Richard O. Cannon

Markers of Inflammation and Cardiovascular Disease Application to Clinical and Public Health Practice: A Statement for Healthcare Professionals From the Centers for Disease Control and Prevention and the American Heart Association

In 1998, the American Heart Association convened Prevention Conference V to examine strategies for the identification of high-risk patients who need primary prevention. Among the strategies discussed was the measurement of markers of inflammation.1 The Conference concluded that “many of these markers (including inflammatory markers) are not yet considered applicable for routine risk assessment because of: (1) lack of measurement standardization, (2) lack of consistency in epidemiological findings from prospective studies with endpoints, and (3) lack of evidence that the novel marker adds to risk prediction over and above that already achievable through the use of established risk factors.” The National Cholesterol Education Program Adult Treatment Panel III Guidelines identified these markers as emerging risk factors,1a which could be used as an optional risk factor measurement to adjust estimates of absolute risk obtained using standard risk factors. Since these publications, a large number of peer-reviewed scientific reports have been published relating inflammatory markers to cardiovascular disease (CVD). Several commercial assays for inflammatory markers have become available. As a consequence of the expanding research base and availability of assays, the number of inflammatory marker tests ordered by clinicians for CVD risk prediction has grown rapidly. Despite this, there has been no consensus from professional societies or governmental agencies as to how these assays of markers of inflammation should be used in clinical practice. On March 14 and 15, 2002, a workshop titled “CDC/AHA Workshop on Inflammatory Markers and Cardiovascular Disease: Applications to Clinical and Public Health Practice” was convened in Atlanta, Ga, to address these issues. The goals of this workshop were to determine which of the currently available tests should be used; what results should be used to define high risk; which patients should be tested; and the indications for which the tests would be most useful. These …

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 μmol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Imipramine in patients with chest pain despite normal coronary angiograms

BACKGROUND Ten to 30 percent of patients undergoing cardiac catheterization because of chest pain are found to have normal coronary angiograms. Because these patients may have a visceral pain syndrome unrelated to myocardial ischemia, we investigated whether drugs that are useful in chronic pain syndromes might also be beneficial in such patients. METHODS Sixty consecutive patients underwent cardiac, esophageal, psychiatric, and pain-sensitivity testing and then participated in a randomized, double-blind, placebo-controlled three-week trial of clonidine at a dose of 0.1 mg twice daily (20 patients), imipramine at a dose of 50 mg nightly with a morning placebo (20 patients), or placebo twice daily (20 patients); this treatment phase was compared with an identical period of twice-daily placebo for all patients (placebo phase). RESULTS Thirteen (22 percent) of the 60 patients had ischemic-appearing electrocardiographic responses to exercise, 22 of the 54 tested (41 percent) had abnormal esophageal motility, 38 of 60 (63 percent) had one or more psychiatric disorders, and 52 of 60 (87 percent) had their characteristic chest pain provoked by right ventricular electrical stimulation or intracoronary infusion of adenosine. During the treatment phase, the imipramine group had a mean (+/- SD) reduction of 52 +/- 25 percent in episodes of chest pain, the clonidine group had a reduction of 39 +/- 51 percent, and the placebo group a reduction of 1 +/- 86 percent, all as compared with the placebo phase of the trial. Only the improvement with imipramine was statistically significant (P = 0.03). Repeat assessment of sensitivity to cardiac pain while the patients were receiving treatment showed significant improvement only in the imipramine group (P = 0.01). The response to imipramine did not depend on the results of cardiac, esophageal, or psychiatric testing at base line, or on the change in the psychiatric profile during the course of the study, which generally improved in all three study groups. CONCLUSIONS Imipramine improved the symptoms of patients with chest pain and normal coronary angiograms, possibly through a visceral analgesic effect.

Impaired Endothelium-Dependent Vasodilation in Patients With Essential Hypertension Evidence That Nitric Oxide Abnormality Is Not Localized to a Single Signal Transduction Pathway

BACKGROUND Patients with essential hypertension have abnormal endothelium-dependent vascular relaxation, largely related to reduced bioactivity of nitric oxide (NO). The purpose of the present investigation was to determine whether this defect is due to a deficit at the specific intracellular signal-transduction pathway level or is a consequence of a more generalized endothelial abnormality. METHODS AND RESULTS The responses of the forearm vasculature to acetylcholine and bradykinin (endothelium-dependent agents that act through different signal transduction pathways) and to sodium nitroprusside (a direct dilator of vascular smooth muscle) were studied in 10 hypertensive patients (5 men, 5 women; aged 48 +/- 9 years old [mean +/- SD]) and 12 control subjects (6 men, 6 women; aged 48 +/- 7 years old). To determine the contribution of NO to bradykinin-induced vasodilation, the vascular responses to bradykinin were also measured after administration of NG-monomethyl-L-arginine, an arginine analogue that inhibits the synthesis of NO. Drugs were infused into the brachial artery, and forearm blood flow was measured by strain-gauge plethysmography. The response to acetylcholine was significantly blunted in hypertensive patients (maximal blood flow, 7.5 +/- 2 versus 16.6 +/- 8 mL.min-1.100 mL-1 in control subjects [mean +/- SD]; P < .005). Similarly, the vasodilator effect of bradykinin was significantly reduced in hypertensive patients compared with control subjects (maximal blood flow, 8.7 +/- 2 versus 15.8 +/- 6 mL.min-1.100 mL-1 in control subjects; P < .005). A significant correlation was found between the maximal blood flow with acetylcholine and that with bradykinin (r = .89). No significant differences were found between the two groups for vascular response to sodium nitroprusside. NG-monomethyl-L-arginine significantly blunted the response to bradykinin in control subjects (maximal blood flow decreased from 15.8 +/- 6 to 10.1 +/- 2 mL.min-1.100 mL-1, P < .003). In contrast, inhibition of NO synthesis did not modify the response to bradykinin in hypertensive patients (maximal blood flow, 8.7 +/- 2 and 8.5 +/- 3 before and during infusion of NG-monomethyl-L-arginine, respectively; P = NS). As a consequence, the response to bradykinin after inhibition of NO synthesis was not significantly different between the two groups. CONCLUSIONS Patients with essential hypertension have impaired endothelium-dependent vasodilator responses to both acetylcholine and bradykinin. These findings indicate that the endothelial dysfunction in this condition is not related to a specific defect of a single intracellular signal-transduction pathway and suggest a more generalized abnormality of endothelial vasodilator function.

Angina Due to Coronary Microvascular Disease in Hypertensive Patients without Left Ventricular Hypertrophy

When angina occurs in patients with hypertension, it is usually attributed to coronary artery disease or left ventricular hypertrophy. To determine the contribution of coronary microvascular abnormalities to angina in patients with hypertension, we evaluated hypertensive patients without coronary artery disease or left ventricular hypertrophy by measuring the coronary responses to rapid atrial pacing before and after administration of ergonovine. We compared 12 hypertensive patients who had pacing-induced angina with 13 normotensive subjects without such angina. The two groups had similar coronary flow (in the great cardiac vein) at rest; however, pacing increased coronary flow less in hypertensive patients with angina than in normotensive subjects (48 vs. 83 percent; P = 0.05). In the hypertensive patients with angina, pacing after ergonovine increased coronary flow by only 32 percent (as compared with 48 percent before ergonovine; P less than 0.05) and decreased coronary resistance by 15 percent (as compared with 28 percent before ergonovine; P less than 0.05), indicating the presence of ergonovine-induced vasoconstriction. In normotensive subjects, in contrast, cardiac pacing after ergonovine increased coronary flow by 112 percent (P less than 0.001), and its effect on coronary resistance was not different from that of pacing before ergonovine. The hypertensive patients with angina had a significant increase in myocardial oxygen extraction during pacing after ergonovine and less of an increase in myocardial lactate consumption - a response consistent with the presence of myocardial ischemia. Thus, angina in hypertensive patients without epicardial coronary disease may be caused by myocardial ischemia, which appears to be due to an abnormally elevated resistance of the coronary microvasculature.

Effects of Hormone-Replacement Therapy on Fibrinolysis in Postmenopausal Women

Background Plasma levels of plasminogen-activator inhibitor type 1 (PAI-1), an essential inhibitor of fibrinolysis in humans, increase in women after menopause, and this may contribute to the risk of cardiovascular disease. We studied the effects of hormone-replacement therapy on PAI-1 levels. Methods In a randomized, crossover study, we investigated the effects of oral conjugated estrogen (0.625 mg per day) in 30 postmenopausal women and transdermal estradiol (0.1 mg per day) in 20 postmenopausal women, either alone or in combination with medroxyprogesterone acetate (2.5 mg daily) for one month, on plasma PAI-1 antigen levels. Degradation products of cross-linked fibrin (D-dimer) were measured in serum as an index of fibrinolysis. Results PAI-1 levels were inversely associated with D-dimer levels at base line (r = -0.540, P = 0.002). Conjugated estrogen, both alone and in combination with medroxyprogesterone acetate, reduced mean (±SD) plasma levels of PAI-1 from 32±34 ng per milliliter to 14±10 ng per m...

Phenotype and course of Hutchinson-Gilford progeria syndrome

BACKGROUND Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal dominant syndrome that involves premature aging, generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. The genetic basis of most cases of this syndrome is a change from glycine GGC to glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic splice donor site to produce abnormal lamin A; this disrupts the nuclear membrane and alters transcription. METHODS We enrolled 15 children between 1 and 17 years of age, representing nearly half of the worlds known patients with Hutchinson-Gilford progeria syndrome, in a comprehensive clinical protocol between February 2005 and May 2006. RESULTS Clinical investigations confirmed sclerotic skin, joint contractures, bone abnormalities, alopecia, and growth impairment in all 15 patients; cardiovascular and central nervous system sequelae were also documented. Previously unrecognized findings included prolonged prothrombin times, elevated platelet counts and serum phosphorus levels, measured reductions in joint range of motion, low-frequency conductive hearing loss, and functional oral deficits. Growth impairment was not related to inadequate nutrition, insulin unresponsiveness, or growth hormone deficiency. Growth hormone treatment in a few patients increased height growth by 10% and weight growth by 50%. Cardiovascular studies revealed diminishing vascular function with age, including elevated blood pressure, reduced vascular compliance, decreased ankle-brachial indexes, and adventitial thickening. CONCLUSIONS Establishing the detailed phenotype of Hutchinson-Gilford progeria syndrome is important because advances in understanding this syndrome may offer insight into normal aging. Abnormal lamin A (progerin) appears to accumulate with aging in normal cells. (ClinicalTrials.gov number, NCT00094393.)

Angina caused by reduced vasodilator reserve of the small coronary arteries

To study the mechanism of chest pain in patients vvith insignificant large vessel coronary artery disease, 22 patients underwent great cardiac vein flow, coronary resistance and lactate determinations at rest and with coronary sinus pacing followed by coronary arteriography. Nine patients experiencing chest pain with pacing demonstrated significantly lesser increase in flow from base-line values (45 ± 30versus86 ± 50% [mean ± standard deviation], p After administration of ergonovine, 0.15 mg intravenously, 2 of 20 patients experienced spontaneous chest pain, which was associated with the greatest decrease in flow ( - 13 and -21%, respectively) and increase in resistance ( + 26 and +39%, respectively) of any patient. Pacing resulted in chest pain in 10 of the remaining 18 patients, including 5 patients who had not experienced chest pain during the control pacing study. Although none of these patients exhibited significant epicardial coronary artery narrowing on arteriography, their flow increased less (38 ± 12 versus 126 ± 68%, p These findings suggest that some patients with atypical chest pain have inappropriate coronary arteriolar or small coronary artery constriction with abnormal vasodilator reserve in response to atrial pacing. The abnormality can be unmasked or exacerbated in some patients by vasoconstrictor stimuli, but cannot be identified with standard angiographic assessment of “spasm” because abnormal vasoconstriction occurs in vessels too small to be visualized by angiography.

Contribution of Nitric Oxide to Metabolic Coronary Vasodilation in the Human Heart

BACKGROUND The vascular endothelium contributes to smooth muscle relaxation by tonic release of nitric oxide. To investigate the contribution of nitric oxide to human coronary epicardial and microvascular dilation during conditions of increasing myocardial oxygen requirements, we studied the effect of inhibiting nitric oxide synthesis with NG-monomethyl-L-arginine (L-NMMA) on the coronary vasodilation during cardiac pacing in patients with angiographically normal coronary arteries with and without multiple risk factors for coronary atherosclerosis. METHODS AND RESULTS In 26 patients with angiographically normal or near-normal epicardial coronary arteries, metabolic vasodilation was assessed as a change in coronary vascular resistance and diameter during cardiac pacing (mean heart rate, 141 beats per minute). Endothelium-dependent vasodilation was estimated with intracoronary acetylcholine and endothelium-independent dilation with intracoronary sodium nitroprusside and adenosine. These measurements were repeated after 64 mumol/min intracoronary L-NMMA. At rest, L-NMMA produced a 16 +/- 25% (mean +/- SD) increase in coronary vascular resistance (P < .05) and an 11% reduction in distal epicardial coronary artery diameter (P < .01), indicating tonic basal release of nitric oxide from human coronary epicardial vessels and microvessels. Significant inhibition of pacing-induced metabolic coronary vascular dilation occurred with L-NMMA, coronary vascular resistance was 38 +/- 56% higher (P < .03), and epicardial coronary dilation during control pacing (9 +/- 13%) was converted to constriction after L-NMMA and pacing (-6 +/- 9%, P < .04). L-NMMA specifically inhibited endothelium-dependent vasodilation with acetylcholine (coronary vascular resistance was 72% higher [P < .01]) but did not alter endothelium-independent dilation with sodium nitroprusside and adenosine. Nine patients had no major risk factors for atherosclerosis, defined as serum cholesterol > 240 mg/dL, hypertension, or diabetes. The remaining 17 patients with one or more of these risk factors had depressed microvascular vasodilation during cardiac pacing (coronary vascular resistance decreased by 13% versus 36% in those without risk factors, P < .05). The inhibitory effect of L-NMMA on pacing-induced coronary epicardial and microvascular vasodilation was observed only in patients without risk factors, whereas those with risk factors had an insignificant change, indicating that nitric oxide contributes significantly to pacing-induced coronary vasodilation in patients free of risk factors and without endothelial dysfunction. Patients with risk factors also had reduced vasodilation with acetylcholine (40 +/- 28% versus 68 +/- 8% decrease in coronary vascular resistance, P < .01), but the responses to sodium nitroprusside were similar in both groups. CONCLUSIONS During metabolic stimulation of the human heart, nitric oxide release contributes significantly to microvascular vasodilation and is almost entirely responsible for the epicardial vasodilation. This contribution of nitric oxide is reduced in patients exposed to risk factors for coronary atherosclerosis and leads to a net reduction in vasodilation during stress. An important implication of these findings is that reduced nitric oxide bioavailability during stress in patients with atherosclerosis or risk factors for atherosclerosis may contribute to myocardial ischemia by limiting epicardial and microvascular coronary vasodilation.

Cardiac Sympathetic Denervation in Parkinson Disease

Orthostatic hypotension is common in Parkinson disease (1). Although earlier studies implicated L-dopa treatment as the cause (2), more recent studies have shown that orthostatic hypotension may result from deficient cardiovascular reflexes that depend on release of the sympathetic neurotransmitter norepinephrine in the heart and blood vessels (3-5). We call this phenomenon sympathetic neurocirculatory failure. Several recent studies have reported decreased myocardial concentrations of radioactivity after injection of the sympathoneural imaging agent 123I-metaiodobenzylguanidine (123I-MIBG) in patients with Parkinson disease (6-13). This finding is consistent with but does not prove cardiac sympathetic denervation. In addition, studies have not specifically considered the possible association between cardiac sympathetic denervation and sympathetic neurocirculatory failure in Parkinson disease. Measures of autonomic function have included blood pressure during tilt-table testing (abnormalities of which can have several causes), heart rate responses to the Valsalva maneuver (which are determined mainly by changes in parasympathetic cholinergic outflow to the heart), or skin conductance or sweating responses (which are determined mainly by alterations in sympathetic cholinergic outflow to the skin). These measures may not allow assessment of sympathetic noradrenergic function. One way to detect sympathetic neurocirculatory failure in a patient with orthostatic hypotension is by analyzing beat-to-beat blood pressure associated with performance of the Valsalva maneuver (Figure 1). In patients with sympathetic neurocirculatory failure, blood pressure decreases progressively during phase II of the maneuver, whereas normally blood pressure plateaus or increases at the end of phase II (phase II-L). In patients with sympathetic neurocirculatory failure, phase IV blood pressure increases slowly back to baseline after release of the maneuver, whereas normally blood pressure overshoots. These abnormalities are a direct result of deficient cardiovascular reflexes that depend on sympathetically mediated release of norepinephrine. In our study, we defined sympathetic neurocirculatory failure as chronic, reproducible orthostatic hypotension associated with abnormal blood pressure responses in both phase II-L and phase IV of the Valsalva maneuver. Figure 1. Heart rate and blood pressure responses to the Valsalva maneuver in a control patient with a history of neurocardiogenic syncope ( left ) and a patient with Parkinson disease and orthostatic hypotension ( right ). Previous studies also have not independently confirmed that a low myocardial concentration of 123I-MIBGderived radioactivity actually reflects cardiac sympathetic denervation in Parkinson disease. Neurochemical findings indicating decreased norepinephrine release, neuronal uptake, turnover, and synthesis in the heart could provide such confirmation. In humans, 6-[18F]fluorodopamine can be used to visualize cardiac sympathetic innervation by positron emission tomographic (PET) scanning (14), which provides excellent spatial and temporal resolution. Since 6-[18F]fluorodopamine is a catecholamine handled in the heart in a manner similar to the way in which norepinephrine is handled (15), PET scanning may allow functional and anatomic assessments of sympathetic cardiac innervation (16). We used PET scanning after injection of 6-[18F]fluorodopamine and neurochemical measurements during cardiac catheterization to answer the following questions: 1) What proportions of patients with Parkinson disease, with or without sympathetic neurocirculatory failure, have decreased myocardial 6-[18F]fluorodopaminederived radioactivity? 2) Does decreased myocardial 6-[18F]fluorodopaminederived radioactivity in Parkinson disease actually reflect cardiac sympathetic denervation, as identified by indices of cardiac norepinephrine release, neuronal uptake, turnover, and synthesis? 3) Does the frequency of cardiac sympathetic denervation differ between groups of patients with Parkinson disease who have sympathetic neurocirculatory failure and those who do not? 4) Does cardiac sympathetic denervation also occur in patients with multiple-system atrophy, a progressive neurodegenerative disease of adults that features autonomic dysfunction and has parkinsonian, cerebellar, or mixed forms (17)? [The diagnosis of multiple-system atrophy is clinical and, except for a typically poor response to L-dopa treatment, can be difficult to distinguish from Parkinson disease.] 5) Is cardiac sympathetic denervation in patients with Parkinson disease related to L-dopa treatment or to disease duration or severity? Methods The Intramural Research Board of the National Institute of Neurological Disorders and Stroke approved the study protocol. All participants provided written informed consent. Participants We included patients with Parkinson disease or multiple-system atrophy who were studied at the National Institutes of Health Clinical Center in Bethesda, Maryland. Twenty-nine patients had Parkinson disease, including 10 who were not receiving or had never received L-dopa. Twenty-four patients had multiple-system atrophy, including 8 who were taking L-dopa at the time of evaluation. For comparison, we used 6-[18F]fluorodopamine PET scan data and, in most cases, cardiac neurochemical data from 7 patients with pure autonomic failure (5 men, 2 women [mean age SE, 60 6 years]) and 33 controls. Of these 33 controls, 22 had a history of neurocardiogenic syncope (4 men, 18 women [mean age, 35 3 years]) and 11 had a history of postural tachycardia syndrome (1 man, 10 women [mean age, 42 4 years]). 6-[18F]fluorodopamine PET scan data were also obtained from 19 normal volunteers. All patients with Parkinson disease were referred by neurologists or movement disorder clinics and fulfilled accepted clinical criteria (18). Parkinson disease was staged by using the HoehnYahr classification. All affected patients had bradykinesia, cogwheel rigidity, and one or more additional parkinsonian features (pill-roll tremor, stooped posture, festinating gait, difficulty initiating movement, masklike face, micrographia, or marked improvement in motor function during treatment with L-dopa). Patients with multiple-system atrophy had at least two parkinsonian features but were not classified in terms of cerebellar, parkinsonian, or mixed subtypes (17). All had gradually progressive parasympathetic failure (manifested by impotence in men, urinary retention or incontinence, constipation, or constant pulse rate) and had one or more additional features of multiple-system atrophy (heat or cold intolerance and decreased sweating, intention tremor or other evidence of cerebellar dysfunction, slurred speech or a history of aspiration, or no or only slight improvement during an adequate trial of L-dopa treatment). Sympathetic neurocirculatory failure was defined as reproducible, chronic orthostatic hypotension (decrease in diastolic pressure of at least 10 mm Hg and in systolic pressure of at least 20 mm of Hg after 3 to 5 minutes of standing), coupled with abnormal responses of beat-to-beat blood pressure associated with the Valsalva maneuver (19). As noted previously, patients with sympathetic neurocirculatory failure usually exhibit a progressive decrease in blood pressure in phase II-L of the maneuver and an absence of a pressure overshoot in phase IV after release of the maneuver. Valsalva Maneuver For the Valsalva maneuver, the patient lay supine with his or her head on a pillow and blew into a plastic or rubber tube connected to a sphygmomanometer, keeping a pressure of 30 mm Hg for 10 to 12 seconds. The response of beat-to-beat blood pressure during phase II-L of the Valsalva maneuver was considered to be normal if the diastolic and mean arterial pressure increased before the end of the straining and abnormal if they decreased. The response during phase IV was considered to be normal if the systolic blood pressure increased progressively to a value exceeding the baseline (measured just before the patient inhaled and then began straining) and abnormal if the systolic pressure did not exceed the baseline. Sympathetic Neuroimaging Patients were positioned in a GE Advance scanner (General Electric, Milwaukee, Wisconsin), with their thoraxes in the gantry. 6-[18F]fluorodopamine (specific activity, 7.4 to 37 MBq/mmol; dose in most cases, 0.037 MBq) was dissolved in approximately 10 mL of normal saline and infused intravenously at a constant rate for 3 minutes. Thoracic PET scanning was performed for up to 3 hours. The tomographic data were divided into intervals of 5 to 30 minutes. Data acquisition was not gated to the electrocardiogram. In most patients, PET scanning was also used to delineate the left ventricular myocardium and assess myocardial perfusion after administration of the perfusion imaging agent 13 N-ammonia. Intravenously injected 13 N-ammonia exits the bloodstream rapidly and enters cells nonspecifically. A few minutes after the injection, the concentration of 13 N-ammoniaderived radioactivity in the left ventricular myocardium exceeds that in the left ventricular chamber, enabling visualization of the myocardium. Myocardial tissue concentrations of 13 N-ammoniaderived radioactivity depend on local perfusion (20). Neurochemical Testing Patients underwent right-heart catheterization for measurements of norepinephrine spillover into coronary sinus plasma and of venousarterial differences in plasma levels of dihydroxyphenylglycol (DHPG) and L-dopa. After placement of arm and right internal jugular venous catheters (the latter advanced into the coronary sinus), a tracer amount of [3H]norepinephrine (levo- [2, 5, 6] [3H]norepinephrine, New England Nuclear, Boston, Massachusetts) was infused intravenously. Coronary sinus blood flow was measured by thermodilution, and arterial and great cardiac venous or coronary sinus blood was sampled