal coronary vascular resistance. In both regions, arteriolar DNA synthesis was activated between days 4 and 7 after MI. Whereas in the LVFW the degree of DNA synthesis declined between days 11 and 14 post-MI, it continued to rise in the septum, and at day 14, the percentage of the arterioles undergoing DNA synthesis was comparable in the LVFW and the septum (9.7 +/- 1.6 and 7 +/- 2.1%, respectively). Arteriolar DNA synthesis was mainly associated with upregulation of Ang-2 and Tie-2 in both LV regions. Although 4 wk after MI the arteriolar beds in the LVFW and the septum expanded to the size of sham-operated rats, this growth did not compensate for the greater minimal coronary vascular resistance in the former. Thus our findings suggest that 1) the dynamics in adaptive arteriolar growth were similar between the two regions, despite a delay in the septum; and 2) the perfusion deficit in post-MI rats cannot be accounted for by inadequate adaptive growth of arterioles.

Abstract: Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

Abstract: Recent studies have provided insights into specific events that contribute to vasculogenesis and angiogenesis in the developing coronary vasculature. This study focused on the developmental progression of coronary vascularization beginning with tube formation and ending with the establishment of a coronary arterial tree. We used electron microscopy, histology of serial sections, and immunohistochemistry in order to provide a comprehensive view of coronary vessel formation during the embryonic and fetal periods of the quail heart, a species that has been used in a number of studies addressing myocardial vascularization. Our data reveal features of progenitor cells and blood islands, tubular formation, and the anatomical relationship of a transformed periarterial tubular network and sympathetic ganglia to the emergence and branching of the right and left coronary arteries. We have traced the pattern of coronary artery branching and documented its innervation. Finally, our data include the relationship of fibronectin, laminin, and apoptosis to coronary artery growth. Our findings bring together morphological events that occur over the embryonic and fetal periods and provide a baseline for studies into the mechanisms that regulate the various events that occur during these time periods.

Abstract: BACKGROUND: Despite an appreciable increase in basal coronary blood flow in cyanotic congenital heart disease, flow reserve remains normal. We hypothesized that preservation of flow reserve resides in remodeling of the coronary microcirculation. Microcirculatory morphometric analyses were performed to test this hypothesis. METHODS AND RESULTS: Necropsy specimens from 4 sources were studied: (1) hearts from patients with Eisenmenger's syndrome (A; n=5), (2) structurally abnormal hearts with ventricular hypertrophy (B; n=8), (3) structurally normal hearts with ventricular hypertrophy (C; n=6), and (4) normal hearts (D; n=5). To compare responses of the microcirculation to hypoxia versus hypertrophy, sections were taken from the left ventricular free wall, which in group A, was hypoxemic but not hypertrophied; in groups B and C, was hypertrophied but not hypoxemic; and in group D, was neither hypertrophied nor hypoxemic. Coronary arterioles were immunolabeled for smooth muscle alpha-actin. Measured morphometric parameters included long and short axes, area, and perimeter. Arteriolar length, volume and surface densities were calculated. There was a significant intergroup difference for arteriolar length density (P=0.03) and diameter (P=0.03). Total length density in group A hearts was markedly lower, but mean arteriolar diameter was significantly greater (34%) compared with group B (P=0.03). Arteriolar volume density was similar to that in the other groups. CONCLUSIONS: Remodeling of the coronary microcirculation is the key mechanism for preservation of flow reserve in cyanotic congenital heart disease. The increase in short axis (diameter) compensated for lower arteriolar length density and was the principal anatomic basis for maintenance of normal flow reserve.

Abstract: Adequate growth of coronary vasculature in the remaining left ventricular (LV) myocardium after myocardial infarction (post-MI) is a crucial factor for myocyte survival and performance. We previously demonstrated that post-MI coronary angiogenesis can be stimulated by bradycardia induced with the ATP-sensitive K(+) channel antagonist alinidine. In this study, we tested the hypothesis that heart rate reduction with beta-blockade may also induce coronary growth in the post-MI heart. Transmural MI was induced in 12-mo-old male Sprague-Dawley rats by occlusion of the left anterior descending coronary artery. Bradycardia was induced by administration of the beta-adrenoceptor blocker atenolol (AT) via drinking water (30 mg/day). Three groups of rats were compared: 1) control/sham (C/SH), 2) MI, and 3) MI + AT. In the MI + AT rats, heart rate was consistently reduced by 25-28% compared with C/SH rats. At 4 wk after left anterior descending coronary ligation, infarct size was similar in MI and MI + AT rats (67.1 and 61.5%, respectively), whereas a greater ventricular hypertrophy occurred in bradycardic rats, as indicated by a higher ventricular weight-to-body weight ratio (3.4 +/- 0.1 vs. 2.8 +/- 0.1 mg/g in MI rats). Analysis of LV function revealed a smaller drop in ejection fraction in the MI + AT than in the MI group ( approximately 24 vs. approximately 35%). Furthermore, in MI + AT rats, maximal coronary conductance and coronary perfusion reserve were significantly improved compared with the MI group. The better myocardial perfusion indexes in MI + AT rats were associated with a greater increase in arteriolar length density than in the MI group. Thus chronic reduction of heart rate induced with beta-selective blockade promotes growth of coronary arterioles and, thereby, facilitates regional myocardial perfusion in post-MI hearts.

Abstract: Little is known about the biological properties of myogenic satellite cells during post-denervation muscle atrophy. The present study investigated the differentiative capacity of satellite cells and their involvement in the compensatory regenerative process in long-term denervated rat muscle. Electron microscopy and immunocytochemical labeling of muscle tissue 1-18 months following denervation demonstrated that despite activation of satellite cells, myogenesis in denervated muscle is abortive and does not lead to the formation of normal muscle fibers. Small sizes, poor development of the contractile system in newly formed denervated myotubes, and the absence of satellite cells on the surface indicate that their differentiation typically does not progress to terminal stages. Many immature myotubes degenerate, and others survive but are embedded in a collagen lattice near their parent fibers. Interestingly, newly formed myotubes located on the surface of parent muscle fibers beneath the basal lamina typically did not contain developed myofibrils. This suggests that the contacts of daughter and parent muscle fibers block myofibrillogenesis. Assembly of sarcomeres in most cases occurs following complete spatial separation of daughter and parent muscle fibers. Another manifestation of the involvement of myogenic precursors in abortive myogenesis is the formation of clusters of underdeveloped branching myotubes surrounded by a common basal lamina. We found that myoblasts can also fuse directly with differentiated muscle fibers. The presence of satellite cells near the openings in the basal lamina and in the interstitial space indicates that myogenic precursors can migrate through the basal lamina and form myotubes at a distance from parent fibers. Our data may explain why long-term denervated skeletal muscle has a poor capacity for regeneration and functional restoration.

Abstract: Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.

Abstract: We analyzed the level of protein expression of two myogenic regulatory factors (MRFs), MyoD and myogenin, in senile skeletal muscles and determined the cellular source of their production in young adult (4 months old), old (24, 26, and 28 months old), and senile (32 months old) male rats. Immunoblotting demonstrated levels of myogenin approximately 3.2, approximately 4.0, and approximately 5.5 times higher in gastrocnemius muscles of 24-, 26-, and 32-month-old animals, respectively, than in those of young adult rats. Anti-MyoD antibody recognized two major areas of immunoreactivity in Western blots: a single MyoD-specific band (approximately 43-45 kDa) and a double (or triple) MyoD-like band (approximately 55-65 kDa). Whereas the level of MyoD-specific protein in the 43- to 45-kDa band remained relatively unchanged during aging compared with that of young adult rats, the total level of MyoD-like immunoreactivity within the 55- to 65-kDa bands was approximately 3.4, approximately 4.7, approximately 9.1, and approximately 11.7 times higher in muscles of 24-, 26-, 28-, and 32-month-old rats, respectively. The pattern of MRF protein expression in intact senile muscles was similar to that recorded in young adult denervated muscles. Ultrastructural analysis of extensor digitorum longus muscle from senile rats showed that, occasionally, the area of the nerve-muscle junction was partially or completely devoid of axons, and satellite cells with the features of activated cells were found on the surface of living fibers. Immunohistochemistry detected accumulated MyoD and myogenin proteins in the nuclei of both fibers and satellite cells in 32-month-old muscles. We suggest that the up-regulated production of MyoD and myogenin proteins in the nuclei of both fibers and satellite cells could account for the high level of MRF expression in muscles of senile rats.

Abstract: PURPOSE: This study was designed to determine whether the quantitative relationship between the levels of the eEF1A-1(developmental) and eEF1A-2/S1 (adult) isoforms of peptide elongation factor remains stable after denervation of skeletal muscle or whether in response to denervation the relative amount of the developmental form would increase. In normal postnatal rat muscle, eEF1A-2/S1 is the dominant form represented, and levels of eEF1A-1 are extremely low. METHODS: One hind limb in young adult rats was permanently denervated. Denervated and corresponding contralateral control muscles were removed for biochemical and morphological analysis from 2 days to 25 months after denervation. RESULTS: By one month after denervation, relative levels of eEF1A-1 rose dramatically in relation to those of eEF1A-2/S1, and they remained high throughout the remainder of the 25-month denervation period. Ultrastructural analysis showed a complex mix of muscle fiber atrophy, dying muscle nuclei and muscle fibers, and newly forming muscle fibers in the same tissue. CONCLUSIONS: As during muscle regeneration, levels of the developmental eEF1A-1 isoform of peptide elongation factor greatly increased relative to those of the adult eEF1A-2/S1 adult isoform following denervation in rat muscles. However, in contrast to regeneration, the elevated level of eEF1A-1 did not return to the basal minimal level. Since switching from eEF1A-1 to eEF1A-2/S1 is an indicator that terminal differentiated is completed, the failure of eEF1A-1 to return to basal level may be indicative of the persistence of an unstable tissue environment that includes muscle fiber atrophy, degeneration and neomyogenesis. The specific cellular basis for the increase in eEF1A-1 could not be determined from this study.

Abstract: We investigated the dynamics of muscle fiber atrophy in denervated fast and slow muscles of young and old rats. Hind limbs of 4-month-old and 24-month-old male rats were denervated, and soleus and tibialis anterior muscles were examined morphometrically 1 and 2 months after denervation. In all denervated muscles, type II muscle fibers underwent rapid atrophy, although muscle-specific differences in rate were observed. In both young and old denervated soleus muscles, the type I fibers underwent a pattern of atrophy closely paralleling that of the type II fibers, but in the tibialis anterior muscle, the mean cross-sectional area of the type I fibers actually increased during the first 2 months postdenervation. This study has shown that, among different muscles and between young and old rats, there is considerable variation in the response of the muscle fibers to denervation and that one cannot generalize from one muscle or one age to another.

Abstract: Satellite cells (SCs) are the main source of new fibers in regenerating skeletal muscles and the key contributor to extra nuclei in growing fibers during postnatal development. Aging results in depletion of the SC population and in the reduction of its proliferative activity. Although it has been previously determined that under conditions of massive fiber death in vivo the regenerative potential of SCs is not impaired in old muscle, no studies have yet tested whether advanced age is a factor that may restrain the response of SCs to muscle denervation. The present study is designed to answer this question, comparing the changes of SC numbers in tibialis anterior (TA) muscles from young (4 months) and old (24 months) WI/HicksCar rats after 2 months of denervation. Immunostaining with antibodies against M-cadherin and NCAM was used to detect and count the SCs. The results demonstrate that the percentages of both M-cadherin- and NCAM-positive SCs (SC/Fibers x 100) in control TA muscles from young rats (5.6 +/- 0.5% and 1.4 +/- 0.2%, respectively) are larger than those in old rats (2.3 +/- 0.3% and 0.5 +/- 0.1%, respectively). At the same time, in 2-month denervated TA muscles the percentages of M-cadherin and NCAM positive SC are increased and reach a level that is comparable between young (16.2 +/- 0.9% and 7.5 +/- 0.5%, respectively) and old (15.9 +/- 0.7% and 10.1 +/- 0.5%, respectively) rats. Based on these data, we suggest that aging does not repress the capacity of SC to become activated and grow in the response to muscle denervation.

Abstract: Caveolin-3, a muscle-specific member of the caveolin family, is strongly localized to the neuromuscular junction (NMJ) in adult rat muscle fibers, where it co-localizes with alpha-bungarotoxin staining. In 24-month-old rats, less distinct staining corresponds with the normal aging changes in the NMJ. After denervation, the pattern and intensity of staining begin to break up as early as 3 days, and by 10 days little staining remains. The functional implications of this concentration of caveolin-3 at the NMJ remain obscure, but it is possible that its absence could account for some of the phenotypic characteristics of individuals with caveolin-3 mutations.

Abstract: In denervated skeletal muscles, atrophy of muscle fibers and interstitial fibrosis are associated with alterations within the vascular bed. Our study has placed particular emphasis on changes occurring in resistance vessels and the microcirculatory bed of rat hindlimb skeletal muscles that had been denervated for 25 months. We found that the tunica media of the majority of long-term denervated resistance vessels undergoes deterioration. In small intramuscular arteries and arterioles, atrophic vascular smooth muscle cells (vSMCs) enclosed in a thick basal lamina are separated by expanded extracellular space. The remodeling and sclerotic changes in the arterial wall occasionally result in deformation of the lumen. It was also found that the microcirculatory bed undergoes significant alterations. In 25-month denervated extensor digitorum longus muscle, the capillary-to-fiber ratio is only 0.13 +/- 0.01 and the mean number of capillaries per fascicle decreases almost ninefold compared to contralateral control muscle. Ultrastructural findings demonstrate that 24.67 +/- 0.48% of capillaries examined in the chronically denervated fascicles show structural features typical for capillary regeneration. In addition, long cytoplasmic extensions of pericytes might develop a layer completely encircling the capillary endothelium. In pre- and postcapillary segments of the microcirculatory bed, some perivascular cells possess a phenotype that is intermediate between that of pericytes and atrophic vSMCs. RT-PCR and/or Western blot analyses showed that molecules participating in angiogenesis are detected in 25-month denervated skeletal muscle. We hypothesize that despite the fact that the microcirculatory bed of chronically denervated muscle undergoes significant reduction it still sustains the capacity for reparative capillary growth.

Abstract: Mammalian skeletal muscle undergoes profound atrophy after denervation. The functional restoration of denervated muscle is a significant clinical problem, and the success of restorative attempts decreases substantially after several months of denervation. Rat extensor digitorum longus muscles are capable of excellent restoration for the first 2-3 months after denervation, but after that time the level of restoration upon reinnervation decreases dramatically. Severe atrophy precedes the loss of restorative capacity. Attempts to understand the basis for the reduced restorative ability have led to an intensive analysis of the biology of long-term denervated muscle. In fast muscles, the satellite cell population undergoes a major increase over the first 2 months after denervation, and thereafter it steadily declines. Atrophying muscle fibers lose nuclei through apoptosis, and some degenerate. New muscle fibers form either alongside atrophying muscle fibers or in place of degenerated ones. The microcirculation undergoes a tenfold diminution over the first year after denervation, and over time denervated muscle is characterized by increasing amounts of interstitial collagen. Various barriers to reinnervation are discussed. Attempts to improve the restoration of long-term denervated muscle have included the stimulation of regeneration and removal of interstitial collagen. Both of these have resulted in significant improvement in the level of functional restoration. Although chronic electrical stimulation maintains an excellent degree of mass and force in a denervated muscle, grafts of such muscles undergo no better restoration than grafts of denervated muscles.

Abstract: It is well established that over time Schwann cells disappear from the endoneurial space of the distal stump of a chronically transected sciatic nerve trunk. Nevertheless, the status of the Schwann cells within terminal branches of the transected sciatic nerve remains poorly understood. To elucidate this issue we examined the endoneurial space of the intramuscular nerves in rat hindlimb skeletal muscles, which had been denervated for a 25-month period. Based on specific ultrastructural characteristics, we identified a small population of viable Schwann cells within the intramuscular nerve trunks. The surviving Schwann cells continued to be immunopositive for both S-100 protein and neural cell adhesion molecule. In addition, reverse transcription-polymerase chain reaction and/or Western blot analyses have shown that at least two molecules, brain-derived neurotrophic factor and a non-catalytic truncated form of tyrosine protein kinase receptor B, which could potentially participate in the process of nerve repair, were detectable in chronically denervated skeletal muscle. Our results demonstrate that Schwann cells can survive inside the intramuscular nerve trunks of denervated skeletal muscle for a 25-month period without axonal contact.

Abstract: We compared the reactions to denervation of limb muscles between young adult and old rats. After denervation for up to 4 months in 24-month-old rats, limb muscles were removed and analyzed for contractile properties, morphology, and levels of several key molecules, including the peptide elongation factors eEF1A-1 and eEF1A-2/S1, myogenin, gamma-subunit of the acetylcholine receptor, and cyclin D3. The principal difference between denervated old and young muscle is a somewhat slower rate of atrophy in denervated older muscle, especially among the type II fibers. Expression levels of certain molecules were higher in old than in young control muscle, but after denervation, levels of these molecules increased to the same absolute values in both young and old rats. Although many aspects of postdenervation reactions do not differ greatly between young and old animals, the lesser degree of atrophy in the old rats may reflect significant age-based mechanisms.

Abstract: This study was undertaken to assess the regenerative capacity of skeletal muscle in rats near the end of their normal life span. Two experiments were performed. In the first, extensor digitorum longus (EDL) muscles were cross-age transplanted from 32-month-old male inbred Wistar (WI/HicksCar) rats in place of an EDL muscle in 4-month-old hosts. The other EDL muscle in the hosts was autotransplanted. After 60 days, the old-into-young muscle transplants regenerated as well as the young-into-young autotransplants. In the second experiment, EDL muscles in young adult (4 months) and old rats (32 and 34 months) of WI/HicksCar and Brown Norway (BN) were injected with a local anesthetic, bupivacaine, and allowed to regenerate for 41 days. In all cases, the masses and absolute maximum tetanic force of the regenerates equaled or exceeded those of untouched contralateral control muscles. These experiments showed that under appropriate conditions, very old muscles can regenerate to equal or exceed the contralateral control values, which in old rats are much less than those in muscles of young rats.

Abstract: Little is known concerning the time-course and structural dynamics of reactivation of compensatory myogenesis in denervated muscle, its initiating cellular mechanisms, and the relationship between this process and the progression of postdenervation atrophy. The purpose of this study was to investigate the interrelations between temporal and spatial patterns of the myogenic response in denervated muscle and progressive atrophy of muscle fibers. Another objective was to study whether reactivation of myogenesis correlates with destabilization of the differentiated state and death of denervated muscle cells. It has remained unclear whether muscle fiber atrophy was the primary factor activating the myogenic response, what levels of cellular atrophy were associated with its activation, and whether the initiation and intensity of myogenesis depended on the local and individual heterogeneity of atrophic changes among fibers. For this reason, our objective was also to identify the levels of atrophic and degenerative changes in denervated muscle fibers that are correlated with activation of the myogenic response. We found that the reactivation of myogenesis in the tibialis anterior and extensor digitorum longus muscles of the rat starts between days 10-21 following nerve transection, before atrophy has attained advanced level, long before dead cells are found in the tissue. Formation of new muscle fibers reaches its maximum between 2 and 4 months following denervation and gradually decreases with progressive postdenervation atrophy. The myogenic response is biphasic and includes two distinct processes. The first process resembles the formation of secondary and tertiary generations of myotubes during normal muscle development and dominates during the first 2 months of denervation. During this period, activated satellite cells form new myotubes on live differentiated muscle fibers. Most of the daughter myotubes in 1- and 2-month denervated muscle develop on the surface of fast type parent muscle fibers, and some of the newly formed muscle fibers express slow myosin. Some fast type parent fibers are weakly or, more rarely, moderately immunopositive for embryonic isomyosin. This indicates that reactivation of myogenesis may also depend on the fiber type. The level of atrophy, destabilization of the differentiated myofiber phenotype, and degenerative changes of individual fibers in denervated muscle are very heterogeneous. The myogenic response of the first type is associated predominantly with fibers of average and higher than average levels of atrophy. Muscle cells that undergo a lesser degree of atrophy also form daughter fibers, although with a lower incidence. We did not find any correlation between the size of newly formed fibers and the level of atrophy of parent fibers. The topographical distribution of new myotubes both in the peripheral and central areas of the mid-belly equatorial sections at the early stages following nerve transection indicates that myogenesis of the first type represents a systemic reaction of muscle to the loss of neural control. These data indicate that activation of the myogenic response does not depend on cell death and degenerative processes per se. The second type of myogenesis is a typical regenerative reaction that occurs mainly within the spaces surrounded by the basal laminae of dead muscle fibers. Myocytes of different sizes are susceptible to degeneration and death, which indicates that cell death in denervated muscle does not correlate with levels of muscle cell atrophy. The regenerative process frequently results in development of abnormal muscle cells that branch or form small clusters. Replacement of lost fibers becomes activated between 2 and 4 months following nerve transection, i.e., mainly at advanced stages of postdenervation atrophy, when cell death becomes a contributing factor of the atrophic process. In long-term denervated muscle, the first and second types of myogenesisoccur concurrently, and the topographical distribution of the myogenic response becomes more heterogeneous than during the first weeks following denervation. Thus, our data demonstrate differential temporal and spatial expression of two patterns of myogenesis in denervated muscle that appear to be controlled by different regulatory mechanisms during the postdenervation period. (c) 2001 Wiley-Liss, Inc.

Abstract: This study, conducted on 25-month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long-term denervated muscles. Despite a approximately 97.6% loss in mean cross-sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing approximately 1.5 times more fibers than age-matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end-plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural-cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long-term denervated muscles, as compared with age-matched control muscles. In 25-month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long-term denervated muscle.

Abstract: Morpho-functional changes of left ventricle myocardium were studied in 30 adult male rats by the method of subcutaneous implantation of diffusion chambers. The transplants were examined in 1, 3, 6, 8 and 10 days after the beginning of cultivation. The method of light microscopy at the 1-2 microm sections was used after histological processing of specimens for the identification of muscle and non-muscle cells and studying of their particular changes. In the muscular fibres there were cardiomyocytes of the destructive, survived and reconstructed forms. It was shown that smooth muscle cells and periocytes are separated from blood microvessels and migrated into the interstitial space. Multinuclear drawing rods formated from activational endotheliocytes were detected between the muscular fibers. Furthermore the cells containing large amount of lipid granules-¿lypophags¿ and the two types of macrophage-like cells were demonstrated. The area of growth including fibroblast-like and spindle-shaped cells were observed around the margins of transplants on the late stages of implantation.

Abstract: 60 implantes of the left atrium myocardium were studied on the 1st, 3rd and 6th days of in vivo culture according to F. M. Lazarenko method (outbred laboratory male rats of 200-250g. body weight were used both as donors and recipients). Reactive changes of the cultured cardiomyocytes were assessed by methods of light and electron microscopy. Polymorphic nature of the cardiomyocyte reactive changes with the preservation of its tissue pecific determination was established. Myocardium implant did not form myosymplastic elements, cellular characteristics of organization remained in its implanted pieces. Data concerning degenerating and surviving cardiomyocytes are presented as well as those on cellular forms, identification of which is difficult on the ultrastructural level.

Abstract: The field of angiogenesis continues to rapidly evolve in many different directions. Initial discoveries of a few angiogenic growth factors, such as vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF) and angiopoietins, gave an impression of a relatively uncomplicated system with a few straightforward regulatory mechanisms revolving around hypoxia and inflammation. However, failures of therapeutic approaches based on brute force stimulation or inhibition of vessel growth combined with discoveries in fields as diverse as developmental biology, signal transduction and neurosciences began painting a much more nuanced and complex system. The existence of a great number of checks and balances involved in growth and maintenance of the vasculature, a great diversity of angiogenic growth factors and inhibitors, and in particular the concepts of vascular guidance and participation of numerous proteins capable of modifying growth factor activities were some of the more prominent discoveries of the last decade. It would be utterly impossible to summarize all these developments in one volume. Therefore, we have chosen to focus on a few select developments in the field of therapeutic angiogenesis that have significantly altered our understanding of vascular biology and pathology. The first part of the book deals with key components of the angiogenic cascade. Semaphorins, plexins and neuropilins have emerged as important regulators of vascular growth by both providing guidance clues and transmitting signaling to the endothelium. The diversity of these families, their structure and interactions, as well as our current understanding of their roles are discussed in Chapter 1 by G. Neufeld and colleagues. Ephrins and their Eph receptors play an important and still poorly understood bidirectional signaling function in various cell-cell interactions that regulates processes as diverse as vascular sprouting, blood and lymphatic vessel morphogenesis and remodeling as well as arterial-venous fate decisions. These and other roles played by numerous members of this family are addressed in Chapter 2 by E. Pasquale. FGF were the first angiogenic growth factors isolated, yet their function in the vasculature still remains mysterious and poorly appreciated. Newinsights intoFGFbiology are presented in Chapter 3 by P.Auguste and A. Bikfalvi. The discovery of various ways in which the nervous system participates in regulation of angiogenesis has been one of the most intriguing recent developments in vascular biology. In Chapter 4, J. Kitlinska and Z. Zukowska address the role of neuropeptideY(NPY) in regulation of blood vessel growth. The extracellular matrix (ECM) plays a critical role in modulating signaling of various growth factors. One relatively little studied component of the ECM is the heparan sulfate matrix that affects signaling of various heparin-binding growth factors. Recent advances in this field are addressed in Chapter 5 by N. Shworak. The second part of the book deals with various processes that affect the angiogenic cascade. The concept of arterial guidance is addressed in Chapter 6 by A. Horowitz. The chapter addresses various guidance systems starting with semaphorins, neuropilins and plexins, and progressing to ephrins/Eph receptors, and then to netrins. New development in our understanding of the role of HIF signaling are discussed in Chapter 7 by G. Semenza. The reactive oxygen species have long been studied in terms of their contribution to vascular wall injury. But new discoveries in this field demonstrate an important role of ROS in regulation of various aspects of endothelial signaling. These and other new ideas about ROS function are examined in Chapter 8 by M. Ushio-Fukai and W. Alexander. The growth of new vessels may affect normal organ function and, conversely, organ growth per se can apparently induce an angiogenic program in the absence of hypoxia by using mechanical stretch as a stimulus. These new insights are discussed in the context of myocardial hypertrophy by R. Tomanek and E. Dedkov. The growth of new vessels may affect microvascular milieu in a number of ways, including changes in microvasculature response to various agonists and antagonists. These microvascular physiological aspects of angiogenesis are addressed in Chapter 10 by F. Sellke and colleagues. The third part of the book deals with new insights into therapeutic applications of discoveries in the field of angiogenic biology. Perhaps one of the most anticipated applications has been the development of various tyrosine kinase inhibitors. This side of therapeutic antiangiogenesis is discussed in Chapter 11 by K.-H. Thierauch. To date, application of angiogenic therapies to cardiovascular diseases have not fulfilled lofty expectations of the past decade. The reasons for this lack of progress and potential paths forward are addressed in Chapter 12 by M. Murakami and M. Simons. Perhaps some of the more exciting recent therapeutic applications in the angiogenic growth factor field have involved the hepatocyte growth factor (HGF). HGF biology and biological role as well as its therapeutic applications are discussed in Chapter 13 by R. Morishita and T. Ogihara. Nitric oxide, a recent Science magazine molecule of the year, has always been at the center of endothelial biology. New advances in the NO field and their potential therapeutic applications are discussed in Chapter 14 by G. Rubanyi. The composition of amulti-author monograph is never a simple process and many thanks are due. First, we would like to thank all the contributors for a thorough review of their respective areas. The editors at the Imperial College Press and especially Joy Quek have been highly professional and patient in dealing with us and helping us to put together the best book possible. Finally, we would like to thank our colleagues for their support and advice in this project. M. Simons G. M. Rubanyi (May 2007)

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Abstract: This study tested the hypothesis that selective blockade of neuropeptide Y (NPY) receptors Y1 and/or Y2 will affect the embryonic formation of coronary vessels induced by VEGF using an in vitro heart explant model. The ventricular apices from the hearts of C57BL/6 mouse embryos (E13) were placed on collagen I gel and cultured for 96 hrs in medium with 10% FBS. For the next 72 hrs the explants were cultured in medium with 1% FBS and treated with the Y receptor selective antagonists (10 nmol/µL) in the presence of NPY (1 nmol/µL) and/or VEGF (50 ng/mL). The explants were stained with Alexa Fluor 594-labeled GS-I Isolectine B4 and the total length of endothelial outgrowths (TLO) was quantified using Image-Pro Plus software. In each explant TLO was normalized by the explant perimeter and finally TLO of experimental explants was expressed as a percentage of TLO in control (VEGF-treated) explants. The VEGF-induced formation of endothelial outgrowths was not modified by stimulation of either Y receptor. However, selective blockade of Y2 receptors, but not of Y1 receptors, showed significant reduction in formation of VEGF-induced cord-like endothelial structures by ~68% (P<0.001). In addition, Y2 receptor blockade seemed to decrease the number of endothelial cells within the area surrounding the explants. Our data demonstrates that the NPY/Y2 signaling system plays a modulatory role in VEGF-induced coronary vessel formation.

Abstract: The current study investigates the expression patterns of the NPY-Y1 receptor (R)/Y2R system and VIP-VPAC1R/VPAC2R system in the developing coronary vessels of the quail embryonic heart. The hearts were collected from embryonic day (ED) 8 to ED16, fixed in 4% PFA/PBS and processed for cryosectioning. The serial histological cross-sections of the hearts were immunostained with rabbit polyclonal antibodies against NPY, Y1R, Y2R, VIP, VPAC1R, and VPAC2R. Peroxidase-conjugated secondary antibodies and DAB substrate were used for visualization. We found that between ED9 and ED16, the endothelial cells (ECs) of main coronary arteries and veins were positively stained for NPY, Y1R, Y2R, VIP, and VPAC2R, while only VIP/VPAC2R-positive ECs were detected on ED8. At the same time, the smooth muscle cells of the arteries were always VPAC2R-positive, whereas they revealed a unique temporary sequence of NPY receptor expression: Y2R from ED9 to ED12 and Y1R from ED12 to ED16. The coronary vessels remained VPAC1R-immunonegative during all embryonic stages. Furthermore, in contrast to the later developmental phases, the cells forming the early coronary vessels showed a great degree of variability in the level of protein expression. Our findings demonstrate that NPY/Y and VIP/VPAC receptor systems exist within the coronary vasculature and may play a vital regulatory role during development of vessels in the embryonic heart.

Abstract: Increasing evidence indicates that sex can influence left ventricular (LV) remodeling after myocardial infarction (MI). The current study addressed coronary vasculature adaptations during post-infarction LV remodeling in 12-month-old female rats (MI-F) and compared the data with previous findings on age-matched males (MI-M). MI was induced by left coronary artery ligation and sham-operated rats served as a control. For a final analysis, 4 weeks after MI, we selected the MI-F rats with the infarct sizes similar to that in MI-M rats (67.2% vs. 63.8% of the LV free wall [LVFW], respectively). We found that in the LVFW of MI-F rats the compensatory angiogenesis, i.e. 22.1% (p<0.05) increase in capillary to myocyte ratio, had exclusively occurred in endomyocardium where it associated with the 58.7% (p<0.01) enlargement of myocyte cross-sectional area. In contrast, MI-M rats have demonstrated significant compensatory angiogenesis associated with myocyte hypertrophy only in epimyocardium. The intraventricular septum remained unaffected in both groups of MI rats. Although, post-MI remodeling of LVFW led to a significant increase in arteriolar length density in both MI-F and MI-M rats (27.6% vs. 22.4%, respectively [p<0.05]), only MI-M rats demonstrated the notable increase (28.1%, p<0.05) in arteriolar growth in the intraventricular septum. Our data provided the first evidence documenting the existence of sex-related differences in regional adaptation of the coronary vasculature during 4 weeks of post-MI remodeling. Supported by NIH RO1-HL62587.

Abstract: Background: Cardiac remodeling after MI is associated with an increased incidence of heart failure. A number of previous animal studies have established that sex influences the severity of LV remodeling, suggesting a cardioprotective role of female sex hormones. Because most of these studies employed young or youngadult animals the data obtained may not be relevant to humans in which MI occurs primarily in middle-aged and senescent individuals. Accordingly, we designed our study to determine whether the post-MI middle-aged male and female rats demonstrate the sex-specific differences in LV remodeling. Methods: A large MI (at least 50% of the LV free wall) was induced in 12-month-old male (M-MI) and female (F-MI) Sprague-Dawley rats by LCA ligation. Sham-operated rats served as a control. Four weeks after MI, the various LV parameters, including maximum coronary conductance (blood flow/pressure), were assessed. Results: We found that while the LV of F-MI rats had significantly lower weight compared to age-matched M-MI rats (830.5 ± 13.3 vs. 1074.2 ± 52.2 mg, p < 0.01), the diastolic diameter and the regional wall thickness were similar between females and males, indicating a better LV wall compliance in F-MI rats. This was also supported by the absence of cardiac myocyte hypertrophy in the epimyocardium of F-MI rats (221.2 ± 18.6 vs. 360.5 ± 28.5 μm2, p < 0.01) and a lower content of interstitial collagen compared to M-MI rats (9.7 ± 0.7 vs. 13.9 ± 0.4 % in LV free wall and 9.5 ± 0.6 vs. 14.5 ± 0.9 % in intraventricular septum, p < 0.001). In addition, we found that although the F-MI rats had a significantly smaller arteriolar volume density (0.63 ± 0.06 vs. 1.54 ± 0.15 % in LV free wall and 0.61 ± 0.13 vs. 1.38 ± 0.11 % in intraventricular septum, p < 0.001) the level of maximum myocardial perfusion in both LV regions markedly exceeded (p < 0.01) those of M-MI rats, indicating a greater vasodilatory ability in females. Conclusions: Our data demonstrate that while there are no evident sex-related differences in global LV remodeling in middle-aged post-MI rats a lesser degree of regional cardiac myocyte hypertrophy and interstitial fibrosis together with better myocardial perfusion may help to attenuate a progression to heart failure in female rats.

Abstract: We have previously demonstrated that VIP receptors are morphogenic modulators of embryonic coronary vessel tube formation in vitro. This study tested the hypothesis that a selective blockade of VIP receptors will affect formation and development of the coronary vessels in vivo. At embryonic day (ED) 5, 6, 7, and 8, a selective VIP receptor antagonist, VIP (6-28), was injected in ovo into the vitelline vein of the Japanese quail embryos at a final concentration of 10 µmol/L. Six embryos per each ED were sacrificed 4 days after the injection and the base of their hearts was fixed in 4% paraformaldehyde/PBS, and processed for frozen or paraffin-embedded histological cross-sections. The serial sections through the origin of the coronary vessels were studied by H&E staining or by immunostaining with the antibodies against alpha-smooth muscle actin and quail endothelium (QH1). We found that the blockade of VIP receptors did not alter the formation of left and right coronary arteries in all experimental embryos. However, when the injection was done on ED8 (the time when multiple endothelial channels penetrate the aortic wall in all three aortic sinuses), an additional coronary artery originating from a non-coronary sinus had been detected on ED12. Furthermore, the embryos from this experimental group showed a delay in the maturation of the tunica media of coronary arteries. Our findings demonstrate that VIP receptors are involved in morphogenic modulation of coronary artery development in the embryonic quail heart.

Abstract: Background: We have previously demonstrated that long-term heart rate reduction (HRR) with ivabradine, a selective cardiac pacemaker I f current inhibitor, prevents myocardial fibrosis in post-infarcted middle-aged rats via limitation of the renin-angiotensin system (RAS). However, the effect of the more commonly used HRR drug atenolol on fibrosis and activity of the RAS in post-MI middle-aged rats remains to be clarified. Accordingly, we tested the hypothesis that atenolol-induced long-term HRR in middle-aged post-MI rats can attenuate the RAS and prevent myocardial fibrosis. Methods: MI was induced in 12-month-old male Sprague-Dawley rats by left coronary artery ligation. Atenolol was administered to post-MI rats in drinking water (0.6 mg/ml) for 4 weeks (MI+AT). Sham-operated rats (Shams) served as an age-matched control. Blood and tissue samples were obtained at 7 and 14 days after MI. Four weeks after MI, the various LV parameters were assessed. Results: During 4 weeks of treatment, heart rate in MI+AT rats was consistently reduced by ~19% compared to Shams (p< 0.001). Atenolol treatment did not affect infarct size, compensatory myocardial hypertrophy and post-MI enlargement of the left ventricle (LV). However, 4 weeks post-MI, the interstitial collagen content was significantly lower in the remote area of LV myocardium (intraventricular septum) in MI+AT rats compared to MI rats (5.3+/-0.3% vs. 9.2+/-1.8%, p < 0.001). The lower value of interstitial collagen in LV myocardium of the atenolol-treated group was associated with a markedly reduced plasma level of angiotensin II (pg/mL) 1 week post coronary artery ligation compared to MI rats (9.8 +/-1.0 vs. 35.3 +/-6.0, p < 0.01). At the same time, atenolol treatment had also prevented the increase in myocardial expression of angiotensin II type 1 receptor and TGFbeta1 proteins characteristic of non-treated MI rats. Conclusions: Our data demonstrate that long-term HRR induced in post-MI middle-aged rats by atenolol limits the activity of the RAS and attenuates myocardial fibrosis during post-infarction remodeling. We believe that the post-MI antifibrotic effect involving inhibition of elements of the circulating and local RAS may be an integral part of the regulatory response initiated uniformly by long-term HRR.

Abstract: We tested the hypothesis that VIP receptors, VPAC1 and VPAC2, are involved in morphogenic modulation of embryonic coronary vessel tube formation. Fertile Japanese quail eggs were used. At the developmental stage HH29, the hearts were excised and the ventricular apices were placed on collagen I gels in a 10% FBS medium. After 2 days, the explants were treated with VIP receptor selective agonists (1 nmol/ll) and/or antagonists (10 nmol/ll) for 48 h in the presence or absence of FGF2 (50 ng/ml). At the end of experiments, the explants were immunolabeled with the quail-specific anti-endotheliumantibody (QH1), and the total length ofQH1-positive tubule outgrowth (TLT) was quantified using ImagePro Plus software. In each explant, TLT was normalized by the explant perimeter and, finally, TLT in treated explantswas expressed as a percent fromTLT in controls (untreated explants). We found that the concurrent stimulation of VPAC1 and VPAC2 did not affect tube formation, while the simultaneous inhibition of VIP receptors suppressed tubulogenesis by 32% (P < 0.05 vs. control). Neither selective stimulation nor selective blockade of VPAC1 affected the tube formation. In contrast, selective stimulation of VPAC2 increased tubulogenesis by 34% (P < 0.01 vs. control). The treatment of the explants with FGF2 induced a significant formation of tubules, but when the stimulatory effect of FGF2 was combined with the blockade of VIP receptors the tubulogenesis was enhanced by 70% (P < 0.05 vs.FGF2 only).Our datademonstrate thatVIPreceptors are involved in morphogenic modulation of embryonic coronary vessel tube formation and,moreover, that their function is related to the tubulogenic effect of FGF2.

Abstract: The inadequate growth of coronary resistance vessels (arterioles) during compensatory hypertrophy has been commonly considered the prime cause of impaired maximal perfusion in the LV myocardium after MI. However, our previous studies have shown that maximal coronary conductance (flow/pressure) was also reduced in the remote region of post-MI hearts that did not undergo compensatory hypertrophy. Accordingly, we investigated the adaptive arteriolar growth in the septum of 12-mo-old Sprague-Dawley rats during 4 wks of post-MI remodeling. We found that between day 7 and 14 after MI arterioles demonstrated a significant increase in DNA synthesis, mainly in vascular smooth muscle cells (VSMC), which was associated with up-regulation of Ang-2 and Tie-2 protein expression. Although VSMC proliferation occurred throughout the entire arteriolar tree it was higher in larger arterioles (25–54 µm in diameter). As a result, 4 wks after MI we detected a significant increase in arteriolar length density in this segment of the arteriolar tree as compared to sham rats (P< 0.05). Mean arteriolar diameters and periarteriolar collagen content were similar in post-MI and sham rats. Therefore, we suggest that despite significant arteriogenesis the perfusion deficit in the septum of post-MI hearts of middle-aged rats might be caused by geometric changes in the resistance vessels (length, tortuosity, etc.) or some functional deficit in their maximal vasodilation.

Abstract: We compared the effects of heart rate reduction (HRR) by the If channel inhibitor, ivabradine (MI+IVA) and the β-blocker, atenolol (MI+ATEN) on ventricular remodeling and perfusion after myocardial infarction (MI). MI was induced in 12-mo.-old male Sprague-Dawley rats by left coronary artery ligation. Sham-operated rats (Sham) served as age-matched controls. Rats were treated for 1-4 weeks and the drug doses were adjusted so that HRR would be comparable in the two groups; mean HRR + SEM was 19.5 +/- 1.1% for MI + IVA and 19.0 +/- 0.8% for MI + ATEN. Infarct areas (% of LV free wall) were not significantly different between groups: MI 64 +/- 3; MI + IVA 56 +/- 3; MI + ATEN 62 +/- 3. Coronary reserve (fold increase in conductance during maximal vasodilation in response to dipyridamole), volume to mass ratio and ejection fraction (determined by echocardiograms 4 weeks after ligation) revealed the following values (*p<0.05 vs. MI): Group Coronary Reserve LV-Free Wall Septum VOL/MASS Ejection Fraction (μL/mg) MI (n=10) 2.59+0.18 2.79+0.25 1.49±0.14 0.31±0.02 MI + IVA (n=12) 3.49+0.30* 3.14+0.23 1.10±0.13* 0.40±0.03* MI + ATEN (n=15) 2.79+0.15 2.79+0.15 1.47±0.11 0.33±0.02 Plasma levels of Ang II (pg/ml) 2 weeks after coronary artery ligation were significantly lower in both treatment groups compared to MI rats: Sham, 43 +/- 7; MI 70 +/- 10; MI+IVA 42 +/- 8; MI+ATEN 21 +/- 5 in the free wall. Ivabradine, but not atenolol, preserves coronaryreserve, attenuates the decline in ejection fraction and the increase in left ventricular volume/mass ratio after myocardial infarction. These favorable modifications with ivabradine therapy occur despite similar increases in LVEDV after either HRR treatment. However, both ivabradine and atenolol are effective in decreasing plasma levels of Ang II.

Abstract: In order to maintain a normal coronary reserve following a myocardial infarction, the surviving hypertropic myocardium must undergo vascular growth. This response requires upregulation of multiple growth factors. Although gene therapy has some promise in this regard, this approach usually focuses on a single growth factor. This presentation reviews the evidence that mechanical factors are key primary stimulators of angiogenic growth factors and their receptors, and consequently, vascular growth that compensates for the cardiomyocyte hypertrophy of the surviving myocardium. Lowering heart rate increases the diastolic interval and enhances the magnitude of ventricular stretch. We have documented that stretch, in vivo and in vitro results in upregulation of key growth factors and receptors. This response occurs in both cardiac myocytes and microvascular endothelial cells. Both paracrine and autocrine signaling are activated. Our data indicate that lowering heart rate with a bradycardic drug can result in arteriolar growth, which compensates for the cardiomyocyte hypertrophy and thereby preserves coronary reserve. This non-invasive post-infarction therapy upregulates several growth factors and receptors, including VEGF, Angiopoietin-2, FGF-2 and VEGFR-1. Most importantly, coronary reserve is preserved and perivascular collagen is decreased after 4 weeks of heart rate reduction. These effects are associated with attenuation of the decline in ejection fraction and a smaller increase in left ventricular volume/mass ratio.

Abstract: We tested the hypothesis that heart rate reduction induced in middle-aged post-MI rats can improve perfusion in the surviving LV myocardium by attenuating periarteriolar fibrosis. MI was induced in 12-month-old Sprague-Dawley rats, which were then treated either with ivabradine (MI+IVA) or placebo (MI) via osmotic pumps for 4 weeks. In addition, blood and tissue samples were obtained at 3, 7 and 14 days after MI. Four weeks of IVA treatment did not affect infarct size, cardiac hypertrophy, arteriolar or capillary growth. However, perivascular collagen content in MI+IVA rats was 37% and 40% lower in the LV free wall and septum, respectively, and was associated with greater (26%) maximal myocardial perfusion. Tissue expression of ANG II type 1 receptor and TGFß1 proteins, as well as the plasma level of ANG II peptide, were higher in untreated compared to IVA-treated rats 14 days after MI. Thus, the data indicate that improved myocardial perfusion in MI+IVA rats was the result of the lower periarteriolar collagen content rather than to arteriolar growth. We suggest that an inhibition of the RAS may be a mechanism by which IVA-induced heart rate reduction affects the myocardial collagen of post-MI rats.

Abstract: Background: Heart rate reduction (HRR) after myocardial infarction (MI) in young rats stimulates angiogenesis, attenuates the decline in left ventricular (LV) function and coronary perfusion. However, the impact of lowering heart rate on older rats needs to be clarified. Accordingly, we tested the hypothesis that HRR in middle-aged post-MI rats induced with ivabradine (IVA), the first and selective inhibitor of cardiac pacemaker If current, can improve cardiac remodeling and preserve myocardial perfusion in the surviving LV myocardium by attenuating periarteriolar fibrosis Methods: MI was induced in 12-month-old male Sprague-Dawley rats by left coronary artery ligation. Post-MI rats received IVA, 10.5 mg/kg/day (MI+IVA) or placebo (MI) via i.p. osmotic pumps, for 4 weeks. Sham-operated rats (Shams) served as an age-matched control. Blood and tissue samples were obtained at 3, 7 and 14 days after MI. Four weeks after MI, treatment was interrupted (2 days) and the various LV parameters were assessed Results: Over the 4 weeks of treatment period, heart rate was consistently reduced by 25% (P<0.001) in MI+IVA rats compared to Shams. Ejection fraction in MI+IVA rats was 25% higher (P<0.05) compared to the non-treated MI rats. IVA treatment did not affect infarct size, cardiac hypertrophy, arteriolar and capillary growth (arteriolar length and volume densities were similar in MI+IVA and MI rats). However, MI+IVA rats demonstrated significantly reduced perivascular and interstitial collagen content compared to non-treated MI rats -37% (P<0.01) and -22% (P<0.01) in free wall, respectively, and -40% (P<0.01) and -33% (P<0.01) in septum, respectively. These beneficial effects were associated with a greater maximal coronary conductance +35% (P<0.01) in LV free wall and +28% (P<0.05) in septum compared to non-treated MI rats. Moreover, IVA prevented the increase in tissue expression of angiotensin II type 1 receptor and TGFβ1 protein observed in LV myocardium of untreated MI rats. In addition, IVA treatment also prevented on day 14, the increase in plasma level of angiotensin II (P<0.05) observed in MI rats. Conclusions: These data indicate that heart rate reduction induced by IVA in post-infarcted middle-aged rats improved LV function and that improvement of myocardial perfusion in MI+IVA rats was the result of the lower periarteriolar and interstitial collagen content rather than to arteriolar growth. We suggest that a limitation of the renin-angiotensin-system may be a mechanism by which ivabradine affects the myocardial collagen of post-MI rats.

Abstract: This study assessed the impact of pure heart rate reduction (HRR) induced with ivabradine (IVA), a selective inhibitor of cardiac pacemaker If current, on left ventricular (LV) function, angiogenesis and coronary perfusion, in middle-aged postmyocardial infarction rats. MI was induced in 12-month-old male Sprague–Dawley rats by left coronary artery ligation. Post-MI rats received IVA (10.5 mg/kg/day, 1month, MI+IVA) or placebo (MI) via i.p. osmotic pumps. Sham-operated older rats (Shams) served as control. In MI+IVA rats, HR was consistently reduced by 25% (P<0.001) compared to Shams. One month after MI and treatment cessation, ejection fraction in MI+IVA rats was 34% higher (P<0.05 vs. MI rats). Maximal coronary conductance in LV free wall and septum was greater in MI+IVA rats by 35% (P<0.01) and 28% (P<0.05), respectively. Arteriolar length and volume densities were similar in MI+IVA and MI rats but MI+IVA rats demonstrated significantly reduced interstitial and perivascular collagen content compared to non-treated MI rats by 21.6% (P<0.01) and 37% (P<0.01) in free wall and by 33.1% (P<0.01) and 35.7% (P<0.01) in septum, respectively. IVA prevented the increase in plasma level of angiotensin II, angiotensin II type 1 receptor and TGFβ1 protein expression in the non-infarcted LV myocardium observed in MI rats (P<0.05 vs. MI rats). In conclusion, HRR induced with IVA in post-MI middle-aged rats substantially reduces both interstitial and perivascular fibrosis via inhibition of renin–angiotensin system. This effect may be the anatomical basis for the preservation of maximal coronary conductance and possibly the improvement of LV function.

Abstract: This study tested the hypothesis that selective activation or blockade of VIP1 and VIP2 receptors will affect embryonic coronary vessel tube formation. The in vitro quail heart explant model was used. The ventricular apices from the hearts (stage HH29) were placed on collagen I gel and cultured for 48 hr in the medium with 10% FBS. For the next 48 hr, the explants were treated with the VIP receptor selective agonists (1 nmol/µL) and/or antagonists (10 nmol/µL). The explants were fixed and immunolabeled with the quail-specific anti-endothelium antibody. The total length of tubulo-like outgrowth (TLT) was quantified using ImagePro Plus software. In each explant, TLT was normalized by the explant perimeter and, finally, TLT in treated explants was expressed as a percent from TLT in untreated controls (UC). The concurrent stimulation of VIP1 and VIP2 induced only an 8% increase in TLT, but when VIP1 and VIP2 was activated independently the TLT was increased by 32% and 24%, respectively (P<0.05 vs. UC). The simultaneous blockade of VIP1 and VIP2 inhibited TLT by 29% (P<0.01 vs. UC), but a selective blockade of VIP1 alone reduced TLT only by 18% (P<0.01 vs. UC). Our data demonstrate that the interaction between VIP1 and VIP2 receptors exerts positive as well as negative modulatory effects on embryonic coronary vessel tube formation.

Abstract: Background: Basal coronary blood flow is increased in adults with cyanotic congenital heart disease (CCHD), but hyperemic flow and flow reserve remain normal. Since the extramural coronary arteries in CCHD are maximally dilated, we hypothesized that preservation of hyperemic flow and flow reserve resides in the coronary microvasculature. Methods: To distinguish coronary microvascular adaptive responses to hypoxemia vs cardiac hypertrophy, the apical left ventricular free wall was studied in four patient groups: 1) Eisenmenger syndrome (hypoxemic, non-hypertrophied); 2) structurally abnormal hearts with hypertrophy (non-hypoxemic); 3) structurally normal hearts with hypertrophy (non-hypoxemic); and 4) structurally normal hearts (non-hypertrophied, non-hypoxemic). Immunolabeling against anti-smooth muscle -actin was used on histological sections to examine coronary arterioles (vessels with external diameters of 6 to 50 µm). Morphometric and stereologic analyses determined arteriolar diameter, length, volume and surface densities. Data were log transformed to remove significant right tail skews. Results: Initial analysis of variance model demonstrated a significant inter-group difference for arteriolar length (p = 0.03) and vessel diameter (p = 0.03), but not for arteriolar volume and surface densities. Sub-analysis by Tukey-Kramer multiple comparison procedure disclosed that total arteriolar length density in Group A was markedly reduced (by 60%) compared to Group B. Lower arteriolar length density in Eisenmenger hearts signified fewer terminal arterioles (6–15 µm) than in structurally abnormal hearts with ventricular hypertrophy (p = 0.01). By contrast, mean arteriolar diameter in Group A was significantly greater (by 34%) than in Group B (p = 0.03). Moreover, a two-sample t-test comparison of Group A with the mean of the other three groups revealed a higher arteriolar diameter in Group A (p = 0.008). Conclusions: Remodeling of the coronary microcirculatory bed rather than angiogenesis is the key mechanism for preservation of hyperemic flow and flow reserve in CCHD. The increase in vessel diameter compensated for lower arteriolar length density, and was the principal anatomical basis for maintenance of normal flow in CCHD.

Abstract: This study tested the hypothesis that long-term heart-rate reduction induced by Ivabradine (IVA) promotes growth of coronary resistance vessels after experimental myocardial infarction (MI) in middle-aged rats. Three groups of 12-month-old Sprague- Dawley rats were used: 1) Sham (SH), 2) MI, 3) MI+IVA. Antero-apical MI of the left ventricle (LV) was induced by proximal occlusion of the LAD artery. On the 2nd day after MI, an osmotic pump with IVA (10.5 mg/kg/day) was placed in the peritoneal cavity for 4 weeks. In MI+IVA rats heart rate was consistently lower by 26-27% (P<0.0001) compared to SH rats. At the end of experiment, infarct size was similar between MI and MI+IVA rats (~ 67%), whereas the LV/body weight ratio was higher in IVA-treated animals (2.41±0.1 vs. 1.85±0.01 mg/g in MI rats, P<0.05). Maximal coronary conductance (flow/pressure, ml/mmHg · min ·100g), assessed using the neutron-activated microspheres technique after dipyridamole infusion, was higher in the LV free wall (FW) and septum (S) of MI+IVA rats compared to MI rats (14.8±1.1 vs. 11.2±0.8 in FW and 14.5±1.1 vs. 11.9±0.9 in S, P<0.05). These data indicate that long-term reduction of heart rate in post-MI hearts with IVA promotes growth of coronary resistance vessels in the surviving portion of LV myocardium.

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Abstract: To elucidate function of synectin (PDZ2 scaffold protein) in vivo, we have generated synectin-/- mice using an exon trap approach. The synectin-/- mice are viable, but are significantly smaller than wild type controls (Table). Coronary angiography demonstrated reduced branching of epicardial coronary arteries in the synectin-/- mice compared to wild type (WT). To further address the issues of intramural arterial branching, ventricular cross-sections from the WT and synectin-/- mice were stained with anti-alpha-smooth muscle actin antibody. The volume (Vv) and surface (Sv) density of total alpha smooth muscle actin positive profiles were measured per whole cross sections of left ventricle. In agreement with angiography, quantification of vascular density parameters demonstrated a significant reduction in vascular density in synectin-/- mice. To assess the impact of this reduced vascularity on myocardial function, left ventricular (LV) fractional shortening was examined using 2-D echocardiography. We observed a significant reduction in LV function at rest. In conclusion, synectin gene disruption results in reduced formation of myocardial coronary arteries, decreased heart size and impaired left ventricular function. These results suggest that synectin could play an important role in arterial branching synectin-/- WT P value Body weight (g) 16.7±1.3 19.9±0.5 0.03 Heart/body weight ratio 0.41±0.05 0.53±0.02 0.03 Vv (%) 0.57±0.02 0.87±0.06 0.01 Sv (%) 0.13±0.01 0.17±0.003 0.01 Fractional shortening (%) 56.8±4.2 66.9±1.8 0.04.

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Abstract: This study tested the hypothesis that chronic bradycardia induced by Atenolol (AT) may trigger compensatory coronary angiogenesis after acute experimental myocardial infarction (MI). Three groups of adult Sprague-Dawley rats were used: 1) control, 2) MI, 3) MI/AT-treated. MI was induced by surgical occlusion of the anterior descending branch of left coronary artery. AT was administered in drinking water (30 mg/rat per day) for 4 weeks, beginning 3 days after MI. AT induced heart rate reduction by 29% and 28% (P<0.0001) compared to control and MI rats, respectively. Infarct size was relatively similar in MI/AT-treated and MI rats (68.5±6.1% and 60.9±4.7%, respectively), whereas the ventricle/body weight ratio was slightly higher in MI/AT-treated rats (0.350±0.013 vs. 0.279±0.012 mg/g, P<0.05). To determine the changes in the coronary microvascular bed we measured the maximal myocardial perfusion in the left ventricular free wall (FW) and septum (S) after dipyridamole infusion using neutron-activation microspheres. Regional myocardial perfusion, expressed as conductance (flow/pressure), in MI/AT-treated rats was significantly higher compared to control and MI hearts: 181.8±7.5% (P<0.001) and 174.2±7.1% (P<0.05), respectively, for FW, and 168.3±19.4% (P<0.05) and 151.5±17.5%, respectively, for S. Our data indicate that chronic beta-1-receptor blockade with AT stimulates angiogenesis in the infarcted heart by promoting resistance vessel growth.

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Abstract: The aim of our study was to determine the effect of innervation on the mitotic activity in uMPCs associated with new fibers in the regenerating EDL muscles. Two groups of 4-month-old rats were used. In both groups, muscle regeneration was induced by the injection of Marcaine. In one group, muscle regeneration was combined with denervation caused by transection of the sciatic nerve. Innervated and non-innervated regenerating muscles were examined at days 2, 3, 4, 6, 12 after surgery. Both uMPCs and myoblasts were determined by immunostaining for M-cadherin. At the same time, the mitotic figures in uMPCs were visualized by nuclear staining with DAPI and myoblasts were verified by presence of either MyoD+ or myogenin+ nuclei. We have found that the sequence of myogenic events was similar in regenerating muscles between both groups of rats. At days 2 and 4 of regeneration, uMPCs proliferated inside the basal lamina of degenerated fibers, transformed in myoblasts, which then fused in myotubes. At days 6 and 12 of regeneration, myoblasts were mostly fusing with new fibers. In denervated muscles fusion was less evident and a large number of myoblasts were situated independently on the surface of new fibers. At the same time, the uMPCs associated with either innervated or non-innervated fibers showed no mitotic activity. We suggest that either a direct interaction between uMPCs and fibers or local humoral factors suppresses the mitotic activity in uMPCs.

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Abstract: The aims of our study were to determine a cellular source of elevated MyoD and myogenin protein expression in senile skeletal muscles and to provide a potential explanation for this phenomenon. Our research was conducted on WI/HicksCar rats 4 (young), 24 (old), and 32 (senile) months of age. Compared to young animals, protein levels of MyoD and myogenin are increased in gastrocnemius muscles of old rats and are sharply elevated in 32-month-old rats. Electron microscopy of senile EDL muscles demonstrated the presence of degenerative alterations in the areas of nerve-muscle junctions, which often were devoid of axon terminals. Activated satellite cells (SCs) were also found in 32-month-old muscles. Immunostaining on sections from senile EDL and TA muscles revealed that MyoD and myogenin protein expression were restricted to myonuclei of atrophied NCAM positive fibers as well as to nuclei of activated M-cadherin positive SCs. We conclude that the up-regulation of the MyoD and myogenin protein expression in senile skeletal muscle is associated with at least of two coexisting processes: 1) motor denervation of muscle fibers and 2) compensatory activation of SCs.

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Abstract: The purpose of the present study was to investigate the dependence of remodeling of intercalated discs (ID) and assembly of myofibrils (AM) in cardiac muscle on the presence of myocyte-myocyte contacts. We studied these processes in two experimental models: (1) long-term cell cultures of neonatal and adult rat cardiomyocytes plated at densities that ensured or prevented the formation of contacts between muscle cells and (2) in groups of myocytes developing intercellular contacts and in single solitary isolated myocytes located in the perinecrotic zone 3, 7, 14 and 21 days after myocardial infarction in adult rats. We found that the temporal patterns of remodeling of ID and AM were similar in vitro and in vivo and depended on the presence of myocyte-myocyte contacts. Breakdown of contacts between muscle cells resulted in resorption of protein components of fasciae adherentes, detachment of myofibrils from the sarcolemma and more or less complete disassembly of sarcomeres in these areas. Formation of elongating finger-like cytoplasmic processes and ruffles was followed by intense AM and led to reestablishment of intercalated discs. Unlike contacting muscle cells, AM in solitary myocytes was significantly slower. The areas of their former ID contained mostly immature precursors of myofibrils such as stress fiber-like structures and premyofibrils. During the 2-nd and 3-rd weeks after disruption of ID, cytoplasmic processes of single myocytes unable to reach other muscle cells started to form contacts between themselves. Subsarcolemmal areas of such contacts were immunopositive for desmin and alpha-actinin, but did not contain desmoplakins, which indicates their similarity to fasciae adherentes. In these areas, myofibrillar precursors anchored to the sarcolemma and underwent complete AM. Our results show that the presence of ID is necessary for AM and functional maturation and stability of the contractile system in cardiac muscle cells.

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