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Background: The present study examined the survival and rate of contraction of (1) cardiomyocytes cultured from cryopreserved fetal rat myocardium and (2) cryopreserved cultured cardiomyocytes. In addition, the effects of transplantation of cryopreserved fetal cardiomyocytes were evaluated., Methods: Segments of fetal rat myocardial tissue (0.2, 2.0, and 6.0 mm(3) mince size) and cultured cardiomyocytes were cryopreserved in liquid nitrogen for 1, 2, and 4 weeks. After cryopreservation, the tissue samples and cultured cardiomyocytes were thawed at 37 degrees C and cultured, and cell proliferation and rate of contraction were determined. Cultured cryopreserved (n = 5) and noncryopreserved (control, n = 5) fetal cardiomyocytes were transplanted into the subcutaneous tissue and into a transmural left ventricular free wall scar of Sprague-Dawley rats (n = 3). The survival and rate of contraction of these transplanted cells were also examined., Results: Cryopreservation of cultured fetal cardiomyocytes resulted in viable and functional cardiomyocytes although the cell number and percentage of beating cells were diminished. Survival of cardiomyocytes isolated from cryopreserved fetal myocardium was a function of tissue size before cryopreservation; the lowest survival was recorded in tissues with the largest mince size (6.0 mm(3)). The subcutaneous transplants contracted spontaneously and regularly with an idioventricular rhythm. In addition, the transplanted cardiomyocytes were elongated and formed a myocardium-like pattern with blood vessels present within the contractile tissue. In the transmural left ventricular scar, both control and experimental fetal cardiomyocyte transplants formed myocardium-like tissue., Conclusions: The present study uncovers the following key observations: (1) cryopreservation of fetal cardiomyocytes and cardiomyocytes isolated from cryopreserved myocardial tissue results in viable and functional cells, (2) cryopreserved fetal cardiomyocytes can be successfully transplanted into subcutaneous and myocardial scar tissue, and (3) improvements in cryopreservation techniques are required to augment the rates of cardiomyocyte survival observed in the study.

This study was designed to evaluate the effect of smooth muscle cell transplantation into myocardial ventricular scar formed by cryo-necrosis. The left ventricular free wall (LVFW) of adult rats was cryo-necrosed. At 4 weeks after cryo-injury cultured fetal rat stomach smooth muscle cells (transplanted group, n = 10) or culture medium (control, n = 10) were transplanted. Sham animals (n = 8) were similarly operated but without cryo-necrosis and transplantation. The animals were administered a daily maintenance dose of cyclosporin A (5 mg/kg). At 8 weeks after cryo-injury, heart function was evaluated using a Langendorff preparation. Myocardial scar and transplanted cells were assessed histologically. Transplanted smooth muscle cells survived and formed smooth muscle cell tissue, as assessed by immunostaining against smooth muscle cell actin, within the myocardial scar. In the control hearts no muscle tissue was found in the scar. Angiogenesis occurred (P < 0.001) in the transplanted scar compared to the control scar. The transplanted cells increased the scar thickness (P < 0.01) by hyperplasia and prevented (P < 0.001) the dilatation of the ventricular chamber size compared to the controlled hearts. For physiological left ventricular volumes of 0.04 to 0.28 ml, the systolic and developed pressures in the transplanted group were greater (P < 0.001) than the control group, but less (P < 0.001) than those of the sham group. Transplanted smooth muscle cells formed smooth muscle tissue in myocardial scar tissue and improved contractile function compared to control hearts.

We showed previously that 20 min of low-volume anoxia ("ischemia") and 20 min of "reperfusion" preconditions quiescent pediatric myocyte cultures against damage resulting from 90 min of subsequent prolonged ischemia and 30 min of reperfusion. The purpose of this study was to assess the roles of adenosine and protein kinase C (PKC) in this preconditioning model. Our results suggest that 1) preconditioned myocytes secrete a protective mediator(s) into the "ischemic" supernatant that is transferable to other cells, and adenosine is released into the supernatant in quantities sufficient for adenosine-receptor activation (2) preconditioning is inhibited by adenosine-receptor antagonism, and myocyte protection similar to preconditioning can be achieved with exogenously administered adenosine or adenosine-receptor stimulation; (3) brief ischemic and adenosine-induced myocyte preconditioning is mimicked by the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PKC agonist) and inhibited by PKC antagonists; and (4) brief ischemic and adenosine-induced myocyte preconditioning both induce PKC translocation to myocyte membranes and increase the PKC phosphorylation rate. These data suggest that adenosine released from ischemic human pediatric myocytes mediates preconditioning through activation of PKC.

Background: Transplantation of cultured cardiomyocytes into myocardial scar tissue may prevent heart failure., Methods: Scar tissue was produced in the left ventricular free wall of 15 rats (weight, 450 g) by cryoinjury. Seven animals had operation only and survived for 8 weeks (sham group). Four weeks after cryoinjury, cultured fetal rat cardiomyocytes or culture medium was injected into the scar tissue of transplantation (n = 5) and control (n = 5) animals, respectively. Five other rats were sacrificed for scar assessment. Eight weeks after cryoinjury heart function in the transplantation, control, and sham groups was measured using a Langendorff preparation. Histologic studies were performed to quantify the extent of the scar and the transplanted cells., Results: Four weeks after cryoinjury, 36% +/- 4% (mean +/- 1 standard error) of the left ventricular free wall surface area was scar tissue. At 8 weeks, the scar size had increased (p < 0.01) to 55% +/- 3% in the control group. Although the scar size (43% +/- 2%) in the transplantation group at 8 weeks was not significantly different from that at 4 weeks, it was less (p < 0.05) than that in the control group. Hearts in the sham group had no scar tissue. The transplanted cardiomyocytes had formed cardiac tissue within the myocardial scar. Systolic and developed pressures in the transplantation group hearts were greater (p = 0.0001) than in the control group hearts but less (p < 0.01) than those in the sham group hearts., Conclusions: The transplanted cardiomyocytes formed cardiac tissue in the myocardial scar, limited scar expansion, and improved heart function compared with findings in the control hearts.

Background: Previous investigations from this institution and others support the role of University of Wisconsin solution for prolonged hypothermic cardiac storage. Modification of the divalent cation concentrations has been beneficial for cardioplegic investigations and may enhance cardiac recovery after extended preservation., Methods: To investigate this hypothesis, isolated rodent hearts were obtained from Sprague-Dawley rats and mounted on a Langendorff apparatus. Rat hearts were flushed (15 ml/kg) and stored (30 ml) for 8 hours at 0 degree C in unmodified University of Wisconsin solution (n = 16/group) or University of Wisconsin solution with calcium concentrations of 0.025 to 10 mmol/L or magnesium concentrations of 10 to 20 mmol/L (six to eight hearts/group). Finally, combinations of calcium and magnesium were examined. Rat hearts were studied before storage and after 45 minutes of reperfusion with an intraventricular balloon., Results: Developed pressure (mean +/- standard deviation) was increased with calcium 0.1 mmol/L (University of Wisconsin solution: 69.2% +/- 7.0%; Ca++ 0.1 mmol/L: 78.9% +/- 6.1%, p < 0.05), whereas only the addition of the highest calcium concentration (10 mmol/L) was significantly harmful (developed pressure: 58.3% +/- 8.4%, p < 0.05; creatine kinase release: 408 +/- 200 versus 170 +/- 104 IU/gm, p < 0.05; lactate dehydrogenase release: 103 +/- 43 versus 37 +/- 26 IU/gm, p < 0.05). Coronary flow recovered to control values with magnesium 15 mmol/L, which was significantly greater than that achieved with unmodified University of Wisconsin solution (97.1% +/- 14.6% versus 72.1% +/- 8.4%, p < 0.05). Of the calcium-magnesium combinations tested, developed pressure was increased compared with unmodified University of Wisconsin solution with calcium 0.1 and magnesium 20 mmol/L (76.8% +/- 6.4%, p < 0.05). Diastolic function was reduced in all groups (p < 0.0001) and not significantly different between groups., Conclusions: The experiments indicated that recovery after storage with University of Wisconsin solution is enhanced with the addition of calcium and magnesium. The addition of high concentrations of calcium (> or = 2.5 mmol/L) appears harmful.

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 x 2 factorial experiment (n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 mumol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0 degrees C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage (p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase (p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased (p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced (p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution.

Sodium tanshinone IIA sulfonate (STS) is a derivative of tanshinone IIA. The latter is a pharmacologically active component isolated from the rhizome of the Chinese herb Salvia miltiorrhiza. Liquid chromatographically pure STS was found to reduce myocardial infarct size by 53.14 +/- 22.79% relative to that in the saline control in a rabbit 1 hr-ischemia and 3 hr-reperfusion model. This effect was comparable to that of Trolox (a better characterized antioxidant serving as a reference cytoprotector), which salvaged the myocardium in the same infarct model by 62.13 +/- 18.91%. Also, like Trolox, STS did not inhibit oxygen uptake by xanthine oxidase (XO), a key enzyme in free radical generation. However, in contrast to Trolox, STS significantly prolonged the survival of cultured human saphenous vein endothelial cells but not human ventricular myocytes in vitro when these cells were separately exposed to XO-generated oxyradicals. Note that the endothelium is recognized to be a key site of oxidant generation and attack. Our findings in vitro and in vivo support the interpretation that STS is a cardioprotective substance, and that it may exert a beneficial effect on the clinically important vascular endothelium.

One hundred seven patients undergoing coronary artery bypass grafting were randomized to receive warm antegrade (n = 21), warm retrograde (n = 22), cold antegrade (n = 20), cold retrograde (n = 22), or intermittent cold antegrade (n = 22) blood cardioplegia. Myocardial oxygen consumption and lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during the operation, and creatine kinase-MB release was assessed postoperatively. Warm cardioplegia resulted in greater myocardial lactate production than cold cardioplegia (p = 0.048). Retrograde cardioplegia was associated with greater lactate production than antegrade cardioplegia (p = 0.015). Adenosine triphosphate depletion was similar among groups. However, poorly diffusible metabolites of adenosine triphosphate accumulated to the greatest extent in the intermittent cold group. Levels of hypoxanthine were highest after warm retrograde cardioplegia. Operative mortality and morbidity were low and were not different among groups. In summary, none of the five techniques of cardioplegia evaluated in this study was able to completely prevent myocardial ischemia. Anaerobic lactate production was minimized with cold cardioplegia and with antegrade cardioplegic delivery. Hypothermia may have impaired regeneration of adenosine triphosphate, however, particularly in association with inadequate or intermittent cardioplegic flow.

The cardiac surgeon is faced with RV failure in two main situations: in isolation or in patients with left-sided cardiac assist. Adequate volume loading, correction of acidosis and oxygenation, cardiac pacing, pharmacologic agents, and systemic intra-aortic balloon pumping allow stabilization in most of these patients. When these measures fail, some form of mechanical assistance of the right ventricle becomes necessary. Balloon counterpulsation in the pulmonary artery improves RV output but does not restore the systemic perfusion if the right ventricle is profoundly depressed. When the right ventricle is profoundly depressed, a mechanical assist pump is the only device capable of restoring systemic perfusion. Like the left ventricle, the right ventricle, given time and support, can recover enough function to allow weaning from the assist device and survival.

Conventional cardioplegic arrest results in persistent atrial electrical and mechanical activity. This activity has been postulated to result in atrial ischemia which can induce postoperative arrhythmias and impair the transport function of the atrium. In this study, the effects of simple cardiopulmonary bypass (CPB) (seven pigs) and conventional cardioplegic arrest (CCA) (seven pigs) on right atrial function were evaluated. Function was assessed in an isolated right atrial preparation with a compliant balloon inserted via the superior vena cava. CCA for 1 hr produced significant deterioration in right atrial function (developed pressure 14.1 +/- 0.7 vs 18.9 +/- 0.8 mm Hg, P less than 0.05, diastolic pressure 10.0 +/- 1 vs 4.5 +/- 1.4 mm Hg, P less than 0.05, dP/dt 134 +/- 25 vs 187 +/- 19 mm Hg/sec, P less than 0.05 at a balloon volume of 20 ml after 1 hr of reperfusion). CPB alone caused no alteration in pressures in the right atrium but was associated with a late decrease in dP/dt (developed pressure 19.3 +/- 1.8 vs 18.9 +/- 0.8 mm Hg, diastolic pressure 4.0 +/- 1.2 vs 4.5 +/- 1.4 mm Hg, dP/dt 148 +/- 18 vs 187 +/- 19, P less than 0.05 at a balloon volume of 20 ml at a time corresponding to 1 hr of reperfusion in the CCA group). These results are consistent with the postulate that conventional techniques of cardioplegic arrest are associated with ischemic dysfunction of the right atrium.

The ability of cold potassium cardioplegia (CPC) to preserve cardiac metabolism and performance was evaluated in 68 patients undergoing anoxic arrest for aortocoronary bypass. Forty-five patients (group I) had a single dose of CPC inducing a mean myocardial temperature (MMT) of 32 degrees C/min. Twenty-three patients had multiple doses of CPC and systemic hypothermia to achieve a MMT of 22 degrees C/min. Arterial and coronary sinus sampling 38 minutes after aortic clamp removal permitted calculation of cardiac oxygen extraction, lactate production, CPK and CPK-MB release. Group I patients extracted less oxygen, produced more lactate, and released more CPK and CPK-MB. These indices of cardiac metabolism were found to correlate with anoxic times exceeding 30 minutes, and demonstrated more cardiac damage in group I patients at longer anoxic times. Serial measurements of cardiac output (thermodilution) and left atrial pressure during volume loading permitted construction of myocardial performance curves. Group I patients had a diminished response to volume loading postoperatively. Both the upslope and the highest stroke work attained were lower in group I and inversely related to the anoxic time. All patients made an uneventful recovery, indicating the insensitivity of clinical parameters of myocardial protection. Coronary sinus sampling and hemodynamic monitoring during volume loading permit an objective assessment of myocardial preservation.

The cardiac response to volume loading was evaluated in fifty severely septic patients. After a rapid infusion of albumin or whole blood the cardiac index (CI) and left ventricular stroke work index (LVSWI) were recorded as the pulmonary arterial wedge pressure (PAWP) increased. Initial values of PAWP, CI, and LVSWI were similar in both the nineteen surviving and thirty-one nonsurviving patients. Surviving patients, however, demonstrated greater increases in CI and LVSWI as PAWP rose. Nearly half of both patient groups developed decreases in CI and LVSWI as the PAWP continued to increase. These downslopes occurred at relatively low PAWP and are taken as evidence of an abnormality of myocardial function in both survivors and nonsurvivors. The lower upslope of the performance curves in nonsurvivors indicates myocardial depression or a negative inotropic effect. Cardiac ischemia, acute respiratory failure, and high affinity red cells were found to diminish the cardiac response to volume loading, whereas hepatic and renal failure were associated with a good CI and LVSWI response.

Although blood cardioplegia preserves perioperative ventricular function better than crystalloid cardioplegia, late results are uncertain. Nuclear ventriculograms were used to assess ventricular function in 47 patients undergoing coronary bypass surgery who were randomly assigned to receive blood (23 patients) or crystalloid cardioplegia (24 patients). Data were acquired at rest and during maximal exercise (bicycle ergometer) 1 month before surgery (PRE), 5 months after surgery (POST), and perioperatively at rest 3 to 5 hr after operation (PERI). Perioperatively, blood cardioplegia decreased ischemic injury (less elevation in creatine kinase-MB fraction and aspartate aminotransferase; p less than .05), preserved ventricular performance (lower stroke work index at higher left ventricular end-diastolic volume index after crystalloid than blood cardioplegia; p = .02 by analysis of covariance [ANOCOVA]) and preserved systolic function (higher left ventricular end-systolic volume index [LVESVI] at similar systolic blood pressure after crystalloid than blood cardioplegia; p = .02 by ANOCOVA). Postoperatively, resting ventricular performance and systolic function were not different with blood and crystalloid cardioplegia and were similar to preoperative measurements. Postoperatively, the response to exercise was similar between the two groups and was improved compared with that at PRE. Postoperative systolic function at exercise was similar between the two groups but was better than that at PRE (higher systolic blood pressure at similar LVESVI; p = .01 by ANOCOVA). The type of cardioplegic solution influenced perioperative but not late postoperative function after elective coronary artery bypass surgery.

The low mortality and perioperative infarction rates for aortocoronary bypass (ACB) make them unsuitable for evaluating the adequacy of myocardial protection. Enzymatic and functional measurements were found to be sensitive and specific indicators of myocardial injury. A prospective concurrent study of 78 patients undergoing triple ACB was conducted to evaluate the effectiveness of three popular methods of myocardial protection. Group I (32 patients) had a single dose of cold (4 degrees C) potassium cardioplegic (CPC) solution infused inducing a mean myocardial temperature (MMT) of 31 +/- 4 degrees C/min. Group II (23 patients) had multiple doses of CPC solution 8nducing a MMT of 22 +/- 2 degrees C/min. Group III (23 patients) had intermittent anoxic arrest at a MMT of 28 +/- 1 degrees C. The groups were not randomized but had comparable clinical symptoms and catheterization findings. Serial measurements of cardiac specific creatine kinase (CK-MB) revealed a peak in enzymatic activity occurring 60 minutes following ACB. The highest CK-MB was significantly (P less than 0.01) lower in group II (25 +/- 8 IU/liter) than group I (50 +/- 8 IU/liter), or group III (68 +/- 14 IU/liter). Myocardial performance was evaluated after ACB by serially measuring left ventricular stroke work index (SW) and left atrial pressure (LAP) in response to volume loading. The rise in SW was significantly (P less than 0.01) greater in group II (3.0 +/- 0.7 gm.m/sq m/mm Hg) than in group I (1.4 +/- 0.7) or group III (1.8 +/- 0.9). The highest SW attained was higher (P less than .01) in group II (43 +/- 7 gm.m/sq m) than group I (19 +/- 6) or group III (34 +/- 8) at comparable LAP values (group I: 20 +/- 5 mm Hg; group II: 18 +/- 3; group III: 18 +/- 4). Post-operative clinical evaluation failed to differentiate among the three groups. The more sensitive indices, however, demonstrated the superiority of cold, multidose cardioplegia in providing optimal myocardial protection.

Coronary bypass performed with moderate systemic hypothermia (25 degrees C) and cold-potassium cardioplegia was associated with a fall and subsequent rise in core (pulmonary arterial) temperature. Serial hemodynamic measurements during rewarming and recovery revealed a decrease in cardiac index (CI) without a decrease in the left atrial pressure (LAP) of 17 patients recovering from uneventful coronary bypass surgery. Nuclear ventriculograms performed during rewarming demonstrated a decrease in left ventricular end-diastolic volume index (EDVI, calculated from the thermodilution stroke index divided by the nuclear ejection fraction) without a change in LAP. Volume loading during both mild hypothermia (35 +/- 5[SD]degrees C) and normothermia revealed that myocardial performance (the relation between CI and EDVI) was unchanged, but diastolic compliance (the relation between LAP and EDVI) decreased with rewarming. LAP was a poor indicator of left ventricular preload (EDVI) during rewarming, and volume loading was required to maintain preload and prevent hypoperfusion.

Twenty-two matched coronary bypass surgery (CABS) patients were randomly divided into two groups; 11 patients in the control group received CPD-stored or fresh blood (mean age, 5.5 days) containing 70 percent of normal 2-3 diphosphoglycerate (2-3 DPG) and 11 patients received 2-3 DPG-enriched previously frozen, washed, concentrated red cells (2-3 DPG 150 percent of normal). Coming off cardiopulmonary bypass, when given a volume load, the high 2-3 DPG patients had a significant increase in cardiac index (2.95 L. per minute vs. 2.18 L. per minute, p smaller than 0.001) at similar filling pressures. At this time body oxygen consumption, in vivo P50, red cell 2-3 DPG, and arterial-venous oxygen content difference were all increased, but P minus vo2 was normal. Improved oxygen delivery occurred without decreasing mixed venous oxygen tension. The results suggest that, with volume loading, function in the heart with coronary artery disease is limited in part by available oxygen. By decreasing oxygen affinity for hemoglobin by altering red blood cells biochemically, myocardial performance can be improved safely.

Right coronary artery stenoses limit cardioplegic delivery to the right ventricle and may contribute to postoperative right ventricular dysfunction. Right ventricular function was evaluated in 39 patients with right coronary artery stenoses following elective coronary bypass operations. Hemodynamic and nuclear ventriculographic measurements, made between 3 and 6 hours postoperatively, revealed a progressive increase in pulmonary arterial pressure, pulse rate, and right ventricular ejection fraction (p less than 0.05). Right ventricular end-diastolic volume index (calculated from the thermodilution stroke index divided by the nuclear ejection fraction) decreased, but right atrial pressure increased (suggesting a decrease in compliance). The response to the infusion of 2 units of plasma (volume loading) was evaluated 3 hours postoperatively (EARLY) and again 5 hours postoperatively (LATE) in 21 patients. Right ventricular performance (the relation between cardiac index or right ventricular stroke work index and right ventricular end-diastolic volume index) and right ventricular systolic function (the relation between systolic pulmonary arterial pressure and right ventricular end-systolic volume index) were depressed EARLY and improved LATE (p less than 0.01 in analysis of covariance). Left ventricular performance (the relation between cardiac index or left ventricular stroke work index and left ventricular end-diastolic volume index) and left ventricular systolic function (the relation between systolic blood pressure and left ventricular end-systolic volume index) were similar EARLY and LATE. Right ventricular diastolic function (the relation between right atrial pressure and right ventricular end-diastolic volume index) and left ventricular diastolic function (the relation between left atrial pressure and left ventricular end-diastolic volume index) were significantly greater LATE than EARLY. Right, but not left, ventricular performance and systolic function were transiently depressed, and right and left ventricular diastolic stiffness were transiently decreased in the EARLY postoperative period. In patients with right coronary artery stenoses, current methods of cardioplegia may inadequately protect the right ventricle, but further studies are required to establish the relation between intraoperative protection and postoperative function.