Large- and Medium-sized Arteries Remaining in Transmural Scar Distal to Permanent Coronary Ligation Undergo Neointimal Hyperplasia and Inward Remodeling.

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.

Preservation of Functional Microvascular Bed Is Vital for Long-Term Survival of Cardiac Myocytes Within Large Transmural Post-Myocardial Infarction Scar.

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.

Reduced epicardial vagal nerve density and impaired vagal control in a rat myocardial infarction-heart failure model.

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/mm2 versus 1.68±0.46 µm/mm2, P=.001) and branching point-density (1.24±0.25 points/mm2 versus 0.66±0.18 points/mm2, P<.001) were lower in MI-HF rats. The chemically stained epicardial nerve bundles contain both sympathetic (tyrosine hydroxylase positive) and vagal (choline acetyltransferase positive) fibers. However, within the stained nerve bundle, the chemical color corresponds mainly with the vagal fibers. 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.

Sex-related differences in intrinsic myocardial properties influence cardiac function in middle-aged rats during infarction-induced left ventricular remodeling.

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 < 0.05), SW index (40.4 ± 2.1 mmHg • mL/kg vs. 20.2 ± 3.3 mmHg • mL/kg, P < 0.001), and CI (139.2 ± 7.9 mL/min/kg vs. 74.9 ± 14.7 mL/min/kg, P < 0.01). The poorer pumping capacity in M-MI hearts was associated with markedly reduced LV compliance and prolonged relaxation. On the tissue level, F-MI rats revealed a higher, than in M-MI rats, density of cardiac myocytes in the LV free wall (2383.8 ± 242.6 cells/mm(2) vs. 1785.7 ± 55.9 cells/mm(2), P < 0.05). The latter finding correlated with a lower density of apoptotic cardiac myocytes in residual LV myocardium of F-MI rats (0.18 ± 0.08 cells/mm(2) vs. 0.91 ± 0.30 cells/mm(2) in males, P < 0.01). Thus, our data suggested that F-MI rats had markedly attenuated decline in cardiac performance compared to males due to ability of female rats to better retain functionally favorable intrinsic myocardial properties.

Effect of Chronic Heart Rate Reduction by I(f) Current Inhibitor Ivabradine on Left Ventricular Remodeling and Systolic Performance in Middle-Aged Rats With Postmyocardial Infarction Heart Failure.

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.

Thyroid hormone replacement therapy attenuates atrial remodeling and reduces atrial fibrillation inducibility in a rat myocardial infarction-heart failure model.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Coronary vessels and cardiac myocytes of middle-aged rats demonstrate regional sex-specific adaptation in response to postmyocardial infarction remodeling.

BACKGROUND: An increasing body of evidence indicates that left ventricular (LV) remodeling, especially the degree of reactive myocardial hypertrophy after myocardial infarction (MI), differs in males and females. Surprisingly, to date, the sex-specific post-MI alterations of the coronary vasculature remain undetermined. Therefore, we tested the hypothesis that adaptive coronary arteriolar and capillary modifications occurring in response to reactive myocyte hypertrophy differ between middle-aged male and female post-MI rats. METHODS: 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 surgery, rats with transmural infarctions, greater than 50% of the LV free wall (FW), were evaluated. Sham-operated male (M-Sham) and female (F-Sham) rats served as an age-matched controls. RESULTS: F-MI and M-MI rats had similar sized infarcts (61.3% ± 3.9% vs. 61.5% ± 1.2%) and scale of LV remodeling, as indicated analogous remodeling indices (1.41 ± 0.11 vs. 1.39 ± 0.09). The degree of reactive post-MI myocardial hypertrophy was adequate to normalize LV weight-to-body weight ratio in both sexes; however, the F-MI rats, in contrast to males, showed no myocyte enlargement in the LVFW epimyocardium. At the same time, a greater than 50% expansion of myocyte area in the male epimyocardium and in the female endomyocardium was accompanied by a 23% (P < 0.05) increase in capillary-to-myocyte ratio, indicative of adaptive angiogenesis. Based on arteriolar length density in post-MI hearts, the resistance vessels grew in the male LVFW as well as the septum by 24% and 29%, respectively. In contrast, in females, a significant (30%) expansion of arteriolar bed was limited only to the LVFW. Moreover, in F-MI rats, the enlargement of the arteriolar bed occurred predominantly in the vessels with diameters <30 µm, whereas in M-MI rats, a substantial (two- to threefold) increase in the density of larger arterioles (30 to 50 µm in diameter) was also documented. CONCLUSION: Our data reveal that while both sexes have a relatively similar pattern of global LV remodeling and adaptive angiogenesis in response to a large MI, male and female middle-aged rats differ markedly in the regional scale of reactive cardiac myocyte hypertrophy and adaptive arteriogenesis.

Both hypothyroidism and hyperthyroidism increase atrial fibrillation inducibility in rats.

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<0.05). Hypothyroidism increased atrial interstitial fibrosis, but connexin 43 was not affected. 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.

Structural composition of myocardial infarction scar in middle-aged male and female rats: does sex matter?

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.

Postmyocardial infarction remodeling and coronary reserve: effects of ivabradine and beta blockade therapy.

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.

Synectin/syndecan-4 regulate coronary arteriolar growth during development.

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.

Preservation of coronary reserve by ivabradine-induced reduction in heart rate in infarcted rats is associated with decrease in perivascular collagen.

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.

Formation of leptofibrils is associated with remodelling of muscle cells and myofibrillogenesis in the border zone of myocardial infarction.

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.

Vascular patterning of the quail coronary system during development.

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.

The coronary microcirculation in cyanotic congenital heart disease.

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.

Compensatory growth of coronary arterioles in postinfarcted heart: regional differences in DNA synthesis and growth factor/receptor expression patterns.

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.

Differential healing activities of CD34+ and CD14+ endothelial cell progenitors.

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.

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture.

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.

Abortive myogenesis in denervated skeletal muscle: differentiative properties of satellite cells, their migration, and block of terminal differentiation.

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.

Reduction of heart rate by chronic beta1-adrenoceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted heart.

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.