Myocardial hypothermia induced after reperfusion does not prevent adverse left ventricular remodeling nor improve cardiac function.

AIMS: The purpose of the study was to determine whether late therapeutic hypothermia (LTH), administered after reperfusion, could prevent adverse left ventricular (LV) remodeling and improve cardiac function in the rat myocardial ischemia/reperfusion model. MAIN METHODS: Rats were randomized to normothermia (n = 10) or LTH (initiated at 1 min after coronary artery reperfusion, n = 10) and subjected to 30 min of coronary occlusion followed by 6 weeks of reperfusion. Hypothermia was induced by pumping cold saline over the anterior surface of the LV until the temperature cooled to <32 °C. In the normothermic group, the heart was bathed in saline at 38 °C. KEY FINDINGS: After 6 weeks of recovery, fractional shortening of the LV was comparable in the LTH (20.2 ±â€¯0.6%) and normothermic group (20.0 ±â€¯2.1%; p = 0.918). Postmortem LV volume (0.47 ±â€¯0.04 ml in LTH and 0.44 ±â€¯0.05 ml in normothermic group) and lung wet/dry weight ratio were similar in both groups. There were no significant differences in scar size, scar thickness, infarct expansion index, LV cavity or transmurality (%) between groups. This data contrasts with our previous study showing that hypothermia administered during the ischemic phase significantly reduced the scar size; decreased LV cavity, infarct expansion index and transmurality (%), and improved the scar thickness. SIGNIFICANCE: LTH did not prevent adverse LV remodeling nor improve cardiac function in the rat myocardial ischemia/reperfusion model. To have a long term benefit on remodeling, hypothermia must be administered during the ischemic phase and not just the reperfusion phase.

The Therapeutic Effect of Cell Transplantation Versus Noncellular Biomaterial Implantation on Cardiac Structure and Function Following Myocardial Infarction.

Although numerous studies demonstrated that localized delivery of either cells or biomaterials improved postinfarction cardiac function, the underlying mechanisms for this effect remain unclear. We performed a comparison of the effects of fetal, neonatal, and human embryonic stem cell-derived cardiac cell as well as mesenchymal stem cell transplantation versus biomaterial (collagen/extracellular matrix) implantation therapy in rat myocardial infarction model in our laboratory, specifically comparing their effects on infarct wall thickness, neovascularization, infarct wall motion, and left ventricular ejection fraction (LVEF). Both cell and biomaterial treatment had similar beneficial effects on cardiac structure (increasing infarct wall thickness and preventing infarct expansion) and function (preventing paradoxical LV systolic bulging and improving LVEF). In this review, we also discussed the underlying mechanisms of cell and biomaterial therapies, their advantages and disadvantages, and future research directions in the field of regenerative cardiology.

Intramyocardial injection of heart tissue-derived extracellular matrix improves postinfarction cardiac function in rats.

AIMS: We determined whether implantation of heart tissue-derived decellularized matrix, which contains native biochemical and structural matrix composition, could thicken the infarcted left ventricular (LV) wall and improve LV function in a rat myocardial infarction model. METHODS AND RESULTS: Myocardial infarction was induced by left coronary ligation in Fischer rats. One week later, saline (75 µL, n = 17) or matrix (75 µL, n = 19) was directly injected into the infarcted area. At 6 weeks after injection, cardiac function was assessed by left ventriculogram, echocardiography, and Millar catheter. The hearts were pressure fixed to measure postmortem LV volume and processed for histology. Left ventriculogram demonstrated that LV ejection fraction (EF) was significantly greater in the matrix-treated (56.7% ± 1.4%) than in the saline-treated group (52.4% ± 1.5%; P = .043), and paradoxical LV systolic bulging was significantly reduced in the matrix-treated group (6.2% ± 1.6% of the LV circumference) compared to the saline-treated group (10.3% ± 1.3%; P = .048). Matrix implantation significantly increased the thickness of infarcted LV wall (0.602 ± 0.029 mm) compared to the saline-treated group (0.484 ± 0.03 mm; P = .0084). Infarct expansion index was significantly lower in the matrix-treated group (1.053 ± 0.051) than in the saline-treated group (1.382 ± 0.096, P = .0058). Blood vessel density and c-kit positive staining cells within the infarct area were comparable between the 2 groups. CONCLUSIONS: Implantation of heart tissue-derived decellularized matrix thickens the LV infarcted wall, prevents paradoxical LV systolic bulging, and improves LV EF after myocardial infarction in rats. This benefit was not dependent on the enhanced angiogenesis or the recruitment of endogenous stem cells to the injury site.

Experience from experimental cell transplantation therapy of myocardial infarction: what have we learned?

During the past 15 years, our research group has transplanted fetal/neonatal cardiomyocytes, mesenchymal stem cells, and embryonic stem cell-derived cardiomyocytes into infarcted myocardium in a rat myocardial infarction model. Our experimental data demonstrated that cell transplantation therapy provides a potential approach for the treatment of injured myocardium after myocardial infarction based on the reported positive effects upon histological appearance and left ventricular function. However, the underlying mechanisms of the benefits from cell transplantation therapy remain unclear and may involve replacement of scar tissue by transplanted cells, induced neoangiogenesis and paracrine effects of factors released by the transplanted cells. In this review, we summarize our experiences from experimental cell transplantation therapy in a rat myocardial infarction model and discuss the controversies and questions that need to be addressed in future studies.

Effect of mild renal dysfunction (s-crea 1.2-2.2 mg/dl) on presentation characteristics and short- and long-term outcomes of on-pump cardiac surgery patients.

OBJECTIVES: The objective of this study is to evaluate differences in patient presentation and short- and long-term outcomes between patients dichotomized by the level of preoperative s-creatinine (s-crea) without renal failure and to use EuroSCORE (ES) risk stratification for validating differences and for predictive purposes. METHODS: A thousand consecutive cardiac surgery patients from January 1999 through May 2000 were analyzed. Patients with off-pump surgery or s-crea >200 micromol/l (>2.2 mg/dl) were excluded leaving 885 patients for analysis. Group 1 (n=703) had s-crea 0.5-1.2 mg/dl and Group 2 (n=182) had elevated s-crea 1.3-2.2 mg/dl but no renal insufficiency. RESULTS: Group 2 patients were older (P<0.0001), had a higher percentage of males (P=0.008), had lower left ventricular ejection fraction (LVEF) (P=0.001), had higher New York Heart Association (NYHA) classification (P<0.0001), had more diabetics (P=0.001) and had more patients with a history of congestive heart failure (CHF) (P<0.0001). Both additive ES (AES) and logistic ES (LES) variables were higher in Group 2 patients, AES 8.45+/-4.28% vs. 6.05+/-3.80% (P<0.0001) and LES 17.7+/-19.1% vs. 9.57+/-13.3% (P<0.0001). Proportions of emergency operations and use of intra-aortic balloon pulsation (IABP) support did not differ. There were more coronary artery bypass grafting (CABG) with or without concomitant procedures in Group 1 but otherwise the procedures performed were similar. Cardiopulmonary bypass (CPB) times did not differ (P=0.1). Operative mortality was similar (P=0.06) but hospital mortality was higher in Group 2: 19/10.4% vs. 25/3.6% (P<0.0001), odds ratio (OR) 3.16. Total length of stay (LOS) and length of stay in the postoperative intensive care unit (ICU) did not differ. Postoperative renal failure (PORF) (s-crea increase to >2.25 mg/dl or >200 micromol/l) developed in 38/4.5% patients in Group 1 and in 41/22.5% patients in Group 2 (P<0.0001), OR=5.08. Follow-up all-cause mortality was higher in Group 2: 68/37.4% vs. 167/23.8% (P<0.0001), OR=1.91. Both ES definitions predicted hospital mortality, LOS, ICU, PORF and long-term mortality well, while increased s-crea predicted PORF and long-term mortality in both groups. CONCLUSIONS: Mild increase in s-crea is a marker for patients with increased cardiac risk factors and the risk for poor outcomes. Both ES definitions are highly predictive of the outcomes.

Cardioprotective effects of angiotensin II type 1 receptor blockade with olmesartan on reperfusion injury in a rat myocardial ischemia-reperfusion model.

We determined the effects of olmesartan on infarct size and cardiac function in a rat ischemia/reperfusion model. Rats underwent 30 min of left coronary artery (CA) occlusion followed by 2 h of reperfusion. In protocol 1, the rats received (by i.v.) 1 mL of vehicle at 10 min after CA occlusion (Group 1, n = 15); olmesartan (0.3 mg/kg) at 10 min after CA occlusion (Group 2, n = 15); 1 mL of vehicle at 5 min before CA reperfusion (Group 3, n = 15); or olmesartan (0.3 mg/kg) 5 min before CA reperfusion (Group 4, n = 15). In protocol 2, the rats received (by i.v.) 1 mL of vehicle at 5 min before CA reperfusion (Group 5, n = 21); or olmesartan (3 mg/kg) at 5 min before CA reperfusion (Group 6, n = 21). Systemic hemodynamics, left ventricular (LV) function, LV ischemic risk zone, no-reflow zone, and infarct size were determined. In protocol 1, olmesartan (0.3 mg/kg) did not affect blood pressure (BP), heart rate, LV +/- dp/dt or LV fractional shortening during the experimental procedure, and did not alter no-reflow or infarct size. In protocol 2, olmesartan (3 mg/kg) significantly reduced infarct size to 21.7 +/- 4.1% from 34.3 +/- 4.1% of risk zone in the vehicle group (P= 0.035), but did not alter the no-reflow size. Prior to CA reperfusion, olmesartan (3 mg/kg) significantly reduced mean BP by 22% and LV +/-dp/dt, but did not affect heart rate. At 2 h after reperfusion, olmesartan significantly decreased heart rate by 21%, mean BP by 14%, and significantly increased LV fractional shortening from 54.1 +/- 1.4% to 61.3 +/- 1.6% (P= 0.0018). Olmesartan significantly reduced myocardial infarct size and improved LV contractility at a dose (3 mg/kg) with systemic vasodilating effects but not at a lower dose (0.3 mg/kg) without hemodynamic effects.

Measured posterior annuloplasty for repair of non-ischemic mitral regurgitation. A single unit follow-up.

UNLABELLED: The aim of this report is to evaluate short- and long-term outcomes of annuloplasty method of our choice: measured posterior annuloplasty (MPA). MPA is a piece of a Duran ring cut to the length of free-edge of anterior mitral leaflet (AML) and anchored with multiple pledgeted U-sutures from trigone to trigone into the posterior annulus. MATERIAL AND METHODS: From 1988 to 2000, 103 consecutive patients with non-ischemic mitral regurgitation were scheduled preoperatively to be repaired by MPA. RESULTS: Preoperative mitral valve regurgitation (MR) grade was 3.8+/-0.5 and decreased to 0.1+/-0.3 (P<0.0001) after repair. One patient was converted to insertion of mechanical prosthesis after grade 3 MR persisted after septal myectomy and MPA. Three patients needed instant revision of the repair one due to SAM and two due to stenosis. No patient had a stenosis or unacceptable (>1) MR after the procedure. There was one operative death (1.0%) and 3 hospital/30-day deaths (2.9%). Sixteen patients (16.3%) expired during the follow-up to 91 months (mean 57.4+/-19.5, median 60 months) none due to failure of MPA. There were no reoperations due to failure of MPA. Three patients had a reoperation, one for dehiscence of reconstruction after P2 resection and two patients due to progression of anterior leaflet degeneration and calcification with 4+ MR. New York Heart Association (NYHA) functional classification decreased from 2.3+/-0.8 to 1.4+/-0.6 (P<0.0001) and only one patient had an increase from II to III. Eighty-eight patients (96.7%) were in NYHA class I-II. Ten patients had an increase of MR from 0 to trace or 1 and one from 0 to 2. Two patients were diagnosed with mild stenosis without need of reoperation. CONCLUSIONS: MPA is a durable and stable alternative for repair of non-ischemic mitral regurgitation of different etiologies. The technique gives an objective measure of the length of the band and no patient is left with a significant MR or mitral valve stenosis (MS). First-time success rate is very high and instant repairs few and minor. Freedom of MPA related reoperations is 100%.

Delivering stem cells to the heart in a collagen matrix reduces relocation of cells to other organs as assessed by nanoparticle technology.

AIM: A limitation of cell therapy for heart disease is the fact that stem cells injected directly into the myocardium are capable of entering the vasculature and migrating to remote organs. We determined whether retention of mesenchymal stem cells (MSCs) in the infarcted myocardium could be improved by implanting the cells in a collagen matrix. METHODS: A myocardial infarction was induced by ligation of the left anterior descending coronary artery in Fischer rats. A total of 7 days after myocardial infarction, saline (n = 12), saline plus 2 million bone marrow-derived rat MSCs labeled with isotopic colloidal nanoparticles containing europium (n = 13), collagen (n = 13) or collagen plus 2 million labeled MSCs (n = 13) were directly injected into the infarcted myocardium. Tissues from the infarcted myocardium, noninfarcted myocardium, lung, liver, spleen and kidney were sampled 4 weeks later. Distribution of grafted MSCs was quantitatively analyzed by measuring the nanoparticle radioactivity in these tissues. Cardiac function was assessed by left ventriculography. RESULTS: There were zero nanoparticles detected in the tissues that received saline or collagen alone into the heart. Nanoparticles were detected in the heart and remote organs in the saline plus MSC group. Labeled cells (expressed as cell number/g tissue weight) were present in three out of 13 lungs (mean of 12,724 +/- 7060 cells/g), four out of 13 livers (12,301 +/- 5924 cells/g), 11 out of 13 spleens (57,228 +/- 11,483 cells/g), zero out of 13 kidneys, 13 out of 13 infarcted myocardium (8,006,835 +/- 1,846,462 cells/g) and nine out of 13 noninfarcted myocardium (167,331 +/- 47,007 cells/g). However, compared with the saline plus MSC group, nanoparticles were detected to a lesser extent in remote organs in collagen plus MSC group. Nanoparticles were detected in two out of 13 lungs (4631 +/- 3176 cells/g; p = NS), zero out of 13 livers (0 cells/g; p <0.05 vs saline plus MSC), four out of 13 spleens (24,060 +/- 17,373 cells/g; p <0.05), zero out of 13 kidneys (p = NS) and five out of 13 noninfarcted myocardium (51,522 +/- 21,548 cells/g; p <0.05). In the collagen plus MSC group, nanoparticles were detected in 12 out of 13 infarcted myocardium (4,830,050 +/- 592,215 cells/g), which did not significantly differ from that in the saline plus MSC group (p = NS). Both saline plus MSCs and collagen alone improved left ventricular ejection fraction compared with saline treatment. However, collagen plus MSCs failed to improve cardiac function. CONCLUSIONS: Collagen matrix as a delivery vehicle significantly reduced the relocation of transplanted MSCs to remote organs and noninfarcted myocardium.

Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts.

Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.

Directed retrograde cerebral protection during moderate hypothermic circulatory arrest.

There are many choices for neurologic protection for aortic arch surgery. Although numerous investigators have challenged the efficacy of retrograde cerebral perfusion, we have had good results with our application of this technique. We performed a retrospective review of 8 consecutive patients who underwent surgery from 1 June 2001 through 31 March 2003; the age range was 33 to 97 years. All patients required circulatory arrest and underwent retrograde cerebral perfusion with use of a tourniquet on the patients' left and right arms above the elbow to direct retrograde flow to the brain. Moderate hypothermia (around 24 degrees C nasopharyngeal) was used; circulatory arrest time ranged from 27 to 63 minutes. There was 1 late hospital death due to multiple-organ system failure. There were no neurologic complications (stroke or temporary neurologic dysfunction). There was no substantive neurologic or renal dysfunction in this cohort, in which moderate hypothermia was used. These results are comparable to those reported in the literature for similar patients. We conclude that, for patients who require circulatory arrest, directed retrograde cerebral perfusion at moderate nasopharyngeal hypothermia gives results comparable to those reported with other techniques.