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This study aimed to investigate the structural integrity and dynamic changes in chronically occluded residual arteries found in post–myocardial infarction (MI) scar. A transmural MI was induced in middle-aged, male Sprague-Dawley rats by left coronary artery ligation. The rats were euthanized 3 days and 1, 2, 4, 8, and 12 weeks after MI, and their hearts were processed into paraffin for histology, immunohistochemistry, and quantitative morphometry. It has been found that large- and medium-sized arteries were able to survive inside the transmural scars for 12 post-MI weeks. Furthermore, most residual arteries preserved their structural integrity for up to 2 weeks post-MI, but gradually all disused vessels had undergone neointimal hyperplasia and inward remodeling at later time periods. In addition, the replacement of vascular smooth muscle cells in the wall of residual arteries by extracellular matrix components led to a disruption of the vessel integrity and progressive obliteration of their lumen between 4 and 12 post-MI weeks. Taken together, this study demonstrate that residual arteries in post-infarcted region were capable of maintaining their structural integrity, including the patent lumen, during two post-MI weeks, suggesting that during this period they can be used as potential conduits for conceivable reflow of arterial blood within the scarred region of the heart

Background: Hypertension (HT)-induced accumulation of myocardial collagen stiffens the left ventricular (LV) wall and, hence, promotes diastolic dysfunction. Considering that a competitive aldosterone receptor antagonist spironolactone (SL) as well as a “pure” heart rate-lowering drug ivabradine (IVA) have both been proven to exert an anti-fibrotic effect during cardiac remodeling, we hypothesized that, in combination, these drugs would complement each other in alleviating HT-induced myocardial fibrosis and, therefore, could better preserve diastolic function. Methods: Sustained HT was induced in 7-week-old male Dahl salt-sensitive rats by feeding them a high-salt (8%) diet for 7 weeks. Then, HT rats were randomly assigned in two experimental groups: 1) IVA (10 mg/kg/day) + SL (20 mg/kg/day)-treated (HT-T) and 2) vehicle only (HT-V). The drugs and the vehicle (a mixture of 50% dimethyl sulfoxide/50% propylene glycol, v/v) were delivered IP by osmotic pumps for 8 weeks. The age-matched rats were used as normotensive controls (NT). Heart rate (HR) and blood pressure (BP) were recorded every other week using a CODA tail-cuff plethysmography system. At the end of the study, the hemodynamic parameters were assessed using a Millar pressure catheter and the hearts were collected for histology and quantitative morphometry. Statistical analysis was performed using Prism 6. Results: We found that during 8-week treatment period, BP had remained comparably elevated in both HT groups by ~26% (P≤0.01) on average vs. NT rats, whereas HR had been persistently reduced in HT-T group by ~38% and ~27% (P≤0.01) on average vs. HT-V and NT rats, respectively. At the end of the study period, all groups revealed a similar level of LV end-diastolic pressure, suggesting the analogous diastolic stiffness of the myocardium. At the same time, morphological examination of the hearts has confirmed that sustained HT caused significant interstitial and perivascular fibrosis (P≤0.01) in both experimental groups compared to NT rats, and that the treatment did not change the extent of fibrillar collagen accumulation as compared to HT-V rats. Conclusions: Taken together, our findings confirmed that a combined treatment with IVA and SL could not alleviate HT-induced myocardial fibrosis.

Background: Gamma-enolase, or neuron-specific enolase, is a multifunctional protein which is primarily found in neurons and neuroendocrine cells. Although the main role of gamma-enolase is associated with enzymatic activity during aerobic glycolysis, recently, it has been implicated in a variety of pathophysiological processes including inflammation, extracellular matrix degradation and cell survival after ischemic insult. Over the last decades, there have been a number of reports demonstrating the expression of gamma-enolase in the heart. Surprisingly, the latter finding has not received much attention in the past. Considering that this protein can play an important role in cellular adaptation to the ischemic environment we decided to examine the expression of gamma-enolase in the heart at different time points following a myocardial infarction (MI). Methods: A transmural MI was induced in male, middle-aged Sprague-Dawley rats by permanent left coronary artery ligation. The rats were euthanized 3 days, 1, 2, 4 and 8 weeks after MI and their hearts were processed into paraffin for immunostaining and quantitative morphometry. Results: We found that in post-MI heart gamma-enolase protein was expressed in cardiac myocytes of the remote myocardium as well as in the muscle cells surviving inside the transmural scar. On a subcellular level, the gamma-enolase expression overlapped with the I-bands of the sarcomeres. Despite the heterogeneity of the gamma-enolase fluorescence intensity among individual cardiac myocytes, there was no evident correlation between the size of the cells and the intensity of the gamma-enolase immunofluorescence in any region of the post-MI heart, although the immunofluorescence intensity of gamma-enolase in myocytes of the remote myocardium was always greater than that in myocytes surviving within the transmural scar. Most importantly, we determined that the intensity of gamma-enolase immunofluorescence recorded in cardiac myocytes has experienced an evident stepwise reduction at 1 and 8 weeks following MI. Conclusions: Taken together, our findings reveal that the cardiac myocytes of the adult rat heart express gamma-enolase and that the expression of this protein has been altered during post-MI cardiac remodeling.

This study was aimed to understand the mechanism of persistent cardiac myocyte (CM) survival in myocardial infarction (MI) scars. A transmural MI was induced in 12-month-old Sprague–Dawley rats by permanent coronary artery ligation. The hearts were collected 3 days, 1, 2, 4, 8, and 12 weeks after MI and evaluated with histology, immunohistochemistry, and quantitative morphometry. Vasculature patency was assessed in 4-, 8-, and 12-week-old scars by infusion of 15-micron microspheres into the left ventricle before euthanasia. The infarcted/scarred area has a small continually retained population of surviving CMs in subendocardial and subepicardial regions. Surprisingly, whereas the transverse area of subepicardial CMs remained relatively preserved or even enlarged over 12 post-MI weeks, subendocardial CMs underwent progressive atrophy. Nevertheless, the fractional volume of viable CMs remained comparable in mature scars 4, 8, and 12 weeks after MI (3.6 ± 0.4%, 3.4 ± 0.5%, and 2.5 ± 0.3%, respectively). Despite the opposite dynamics of changes in size, CMs of both regions displayed sarcomeres and gap junctions. Most importantly, surviving CMs were always accompanied by patent microvessels linked to a venous network composed of Thebesian veins, intramural sinusoids, and subepicardial veins. Our findings reveal that long-term survival of CMs in transmural post-MI scars is sustained by a local microcirculatory bed.

We have reported previously that local, patent microvascular networks support long-term survival of adult cardiac myocytes in subendocardial and subepicardial regions of large transmural post-myoca...

Current understanding of the rewarding and addictive effects of opioids involves mu-opioid receptor (MOR) binding within the nucleus accumbens (NAcc), a region of the ventral striatum. GABAergic neurotransmission in the NAcc potentiates the rewarding response to opioids, and in fact, drugs that stimulate GABAergic activity are also addictive, a phenomenon mediated in part by endogenous opioids. However, the neuroanatomical relationship between opioid and GABA systems is still unclear, and further study of the interaction between these neurotransmitter systems in the reward pathway is warranted. We report evidence supporting the direct interaction between GABAergic and opioidergic neurotransmitter systems within the mouse NAcc. Male and female FVB/NJ mice (12-16 months of age) were euthanized via carbon dioxide inhalation and brains processed for histology and immunohistochemistry (IHC). Coronal cryosections (10-12 um in thickness) were taken through the NAcc at the level of the anterior commissure. A mouse monoclonal antibody against GAD67, an enzyme catalyzing GABA production, was used in conjunction with an anti-mouse rhodamine red-X-labeled secondary antibody to identify GABAergic neurons in the NAcc. Alternating sections were stained for MOR using a fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal anti-MOR1 antibody. DAPI (4′,6-diamidino-2-phenylindole) was used to counterstain the nuclei. As expected, fluorescence microscopy results show that GAD67 staining is localized predominately in the neuronal cytoplasm, with approximately 40% of the population staining. The MOR1-FITC stain showed localization within the cytoplasm and plasma membrane as expected, however, there was also significant staining in the nuclear membrane and neuronal nucleus. MOR1-FITC was present in approximately 35% of the neuronal population. In separate experiments, we used double-immunostaining to study the co-expression of MOR1 and GAD67 within the same NAcc neurons. A similar localization pattern was detected with a small subset, approximately 2%, of neurons expressing both labels. There are few published reports of GAD67 and MOR1 co-expression within neurons of the NAcc. Previous studies of MOR expression show the receptor to be localized to the plasma membrane and, to a smaller degree, intracellularly. Here we found the MOR1 staining to be predominantly in the nucleus and nuclear membrane, with minimal expression on the plasma membrane. Further studies are in progress to validate the nuclear expression of MOR in GABAergic NAcc neurons. Results of the study suggest that a subset of mouse NAcc neurons express both MOR1 and GAD67, providing a direct intracellular link between opioid and GABAergic systems in the reward pathway. Preliminary intranuclear localization of MOR suggests a novel signaling pathway that may be important in fully elucidating neurobiological mechanisms underlying behaviors related to reward and addiction.

Background Autonomic remodeling, characterized by sympathetic activation and vagal withdrawal, contributes to heart failure (HF) progression. However, the exact mechanism(s) responsible for vagal withdrawal in HF remain(s) unclear, and whether HF causes epicardial autonomic nerve remodeling is unknown. Methods and results Myocardial infarction (MI) was produced in 14 Sprague–Dawley rats, and 10 sham surgery rats served as the control. MI–HF was confirmed 2 months after the surgery by echocardiography and hemodynamic measurement. Cervical vagal nerve stimulation was delivered to examine the heart rate slowing effect. Whole heart acetylcholinesterase histochemistry was used to examine the epicardial autonomic nerve remodeling at dorsal ventricles (remote from the infarcted area). Compared with the control animals, the same vagal nerve stimulation had less heart rate slowing effect in MI–HF group. Both epicardial nerve bundle length-density (2.56±0.60 μm/mm 2 versus 1.68±0.46 μm/mm 2 , P =.001) and branching point-density (1.24±0.25 points/mm 2 versus 0.66±0.18 points/mm 2 , P Conclusions Whole heart acetylcholinesterase histochemistry revealed a decreased ventricular epicardial vagal nerve density in MI–HF rats, which may contribute to impaired cardiac vagal control in HF.

The presence of surviving cardiac myocytes in post-myocardial infarction (MI) scars has been repeatedly documented in humans as well as in various laboratory animals, particularly in rats. In the m...

Introduction Chronic cervical vagus nerve stimulation has been introduced as a novel therapy for various diseases, including refractory epilepsy and chronic heart failure. Although it has been show...

Background: A large myocardial infarction (MI) initiates progressive cardiac remodeling that leads to systolic heart failure (HF). Long-term heart rate reduction (HRR) induced by the I f current inhibitor ivabradine (IVA) ameliorates left ventricular (LV) remodeling and improves systolic performance in young post-MI rats. However, the beneficial effects of chronic IVA treatment in middle-aged rats remain to be determined. Methods: A large MI was induced in 12-month-old rats by left coronary artery ligation. Rats were treated with IVA via osmotic pumps intraperitoneal in a dose of 10.5 mg/kg/d (MI + IVA) and compared with MI and sham-operated animals 12 weeks after MI. Results: Heart rate in MI + IVA rats was on average 29% lower than that of rats in the MI group. Left ventricular remodeling was comparable between post-MI groups, although MI + IVA rats did not show the compensatory thickening of the noninfarcted myocardium. Chronic HRR had no effect on transverse cardiac myocyte size and capillary growth, but it reduced the collagen content in noninfarcted myocardium. Left ventricular systolic performance remained similarly impaired in MI and MI + IVA rats. Moreover, abrupt IVA withdrawal led to worsening HF and reduction of coronary reserve. Conclusion: Our data reveal that chronic IVA-induced HRR does not provide sustainable benefits for LV systolic performance in middle-aged rats with post-MI HF.

The present study was designed to determine whether the structural composition of the scar in middle-aged post–myocardial infraction (MI) rats is affected by the biological sex of the animals. A large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague-Dawley rats by ligation of the left coronary artery. Four weeks after the MI, rats with transmural infarctions, greater than 50% of the left ventricular (LV) free wall, were evaluated. The extent of LV remodeling and fractional volumes of fibrillar collagen (FC), myofibroblasts, vascular smooth muscle (SM) cells, and surviving cardiac myocytes (CM) in the scars were compared between the two sexes. The left ventricle of post-MI male and female rats underwent a similar degree of remodeling as evidenced by the analogous scar thinning ratio (0.46 ± 0.02 vs. 0.42 ± 0.05) and infarct expansion index (1.06 ± 0.07 vs. 1.12 ± 0.08), respectively. Most important, the contents of major structural components of the scar revealed no evident difference between M-MI and F-MI rats (interstitial FC, 80.74 ± 2.08 vs. 82.57 ± 4.53; myofibroblasts, 9.59 ± 1.68 vs.9.56 ± 1.15; vascular SM cells, 2.27 ± 0.51 vs. 3.38 ± 0.47; and surviving CM, 3.26 ± 0.39 vs. 3.05 ± 0.38, respectively). Our data are the first to demonstrate that biological sex does not influence the structural composition of a mature scar in middle-aged post-MI rats.

Introduction: Calcification of atherosclerotic plaques is associated with different histopathological and clinical manifestations. Microcalcification promotes inflammation and increases plaque instability, whereas macrocalcification is associated with stable fibroatheroma. The mechanisms of initiation and progression of calcification in atherosclerosis are not completely understood. Hypothesis: Upregulation of tissue-nonspecific alkaline phosphatase (TNAP) in monocytes/macrophages can lead to calcification of atherosclerotic plaques in a mouse model. Methods: TNAP overexpression was induced from a targeted transgene in Hprt locus utilizing Cre recombinase driven by monocyte-specific LyzM gene promoter. Resultant mice overexpressing TNAP in cells of monocyte/macrophage lineage (TNAP-Tg) were bred with animals bearing low density lipoprotein receptor mutation. Hypercholesterolemia was induced by feeding Paigen diet for ten weeks. Average plaque area and calcification (area %) were determined from histological sections of the aortic root (5-10 sections/sample). Blood flow velocity in the ascending aorta and left ventricular function were assessed by echocardiography. Results: In the absence of atherosclerosis, no appreciable soft tissue calcification was observed in TNAP-Tg animals (n=2). During progression of atherosclerosis, calcification was detected in the aortic root lesions of TNAP-Tg mice (4.85% of total plaque area) in significant excess compared to controls (0.02%, p Conclusions: We developed an animal model in which the interaction between early calcium deposition inside atherosclerotic lesions and the stability of such lesions can be studied experimentally.

We compared the effects of heart rate reduction (HRR) by the hyperpolarization-activated pacemaker current ( If) channel inhibitor ivabradine (MI+Iva) and the β1-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 (AT1) receptor and transforming growth factor (TGF)-β1 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.

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 AT1 receptor and transforming growth factor (TGF)-β1 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.

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 -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 m) 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. Developmental Dynamics 236:2004 –2010, 2007. © 2007 Wiley-Liss, Inc.

Our previous study on post-MI male and female middle-aged rats has revealed that although both sexes had a similar scale of LV remodeling, the females showed a greater density of cardiac myocytes in the epimyocardium. However, the mechanism of this structural alteration, as well as its impact on LV function, remained unclear. Our current study was aimed at determining whether the sex-specific difference in the density of cardiac myocytes is related to the rate of their death and whether it affects the cardiac performance in post-MI rats. A large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague-Dawley rats by ligation of the left coronary artery. The cardiac function was monitored for two months post-MI. At the end of the experiment, various LV parameters were compared between sexes. We found that although both groups had a similar pattern of decline in LV function, the F-MI rats exhibited a less deteriorated cardiac performance compared to M-MI rats, as indicated by the values of strok...

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.

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 α-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.

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.

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.

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.

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.

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.

Heart failure (HF) is associated with increased atrial fibrillation (AF) risk. Accumulating evidence suggests the presence of myocardial tissue hypothyroidism in HF, which may contribute to HF development. In a recent report we demonstrated that hypothyroidism, like hyperthyroidism, leads to increased AF inducibility. The present study was designed to investigate the effect of thyroid hormone (TH) replacement therapy on AF arrhythmogenesis in HF.Myocardial infarction (MI) was produced in rats by means of coronary artery ligation. Rats with large MIs (40%) were randomized into L-thyroxine (T4; n = 14) and placebo (n = 15) groups 2 weeks after MI. Rats received 3.3 mg T4 (in 60-day release form) or placebo pellets for 2 months. Compared with the placebo, T4 treatment improved cardiac function and decreased left ventricular internal diameters as well as left atrial diameter. T4 treatment attenuated atrial effective refractory period prolongation (45 ± 1.5 ms in placebo group vs 37 ± 1.6 ms in T4 group; P.01) and reduced AF inducibility (AF/atrial flutter/tachycardia were inducible in 11/15 rats [73%] in the placebo- vs 4/14 rats [29%] in the T4-treated group; P.05). Arrhythmia reduction was associated with decreased atrial fibrosis but was not associated with connexin 43 changes.To our knowledge this is the first study demonstrating that TH replacement therapy in HF attenuates atrial remodeling and reduces AF inducibility after MI-HF. Clinical studies are needed to confirm such benefits in human patients.

Our previous study on post-MI male and female middle-aged rats has revealed the regional sex-specific differences in the adaptive changes of the left ventricular (LV) cardiac myocytes and coronary ...

Background— Evidence indicates that cardiac hypothyroidism may contribute to heart failure progression. It is also known that heart failure is associated with an increased risk of atrial fibrillation (AF). Although it is established that hyperthyroidism increases AF incidence, the effect of hypothyroidism on AF is unclear. This study investigated the effects of different thyroid hormone levels, ranging from hypothyroidism to hyperthyroidism on AF inducibility in thyroidectomized rats. Methods and Results— Thyroidectomized rats with serum-confirmed hypothyroidism 1 month after surgery were randomized into hypothyroid (N=9), euthyroid (N=9), and hyperthyroid (N=9) groups. Rats received placebo, 3.3-mg l -thyroxine (T4), or 20-mg T4 pellets (60-day release form) for 2 months, respectively. At the end of treatment, hypothyroid, euthyroid, and hyperthyroid status was confirmed. Hypothyroid animals showed cardiac atrophy and reduced cardiac systolic and diastolic functions, whereas hyperthyroid rats exhibited cardiac hypertrophy and increased cardiac function. Hypothyroidism and hyperthyroidism produced opposite electrophysiological changes in heart rates and atrial effective refractory period, but both significantly increased AF susceptibility. AF incidence was 78% in hypothyroid, 67% in hyperthyroid, and the duration of induced AF was also longer, compared with 11% in the euthyroid group (all P Conclusions— Both hypothyroidism and hyperthyroidism lead to increased AF vulnerability in a rat thyroidectomy model. Our results stress that normal thyroid hormone levels are required to maintain normal cardiac electrophysiology and to prevent cardiac arrhythmias and AF.

Background: We have previously shown that 1-month treatment with ivabradine (IVA), the selective cardiac pacemaker I f current inhibitor, preserved myocardial perfusion and coronary perfusion reserve in post-MI middle-aged rats. However, the persistence of this cardioprotective effect after a prolonged period of IVA treatment remains to be determined. Methods: Acute MI was induced in 12-month-old male Sprague-Dawley rats by left coronary artery ligation. Twenty four hours later, the rats with a confirmed large transmural MI (>50% of the left ventricular (LV) free wall) were randomly assigned in two experimental groups. In a first group, rats were treated with IVA i.p. via osmotic pumps in a dose of 10.5 mg/kg/day for 3 months (MI+IVA). In a second group, rats received placebo treatment (5% dextrose) during the same time period (MI). Sham-operated rats served as an age-matched control. At the end of experimental period, myocardial perfusion (baseline and maximal coronary conductance per 100g of tissue) and coronary perfusion reserve (fold increase between baseline and maximal coronary conductance) were determined in non-infarcted LV free wall and interventricular septum by using the neutron-activated stable isotope-labeled microsphere technique. Results: During 3 months of IVA treatment, heart rate in MI+IVA rats was consistently reduced compared to untreated MI rats by mean of 30.6%. Nevertheless, we found that the infarct size and the extent of LV remodeling were relatively comparable between MI and MI+IVA rats three months after surgery. Moreover, the levels of baseline and maximal coronary conductance were similar in LV free wall and septum between two experimental groups. Consequently, IVA-treated rats revealed no difference in coronary perfusion reserve as compared to untreated post-MI animals (2.22±0.46 vs. 2.59±0.41 in LV free wall and 2.30±0.59 vs. 2.68±0.44 in septum, respectively). However, the rats of both post-MI groups had markedly reduced levels of maximal coronary blood flow as compared to non-infarcted controls (p≤0.01). Conclusion: Our data demonstrate that long-term IVA treatment does not provide sustainable improvement in LV myocardial perfusion and coronary perfusion reserve in middle-aged rats following large MI.

A large myocardial infarction (MI) initiates progressive structural remodeling that leads to a decline in left ventricular (LV) performance. Four weeks of heart rate reduction (HRR) in post-MI rats...

The current study investigates the spatio-temporal pattern of neuropeptide Y protein expression in coronary vessels and cardiac nerves during development of the mouse heart. The hearts of the C57BL/6 mice were collected on embryonic days 16 to 19 and on postnatal days 3 to 21. They were immersion-fixed with 4% PFA/PBS and processed for cryo-sectioning. The serial histological cross-sections through the heart ventricles were immunostained with an antibody against NPY alone or in combination with Griffonia simplicifolia IB4 lectin, the antibodies against α-smooth muscle (SM) actin and 150 kDa neurofilaments. We found that during embryonic and postnatal development, the coronary vessels demonstrated a high level of variability in the intensity of NPY immunostaining. Furthermore, whereas during embryogenesis, NPY was primarily expressed in the endothelial cells (ECs) of the coronary vessels, in postnatal period, the NPY immunostaining was typically detected in vascular SM cells. More importantly, the disappearance of NPY expression in ECs closely correlated with a significant expansion of NPY-containing perivascular nerves. Therefore, our findings suggest that NPY has distinct dynamics of expression within the cells of developing coronary vessels and perivascular nerves, indicating its potential involvement in regulation of coronary vessel formation and/or function during development of the mouse heart. Grant Funding Source: Internal Award from the NYCOM Office of ResearchThe dynamics of neuropeptide Y protein expression in the developing mouse heart -- Sverkunova... Page 1 of 2http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/529.6?sid=a3f27a37-f... 4/11/2013

We previously determined that residual left ventricular (LV) myocardium of middle‐aged rats had sex‐related differences in regional tissue properties 4 weeks after a large myocardial infarction (MI). However, the impact of such differences on cardiac performance remained unclear. Therefore, our current study aimed to elucidate whether sex‐related changes in MI‐induced myocardial remodeling can influence cardiac function. A similar‐sized MI was induced in 12‐month‐old male (M‐MI) and female (F‐MI) Sprague–Dawley rats by ligation of the left coronary artery. The cardiac function was monitored for 2 months after MI and then various LV parameters were compared between sexes. We found that although two sex groups had a similar pattern of MI‐induced decline in LV function, F‐MI rats had greater cardiac performance compared to M‐MI rats, considering the higher values of EF (39.9 ± 3.4% vs. 26.7 ± 7.7%, P

Background: In recent years, the use of middle-aged and old animals of both sexes has become preferential in experimental studies involving the models of cardiovascular diseases to better reflect a human population. However, the evidence showing the extent of sex-related differences in cardiac aging of laboratory animals remains obscure. Accordingly, we designed our study to determine whether the male and female rats differ during aging with regard to left ventricular (LV) geometry and systolic function. Methods: We performed transthoracic echocardiographic examinations in 12-month-old (12mo) and 24 month-old (24mo) male and female Sprague-Dawley rats (Charles River, Wilmington, MA) under 1.5% isoflurane anesthesia using a linear (Matrix) array transducer probe GE M12L (5.5-14.0 MHz) and a GE Vivid 7 Dimension ultrasound system. Two-dimensional short-axis views and M-mode tracings of the LV at the papillary muscle level were recorded and used to estimate various LV parameters. Results: We found that 24mo-Female rats had a significant increase in LV chamber dimensions associated with a reduction in its systolic performance as compared to 12mo-Female rats (Table 1). In contrast, LV parameters remained similar between 12mo- and 24mo-Male rats. Table 1. Echocardiographic parameters of LV chamber geometry and systolic performance Values are means ± SE. Arrow indicates a significant difference between age groups ( P ≤ 0.05). Conclusion: Our data demonstrate that during cardiac aging female rats have a different pattern of LV changes compared to males that must be considered in the design of future experimental studies.

D-Dopachrome tautomerase is an enzyme related by amino acid sequence and catalytic activity to macrophage migration inhibitory factor. Both of these small molecules are pro-inflammatory cytokines mediating broad innate immune responses. Although it is well established that the gene product of D-dopachrome tautomerase is widely expressed in liver and kidney cells, no study has mapped the distribution pattern of this tautomeric enzyme in the mammalian nervous system. Here, we address this void by characterizing the cellular localization of D-dopachrome tautomerase in the adult mouse brain. Two well-characterized polyclonal antibodies were used for Western blotting and immunohistochemical localization of the endogenous tautomeric enzyme. Our results show that D-dopachrome tautomerase is present throughout the brain parenchyma with a large fraction of heterogeneous interneurons harboring a stable and robust expression of the enzyme. These data point to a potential involvement of D-dopachrome tautomerase activity in the mature mouse brain, and suggest some functional and evolutionary relationship between innate immunity and tautomerization of D-dopachrome in mammalian species.

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...

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.

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.

SummaryBranching 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.

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.

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 α-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.

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 × 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.

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.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.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.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.

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.

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.

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.