Muscle necrosis has been frequently observed in cardiomyoplasty patients and in experimental animal studies. The purpose of this study was to determine if heat shock could provide protection to skeletal muscle as has been shown in cardiac muscle. A 15-minute heat shock at 42 degrees C resulted in an immediate increase in HSP72 mRNA and was followed within 3 hours by a two-fold increase in HSP72. Surgical dissection of the latissimus dorsi muscle (LDM) followed by an ischemic period resulted in a two-fold increase in HSP72 in control LDM, whereas the already high levels in the heat-shocked LDM increased only slightly with surgery and ischemia. Citrate synthase activity and tissue histology indicated that heat shock did not protect the LDM from the imposed surgical trauma and ischemia insults used in this study.

Muscle soreness, serum creatine kinase (CK), and serum and urinary hydroxyproline levels were examined following muscle over-exertion and resistance training. Seven untrained men performed high intensity leg-extension exercises at a resistance at which they could initially accomplish 90% of their 10 repetitions maximum (10 RM), for 30 minutes to induce muscle over-exertion. In Phase 1 a single bout of muscular over-exertion was followed by a week of rest. Phase 2, the training period, was a repeat of the over-exertion workload of Phase 1 for five consecutive days. Phase 3 was a single bout of over-exertion at a higher 10 RM workload. Normo-responsive and hyper-responsive changes in serum CK were exhibited by different subjects in Phase 1. Muscle soreness was perceived by both groups but to a greater extent in the hyper-responsive group. Serum CK and muscle soreness values were lower during Phase 2 and 3 than in Phase 1. Neither serum nor urinary hydroxyproline levels changed significantly post-exercise. These findings show that a single bout of intense exercise and a brief period of muscular training reduces serum CK and muscle soreness responses following a subsequent single bout of exercise at a higher intensity.

To meet the demand for an easy and accurate method to identify fast and slow skeletal muscle fiber types in the ongoing clinical trial of cardiomyoplasty, we developed protocols of tissue preparation and immunohistochemical staining using monoclonal antibodies to MHC-I and II. A range of enzymatic and antigen recovery solutions were tested, and an optimal procedure was established for each antibody. Enzyme digestion with 20 μg/ml of proteinase K at 42°C for 30 min was most suited to unveiling MHC-I and Target Unmasking Fluid gave the best results for MHC-II. The staining results were comparable to conventional ATPase staining and immunostaining on unfixed cryostat sections. The modified procedures used in a double irnmunostaining protocol allowed simultaneous demonstration and distinction of the 2 fiber types on the same tissue section. This approach provided the centers, participating in the ongoing cardiomyoplasty clinical trial, a uniform and easy method to assess muscle fiber types and facilitate standardization of tissue protocols and cross-laboratory result comparison. It canilso be used in other experiments requiring identification-of the 2 fiber types of skeletal muscles. (The J Histotechnol 21:19, 1998)

We studied myocardial Ca2+ cycling during cardiopulmonary bypass and cold-blood cardioplegia (CPB/CBC) in patients with coronary heart disease undergoing coronary artery bypass grafting. Right atrial biopsies were taken from 13 patients before and after CPB/CBC: after pericardiotomy, immediately after aortic cross-clamp removal, and following termination of CPB/CBC. Changes in ionized Ca2+ concentration (nM) were monitored with indo 1 during Ca2+ uptake and Ca2+ release by sarcoplasmic reticulum in a medium containing 1% homogenized myocardium. Ryanodine inhibition was used to estimate Ca2+ release channel activity. With CPB/CBC, the initial Ca2+ concentration of reaction media increased 33%, (962 +/- 150 to 1262 +/- 106 nM; mean +/- SD). Ca2+ cycling increased asymmetrically, 108% for Ca2+ uptake (3.91 +/- 1.32 to 8.15 +/- 3.17 nM/s), 197% for Ca2+ release (0.90 +/- 0.80 to 2.73 +/- 1.13 nM/s), and 68% for the ratio of Ca(2+)-release to Ca(2+)-uptake activities (0.22 +/- 0.14 to 0.37 +/- 0.13). The dissociation constant of the Ca2+ pump for Ca2+ was unaltered by CPB/CBC (289 +/- 76 nM). During the time period that was studied post-bypass, Ca(2+)-pump activity remained increased, although the Ca(2+)-channel activity returned to pre-bypass values (all p < 0.05). We conclude that CPB/CBC produces increased myocardial Ca2+ load, twofold increased Ca2+ uptake, and threefold increased Ca2+ release by sarcoplasmic reticulum.

Stress-induced regulation of the 72 kD heat shock protein (HSP 72), the major stress inducible protein in mammalian cells, is mediated by the activation and binding of a heat shock transcription factor (HSF) to a specific sequence in the 5′ region of the promoter termed the heat shock element (HSE). In agreement with this regulation, HSP 72 is absent in most cells under unstressed conditions but is rapidly synthesized following exposure to protein damaging stressors. An exception is the skeletal muscle, where HSP 72 is constitutively expressed in muscles that express the beta myosin heavy chain (β-MHC) protein. Since β-MHC is also expressed in the ventricles of large mammals, we have examined if HSP 72 was also constitutively expressed in β-MHC positive hearts. Chambers of the heart muscle from Yorkshire swine were examined for α-MHC, β-MHC and HSP 72 content. HSF:HSE activation was also assessed by gel shift analyses. In the swine heart, atria and ventricles differed in their α-MHC and β-MHC protein content but all expressed a high HSP 72 content. Gel shift analyses demonstrated no HSF:HSE binding in extracts from unstressed swine hearts. These results indicate that HSP 72 is constitutively expressed in all portions of the swine heart and this expression may not be dependent on an HSF:HSE interaction.

Background: Dynamic cardiomyoplasty has been shown to result in muscle damage and necrosis. The purpose of this study was to compare the effects of intermittent versus continuous stimulation on the latissimus dorsi muscle (LDM) with and without surgical dissection. Methods: Surgically dissected and nondissected goat LDM were either stimulated continuously (24 hours/day) or intermittently (16 hours on/8 hours off/day) for 60 to 75 days. Results: The findings show that the continuous stimulation induced the most complete transformation of myosin isoforms and Ca2 pump isoforms in the nondissected LDM, whereas, intermittent stimulation resulted in less muscle damage in the surgically dissected LDM. Intermittent stimulation of the dissected LDM resulted in larger fiber areas and a lower connective tissue concentration than did the continuous stimulation. Conclusions: It was concluded that surgically dissected muscle responds differently to electrical stimulation and that intermittent stimulation may result in less damage while transforming the LDM pedicle for use in cardiomyoplasty.

This study determined effects of surgical dissection and chronic stimulation on degeneration of the latissimus dorsi muscle (LDM), the muscle used for contractile assistance in cardiomyoplasty. LDMs from 10 goats were allocated into four groups: N-LDM (normal), D-LDM (dissected muscle and collateral vessels ligated, muscle remained in original anatomic location), S-LDM (electrically stimulated for 65-75 days), and DS-LDM (dissected and stimulated). S-LDM had nearly a complete transformation to type I fibers throughout the lengths of the muscle. Both groups of dissected muscles (D-LDM and DS-LDM) showed lesser transformation and significant damage. Type I myosin heavy chain and citrate synthase activity were less in the distal compared with the proximal LDM. Morphology of the N-LDM and S-LDM was normal, whereas dramatic morphological abnormalities were observed in the D-LDM and DS-LDM, including lipid-containing ghostlike fibers, atrophied and hypertrophied fibers within the same fascicle. In conclusion, muscle degeneration associated with the cardiomyoplasty procedure was caused by surgical dissection, which was exacerbated by chronic stimulation but was not caused by stimulation alone.

Cardiomyoplasty has the potential to become an alternative therapy for congestive heart failure patients and is presently in Phase III clinical trials. In experimental studies, it is necessary to use an animal with muscle characteristics that resemble those of humans. Therefore, the purpose of this study was to compare morphological and biochemical characteristics of the latissimus dorsi muscle (LDM) of three common large mammals with those of human. Of the three mammals studied, the goat had the most overall similarities to the human when comparing mitochondrial capacity, percent fiber types, fiber areas, myofibrillar (MF)-AT-Pase activity, and 72-kDa heat shock protein (HSP) content. The pig was dissimilar to the human in its fiber-type arrangement, glycolytic capacity, percent fiber type, MF-ATPase activity, and HSP-72 content. The dog differed from the human in that it had high-mitochondrial enzyme activity, a fiber-type profile consisting of all high-aerobic fibers, and fiber cross-sectional areas that were nearly half those of humans. These findings show that the LDM of the goat most resembles that of the human.

BACKGROUND:Cardiomyoplasty surgery has been shown to be associated with damage and degeneration of the assisting skeletal muscle. The purpose of this study was to use ischemic (short-term) and thermal (long-term) preconditioning to protect the muscle during surgery and the subsequent ischemia.METHODS:Three 10-minute cycles of ischemia-reperfusion were accomplished noninvasively on goat latissimus dorsi muscle (LDM) immediately prior to surgery. In another experiment, LDM was noninvasively heat shocked for 20 minutes at 42 degrees C 24 hours prior to surgery. LDM damage was evaluated 5 days postsurgery using enzyme activities (beta-glucuronidase, beta-GLN; citrate synthase), hydroxyproline, morphology, and blood flow.RESULTS:The lysosomal enzyme, beta-GLN, was significantly increased (43%, p < 0.05) by surgical dissection and remained high in the ischemic preconditioned LDM (58%, p < 0.05) and in the heat shocked LDM (57%, p < 0.05).CONCLUSION:These findings show that these two protective protocols do not reduce the muscle damage that occurs during surgical preparation of the LDM for cardiomyoplasty.

Latissimus dorsi muscle (LDM) regional blood flows were determined in anesthetized goats by using colored microspheres under noncontracting and contracting conditions, either before or after 8-10 wk of chronic muscle stimulation. Surgical dissection of the LDM, leaving only the thoracodorsal artery to supply the muscle, did not alter regional noncontracting blood flows but significantly reduced the normal hyperemic response to muscle contraction in muscle regions (posterior-medial) furthest from the entrance of the thoracodorsal artery. Eight to 10 wk after acute muscle dissection, posterior-medial hyperemic flows were restored. Chronic stimulation of the LDM for 8-10 wk, in either dissected or nondissected muscles, did not alter regional blood flows in noncontracting muscle; however, it significantly reduced hyperemic flows in all muscle regions, although capillary density was increased and the muscle was transformed into a predominantly type I fiber type. These results, coupled with data from previous experiments, suggest that the muscle damage observed in the posterior-medial regions of the LDM after surgical dissection and chronic stimulation may be related to reduced hyperemic flow responses caused by surgical isolation of the muscle.

BACKGROUND:Dynamic cardiomyoplasty has shown promise as a surgical treatment for congestive heart failure, however, skeletal muscle damage has been reported in the latissimus dorsi muscle flap. Possible etiologies for the muscle damage include surgical dissection of the latissimus dorsi muscle with interruption of collateral blood supply, as well as chronic electrical stimulation of the muscle.METHODS:To investigate these possible etiologies, we conducted a series of experiments using the goat model, evaluating blood flow and muscle morphology following surgical dissection and chronic stimulation of the latissimus dorsi muscle. Four different conditions were evaluated: (1) latissimus dorsi muscle that was neither dissected nor chronically stimulated; (2) latissimus dorsi muscle that was stimulated, but not dissected; (3) latissimus dorsi muscle that was surgically dissected, but not chronically stimulated; and (4) latissimus dorsi muscle that was both surgically dissected and chronically stimulated.CONCLUSION:We concluded that skeletal muscle damage resulted primarily from the surgical dissection, whereby the collateral blood supply to the latissimus dorsi muscle was interrupted and not primarily as a result of chronic electrical stimulation.

1Deborah Research Institute, 2Browns Mills, NJ, and Laboratoire de Génétique Cellulaire et développementale, Université Laval, Ste Foy, Québec, Canada.

The inducible isoform of the 70-kDa heat shock protein (HSP) family, HSP 72, has been shown to protect cells from protein-damaging stressors and has been associated with myocardial protection. Because exercise is capable of increasing HSP 72 content, we determined whether exercise induction of HSP 72 also provided myocardial protection. Twenty-eight rats (n = 7 per group) were divided into control, heat-shocked (15 min at 42 degrees C), and two exercised groups. Exercise consisted of either one or three bouts (on 3 consecutive days) of treadmill running for 60 min at 30 m/min. Twenty-four hours after heat shock or exercise, hearts were placed on a Langendorff apparatus and subjected to 30 min of global ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP), maximal rate of contraction and relaxation (+/- dP/dt, respectively), coronary flow, catalase activity, and HSP 72 content were determined. During reperfusion, hearts from heat-shocked animals and animals subjected to three bouts of exercise recovered a greater percentage of preischemic LVDP and +/- dP/dt compared with controls or animals that exercised only once. Compared with hearts from controls, HSP 72 content was significantly elevated in the hearts of heat-shocked animals and in animals subjected to three bouts of exercise, but not in animals that exercised only once. These results suggest that exercise induction of HSP 72 can confer an enhanced postischemic recovery and may explain, at least in part, the myocardial protection associated with exercise.

Stress-induced transcriptional regulation of the heat-shock proteins (HSP) is mediated by activation and binding of the heat-shock transcription factors (HSF) to the heat-shock element (HSE). Given the similarities between the stressors known to activate the HSF in cultured cells and the physiological stresses known to occur during exercise, HSF activation was examined in the hearts from exercising animals. Sprague-Dawley rats (5 rats/group) were run on a treadmill (24 m/min) for either 0, 20, 40, or 60 min or to exhaustion (102 +/- 7 min). Protein extracts were assessed for HSF activation by mobility-shift gels. Extracts from the hearts of nonrunning rats demonstrated no HSF activation, whereas HSF activation was detected in 80% of the hearts from animals that run for at least 40 min. These results demonstrate that treadmill running is capable of activating the HSF and increasing 70-kDa HSP mRNA in the rat myocardium.

The major mammalian stress-inducible protein, heat shock protein 72, protects cells from certain stresses and rapidly accumulates in cells after ischemia. Heat shock protein 72 is rapidly synthesized in the myocardium of various species in response to ischemia, but it has not been investigated in human heart. To determine if heat shock protein 72 accumulated in the ischemic myocardium of patients undergoing cardiac operations, we obtained sequential right atrial biopsy specimens from 12 patients undergoing repair at three intervals: before bypass, after reperfusion, and after bypass. Immunoblot analysis for heat shock protein 72 demonstrated a high expression in the human heart compared with other mammalian hearts, p(Binomial) = 0.01. Compared with before bypass, heat shock protein 72 contents after reperfusion and after bypass were 98.2% +/- 8.9%, p(signed-rank) = 0.65, and 87.6% +/- 17.1%, p(signed-rank) = 0.28, respectively. Although heat shock protein 72 concentration was unchanged in hearts after reperfusion and after bypass, the initial prebypass level of heat shock protein 72 was high. The high heat shock protein 72 level detected in human hearts may reflect preoperative disease and drug therapy, or inherently high levels may be usual in the human myocardium. These findings indicate that the myocardium of patients undergoing cardiac operations contains relatively high concentrations of heat shock protein 72, which are not increased during the surgical procedure.