John M. Canty

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture.

The in vitro culture of porcine bone marrow‐derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants‐rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell‐cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid β‐galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP‐7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony‐stimulating factor (G‐CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long‐term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.

Preload Induces Troponin I Degradation Independently of Myocardial Ischemia

BackgroundAlthough global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of the amount of myocardium at risk of ischemia. Methods and ResultsIsolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mmHg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4±3.6% versus 4.7±1.9% in immediately excised controls, P <0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 &mgr;mol/L calpeptin (4.3±0.6%). This improved function, with left ventricular systolic pressure increasing from 103±4 mmHg to 137±7 mmHg (P <0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation. ConclusionsCalpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.

Regional Myocardial Sympathetic Denervation Predicts the Risk of Sudden Cardiac Arrest in Ischemic Cardiomyopathy

OBJECTIVES The PAREPET (Prediction of ARrhythmic Events with Positron Emission Tomography) study sought to test the hypothesis that quantifying inhomogeneity in myocardial sympathetic innervation could identify patients at highest risk for sudden cardiac arrest (SCA). BACKGROUND Left ventricular ejection fraction (LVEF) is the only parameter identifying patients at risk of SCA who benefit from an implantable cardiac defibrillator (ICD). METHODS We prospectively enrolled 204 subjects with ischemic cardiomyopathy (LVEF ≤35%) eligible for primary prevention ICDs. Positron emission tomography (PET) was used to quantify myocardial sympathetic denervation ((11)C-meta-hydroxyephedrine [(11)C-HED]), perfusion ((13)N-ammonia) and viability (insulin-stimulated (18)F-2-deoxyglucose). The primary endpoint was SCA defined as arrhythmic death or ICD discharge for ventricular fibrillation or ventricular tachycardia >240 beats/min. RESULTS After 4.1 years follow-up, cause-specific SCA was 16.2%. Infarct volume (22 ± 7% vs. 19 ± 9% of left ventricle [LV]) and LVEF (24 ± 8% vs. 28 ± 9%) were not predictors of SCA. In contrast, patients developing SCA had greater amounts of sympathetic denervation (33 ± 10% vs. 26 ± 11% of LV; p = 0.001) reflecting viable, denervated myocardium. The lower tertiles of sympathetic denervation had SCA rates of 1.2%/year and 2.2%/year, whereas the highest tertile had a rate of 6.7%/year. Multivariate predictors of SCA were PET sympathetic denervation, left ventricular end-diastolic volume index, creatinine, and no angiotensin inhibition. With optimized cut-points, the absence of all 4 risk factors identified low risk (44% of cohort; SCA <1%/year); whereas ≥2 factors identified high risk (20% of cohort; SCA ∼12%/year). CONCLUSIONS In ischemic cardiomyopathy, sympathetic denervation assessed using (11)C-HED PET predicts cause-specific mortality from SCA independently of LVEF and infarct volume. This may provide an improved approach for the identification of patients most likely to benefit from an ICD. (Prediction of ARrhythmic Events With Positron Emission Tomography [PAREPET]; NCT01400334).

18F-2-Deoxyglucose Deposition and Regional Flow in Pigs With Chronically Dysfunctional Myocardium : Evidence for Transmural Variations in Chronic Hibernating Myocardium

BACKGROUND Hibernating myocardium in patients with collateral-dependent myocardium is characterized by relative reductions in resting flow and increases in the uptake of 18F-2-deoxyglucose (FDG) in the fasting state. We performed the present study to examine whether these key physiological alterations could be produced in a porcine model of chronic coronary occlusion and to assess whether the adaptations consistent with hibernation varied across the myocardial wall. METHODS AND RESULTS We chronically instrumented pigs (n = 18) with a fixed occluder on the proximal left anterior descending coronary artery (LAD). Three months later, ventricular function, regional myocardial perfusion, and FDG deposition (by excised tissue counting or positron emission tomography) were assessed in pigs after an over-night fast in the closed-chest anesthetized state. Total LAD occlusion with angiographic collaterals was present in the majority of animals. Left ventriculography showed severe anterior hypokinesis, and resting perfusion was significantly reduced in the hibernating LAD region in comparison with the normal remote regions (subendocardium: 0.80 +/- 0.06 versus 1.07 +/- 0.06 mL.min-1.g-1, P < .001; full-thickness: 0.87 +/- 0.04 versus 0.99 +/- 0.06 mL.min-1.g-1, P < .01). There was a twofold increase in full-thickness fasting FDG uptake in the dysfunctional LAD region (1.8 +/- 0.2 by positron emission tomography versus 1.9 +/- 0.1 by ex vivo counting). Ex vivo tissue counting revealed a pronounced transmural variation in FDG uptake in the hibernating region (LAD/normal), which averaged 2.5 +/- 0.2 in the subendocardium, 1.9 +/- 0.2 in the midmyocardium, and 1.4 +/- 0.1 in the subepicardium. CONCLUSIONS These results demonstrate that pigs instrumented with a proximal LAD stenosis develop hibernating myocardium characterized by relative reductions in resting function and perfusion in association with increased uptake of FDG in the fasting state. The transmural variations in relative resting flow and FDG uptake suggest that myocardial adaptations consistent with hibernation are most pronounced in the subendocardial layers and vary in relation to local coronary flow reserve.

Fast computed tomography detection of coronary calcification in the diagnosis of coronary artery disease. Comparison with angiography in patients < 50 years old.

BACKGROUND The predominant cause of coronary artery calcification is atherosclerosis. Although fast x-ray computed tomography (CT) has been demonstrated to be a sensitive technique to detect coronary calcification, the increasing prevalence of calcification with age has been associated with a low specificity for identifying obstructive atherosclerosis. We hypothesized that the specificity of this test would be improved in a younger patient population, making it more useful in the diagnosis of coronary artery disease. METHODS AND RESULTS We compared fast CT-detected calcification with coronary angiography in 106 patients under the age of 50 years. Nonenhanced fast CT scans consisting of 20 contiguous 3-mm tomograms of the proximal coronary arteries were obtained during a single breath hold. A positive scan was defined as 4 contiguous voxels (> or = 1 mm2) of density > 130 Hounsfield units in the region of the epicardial coronary arteries. Calcification detected by fast CT had an 85% sensitivity to predict patients with significant coronary artery disease (> or = 50% diameter stenosis), with a specificity of 45%. Although the sensitivity to detect multivessel disease was 94%, the sensitivity to detect single-vessel disease was 75%. Changing the threshold for defining a positive fast CT scan from 4 to 2 contiguous voxels produced a small improvement in sensitivity, to 88%, but reduced specificity to 36%. CONCLUSIONS Although the specificity to detect angiographically significant coronary disease with fast CT improves in a younger patient population, it continues to be relatively low. In contrast to older patient populations, a small but significant number of patients < 50 years old with angiographically significant coronary artery disease do not have coronary calcification demonstrated by fast CT. Thus, caution should be used in excluding significant coronary artery disease on the basis of a negative fast CT study.

Profound Apoptosis-Mediated Regional Myocyte Loss and Compensatory Hypertrophy in Pigs With Hibernating Myocardium

BACKGROUND Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. METHODS AND RESULTS Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). CONCLUSIONS These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Coronary pressure-function and steady-state pressure-flow relations during autoregulation in the unanesthetized dog.

The present study was intended to define the interrelation among endocardial flow, endocardial function, and coronary arterial pressure during spontaneous antoregulation in the left ventricle of chronically instrumented unanesthetized dogs. Steady-state sonomicrometric measurements of regional function and epicardial coronary artery pressure were used to determine the lower pressure limit of endocardial autoregulation while global indexes of myocardial demand remained constant. Transmural wall thickening in the circumflex bed remained unchanged (±5% of control values) until coronary pressure fell below 39 ± 5.6 (SD) mm Hg. Endocardial segment shortening was similarly constant until coronary pressure fell below 42 ± 7.4 mm Hg. There was no significant change in endocardial flow as coronary pressure was reduced over the autoregulatory plateau from 84 to 49 mm Hg (1.05–0.99 ml/min/g, p=NS). Below the critical pressure limits, small additional reductions in pressure were associated with marked reductions in both endocardial flow and function. The coronary pressure-function relation was linear as well as steep in this range for both wall thickening (r=0.94 ± 0.05) and segment shortening (r=0.96 ±0.03). Although the relation between endocardial flow and function showed more variability than pressure-function relations at low pressures, wall thickening reductions and endocardial flow reductions related on a nearly one-to-one basis. The present study establishes that the coronary pressure-function relation can be used to define the lower limit of endocardial antoregulation. It also indicates that the lower pressure limit of endocardial autoregulation is considerably less than in anesthetized animals (40 vs. 70 mm Hg) and that steady-state flow above this limit is controlled more tightly. Although these differences may relate to systemic hemodynamics, it seems likely that general anesthesia and/or acute surgical instrumentation alter coronary autoregulation under at least some experimental circumstances.

Reduced regional myocardial perfusion in the presence of pharmacologic vasodilator reserve.

To determine whether reductions in regional myocardial perfusion at reduced coronary arterial pressures reliably indicate maximal vasodilation of the distal vasculature, coronary autoregulation was studied in open-chest dogs at heart rates of approximately 60 beats/min, a level at which metabolic demand, time-averaged systolic compressive forces, and transmural vasodilator reserve approximate those found under usual resting conditions. Circumflex pressure was controlled with a programmable pressure source. Regional circumflex inflow was 0.56 +/- 0.04(SEM) ml . min-1 . g-1 when circumflex pressure equaled spontaneous aortic pressure and fell to 0.34 +/- 0.02 ml . min-1 . g-1 when circumflex pressure was reduced to 35 mm Hg. Reductions were similar in each myocardial layer, with endocardial flow falling from 0.68 +/- 0.04 to 0.39 +/- 0.03 ml . min-1 . g-1. During adenosine-induced vasodilation at 35 mm Hg, full-thickness and endocardial flows rose to 0.92 +/- 0.08 and 1.07 +/- 0.10 ml . min-1 . g-1, respectively. When coronary pressure was reduced to 25 mm Hg and autoregulation was again operative, full-thickness and endocardial flows fell to 0.28 +/- 0.03 and 0.28 +/- 0.04 ml . min-1 . g-1. During adenosine vasodilation at 25 mm Hg endocardial flow did not increase significantly but epicardial reserve remained present. These results indicate that significant reductions in regional myocardial perfusion can occur before pharmacologic vasodilator reserve is exhausted. In the absence of tachycardia, endocardial vasodilator reserve can persist to coronary pressures less than 35 mm Hg, but is ordinarily exhausted before epicardial vasodilator reserve.

Modulation of coronary autoregulatory responses by nitric oxide. Evidence for flow-dependent resistance adjustments in conscious dogs.

The present study tested the hypothesis that nitric oxide production in coronary resistance vessels is an important mechanism affecting the regulation of myocardial perfusion in unanesthetized dogs. We inhibited nitric oxide synthesis with the arginine analogue N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) and maintained the compressive determinants of myocardial blood flow constant by ventricular pacing. L-NAME did not affect resting coronary blood flow and reduced the receptor-mediated increase in flow to intracoronary acetylcholine (100 micrograms/min IC) from 143 +/- 20% (mean +/- SEM) under control conditions to 31 +/- 10% after L-NAME (P < .001). Coronary autoregulatory relations were determined as steady-state coronary pressure was reduced by inflating a hydraulic occluder. Initial resistance adjustments over the autoregulatory plateau were not affected by L-NAME. Closed-loop autoregulatory gain was 0.84 +/- 0.09 under control conditions versus 0.78 +/- 0.07 after L-NAME (P = NS). As coronary pressure was reduced further, however, the critical pressure at which myocardial ischemia began (lower autoregulatory break point) increased from 45 +/- 3 mm Hg under control conditions to 61 +/- 2 mm Hg (P < .001) after L-NAME. In addition, the slope of the coronary pressure-flow relation below the autoregulatory break point was reduced (1.0 +/- 0.2 versus 0.58 +/- 0.09 mL.min-1.mm Hg-1 after L-NAME, P < .05), reflecting a reduction in the maximal conductance recruitable during ischemia. In concert with the effects of L-NAME on autoregulatory responses during ischemia, peak reactive hyperemic flow to a 30-second coronary occlusion was also reduced (from 200 +/- 22 to 166 +/- 24 mL/min after L-NAME, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Hibernating Myocardium Chronically Adapted to Ischemia but Vulnerable to Sudden Death

Abstract— The inability to reproduce spontaneous ventricular fibrillation in an animal model of chronic coronary artery disease has limited advances in understanding mechanisms of sudden cardiac death (SCD). Swine with hibernating myocardium arising from a chronic left anterior descending coronary artery (LAD) occlusion have a high rate of SCD that parallels the poor clinical survival of medically treated patients with hibernating myocardium. Kaplan-Meier analysis (n = 426) demonstrated a cumulative mortality of 49% after 5 months that was almost entirely attributable to spontaneous SCD. Using implantable loop recorders, ventricular fibrillation was documented as the arrhythmic mechanism of death in all animals (n = 10) and was usually preceded by ventricular tachycardia (n = 8). Physiological studies before SCD (n = 7) demonstrated total LAD occlusion and collateral-dependent myocardium (n = 5), excluding acute occlusion as a major trigger of arrhythmia. The physiological substrate of hibernating myocardium was present before SCD, with reductions in LAD perfusion (SCD 0.79±0.13 versus 0.80±0.08 mL/min per g) and wall thickening (SCD 28±3% versus 22±3%) that were similar to survivors (n = 14). Triphenyltetrazolium chloride infarcts among animals with SCD were infrequent (4 of 32) and small, averaging 4.6% of LV mass. Histology (n = 4) showed postmortem changes but no acute inflammation nor contraction band necrosis. These data support the notion that hibernating myocardium is a pathophysiological substrate at high risk of SCD. This is independent of changes in functional stenosis severity, acute myocardial necrosis, or fibrotic scar. Thus, regional adaptations that promote myocyte survival in the setting of chronic repetitive ischemia result in a substrate with enhanced vulnerability to lethal arrhythmias and SCD.