Interest in the use of the latissimus dorsi muscle (LDM) in cardiac surgery appeared after the discovery that a stimulation protocol, which trained the skeletal muscle, would increase fatigue resistance. 1–3 4,5 6–8 9–11 12,13 14–16 17,18
The first step in cardiomyoplasty is subtotal mobilization of the LDM, which leaves the muscle in a severe ischemic state. 19–21 22 23,24
We believe that prevention of ischemia-reperfusion injuries immediately after muscle mobilization, and enhancement of neovascularization (angiogenesis) will be effective in improving cardiomyoplasty results. 25,26
Methods
Animal studies reported here conform to the Guiding Principles Regarding the Care and Use of Animals of the American Physiological Society and to all federal laws and regulations regarding animal use, and were approved by our institution’s Animal Care Committee.
General Anesthesia and Antibiotic Therapy
Adult sheep were operated on as described later in this article. Amoxicillin (15 mg/kg intramuscularly [im]) was administered to all animals 24 hours before surgery and continued for 5 days after surgery to guard against infection. Sterile technique was followed at all times to reduce the potential for infection. All surgical procedures and biopsies were conducted while the animals were under general anesthesia induced with diazepam (5 mg/kg given intravenously [iv]) and thiopental sodium (20–25 mg/kg iv). The animals were intubated, placed on a Drager ventilator (North American Drager, Telford, PA), and maintained on halothane gas anesthesia (1–2% with 4.0 L/min O2 ). Oxygen saturation levels and heart rate were monitored via a pulse oximeter placed on the animal’s tongue.
Surgical Procedures
Creation of Skeletal Muscle Pockets.
Series 1 and 2 each included four animals.
After the animals were put under general anesthesia, they were placed in a lateral position, and a longitudinal skin incision was made from the right axilla toward the costovertebral angle. A 6 ×16 cm flap of subcutaneous adipose tissue was dissected free, leaving the lateral portion connected. The anterior border of the LDM was completely mobilized. Several vessels originating from intercostal arteries that penetrated the muscle were ligated, but vessels entering the LDM from the spinal posterior, the profound posterior, and the superficial anterior areas were not disturbed (Figure 1A ).
Figure 1: Pocket creation. 1, in situ muscle flap (nonischemic LDM); 2, ischemic LDM flap (explained in text); 3, adipose tissue.
Two distinct sections of the LDM were identified (defined by their blood supply): the posterior portion with its undisturbed vascular supply, and the anterior portion, which was still ischemic after partial removal of its blood supply. These sections were separated from each other, exposing the anterior flap to even greater ischemia (simulating the condition inflicted on the LDM after subtotal mobilization for cardiomyoplasty). We were left with three distinct tissue sections: nonischemic LDM in situ ; ischemic LDM; and an adipose tissue flap (Figure 1B ).
The adipose tissue flap was placed on top of the in situ LDM, and the ischemic LDM section was placed on top of the adipose tissue. This “sandwich,” with three tissue layers, was sutured together to form two double pockets (each 3 × 10 cm) in the medial and peripheral regions of the LDM, after which each pocket was divided in half. This gave us two double pockets in the distal and two in the middle areas of the LDM. (Figure 1C ). These regions of the LDM were chosen because the distal portion of the LDM flap undergoes the most dramatic change after muscle mobilization. In four sheep (series 1), two pockets were left without ABG to serve as a control and two pockets were filled with ABG without any other additives; in another four sheep (series 2), two pockets were filled with ABG plus 1000 U/ml of aprotinin (a serine proteinase inhibitor) and two pockets were filled with ABG plus 10 μM of pyrrolostatin (a free radical scavenger).
Subtotal Latissimus Dorsi Muscle Mobilization.
Subtotal latissimus dorsi muscle mobilization was performed in series 3 through 7. Each series included four animals.
With the animal in the lateral position, a left 25-cm cutaneous incision was made at the level of the lateral border of the scapula, from the axillary region to the intersection between the iliac crest and paravertebral muscles. The LDM was dissected from the iliac crest, vertebral, inferior scapular angle, and 9th to 12th rib attachments. Collateral blood vessels arising from intercostal arteries were dissected. Only the neurovascular pedicle was carefully preserved. For investigation of the influence of different regimens of stimulation on morphology and angiogenesis of the mobilized LDM, the LDM was left in situ, with its distal portion resutured back to the iliac crest.
Implantation of Intramuscular Pacing Electrodes.
Implantation of intramuscular pacing electrodes was done in series 3 through 7, each of which included four animals.
Two intramuscular leads (Medtronic, Inc., Minneapolis, MN) were placed according to the standard Medtronic protocol, parallel to one another and perpendicular to the thoracodorsal nerve branches. The cathode electrode was placed first, in the vicinity of the main nerve trunk after it begins to branch into the muscle. The anode electrode was then placed into the muscle approximately 5 cm distal and parallel to the cathode.
Stimulator Implantation.
In series 3 through 7, a Medtronic stimulator was implanted subcutaneously in a separate pocket. To change the electrical stimulation protocol a magnetic programming wand from the computer was placed externally over the area of the implanted myocardiostimulator and the changes made. No sedation was required for this procedure.
Electrical Stimulation Protocol
The electrical stimulation training protocol in series 3 through 7 began 14 days after subtotal LDM mobilization using a single pulse with an amplitude of 5 V, and a frequency of 10 Hz. The protocol continued for 8 weeks with a change in the number of pulses (up to six) per burst every 10 days. Different regimens of stimulation were used for different groups of animals: 15 (series 3), 30 (series 4), and 60 (series 5) contractions per minute. In series 6 (15 CPM) and series 7 (30 CPM), ABG with aprotinin was applied to mobilized LDM.
Histology
Biopsies.
Biopsy specimens for light microscopy, immunohistochemistry, and transmission electron microscopy (TEM) were taken from the LDM before and 3 hours after subtotal mobilization, and on days 14 and 56. At the termination of the experiment, the pockets of the LDM were carefully excised. Samples from the different areas (proximal, middle, and distal) of the LDM were taken for analysis. Samples (3 × 4 mm) for light microscopy and immunohistochemistry were placed in 10% formalin and taken to the hospital’s pathology department for embedding and sectioning.
Light Microscopy.
For histologic examination, samples were taken from each area and fixed as described earlier. Transverse sections were made for conventional histologic (hematoxylin and eosin) staining and for subsequent evaluation. Multiple slides were made of each biopsy sample. Particular attention was paid to evidence of muscle regeneration, thickness and composition of the reparative response, and the density of neovascularization.
Transmission Electron Microscopy.
Biopsy specimens of LDM (approximately 3 × 4 mm) for transmission electron microscopy were placed into Karnovsky’s fixative (2% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) and then minced into smaller (1–2 mm) pieces. The biopsies were postfixed in 1% Osmium Tetroxide, dehydrated through a series of graded alcohols and acetone, and embedded in Spurr resin. Preliminary thick (1 μM) sections were cut and stained with 0.1% Toluidine blue. Longitudinal areas of muscle were selected for ultra-thin sectioning; thin sectioning (60–90 nm) was done with Reichert Ultra-Cut microtome. The thin sections were stained with 5% Uranyl Acetate and Reynolds’s Lead stain (3.5% lead citrate, 2% lead nitrate). Examination and photography of the thin sections was done with a Philips 400T transmission electron microscope (TEM) at an accelerating voltage of 60 kV on Kodak SO-163 image film. Five electron micrographs of adjacent areas in each of two regions were taken for each biopsy at a magnification of 6000×. Photographic enlargements (8 × 10 in) were made of each micrograph, with final magnifications of 16740× and 7812×, respectively.
Conventional Indirect Immunofluorescent En-Face Staining.
To assess angiogenesis, conventional indirect immunoperoxidase staining was used after fixation and proteolytic predigestion of formalin-fixed tissue, followed by incubation with von Willebrand factor (vWF) and PECAM (CD-36) as angiogenic markers. Polyclonal rabbit antibodies to both of these markers (alfa-vWF, DAKO; alfa-PECAM, Dr. P.J. Newman) are available. As determined immunohistochemically by the manufacturers, DAKO rabbit anti-human vWF cross reacts with vWF from sheep. This analysis yields information as to overall angiogenesis and vascularization in the ABG, as well as the adjacent skeletal muscle and myocardium. The degree of vascularization was evaluated by counting the number of vessels per unit area.
Preparation of ABG and Its Application to the Muscle Pockets
Autologous cryoprecipitate was prepared from each animal. Each protein preparation was produced under sterile conditions using a standard procedure (AABB Technical Manual , 10th ed, 1990). Briefly, whole blood was obtained from the animal and centrifuged, and the plasma decanted. After freezing for 24 hours at −80°C, the plasma was thawed at 4°C for 4 hours and then centrifuged. The yellowish-white precipitate was collected and stored at −18°C. The resultant cryoprecipitate contained concentrated fibrinogen, factor XIII, fibronectin, and vitronectin. For formation of the fibrin meshwork an FDA approved thrombin preparation (Johnson & Johnson Patient Care Inc, New Brunswick, NJ) was used. When fibrinogen and thrombin are mixed together, the resulting preparation immediately creates a meshwork of fibrin fibers, known as a fibrin clot. Therefore, when the ABG was applied to the tissue surface, two separate syringes were filled with thrombin and the cryoprecipitate, respectively. An equal amount of each compound was applied at the same time. The total glue volume to the muscle pockets ranged from 15 to 20 ml. Aprotinin was added to the ABG in a concentration of 1000 U/ml. Pyrrolostatin was added to the ABG to create a final concentration of 10 μM.
Results
LDM Condition 3 Hours After Subtotal Mobilization
Light Microscopy.
In the proximal portion of the mobilized LDM flap, the specimen was relatively normal except for minimal peripheral eosinophilia. There were granulocytic pavements and, at the edges of the tissue, alternating areas of swollen and shrunken wavy fibers. In the medial portion, scattered fibers showed degenerative changes, primarily swelling, eosinophilia, or basophilic degeneration. In the peripheral portion of the ischemic LDM, the degenerative process was pronounced, with a high proportion of swollen degenerated cells and some progression to basophilic degeneration and necrosis. Visual inspection suggested significantly more leukocyte margination in the periphery than the medial portion of the LDM.
Immunohistochemistry.
Before LDM mobilization, in samples from medial and peripheral portions of the LDM, capillaries occupied 3.99 ± 0.24% of the area (Table 1 ). Three hours after mobilization the number of capillaries per area did not change.
Table 1: Percent of Capillaries Per Area After Latissimus Dorsi Muscle Mobilization (Without Electrical Stimulation)
Transmission Electron Microscopy.
Endothelial cells contained typical cytoplasmic organelle constituencies, and had prominent nucleoli. There were tightly apposed intercellular junctions between endothelial cells; these cells were abluminally enveloped by a clearly visible and continuous basement membrane, and were often encircled by pericytes. Muscle fibril organization appeared normal, and sparse collagen fibers were present in the interstitium. Within hours after mobilization, the endothelial cells within the LDM vasculature began thickening, becoming rounded or cuboidal in shape, and their plasma membranes appeared disrupted, with discontinuous intercellular margins. In severely damaged cells, cytoplasmic vacuolar accumulation, and mitochondrial swelling and destruction of cristae were other characteristics frequently observed in conjunction with gross tissue edema. Interestingly, apparently normal endothelial cells were often observed directly adjacent to the swollen, damaged endothelial cells, suggesting variable responses among these cells to ischemic conditions. All of these early vascular changes were associated with an increased frequency of neutrophil margination and diapedesis out of the affected vessels, supporting their purported critical role in mediating the early sequelae of ischemia-reperfusion. Within capillaries in the ischemic tissue, TEM revealed various stages of leukocyte-endothelium interaction: leukocytes binding to the endothelium; leukocyte destruction of endothelium; and leukocytes leaving capillaries through gaps in the endothelium. In ischemic tissue, there were many leukocytes, macrophages, and mast cells with fibrous deterioration and swelling (Figure 2 ).
Figure 2: Damage to endothelium 3 hours after subtotal LDM mobilization. (A) Leukocytes binding to the endothelium. (B) Leukocyte destruction of endothelium. (C) Leukocytes leaving capillaries through gaps in the endothelium. (D) Swollen, damaged endothelial cells.
LDM Condition 14 and 56 Days After Subtotal Mobilization: Controls Without Pharmaceutical Support or ABG
The condition of the LDM was examined 14 and 56 days after subtotal mobilization without pharmaceutical agents. These investigations were performed on biopsy specimens taken from control pockets in series 1 animals.
Light Microscopy.
Biopsies on days 14 and 56 produced moderate blood oozing and showed no strong connections between the ischemic LDM flap and the adipose tissue. Leukocyte margination, present on day 14, was absent on day 56. In specimens obtained on both days 14 and 56, various stages of necrosis were discernible; the muscle appeared damaged and edematous. By day 56, some muscle fibers had developed a wrinkled appearance. These changes were more evident in the distal part of the LDM, as compared with the medial section.
Immunohistochemistry.
On day 14, capillaries occupied 3.0 ± 0.9% of the area in ischemic tissue (p > 0.05 vs. control). On day 56, there was no statistically significant change in the fractional area occupied by capillaries between ischemic tissue (3.45 ± 0.26%, p > 0.05 vs. control) and nonischemic tissue (4.12 ± 0.29%, p > 0.05 vs. control). Diameter of the vessels was 44 ± 12μm (Table 1 ).
Transmission Electron Microscopy.
On day 14, there was considerable destruction of a large fraction of the muscle tissue as well as the microcirculation. Endothelial intercellular junctions were disrupted, and the vast majority of the capillary bed appeared necrotic. On day 56, essentially all of the remaining endothelial cells exhibited morphologic degeneration of their intracellular components and alteration of their plasma membranes, characterized by a large frequency of cytoplasmic projections extending into the capillary lumen (Figure 3A ).
Figure 3: Endothelium in the distal part of the LDM 56 days after subtotal mobilization. (A) Without pharmaceutical support, endothelial cells exhibited morphologic degeneration of their intracellular components, cytoplasmic projections extending into the capillary lumen. (B) With ABG and aprotinin: well preserved endothelium. (C) With ABG and pyrrolostatin: well formed new capillaries in the interlayer between ischemic and nonischemic muscle.
Influence of Pharmaceutical Support on the Subtotal Mobilized LDM
After identifying the prominent pathologic events in LDM degeneration due to mobilization, vascular insufficiency, and muscle disuse, we began testing the efficacy of the autologous biologic glue and pharmaceutical agents (aprotinin and pyrrolostatin) in prevention of muscle damage and protection of its angiogenic potential.
Light Microscopy.
There was considerable bleeding from the pocket with only ABG. Strong adhesions had grown through the adipose tissue connecting the ischemic and nonischemic layers of LDM by day 14. Leukocyte margination on day 14 was considerably less than at 3 hours after mobilization. Occasional fiber degeneration was also noted on day 14. Fibrosis and calcified necrosis were less than in control pockets. These changes were also noted in some samples on day 56 (series 1). The same intense bleeding and strong adhesions were present in pockets containing aprotinin and pyrrolostatin (series 2). Granulation tissue invaded from the ischemic part of the LDM into the interface. We found no significant morphologic differences between pockets treated with aprotinin and those treated with pyrrolostatin.
Immunohistochemistry.
With only ABG application (series 1) on day 14, the area occupied by capillaries was 4.1 ± 0.41% in the ischemic muscle (p > 0.05 vs. control) and there were numerous small capillary structures. By day 56, this had increased to 5.57 ± 0.24% (p < 0.05 vs. control). The diameter of the vessels was 49 ± 14 μm (p > 0.05 vs. control). With ABG and aprotinin application (series 2) new vascular structures (i.e., an increased number of capillaries) revealed considerable neovascularization in the ischemic tissue. On day 14, the area occupied by capillaries was 5.21 ± 0.28% (p < 0.05 vs. control). By day 56, this had increased to 8.47 ± 0.72% (p > 0.05 vs control and day 14). Many vessels had diameters larger than 50 μm (75 ± 11μm) (p < 0.05 vs. control). When pyrrolostatin was added to ABG (series 2), the numbers of capillaries on days 14 and 56 were 6.03 ± 0.46% and 9.40 ± 1.24% (p < 0.05 vs. control), respectively (Table 1 ).
Transmission Electron Microscopy.
When glue was applied, as mentioned previously, this fibrin meshwork served as an interlayer between the ischemic and nonischemic muscles. In this study it was necessary for us to determine the status of the endothelial cells in the LDM and the newly formed capillaries found in this interlayer. On day 56, the majority of the capillaries in the LDM were healthy. There was no morphological degeneration of the intracellular components. In the fibrin interlayer, there were many new endothelial cells, which appeared normal in morphology and ultrastructure, especially when aprotinin or pyrrolostatin was added (Figure 3b and c ). In addition to endothelial cells, a variety of other cell types, including fibroblasts, smooth muscle cells, pericytes, and occasional mast cells and leukocytes were seen and likely played an important role in the development of this matrix into a functional tissue. In several sections, longitudinal views of capillaries consisting of three or four tightly adjoined endothelial cells were visible, and these often contained erythrocytes within their lumen, implying their ability to transport oxygen and nutrients to the surrounding tissue.
Influence of Electrical Stimulation on the Subtotally Mobilized LDM
In our preliminary investigation, 27
Light Microscopy.
After 56 days of electrical stimulation at 60 CPM, there were dramatic changes in muscle morphology. Most of the muscle fibers in the distal part of the LDM appeared swollen and shrunken. Some of the muscle was necrotic and calcified. Using 30 CPM, the degenerative process was less evident; however, there was still a high proportion of swollen degenerative cells with some progression to basophilic degeneration. There were also many shrunken wavy fibers. The morphologic state of the LDM was not aggravated using 15 CPM. Although there was some occasional fiber degeneration and fibrosis in the distal region of the LDM, there was no necrosis, calcification, or shrunken wavy fibers.
Immunohistochemistry.
As we have shown in series 2, if mobilized LDM was left in situ without electrical stimulation or pharmacologic treatment, it did not recover completely after 56 days, especially in the distal component. Capillaries occupied only 3.45 ± 0.26% of the area, compared with 3.99 ± 0.24% in the control nonmobilized muscle. Conventional electrical stimulation with 30 CPM did not help to establish the capillary network in the muscle, but actually decreased its density, to 3.02 ± 0.39%. The situation at 60 CPM is dangerous because the percent area occupied by capillaries decreased to 2.15 ± 0.70%. Muscle subjected to 15 CPM had a percent area occupied by capillaries of 5.01 ± 0.56%, which is statistically better than in control muscle (p < 0.05) (Table 2 ). These data confirmed that a carefully applied regimen of stimulation may be beneficial to ischemic skeletal muscle.
Table 2: Percent of Capillaries Per Area After Latissimus Dorsi Muscle Mobilization (With Electrical Stimulation)
Transmission Electron Microscopy.
With a regimen of 30 CPM, many endothelial cells showed morphologic degeneration, with cytoplasmic projections into the capillary lumen. Simultaneously, there were many normal endothelial cells without degeneration of intracellular components. With a regimen of 60 CPM, the majority of endothelial cells were damaged, cuboidal in shape, with discontinuous and disrupted plasma membranes. At 15 CPM, endothelial cells appeared normal in ultrastructure without the degeneration of intracellular components (Figure 4 ).
Figure 4: Endothelium in the distal part of the LDM 70 days after subtotal mobilization and 56 days after beginning of electrical stimulation. (A) Electrical stimulation at 60 CPM: endothelial cells with discontinuous and disrupted plasma membranes. (B) Electrical stimulation at 30 CPM: morphologic degeneration of endothelial cells with cytoplasmic projection into capillary lumen. (C) Electrical stimulation at 15 CPM: normal endothelial cell.
Influence of Combined Electrical Stimulation and Pharmaceutical Support on the Subtotally Mobilized LDM In Situ
In this investigation we combined application of ABG and aprotinin with electrical stimulation of the mobilized LDM at 15 CPM (series 6) and 30 CPM (series 7). We used 30 CPM because this is the conventional rate of stimulation now used for cardiomyoplasty, and 15 CPM because this seemed to be the most beneficial rate, according to data reported previously 28
Light Microscopy.
When 30 CPM and ABG application were used, there were fewer swollen cells with evidence of degeneration, and there were only a few shrunken wavy fibers. When 15 CPM and glue were used, the muscle appeared normal without any evidence of necrosis, calcification, shrunken wavy fibers, or fat deposits.
Immunohistochemistry.
When 30 CPM and ABG with aprotinin were used, the area occupied by capillaries was 4.08 ± 0.40%. This area was less than when only glue was applied (no electrical stimulation) (p < 0.05), but greater than when only electrical stimulation was applied at the same rate (no glue) (3.02 ± 0.39%, p < 0.05). When 15 CPM was used along with the ABG and aprotinin, the area occupied by capillaries was 6.01 ± 0.66% (Table 2 ). This was greater than with electrical stimulation only (5.01 ± 0.56), but statistically insignificant (p > 0.05). These results are very good, but the number of capillaries did not achieve the level attained when ABG with aprotinin was applied to the LDM without electrical stimulation (8.47 ± 0.72%).
Transmission Electron Microscopy.
With 30 CPM, most of the capillaries and endothelial cells had no morphologic degeneration, and the ultrastructure seemed normal. However, a few endothelial cells had altered plasma membranes. With 15 CPM and glue application, a majority of the capillaries were healthy. The endothelial cells were tightly adjoined.
Discussion
Skeletal Muscle Flap Ischemia
As a promising surgical treatment of pre-end-stage heart failure, 6–8 9 i.e., augmentation of systolic function), from indirect myocardial revascularization (i.e., augmentation of the blood supply), or from all three factors. Enhancement of contractile performance of a weak cardiac muscle requires a healthy and strong LDM with an active angiogenic potential that can provide indirect myocardial vascularization.
There are reports of a correlation between lack of improvement in left ventricular ejection fraction and muscle flap ischemia after cardiomyoplasty. 17 29 30 17 31,32
In our studies, we found that 3 hours after mobilization, the distal region of the flap was markedly cyanotic and the medial region slightly cyanotic in all sheep. Significant portions of muscle fibers were swollen and eosinophilic with a high proportion of degenerated cells, some having progressed to basophilic degeneration and necrosis. Ideally, after a period of vascular delay (approximately 2 weeks), adequate revascularization should take place. 22 in situ muscle) (p > 0.05); these data were statistically insignificant, however. It is very important to note that even 2 months later the LDM had not recovered completely from ischemic shock. Various stages of muscle necrosis were discernible, and the muscle appeared damaged and edematous. The percent of capillaries per area was still less than in control muscle (3.45 ± 0.26%). It is doubtful that this compromised muscle would contract adequately.
Ischemia, Reperfusion, and Endothelial Cells
Tissue damage also is worsened when reperfusion occurs after periods of arrested blood flow (as little as 2 to 3 hours), because the cells that have remained viable can be injured by readmission of blood and oxygen. This sudden restoration of blood flow to previously ischemic tissue can lead to irreversible tissue injury, via a process termed the “oxygen paradox” phenomenon. 33 34 35 36 37
The accumulation and activation of adhesion molecules on endothelial cells causes leukocyte-endothelial cell interaction and coagulation. 38
Autologous Biologic Glue
Autologous biologic glue effectively stops bleeding by forming a layer around the traumatized LDM, thereby protecting it from severe damage. On day 14 after mobilization we noted considerably less leukocyte margination, fibrosis, and calcified necrosis in LDM tissue treated with ABG than in untreated ischemic LDM tissue.
Our study also showed that ABG helped to initiate angiogenesis in the interlayer between the ischemic and nonischemic tissue. TEM showed newly formed capillaries by day 56 in the adipose interlayer in ABG-treated pockets. It is important to note that these capillaries grew to the adipose interlayer only from the ischemic portion, not from the nonischemic portion of the LDM. In pockets without ABG, there was no capillary growth from either ischemic or nonischemic LDM. However, we cannot assert that capillaries will never grow from ischemic or nonischemic LDM into the interlayer, because 56 days may be too short a time for this process when angiogenic accelerants are not applied.
Proteinase Inhibitor Protects Against Endothelial Cell Damage
A key event in ischemic muscle damage, proteinase release from leukocytes, may inhibit the active healing process (including angiogenesis), so it seems logical to expect that utilizing proteinase inhibitors may prevent some undesirable leukocyte effects. Aprotinin, a natural inhibitor of serine proteinase, may represent the ideal inhibitor in preventing proteolytic degradation. 39,40 41
In our investigation, light microscopy showed that muscle tissue looked healthier when aprotinin was applied to the mobilized LDM by means of ABG. Although some samples showed degenerative changes, these were considerably less than in ischemic samples from the control group.
The suggested mechanism leading to endothelial cell swelling and detachment, to increased vascular permeability, and to microvascular obstruction by detached cells and cellular debris is proteolytic digestion of endothelial basement membranes by migrating neutrophils. When aprotinin was added to the ABG, we noted considerably decreased damage of the endothelium and muscle tissue.
ABG plus aprotinin also invigorated the process of neovascularization, represented by an increased number of capillaries, and as reflected by the evident vascular structures (most capillaries were > 50 μm in diameter). TEM showed well formed capillaries in the interlayer between ischemic and nonischemic muscle.
Electrical Stimulation and Muscle Ischemia
Different regimens of electrical stimulation may be factors that damage ischemic muscle, or may be beneficial factors that accelerate muscle recapillarization and angiogenesis. If contractions at a rate of 60 CPM are damaging to ischemic muscle, a cautious contraction rate of 15 CPM may improve the morphologic state of the muscle and increase the number of capillaries. Rates of 30 or 60 CPM cause serious damage to the endothelial cells by degenerating intracellular components. Cells in this situation cannot be a resource for angiogenesis. It is important to note that when a slower rate of contraction is used, the endothelial cells appeared normal, without degeneration of intracellular components. Our results allow us to strongly recommend a change in the conventional electrical stimulation protocol used for cardiomyoplasty from a cardiosynchronization rate of 1:2 (approximately 30–40 CPM) to 1:4 (approximately 15–20 CPM).
At this time we do not clearly understand the process of muscle transformation in elderly patients with chronic cardiac failure. The risk of muscle damage in such patients is particularly high. We believe that conventional muscle cardiosynchronized contraction in a 1:2 mode may be too damaging and propose that future stimulation should be in a 1:4 synchronization regimen. In ten patients 1 to 2 years after cardiomyoplasty we performed hemodynamic testing using different ratios of electrical stimulation at the Bakulev Institute of Cardiovascular Surgery in Moscow. 8 2 was obtained using a 1:4 ratio, and 4.1 ± 0.9 L/min/m2 was obtained using a 1:2 ratio. Cardiac output was 4.7 ± 0.5 L/min and 4.4 ± 0.5 L/min, respectively; and stroke index was 51.0 ± 435 ml/m2 and 47.2 ± 3.8 ml/m2 , respectively. Thus, changing the electrical stimulation ratio from 1:2 to 1:4 may be beneficial, especially in elderly patients, and for the long-term preservation of the latissimus dorsi muscle during conditioning for cardiac assistance.
Our hope in this study was to establish an electrical stimulation training regimen that would not aggravate the ischemic state of the muscle. Data from recent studies demonstrated that the LDM suffers not only from ischemic injury during mobilization, but also from the ensuing rest period. 42 et al. 43
Conclusions
There is severe ischemia-reperfusion damage and LDM degeneration after mobilization, especially in the peripheral, working portion of the muscle. Endothelial cells are the first to be injured after mobilization, and, because they have no reserves, they must be protected. Angiogenic potential is severely damaged and must be restored for muscle survival and for future indirect myocardial revascularization. To decrease postmobilization ischemia-reperfusion damage to the LDM, autologous biological glue may be applied to the muscle. To accelerate the muscle’s angiogenic potential, pharmaceutical agents (aprotinin or pyrrolostatin) may be added to the glue.
Conventional electrical stimulation at 30 CPM (1:2 ratio) depresses the angiogenic potential. Electrical stimulation at 60 CPM (1:1 ratio) is dangerous for ischemic muscle. Electrical stimulation at 15 CPM (1:4 ratio), however, prevents capillary damage and accelerates angiogenesis. Both ABG application and electrical stimulation at 15 CPM (1:4 ratio) will prevent the LDM from postmobilization damage, increase angiogenic potential, and improve contractile performance.
Acknowledgment
The authors acknowledge the technical assistance of Brian Miller and Brian Schurer in the preparation of the figures for this manuscript.
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