Cells under pressure - the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis.

Early osteoarthritis (OA), characterised by cartilage defects, is a degenerative disease that greatly affects the adult population. Cell-based tissue engineering methods are being explored as a solution for the treatment of these chondral defects. Chondrocytes are already in clinical use but other cell types with chondrogenic properties, such as mesenchymal stem cells (MSCs), are being researched. However, present methods for differentiating these cells into stable articular-cartilage chondrocytes that contribute to joint regeneration are not effective, despite extensive investigation. Environmental stimuli, such as mechanical forces, influence chondrogenic response and are beneficial with respect to matrix formation. In vivo, the cartilage is subjected to multiaxial loading involving compressive, tensile, shear and fluid flow and cellular response. Tissue formation mechanobiology is being intensively studied in the cartilage tissue-engineering research field. The study of the effects of hydrostatic pressure on cartilage formation belongs to the large area of mechanobiology. During cartilage loading, interstitial fluid is pressurised and the surrounding matrix delays pressure loss by reducing fluid flow rate from pressurised regions. This fluid pressurisation is known as hydrostatic pressure, where a uniform stress around the cell occurs without cellular deformation. In vitro studies, examining chondrocytes under hydrostatic pressure, have described its anabolic effect and similar studies have evaluated the effect of hydrostatic pressure on MSC chondrogenesis. The present review summarises the results of these studies and discusses the mechanisms through which hydrostatic pressure exerts its effects.

Bone block augmentation from the iliac crest for treatment of deep osteochondral defects of the knee resembles biomechanical properties of the subchondral bone.

PURPOSE: Bone block augmentation from the iliac crest can be used for reconstruction of the osteochondral unit to restore the underlying subchondral bone upon restoration of the cartilaginous layer via matrix-induced chondrocyte transplantation. To critically understand the successful restoration of the defect, biomechanical and histological analysis of the implanted bone blocks is required. The aim of the study was to analyse the ability of the bone block technique to restore huge bone defects by mimicking the physiological subchondral zone. METHODS: The experiments were performed using lateral femoral condyles and iliac crest bone grafts from the same cadavers (n = 6) preserved using the Thiel method. CT scans were made to evaluate bone pathology. Bone mineral density of all specimens was evaluated in the femoral head prior to testing. A series of tests were conducted for each pair of specimens. A static compression test was performed using an electro dynamic testing machine with maximal strength and failure behavior analyzed. Biomechanical tests were performed in the medial-lateral direction for iliac crest and for femoral condyles with and without removal of the cartilage layer. Histological analysis was performed on decalcified specimens for comparison of the condyle at lesion site and the graft. RESULTS: No significant difference in failure load could be found for iliac crest (53.3-180.5 N) and femoral condyle samples upon cartilage removal (38.5-175.1 N) (n.s.). The femoral condyles with an intact cartilage layer showed significantly higher loads (118.3-260.4N) compared to the other groups indicating that native or regenerated cartilage can further increase the failure load (p < 0.05). Bone mineral density significantly influenced failure load in all study groups (p < 0.05). Histological similarity of the cancellous bone in the femoral condyle and in the iliac crest was observed. However, within the subchondral zone, there was a higher density of sponge like organized trabeculae in the bone samples from the iliac crest. Tide mark was only detected at the osteochondral interface in femoral condyles. CONCLUSION: This study demonstrated that, bone blocks derived from the iliac crest allow a biomechanical appropriate and stable restoration of huge bony defects by resembling the subchondral zone of the femoral condyle. Therefore, bone augmentation from the iliac crest combined with matrix-induced autologous chondrocyte transplantation seems to be a reasonable method to treat these challenging injuries.

Timing of anterior cruciate ligament reconstruction within the first year after trauma and its influence on treatment of cartilage and meniscus pathology.

PURPOSE: Anterior cruciate ligament (ACL) ruptures are often associated with primary meniscal and cartilage lesions. Late reconstruction of ACL-deficient knees may increase the risk of developing secondary meniscal and cartilage lesions; hence, the timing of ACL repair is of the utmost importance. Because meniscus outcome is also a potential predictor for osteoarthritis (OA), this study compared ACL repair within the first 6 months after injury to that of surgery conducted 7-12 months after injury with regard to the incidence of meniscal and cartilage lesions. METHODS: This prospective cross-sectional study included all complete isolated primary ACL ruptures treated in our institution within 1 year after trauma over a 12-month period. Exclusion criteria were revision ACL, complex ligament injuries, previous knee surgery, and missing injury data. Cartilage lesions were classified according to the score established by the International Cartilage Repair Society (ICRS score) and meniscal tears according to their treatment options. RESULTS: Two hundred and thirty-three of 730 patients (162 men, 71 women) with ACL repair met the inclusion criteria. 86.3 % of surgical interventions were conducted within 6 months and 13.7 % after 6 months of trauma. Severe cartilage lesions grade III-IV did not significantly differ between the different time points of ACL repair (<6 months 39.9 %; >6 months 31.3 %; p = n.s.). Medial meniscus lesions received significantly higher meniscal repair in early compared to delayed ACL repair. Significantly higher rate of meniscal repair of the medial meniscus was seen in cases of early ACL repair compared to delayed (<6 months 77.2 %, >6 months 46.7; p = 0.022). The rate of medial meniscal repair in early ACL repair was significantly higher for women (89.5-0 %; p = 0.002), however, not for men (73.3-53.8 %; p = n.s.). No differences were found for lateral meniscal lesions, with regard to neither the different time points (p = n.s.) nor the sex (p = n.s.). CONCLUSIONS: Because of the significantly higher rate of prognostically advantageous meniscal repair, the recommendation for an ACL reconstruction within 6 months after trauma was made to preserve the meniscus and reduce the risk of developing OA. LEVEL OF EVIDENCE: Prospective cross-sectional cohort study, Level II.

Non-surgical treatment of pubic overload and groin pain in amateur football players: a prospective double-blinded randomised controlled study.

PURPOSE: The incidence of groin pain in athletes is steadily increasing. Symptomatic pubic overload with groin pain and aseptic osteitis pubis represent well-known and frequently misdiagnosed overuse injuries in athletes. This study investigated the benefits of standardised non-surgical treatment for swift return-to-football. METHODS: In a prospective double-blinded controlled study, 143 amateur football players with groin pain as well as radiological signs and clinical symptoms of pubic overload were analysed for 1 year. Two randomised study groups participated in an intensive physical rehabilitation programme, either with or without shock wave therapy. The control group did not participate in any standardised rehabilitation programme but only stopped participating in sports activity. Follow-up examinations took place 1, 3 months and 1 year after the beginning of therapy. Endpoints were visual analogue scale (VAS), functional tests, the time of return-to-football, recurrent complaints and changes in the MR image. RESULTS: Forty-four football players with groin pain and aseptic osteitis pubis were randomised into two study groups; 26 received shock wave therapy, 18 did not. Clinical examination showed pubic overload as a multi-located disease. Players receiving shock wave therapy showed earlier pain relief in the VAS (p < 0.001) and returned to football significantly earlier (p = 0.048) than players without this therapy. Forty-two of 44 players of both study groups returned to football within 4 months after the beginning of therapy and had no recurrent groin pain within 1 year after trauma. Fifty-one players of the control group returned to football after 240 days (p < 0.001), of whom 26 (51%) experienced recurrent groin pain. Follow-up MRI scans did not show any effect of shock wave therapy. CONCLUSION: Non-surgical therapy is successful in treating pubic overload and osteitis pubis in athletes. Shock wave therapy as a local treatment significantly reduced pain, thus enabling return-to-football within 3 months after trauma. Early and correct diagnosis is essential for successful intensive physiotherapy. LEVEL OF EVIDENCE: I.

Resazurin dye as a reliable tool for determination of cell number and viability in mesenchymal stem cell culture.

Human mesenchymal stem cells are a valuable cell source for tissue engineering. Determination of cell number and viability is crucial. However, this can be tested only at the end of cell culture. This study shows that Resazurin dye staining is a reliable tool for evaluation of cell number and viability in culture without cell perturbation.

Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro.

This study examined the effects of low intensity pulsed ultrasound (LIPUS) on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested for histology, immunohistochemistry, RT-PCR, and quantitative DNA and matrix macromolecule analysis. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. However ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. In contrast, 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. This study indicates that LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs. We have found a dependency on the specific treatment parameters. We hypothesize that LIPUS can be used for an improved in vitro preparation of optimized tissue engineering implants for cartilage repair. Furthermore this non-invasive method could also be of potential use in vivo for regenerative therapy of cartilage in the future.

A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to matrix remodeling in disease states such as tumor metastases. Extracellular matrix metalloproteinase inducer (EMMPRIN) has been reported to increase MMP expression, and membrane-type MMP or MT1-MMP has been implicated to activate MMPs. The present study examined whether and to what degree EMMPRIN and MT1-MMP were expressed in human left ventricular (LV) myocardium as well as the association with MMP activity and expression in dilated cardiomyopathy (DCM). METHODS AND RESULTS: LV myocardial zymographic MMP activity increased by >2-fold with both nonischemic DCM (n=21) and ischemic DCM (n=16) compared with normal (n=13). LV myocardial abundance of MMP-9 was increased with both forms of DCM. MMP-2 and MMP-3 were increased with nonischemic DCM. MMP-1 levels were decreased with both forms of DCM. EMMPRIN increased by >250% and MT1-MMP increased by >1000% with both forms of DCM. CONCLUSIONS: Increased LV myocardial MMP activity and selective upregulation of MMPs with nonischemic and ischemic forms of DCM occurred. Moreover, a local MMP induction/activation system was identified in isolated normal human LV myocytes that was upregulated with DCM. The control of MMP activation and expression in the failing human LV myocardium represents a new and potentially significant therapeutic target for this disease process.

Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target.

A fundamental structural event in the progression of heart failure due to dilated cardiomyopathy is left ventricular (LV) myocardial remodeling. The matrix metalloproteinases (MMPs) are an endogenous family of enzymes which contribute to matrix remodeling in several disease states. The goal of this report is to summarize recent findings regarding the myocardial MMP system and the relation to matrix remodeling in the failing heart. In both experimental and clinical forms of dilated cardiomyopathy (DCM), increased expression of certain species of myocardial MMPs have been demonstrated. Specifically, increased myocardial levels of the gelatinase, MMP-9 has been identified in both ischemic and non-ischemic forms of human DCM. In addition, stromelysin or MMP-3 increased by over four-fold in DCM. The increased levels of MMP-3 in DCM may have particular importance since this MMP degrades a wide range of extracellular proteins and can activate other MMPs. In normal human LV myocardium, the membrane type 1 MMP (MT1-MMP) was detected. These MT-MMPs may provide important sites for local MMP activation within the myocardium. In a pacing model of LV failure, MMP expression and activity increased early and were temporally associated with LV myocardial matrix remodeling. Using a broad-spectrum pharmacological MMP inhibitor in this pacing model, the degree of LV dilation was attenuated and associated with an improvement in LV pump function. Thus, increased LV myocardial MMP expression and activity are contributory factors in the LV remodeling process in cardiomyopathic disease states. Regulation of myocardial MMP expression and activity may be an important therapeutic target for controlling myocardial matrix remodeling in the setting of developing heart failure.

Angiotensin converting enzyme inhibition, AT1 receptor inhibition, and combination therapy with pacing induced heart failure: effects on left ventricular performance and regional blood flow patterns.

BACKGROUND: AT1 receptor activation has been demonstrated to cause increased vascular resistance properties which may be of particular importance in the setting of congestive heart failure (CHF). The overall goal of this study was to examine the effects of ACE inhibition (ACEI) alone, AT1 receptor blockade alone and combined ACEI and AT1 receptor blockade on LV pump function, systemic hemodynamics and regional blood flow patterns in the normal state and with the development of pacing induced CHF, both at rest and with treadmill induced exercise. METHODS AND RESULTS: Pigs (25 kg) were instrumented in order to measure cardiac output (CO), systemic (SVR) and pulmonary vascular (PVR) resistance, neurohormonal system activity, and myocardial blood flow distribution in the conscious state and assigned to one of 4 groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n = 7); (2) ACEI (benazeprilat, 3.75 mg/day) and pacing (n = 7); (3) AT1 receptor blockade (valsartan, 60 mg/day) and rapid pacing (n = 7); and (4) ACEI and AT1 receptor blockade (benazeprilat/valsartan, 1/60 mg/day, respectively) and pacing (n = 7). Measurements were obtained at rest and with treadmill exercise (15 degrees, 3 miles/h; 10 min) in the normal control state and after the completion of the treatment protocols. With rapid pacing, CO was reduced at rest and with exercise compared to controls. ACEI or AT1 blockade normalized CO at rest, but remained lower than control values with exercise. Combination therapy normalized CO both at rest and with exercise. Resting SVR in the CHF group was higher than controls and SVR fell to a similar degree with exercise; all treatment groups reduced resting SVR. With exercise, SVR was reduced from rapid pacing values in the ACEI and combination therapy groups. PVR increased by over 4-fold in the rapid pacing group both at rest and with exercise, and was reduced in all treatment groups. In the combination therapy group, PVR was similar to control values with exercise. Plasma catecholamines and endothelin levels were increased by over 3-fold with chronic rapid pacing, and were reduced in all treatment groups. In the combination therapy group, the relative increase in catecholamines and endothelin with exercise were significantly blunted when compared to rapid pacing only values. LV myocardial blood flow at rest was reduced in the rapid pacing only and monotherapy groups, but was normalized with combination therapy. CONCLUSION: These findings suggest that with developing CHF, combined ACE inhibition and AT1 receptor blockade improved vascular resistive properties and regional blood flow distribution to a greater degree than that of either treatment alone. Thus, combined ACEI and AT1 receptor blockade may provide unique benefits in the setting of CHF.

Dynamic hydrostatic pressure enhances differentially the chondrogenesis of meniscal cells from the inner and outer zone.

This study analyses the influence of dynamic hydrostatic pressure on chondrogenesis of human meniscus-derived fibrochondrocytes and explores the differences in chondrogenic differentiation under loading conditions between cells derived from the avascular inner zone and vascularized outer region of the meniscus. Aggregates of human fibrochondrocytes with cell origin from the inner region or with cell origin from the outer region were generated. From the two groups of either cell origin, aggregates were treated with dynamic hydrostatic pressure (1Hz for 4h; 0.55-5.03MPa, cyclic sinusoidal) from day 1 to day 7. The other aggregates served as unloaded controls. At day 0, 7, 14 and 21 aggregates were harvested for evaluation including histology, immunostaining and ELISA analysis for glycosaminoglycan (GAG) and collagen II. Loaded aggregates were found to be macroscopically larger and revealed immunohistochemically enhanced chondrogenesis compared to the corresponding controls. Loaded or non-loaded meniscal cells from the outer zone showed a higher potential and earlier onset of chondrogenesis compared to the cells from the inner part of the meniscus. This study suggests that intrinsic factors like cell properties in the different areas of the meniscus and their reaction on mechanical load might play important roles in designing Tissue Engineering strategies for meniscal repair in vivo.

Thermodilution right ventricular ejection fraction. Catheter positioning effects.

Right ventricular (RV) ejection fractions have been difficult to estimate clinically. It has been demonstrated recently that RV ejection fractions can be calculated by thermodilution techniques using a rapid response thermistor and computer. This method critically depends on adequate mixing of the thermal bolus and sensing of the rapid response thermistor. This study examined the effects of the thermistor position within the pulmonary artery and injectate site within the right atrium on RV thermodilution ejection fraction measurements. Ten pigs were instrumented with a RV thermodilution catheter in the pulmonary artery, an injectate catheter in the right atrium, an atrial-pacing electrode, and a systemic arterial catheter. The RV ejection fractions were determined using thermodilution in two ways: (1) with incremental increases in pulmonic valve to thermistor distance, and (2) with incremental increases in injectate port to tricuspid valve. These measurements were obtained at a paced rate of 107 +/- 1 beats per minute (bpm) and then repeated with pacing-induced tachycardia (140 bpm). The highest RV ejection fraction with the lowest coefficient of variation was with the thermistor 2 cm from the pulmonic valve (50 +/- 2 percent), with a significant decline from this value at 10 cm (42 +/- 4 percent, p less than 0.05). This reduction in RV ejection fraction values with increased pulmonic valve to thermistor distance became more pronounced with tachycardia where a significant decline in RV ejection fraction occurred at 4 cm from the valve when compared with 0 cm (38 +/- 6 percent vs 47 +/- 3 percent, respectively, p less than 0.05). There was no significant change in RV ejection fraction at any injectate port to tricuspid valve distance at the lower heart rate. With tachycardia, however, a significant decline in RV ejection fraction occurred with the injectate port located 7 cm from the tricuspid valve (p less than 0.05). These results demonstrate that RV ejection fractions can be reliably obtained using thermodilution. Positioning of the thermodilution catheter is an important consideration for obtaining optimal RV ejection fraction measurements. Care should be taken to position the catheter with the thermistor a minimal distance from the pulmonic valve and the injectate port within the central body of the right atrium.

Isolated left ventricular myocyte contractility in patients undergoing cardiac operations.

BACKGROUND: Because of methods required for obtaining isolated left ventricular myocytes, evaluation of the contractile function of isolated left ventricular myocytes in normal human patients has been limited. Accordingly, the goal of the present study was to develop a means to isolate human left ventricular myocytes from small myocardial biopsy specimens collected from patients undergoing elective coronary artery bypass operations and to characterize indices of myocyte contractile performance. METHODS: Myocardial biopsy specimens were obtained from the anterior left ventricular free wall of 22 patients undergoing coronary artery bypass operations. Myocytes were isolated from these myocardial samples by means of a stepwise enzymatic digestion method and micro-trituration techniques. Isolated left ventricular myocyte contractile function was assessed by computer-assisted high-speed videomicroscopy under basal conditions and in response to beta-adrenergic receptor stimulation with isoproterenol. RESULTS: A total of 804 viable left ventricular myocytes were successfully examined from all of the myocardial biopsy specimens with an average of 37+/-4 myocytes per patient. All myocytes contracted homogeneously at a field stimulation of 1 Hz with an average percent shortening of 3.7%+/-0.1% and shortening velocity of 51.3+/-1.3 microm/s. After beta-adrenergic receptor stimulation with isoproterenol, percent shortening and shortening velocity increased 149% and 118% above baseline, respectively (P < .05). CONCLUSION: The unique results of the present study demonstrated that a high yield of myocytes could be obtained from human left ventricular biopsy specimens taken during cardiac operations. These myocytes exhibited stable contractile performance and maintained the capacity to respond to an inotropic stimulus. The methods described herein provide a basis by which future studies could investigate intrinsic and extrinsic influences on left ventricular myocyte contractility in human beings.

Endothelin-1 during and after cardiopulmonary bypass: association to graft sensitivity and postoperative recovery.

OBJECTIVE: Our objectives are 2-fold: (1) to serially measure the release of endothelin and graft-conduit endothelin sensitivity during and after coronary artery bypass grafting and (2) to define potential relationships of changes in endothelin levels to perioperative parameters. METHODS: Endothelin plasma content was measured in patients (n = 105) undergoing bypass grafting from select vascular compartments before operations and at specific intervals up to 24 hours postoperatively. Endothelin sensitivity was determined in isolated internal thoracic artery segments. RESULTS: Systemic arterial and pulmonary arterial endothelin levels were increased by approximately 50% immediately after bypass grafting and increased by another 85% during the first 24 hours postoperatively. Endothelin levels were highest in patients with prolonged ventilatory requirements and extended stays in the intensive care unit (10.2 +/- 0.8 vs 13.2 +/- 1.1 fmol/mL, P =.02, and 9.8 +/- 0.7 vs 13.9 +/- 1.2 fmol/mL, P =.01, respectively. Endothelin sensitivity of the internal thoracic artery was increased in patients requiring prolonged vasodilator support with nitroglycerin. CONCLUSIONS: Systemic and pulmonary arterial endothelin levels remained increased for at least 24 hours postoperatively. Prolonged pharmacologic management and increased intensive care unit stay were associated with increased systemic endothelin release and heightened graft-conduit sensitivity to endothelin.

Pneumoperitoneum to treat air leaks and spaces after a lung volume reduction operation.

Lung volume reduction surgery is an option for the management of end-stage emphysema. The most frequent surgical complication of lung volume reduction is prolonged air leaks. We describe a patient undergoing a lung volume reduction operation complicated by persistent bilateral air spaces with large air leaks. Treatment with recurrent pneumoperitoneum via a peritoneal dialysis catheter along with chemical sclerosis successfully resolved both problems.

Cardiopulmonary bypass in a gravid patient: perioperative changes in endothelin levels.

Transient elevations of the potent vasoconstrictive peptide endothelin have been reported to occur with the institution of cardiopulmonary bypass. We measured plasma endothelin levels in a 24-year-old gravid patient undergoing a mitral valve replacement operation. Plasma endothelin levels increased by more than 250% in the first 24 hours postoperatively and remained elevated above baseline values at 36 hours postoperatively.

Temporal relation of ATP-sensitive potassium-channel activation and contractility before cardioplegia.

BACKGROUND: Pharmacologic treatment using potassium-channel openers (PCOs) before cardioplegic arrest has been demonstrated to provide beneficial effects on left ventricular performance with subsequent reperfusion and rewarming. However, the PCO treatment interval necessary to provide protective effects during cardioplegic arrest remains to be defined. The present study was designed to determine the optimum period of PCO treatment that would impart beneficial effects on left ventricular myocyte contractility after simulated cardioplegic arrest. METHODS: Left ventricular porcine myocytes were assigned randomly to three groups: (1) normothermic control = 37 degrees C for 2 hours; (2) cardioplegia = K+ (24 mEq/L) at 4 degrees C for 2 hours followed by reperfusion and rewarming; and (3) PCO and cardioplegia = 1 to 15 minutes of treatment with the PCO aprikalim (100 micromol/L) at 37 degrees C followed by hypothermic (4 degrees C) cardioplegic arrest and subsequent rewarming. Myocyte contractility was measured after rewarming by videomicroscopy. A minimum of 50 myocytes were examined at each treatment and time point. RESULTS: Myocyte velocity of shortening was reduced after cardioplegic arrest and rewarming compared with normothermic controls (63+/-3 microm/s versus 32+/-2 microm/s, respectively; p < 0.05). With 3 minutes of PCO treatment, myocyte velocity of shortening was improved after cardioplegic arrest to values similar to those of normothermic controls (56+/-3 microm/s). Potassium channel opener treatment for less than 3 minutes did not impart a protective effect, and the protective effect was not improved further with more prolonged periods of PCO treatment. CONCLUSIONS: A brief interval of PCO treatment produced beneficial effects on left ventricular myocyte contractile function in a simulated model of cardioplegic arrest and rewarming. These results suggest that a brief period of PCO treatment may provide a strategy for myocardial protection during prolonged cardioplegic arrest in the setting of cardiac operation.

Beneficial effects of myocyte preconditioning on contractile processes after cardioplegic arrest.

BACKGROUND: Myocardial precondition, which can be achieved through short intervals of ischemia or hypoxia followed by reperfusion, protects the myocardium with subsequent prolonged periods of ischemia. Accordingly, the present study tested the hypothesis that hypoxic preconditioning before cardioplegic arrest would have direct and beneficial effects on myocyte contractile processes with reperfusion. METHODS: Left ventricular porcine myocytes (n = 335) were randomly assigned to one of three treatments: normothermia, maintained in cell media (37 degrees C, 2 hours); cardioplegia, hyperkalemic arrest (24 mEq K+, 4 degrees C, 2 hours) followed by normothermic reperfusion; preconditioning, hypoxia (20 minutes) and reperfusion (20 minutes), and then followed by cardioplegic arrest and rewarming. Myocyte velocity of shortening was measured using computer-assisted videomicroscopy at baseline and with beta-adrenergic receptor stimulation with isoproterenol (25 nmol/L). RESULTS: In the cardioplegia group, myocyte function was reduced at baseline (22 +/- 1 versus 57 +/- 2 microns/s) and with beta-adrenergic receptor stimulation (81 +/- 5 versus 156 +/- 7 microns/s) compared to normothermic controls (p < 0.05). Preconditioning improved myocyte function at baseline (38 +/- 2 microns/s) and with beta-adrenergic receptor stimulation (130 +/- 6 microns/s) compared to the cardioplegic alone group (p < 0.05). CONCLUSIONS: The important findings from this study are twofold. First, preconditioning can be induced directly at the level of the myocyte, independent of nonmyocyte populations and extracellular influences. Second, myocyte preconditioning provides protective effects on myocyte function and beta-adrenergic responsiveness after cardioplegic arrest and rewarming. These findings suggest that preconditioning may provide a novel approach in protecting myocyte contractile processes during cardioplegic arrest.

Differential effects of calcium channel antagonists in the amelioration of radial artery vasospasm.

BACKGROUND: Radial artery (RA) is being used for coronary artery bypass grafting (CABG) with greater frequency. However, RA is prone to post-CABG vasospasm, which may be neurohormonally mediated. Use of the calcium channel antagonist diltiazem has been advocated as a strategy to reduce post-CABG RA vasospasm. However, whether and to what degree different calcium channel antagonists influence neurohormonally induced RA vasoconstriction remains unknown. METHODS: RA segments were collected from patients undergoing elective CABG (n = 13), and isometric tension was examined in the presence of endothelin (10 nM) or norepinephrine (1 microM). In matched RA, endothelin- or norepinephrine-induced contractions were measured in the presence of diltiazem (277 nM), amlodipine (73 nM), or nifedipine (145 nM). These concentrations of calcium channel antagonists were based upon clinical plasma profiles. RESULTS: Endothelin and norepinephrine caused a significant increase in RA-developed tension (0.54+/-0.1 and 0.68+/-0.1 g/mg, respectively; p<0.05). Amlodipine or nifedipine significantly reduced RA vasoconstriction in the presence of endothelin (30+/-6% and 41+/-9%, respectively; p<0.05) or norepinephrine (27+/-8% and 53+/-9%, respectively; p<0.05), whereas diltiazem did not significantly reduce RA vasoconstriction. CONCLUSIONS: These results demonstrate that neurohormonal factors released post-CABG can cause RA vasoconstriction, and that calcium channel antagonists are not equally effective in abrogating that response. Both amlodipine and nifedipine, which have a higher degree of vascular selectivity, appear to be the most effective in reducing RA vasoconstriction.

Endothelin receptor pathway in human left ventricular myocytes: relation to contractility.

BACKGROUND: Increased synthesis and release of the potent bioactive peptide endothelin-1 (ET-1) occurs during and after cardiac surgery. However, the cellular and molecular basis for the effects of ET-1 on human left ventricular (LV) myocyte contractility remains unknown. METHODS: LV myocyte contractility was examined from myocardial biopsies taken from patients (n = 30) undergoing elective coronary artery bypass. LV myocytes (n = 997, > 30/patient) were isolated using microtrituration and contractility examined by videomicroscopy at baseline and after ET-1 exposure (200 pmol/L). In additional studies, myocytes were pretreated to inhibit either protein kinase C (PKC) (chelerythrine, 1 micromol/L), the sodium/hydrogen (Na/H) exchanger (EIPA, 1 micromol/L), both PKC and the Na/H exchanger, or the ET(A) receptor (BQ-123, 1 micromol/L), followed with ET-1 exposure. RESULTS: Basal myocyte shortening increased 37.8 +/- 6.3% with ET-1 (p < 0.05). Na/H exchanger, PKC, and dual inhibition all eliminated the effects of ET-1. Furthermore, ET(A) inhibition demonstrated that ET-1 effects on myocyte contractility were mediated through the ET(A) receptor subtype. CONCLUSIONS: ET-1 directly influences human LV myocyte contractility, which is mediated through the ET(A) receptor and requires intracellular activation of PKC and stimulation of the Na/H exchanger.