Compensatory up-regulation of cardiac SR Ca2+-pump by heat-shock counteracts SR Ca2+-channel activation by ischemia/reperfusion.

We tested the hypothesis that heat-shock protected myocardial Ca2+-cycling by sarcoplasmic reticulum from ischemia and reperfusion (I/R) injury. Twenty-four hours after increasing body temperature to 42 degrees C for 15 min, rat hearts were isolated, Langendorff-perfused, and subjected to 30 min ischemia then 30 min reperfusion. Left ventricles were homogenized and their ionized Ca2+ concentration monitored with indo- during Ca2+-uptake in the presence and absence of the Ca2+-release channel (CRC) modulator ryanodine. Tissue content of heat-shock protein 72 (HSP 72) was analyzed. Exposure to I/R resulted in a 37% enhancement of CRC activity but no effect on Ca2+-pumping activity, resulting in 25% decreased net Ca2+-uptake activity. Pre-exposure to heat-shock resulted in a 10-fold increase in HSP 72, and a 25% enhancement of maximal Ca2+-pumping activity which counteracted the effect of I/R on CRC and net Ca2+-uptake activities. This protection of SR Ca2+-cycling was associated with partial protection of myocardial physiological performance. Net Ca2+-uptake activity was correlated with the left ventricular developed pressure and its rate of change. We conclude that one of the mechanisms by which heat-shock protects myocardium from I/R injury is to upregulate SR Ca2+-pumping activity to counteract the enhanced SR Ca2+-release produced by I/R.

Enhancement of calcium cycling by atrial sarcoplasmic reticulum after cardiopulmonary bypass and cardioplegia during open heart surgery.

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.

Regional blood flows in the goat latissimus dorsi muscle before and after chronic stimulation.

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.

Cardiomyoplasty: studies on goat latissimus dorsi blood flow and muscle damage following surgical dissection and chronic electrical stimulation.

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.

Cardiomyoplasty: transformation of the assisting muscle using intermittent versus continuous stimulation.

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.

Cardiomyoplasty: degeneration of the assisting skeletal muscle.

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.

Preservation of the latissimus dorsi muscle during cardiomyoplasty surgery.

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.

Constitutive expression of HSP 72 in swine heart.

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 (beta-MHC) protein. Since beta-MHC is also expressed in the ventricles of large mammals, we have examined if HSP 72 was also constitutively expressed in beta-MHC positive hearts. Chambers of the heart muscle from Yorkshire swine were examined for alpha-MHC, beta-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 alpha-MHC and beta-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.

Cardiomyoplasty: comparison of latissimus dorsi muscles of three large mammals with that of human.

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.

Enhanced postischemic myocardial recovery following exercise induction of HSP 72.

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.

Activation of heat-shock transcription factor in rat heart after heat shock and 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.

Cardiomyoplasty: preservation of the latissimus dorsi muscle.

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.

Heat shock protein (HSP 72) expression in patients undergoing cardiac operations.

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.

Myofibrillar adaptations during cardiac hypertrophy.

The main purpose of this study was to determine the transmural adaptive changes that occur in cell size, myofibrils, and myosin isoforms from the endocardium (ENDO) to the epicardium (EPI) of the left ventricle (LV) of the rat heart during compensatory hypertrophy. Hypertrophy was induced by supra-renal aortic constriction for periods of 2, 7, 15 and 30 days. Percent left ventricular hypertrophy averaged 63 +/- 9.7% at 30 days following constriction. A significant (p < 0.05) transmural gradient in the V3 myosin isoform (9 +/- 0.7% ENDO vs. 5 +/- 1.8% EPI) was initially observed at 7 days and was still evident by 30 days (25 +/- 3.6% ENDO vs 15 +/- 2.0% EPI). Cell cross-sectional area was also greater (p < 0.05) in the ENDO than in the EPI at 7, 15 and 30 days. MF diameter was determined only at 30 days and was found to be similar to control values in both the hypertrophied ENDO (sham 1.24 +/- 0.05 vs hyp 1.18 +/- 0.09 microns) and EPI (sham 1.17 +/- 0.08 vs hyp 1.06 +/- 0.08 microns). The combined effects of cardiac myocyte hypertrophy with no change in MF diameter resulted in a calculated increase of approximately 70% in the number of myofibrils per myocyte both in the ENDO and EPI. It was concluded that the adaptive strategy of the left ventricular free wall to pressure overload was to initially increase myocyte cross-sectional area and then switch myosin expression from V1 to V3, both of which proceeds transmurally from the sub-endocardium towards the sub-epicardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy status of the rapidly paced canine myocardium in congestive heart failure.

Rapid ventricular pacing (RVP) is used as an experimental model of congestive heart failure (CHF). The purpose of this study was to determine the energy status of the dog myocardium after the development of CHF via chronic RVP. The myocardium had a significantly lower (P < 0.05) energy charge (EC) during CHF (0.63 +/- 0.01) than in sham-operated controls (0.82 +/- 0.02). This was due to significant differences in concentrations in ATP (-48%), ADP (29%), and AMP (275%) in the RVP group. However, the total adenine nucleotide pool was not different between groups. Myocardial lactate concentration was also similar. Glycogen was significantly lower (P < 0.05) by 20% at peak CHF. The adenine nucleotides were similar among the different myocardial layers (endo-, mid-, and epicardium). The administration of enalapril (an inhibitor of angiotension-converting enzyme) to decrease vascular resistance had no effect on the myocardial energy status of CHF dogs. These findings suggest that the lower EC in CHF animals is not the result of subendocardial ischemia. Also, lower EC is not associated with endogenous glycogen depletion or increased lactate concentration. The energy status of the myocardium in RVP-induced CHF is unlike that seen in ischemia-induced heart failure. This suggests that CHF in RVP is not vascular in origin.

Cell size of mammalian myocardia is not related to physiological demand.

Morphological characteristics of myocardial ventricular myocytes have been evaluated from 5 mammalian orders with resting heart rates ranging from 51 to 475 bpm. The purpose was to determine if morphological characteristics of the myocardia are related to the functional demand imposed on the cell as represented by the resting heart rate. Cell size is a constant among mammals of different sizes which have different physiological demands. In contrast, there is more mitochondrial area and less myofibrillar area per cell in animals with rapidly beating hearts than in animals with slower heart rates. Additionally, the mean cross sectional area of individual myofibrils is 30% larger in the cow as compared to the mouse. These findings combined with our previous studies indicate that the different functional requirements of myocardia from different mammalian orders are satisfied by intracellular adaptations of both a structural and biochemical nature.

Contractile and calcium regulating capacities of myocardia of different sized mammals scale with resting heart rate.

The purpose of this study was to determine if selected biochemical parameters representing the contractile and calcium regulating systems of cardiac muscle scaled among mammals having inherently different resting heart rates (RHR). Eight mammalian species with RHR ranging from 51 to 475 beats per minute (bpm) were studied. The oxidative capacity of the myocardium is highly correlated with the RHR. The hypothesis of the present study was that the capacities of the energy utilizing processes of contraction and calcium regulation would also be correlated to the functional demand imposed on the muscle as represented by the RHR. Myosin (M) and myofibrillar (MF) ATPase activities, myosin isoenzyme distribution and sarcoplasmic reticulum (SR) ATPase activity were determined. Animals with RHR above 300 bpm express V1 myosin while animals with lower RHR express primarily V3. M and MF ATPase activities correlated with RHR, but the major difference in activities occurred at the 'threshold' RHR of about 300 bpm at which the switch from V3 to V1 appears to occur. SR ATPase activity per mg of microsomal protein was for the most part constant among different mammals, but the SR ATPase activity per g of heart tissue was significantly correlated with RHR as slower beating hearts tended to yield less SR protein per unit mass. We conclude that both the contractile and calcium regulating systems are scaled to the functional parameter of RHR among different mammals. The contractile system uses a slow myosin ATPase isoform at low resting heart rates whereas above the postulated threshold RHR of about 300 bpm a switch in gene expression to a fast myosin ATPase isoform occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of exercise-trained porcine myocardium.

The purpose of this study was to determine whether cardiac biochemical adaptations are induced by chronic exercise training (ET) of miniature swine. Female Yucatan miniature swine were trained on a treadmill or were cage confined (C) for 16-22 wk. After training, the ET pigs had increased exercise tolerance, lower heart rates during exercise at submaximal intensities, moderate cardiac hypertrophy, increased coronary blood flow capacity, and increased oxidative capacity of skeletal muscle. Myosin from both the C and ET hearts was 100% of the V3 isozyme, and there were no differences between the myosin adenosine triphosphatase (ATPase) or myofibrillar ATPase activities of C and ET hearts. Also, the sarcoplasmic reticulum Ca(2+)-ATPase activity and Na(+)-Ca2+ exchange activity of sarcolemmal vesicles were the same in cardiac muscle of C and ET hearts. Finally, the glycolytic and oxidative capacity of ET cardiac muscle was not different from control, since phosphofructokinase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase activities were the same in cardiac tissue from ET and C pigs. We conclude that endurance exercise training does not provide sufficient stress on the heart of a large mammal to induce changes in any of the three major cardiac biochemical systems of the porcine myocardium: the contractile system, the Ca2+ regulatory systems, or the metabolic system.

Competitive control of myosin expression: hypertrophy vs. hyperthyroidism.

The competition between two opposing influences on the phenotypic expression of skeletal muscle myosin were studied to determine which was the dominant regulator. Experimental hyperthyroidism, which induces fast myosin expression, was produced by subcutaneous implantation of a 40-day constant-time-release triiodothyronine pellet. Compensatory hypertrophy, which induces slow myosin expression, was produced by surgical removal of a synergistic hindlimb muscle. Hyperthyroidism increased the percentage of type II fibers and the fast myosin isoforms in both the plantaris and soleus muscles. Hypertrophy significantly increased the percentage of type I fibers and the slow myosin type in the plantaris and soleus muscles. However, with the simultaneous introduction of hyperthyroidism and hypertrophy, only the hyperthyroid effects were observed. Hyperthyroidism and not physiological demand was found to be the dominant regulator of skeletal muscle myosin expression.