Impairment of local metabolic coronary control involves perfusion-contraction matching not supply–demand imbalance

Abstract

Understanding of local metabolic control of coronary flow remains stifled by debate around data interpretation and anticipated outcomes. To address this question we performed experiments in a cannulated coronary preparation in swine to precisely control flow as myocardial oxygen consumption (MVO₂) and contractile function were modulated by dobutamine (1–10 μg/kg/min, iv), reductions in coronary perfusion pressure (CPP), and the inhibition of voltage-gated K⁺ channels with 4-aminopyridine (4-AP; 1 mM, ic). Reduction of CPP to 40 mmHg diminished coronary flow (~ 55%; P < 0.001) and systolic wall thickening (~ 35%; P < 0.001). 4-AP-mediated reductions in coronary flow (~ 35%; P = 0.01) and wall thickening (~ 40%; P < 0.05) were restored by returning coronary flow to normal baseline levels. Dobutamine increased heart rate and coronary flow ~ 65% (P < 0.001) and coronary flow remained tightly coupled with MVO₂. Inhibition of coronary responses to dobutamine was associated with an ~ 35% reduction in wall thickening and an ~ 50% increase in MVO₂. Reductions in CPP, administration of 4-AP, and diminished flow during dobutamine infusion were associated with proportional decreases in coronary flow and MVO₂. Wall thickening progressively decreased as coronary flow was reduced below ~ 5.0–7.5 μL/g/beat regardless of whether the decrease was due to diminished “supply” (CPP, 4-AP) or limitations during increased “demand” (flow clamp or restriction with dobutamine). These findings demonstrate that impairments in local metabolic control of coronary flow are reliably demonstrated by decreases in contractile function as a consequence of reductions in the volume of myocardial perfusion per beat.

Introduction

Every area of science has its own seemingly unanswerable question that drives the field toward discovery and understanding. This assertion certainly applies to the field of coronary physiology as studies in the early/mid 1900’s by Rein [1], Anrep [2–5], Wiggers [6, 7], Gregg [8, 9] and Eckenhoff et al. [10] established the inextricable relationship between myocardial metabolism, perfusion, and contractile function. Recognition that the heart utilizes “local metabolic” mechanisms to fine-tune myocardial oxygen delivery to match myocardial metabolic requirements for cardiac performance under a wide variety of (patho-)physiologic conditions advanced a basic but fundamental question: How does the heart couple coronary blood flow to myocardial metabolism? Despite over 100 + years of dedicated research, understanding of this essential phenomenon remains poor, with many hypotheses and few, if any generally accepted mechanisms [11–14].

On the surface, the concept of local metabolic control seems simple enough, alterations in cardiac function and myocardial oxygen consumption (MVO₂) produce commensurate changes in coronary microvascular resistance to ensure sufficient oxygen supply to the myocardium [14]. However, precisely how this delicate balance is achieved remains highly contested and confounded by inherent circular interrelationships between key variables that affect myocardial perfusion. This complex interdependence is demonstrated in Fig. 1 wherein under normal physiological conditions the level of coronary blood flow is dictated by changes in MVO₂ (Fig. 1a) and the underlying degree of cardiac contractile function (Fig. 1b), which drive changes in MVO₂ (Fig. 1c); i.e. function and metabolism determine coronary flow (blue line in Fig. 1). Conversely, when oxygen supply is limited (i.e. ischemia), the level of MVO₂ (Fig. 1a) and contractile function (Fig. 1b) are determined by the amount of coronary blood flow. Subsequently, limitations in regional function are governed by reductions in MVO₂ (Fig. 1c); i.e. coronary flow determines function and metabolism (orange line in Fig. 1) [11, 13–17].

Fig. 1.

Schematic diagram of the Gordian knot between coronary blood flow, MVO₂ and contractile function. Under normal physiological conditions the level of coronary blood flow is dictated by changes in MVO₂ (a) and cardiac contractile function (b), which drive changes in MVO₂ (c); i.e. function and metabolism determine coronary flow (blue line). When oxygen supply is limited, the level of MVO₂ (a) and contractile function (b) are determined by the amount of coronary flow, and limitations in function are governed by reductions in MVO₂ (c); i.e. coronary flow determines function and metabolism (orange line)

The consistent interdependence between the variables within these reciprocal physiological relationships (Fig. 1) serves as the framework for investigations into precisely “how” coronary blood flow is coupled to MVO₂. The prevailing paradigm asserts that inhibition of an active local metabolic factor(s)/pathway(s) results in an imbalance between myocardial oxygen delivery (supply) and myocardial metabolism (demand); conventionally evidenced by the reduction/truncation of the relationships between coronary blood flow or coronary venous PO₂ plotted relative to MVO₂ and/or indices of cardiac contractile function [14, 18]. However, data from Ross [19–22] and Heusch [16, 17, 23] argue against the predicted supply/demand mismatch and in support of inherent “perfusion-contraction matching”, as regional wall thickening has been shown to consistently decrease in direct proportion to reductions in coronary blood flow per beat both at rest and during increases in MVO₂ [19, 20]. Critical barriers to synthesizing an integrated understanding of metabolic control of coronary blood flow include a limited number of studies reporting concurrent measures of coronary blood flow, MVO₂, and contractile function, a general lack of consensus regarding interpretation of cause-and-effect relationships amongst a variety of reported coronary/cardiac variables, and an inability to experimentally control the interacting variables in this complex integrated system.

To better understand local metabolic control of coronary blood flow and the seemingly indecipherable “Gordian Knot” between myocardial perfusion, metabolism, and contractile function (Fig. 1) [14], we performed a series of experiments to define coronary interrelationships when blood flow responses to changes in myocardial metabolism and function were either impaired or absent. For this purpose, we utilized a cannulated coronary artery preparation in open-chest swine to precisely control coronary blood flow as MVO₂ and contractile function (systolic wall thickening) were modulated by dobutamine, changes in perfusion pressure, and inhibition of voltage-gated K⁺ channels with 4-aminopyridine (4-AP). We tested the hypothesis that impairments in local metabolic control of coronary blood flow are reliably demonstrated by reductions in contractile function in response to reductions in myocardial perfusion per beat. Results provide much needed insight into anticipated outcomes to guide interpretation of data related to the inhibition of local metabolic control of coronary blood flow.

Methods

This investigation was approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, Revised 2011). Domestic swine (n = 14; ~ 60 kg) were sedated with Telazol, xylazine, and ketamine (5.0, 2.5, and 2.5 mg/kg, im) prior to anesthesia with buprenorphine (0.03 mg/kg, im) and α-chloralose (60 mg/kg, iv). Additional α-chloralose (20 mg/kg, iv) was given hourly to maintain anesthesia. Upon completion of experimental protocols outlined below, anesthetized animals were humanely euthanized by electrical fibrillation and excision of the heart.

Experimental preparation

Swine were anesthetized, intubated and ventilated with O₂-supplemented room air to achieve > 95% oxyhemoglobin saturation and an end tidal PCO₂ of ~ 40 mmHg; measured by aural pulse oximetry and inline capnography. Bilateral femoral cut downs were performed, and catheters placed in both femoral arteries and femoral veins. An incision was made through the sternohyoideus muscle in the neck and the left carotid artery and internal jugular vein were exposed and catheters placed. One femoral artery catheter provided continuous blood pressure and heart rate monitoring and venous catheters were utilized for intravenous drug administration (α-chloralose anesthesia, heparin, dobutamine, 4-AP). The other femoral catheter was used for arterial blood gas sampling. The carotid catheter was used to supply blood to an extracorporeal servo-controlled roller pump system to perfuse the left anterior descending (LAD) coronary artery to allow for control of coronary perfusion pressure (CPP) and clamping of coronary flow at desired levels [24, 25]. CPP was measured proximal to the cannulation site (within the perfusion line). This proximal pressure was continuously measured and used to adjust the servo-controller to maintain the measured pressure at the desired level. For protocols involving constant coronary flow, the servo-controller was turned off and the roller pump system was manipulated to achieve the desired level of flow.

Following the administration of a bolus of succinylcholine (0.5 mg/kg, iv), a left lateral incision in the fifth intercostal space was made and the pericardium was reflected to expose the heart. The LAD was then isolated, and heparin (500 units/kg, iv) was administered before cannulation of the proximal LAD (distal to the first major branch) with a steel-tipped cannula that was fed via the extracorporeal perfusion circuit. Coronary blood flow was continuously measured by an in-line flow transducer (Transonic Systems, Ithaca, NY, USA). The anterior interventricular vein was also catheterized to allow for coronary venous blood sampling from the LAD-perfusion territory.

Experimental protocol

CPP reductions and 4-AP experiments

A total of n = 8 swine were utilized for experimental protocols involving 1) reductions in CPP (n = 6; 4 female, 2 male) and 2) intracoronary 4-AP administration (n = 5; 3 male, 2 female). A subset of these pigs (n = 3) were subjected to both reductions in CPP followed by 4-AP. Following cannulation of the LAD and an 15–30 min stabilization period, CPP was reduced from 100 to 40 mmHg in 20 mmHg decrements via the servo-controlled roller pump system (n = 6). Arterial and coronary venous blood samples were collected and echocardiographic imaging of the left ventricle (LV) obtained once hemodynamic variables were stable at each CPP (~ 5 min time period at each CPP). Following data collection at CPP of 40 mmHg in a subset of these pigs (n = 3), CPP was returned to 100 mmHg and hemodynamics allowed to return to baseline levels over an ~ 15 min time period. Baseline data were collected and echocardiography performed in these plus n = 2 additional swine followed by an intracoronary infusion of the voltage-gated potassium channel blocker 4-AP (1 mM). Once coronary blood flow and hemodynamic variables were stable with 4-AP administration (~ 5 min of infusion), blood samples and echocardiographic images were obtained. Additional data were also recorded following restoration of coronary blood flow to baseline levels during intracoronary administration of 4-AP (1 mM) via manipulation of the extracorporeal perfusion pump system (n = 5).

Dobutamine and flow-clamp experiments

In a separate group of LAD cannulated swine (n = 6; 3 female, 3 male), CPP was maintained at 100 mmHg before and during intravenous infusion of dobutamine at concentrations of 1, 3, and 10 µg/kg/min in the absence and presence of flow-clamped conditions. The same swine (n = 6) were subjected to each experimental condition. As described above, arterial and coronary venous blood samples were drawn and echocardiographic imaging obtained once hemodynamic variables had stabilized at each level (~ 5 min). Once data were recorded at the highest concentration of dobutamine (10 µg/kg/min), the servo-controlled extracorporeal perfusion circuit was switched from constant CPP to constant-flow mode. Coronary blood flow was subsequently reduced by ~ 20% in a stepwise manner and blood samples and echocardiography acquired at each step while dobutamine was continuously infused at a concentration of 10 µg/kg/min. Following these graded reductions, systemic administration of dobutamine was stopped, coronary flow returned to each animal’s initial baseline flow value and hemodynamics allowed to return to untreated control levels over an ~ 15 min recovery period. Baseline data and echocardiography were performed after this recovery period to ensure hemodynamics and wall thickening returned to normal levels; i.e. no myocardial stunning was evident. With coronary blood flow clamped at baseline levels, systemic administration of dobutamine was then repeated at concentrations of 1, 3, and 10 µg/kg/min with blood sampling and echocardiographic imaging obtained at each dose as described above.

Blood gas analyses

Arterial and coronary venous blood samples were collected, sealed and placed on ice for analysis of pH, PCO₂, PO₂, hemoglobin saturation, lactate, and oxygen content using an automated blood gas analyzer with CO-oximetry (Prime Plus Vet, Nova Biomedical). Myocardial oxygen extraction was determined by the difference between arterial and coronary venous oxygen content divided by arterial oxygen content, multiplied by 100. MVO₂ was calculated by multiplying coronary blood flow by the arterial-coronary venous difference in oxygen content. Lactate uptake was calculated by multiplying coronary blood flow by the arterial-coronary venous difference of lactate concentration. LAD perfusion territory was estimated to be 30% of the total heart weight, as previously described by Feigl [26].

Echocardiographic imaging

Acquisition of echocardiographic images was performed using a Philips Epiq-7C model machine along with an X-51 cardiovascular probe to acquire 2D images of the LV anterior wall. Images were obtained in open-chest swine via placement of the probe (placed in a gel-filled sheath) directly on the surface of the heart. 2D images to measure anterior LV wall thickness at the end of diastole (DWT: end of QRS complex) and the end of systole (SWT: end of the T-wave) were acquired and averaged from 5 consecutive beats in the apical short-axis view at each stage of the experimental protocol. Ventricular volumes were calculated using the Simpson method. Systolic wall thickening was calculated as the percent change in wall thickness from end diastole to end systole ((SWT-DWT)/DWT) × 100.

Statistical analyses

A hyperlink to a figshare file containing all individual data from each of the experimental protocols is provided at the end of the manuscript. All reported variables were averaged over a period of ~ 5 cardiac cycles under all experimental conditions in each animal. All values are presented as mean ± SEM. For all statistical comparisons, P < 0.05 was considered statistically significant. Statistical comparisons for data presented in Tables were performed by two-way repeated measures analysis of variance (ANOVA) with Sigma Stat (version 4.0). When significance was found with ANOVA, a Student–Newman–Keuls multiple comparison test was performed to identify differences between conditions. Multiple linear regression analysis was used to compare slopes of relationships based on average data from each experimental group and condition (GraphPad Prism 10). If slopes were equivalent, subsequent analysis of covariance (ANCOVA) was used to test for differences in elevation in the relationship between specific response (y-axis) variables relative to changes in the relevant independent (x-axis) variable. Regression analyses were also performed on the global fits of average data from all groups collectively. Nonlinear regression fits were accomplished by 3rd order polynomial (cubic) fits.

Results

Coronary interrelationships in response to reductions in blood flow

Systemic hemodynamic and cardiac effects of reductions in CPP are provided in Table 1. Reductions in CPP from 100 to 40 mmHg did not affect blood pressure (P = 0.33) or heart rate (P = 0.94) but significantly decreased coronary blood flow (~ 55%; P < 0.001), MVO₂ (~ 40%; P = 0.01), coronary venous PO₂ (~ 30%; P < 0.001), stroke volume (~ 35%; P < 0.001) and cardiac output (~ 35%; P < 0.001) (Table 1). Initial decreases in CPP from 100 to 80 mmHg resulted in a significant increase in myocardial oxygen extraction from 50 ± 5% to 63 ± 5% (P < 0.05; Table 1). Reductions in CPP from 100 to 60 mmHg did not influence either MVO₂ (P = 0.99; Fig. 2a) or systolic wall thickening (P = 0.79; Fig. 2b). However, further reductions in CPP to 40 mmHg were associated with diminished MVO₂ (P = 0.01; Fig. 2a) and systolic wall thickening of the anterior region of the LV (P < 0.001; Fig. 2b). This decrease in regional wall thickening was directly related to reductions in MVO₂ at low CPP (P = 0.01; Fig. 2c).

Table 1.

Hemodynamic and cardiac effects of reductions in coronary perfusion pressure

	CPP 100	CPP 80	CPP 60	CPP 40
Coronary Perfusion Pressure (mmHg)	99 ± 1	79 ± 1*	60 ± 1*	40 ± 1*
Coronary Blood Flow (mL/min/g)	0.76 ± 0.07	0.57 ± 0.10*	0.50 ± 0.09*	0.34 ± 0.07*
Mean Blood Pressure (mmHg)	108 ± 6	110 ± 4	110 ± 5	107 ± 5
Systolic Blood Pressure (mmHg)	132 ± 5	134 ± 4	133 ± 5	130 ± 5
Diastolic Blood Pressure (mmHg)	95 ± 6	98 ± 4	98 ± 5	95 ± 5
Heart Rate (beats/min)	113 ± 9	114 ± 9	114 ± 9	114 ± 10
Hematocrit (%)	35 ± 1	36 ± 1	37 ± 1	36 ± 1
O2 Extraction (%)	50 ± 5	63 ± 5*	64 ± 7	62 ± 10*
Coronary Venous PO2 (mmHg)	33 ± 5	28 ± 4*	26 ± 4*	23 ± 4*
Myocardial Oxygen Consumption (µL O2/min/g)	62 ± 7	62 ± 11	56 ± 11	37 ± 8*
Myocardial Lactate Uptake (µmol/min/g)	0.12 ± 0.28	0.32 ± 0.10*	0.23 ± 0.12*	– 0.02 ± 0.06*
Stroke Volume (mL)	56 ± 3	58 ± 3	54 ± 3	37 ± 5*
Systolic Wall Thickening (%)	26.4 ± 2.0	26.9 ± 1.7	23.2 ± 1.9	16.9 ± 3.7*
Cardiac Output (L/min)	6.3 ± 0.6	6.6 ± 0.5	6.1 ± 0.5	4.2 ± 0.8*

Values are mean ± SE for n = 6 pigs. * P < 0.05 vs. CPP 100

Fig. 2.

Interrelationships between coronary blood flow, MVO₂ and regional systolic wall thickening during reductions in CPP from 100 to 40 mmHg (n = 6) demonstrate decreases in coronary flow are associated with reductions in MVO₂ (a) and wall thickening (b). Decreases in wall thickening were also associated with reductions in MVO₂ (c). Reductions in coronary flow induced by intracoronary administration of 4-AP decreased MVO₂ (d) and wall thickening (e), that was associated with a modest decrease in MVO₂ (f). Restoration of coronary flow to baseline levels (4-AP Rescue) restored each of these variables to their respective control levels (n = 5). * P < 0.05 vs. Baseline

Intracoronary administration of the K_V channel inhibitor 4-AP (1 mM) significantly diminished coronary blood flow from 0.79 ± 0.18 mL/min/g to 0.51 ± 0.11 mL/min/g (Table 2). Systemic blood pressure (P = 0.80) and heart rate (P = 0.09) were not significantly altered by 4-AP, despite decreases in stroke volume (~ 25%; P = 0.06) and cardiac output (~ 40%; P < 0.05) (Table 2). 4-AP-mediated reductions in coronary blood flow were associated with an ~ 20% decrease in MVO₂ (P = 0.15; Fig. 2d) and resulted in an ~ 40% reduction in wall thickening (P < 0.05; Fig. 2e). Thus, the decrease in regional function was associated with a moderate change in MVO₂ (Fig. 2f). Manual manipulation of the extracorporeal perfusion system pump to restore coronary blood flow to baseline levels in the presence of 4-AP administration restored regional wall thickening to normal, pre-4-AP treatment values (P = 0.42; Fig. 2e).

Table 2.

Hemodynamic and cardiac effects of intracoronary administration of 4-aminopyridine and subsequent flow restoration (rescue)

		4- Aminopyridine
	Baseline	1 mM	1 mM Rescue
Coronary Perfusion Pressure (mmHg)	100 ± 1	100 ± 1	184 ± 15*†
Coronary Blood Flow (mL/min/g)	0.79 ± 0.18	0.51 ± 0.11*	0.80 ± 0.18†
Mean Blood Pressure (mmHg)	108 ± 4	111 ± 7	103 ± 9
Systolic Blood Pressure (mmHg)	132 ± 5	134 ± 8	129 ± 9
Diastolic Blood Pressure (mmHg)	96 ± 4	95 ± 7	88 ± 8
Heart Rate (beats/min)	97 ± 13	84 ± 17	79 ± 14
Hematocrit (%)	38 ± 2	40 ± 1	39 ± 1
O2 Extraction (%)	50 ± 7	62 ± 4	47 ± 6
Coronary Venous PO2 (mmHg)	34 ± 4	31 ± 5	34 ± 5
Myocardial Oxygen Consumption (µL O2/min/g)	83 ± 16	66 ± 13	88 ± 17
Myocardial Lactate Uptake (µmol/min/g)	0.18 ± 0.17	0.24 ± 0.12	0.15 ± 0.13
Stroke Volume (mL)	54 ± 1	40 ± 3	54 ± 1†
Systolic Wall Thickening (%)	25.5 ± 1	15.5 ± 2.4*	23.5 ± 1.4†
Cardiac Output (L/min)	4.6 ± 0.2	2.7 ± 0.1	3.6 ± 0.3†

Values are mean ± SE for n = 5 pigs. * P < 0.05 vs. Baseline; † P < 0.05 vs. 1 mM 4-AP

Coronary interrelationships in response to dobutamine

Hemodynamic and cardiac effects of systemic dobutamine administration are provided in Table 3. Dobutamine (1–10 μg/kg/min) increased heart rate ~ 65% (P < 0.001) and cardiac output ~ 50% (P < 0.001), while mean blood pressure was not significantly altered (P = 0.64) (Table 3). These hemodynamic changes were associated with increases in MVO₂ from 65 ± 11 μL O₂/min/g at baseline to 126 ± 11 μL O₂/min/g at the highest dose of dobutamine (10 μg/kg/min) (P < 0.001). Coronary blood flow was tightly coupled to increases in MVO₂ (P < 0.05; Fig. 3a), while systolic wall thickening remained relatively unchanged at the highest (10 μg/kg/min) concentration of dobutamine (P = 0.15; Fig. 3b). Accordingly, dobutamine-induced increases in MVO₂ were not associated with changes in wall thickening (P = 0.14; Fig. 3c).

Table 3.

Hemodynamic and cardiac effects of systemic dobutamine administration with constant coronary pressure vs. constant coronary flow

	Dobutamine
	Baseline	1 µg/kg/min	3 µg/kg/min	10 µg/kg/min
Coronary Perfusion Pressure (mmHg)
Constant Pressure	101 ± 1	101 ± 1	100 ± 1	100 ± 1
Constant Flow	88 ± 5	81 ± 6	61 ± 6*†	47 ± 1†
Coronary Blood Flow (mL/min/g)
Constant Pressure	0.85 ± 0.07	0.94 ± 0.08	1.14 ± 0.14*	1.40 ± 0.15*
Constant Flow	0.84 ± 0.07	0.84 ± 0.07	0.84 ± 0.07†	0.84 ± 0.07†
Mean Blood Pressure (mmHg)
Constant Pressure	108 ± 8	120 ± 8	119 ± 11	111 ± 11
Constant Flow	84 ± 10	107 ± 10	106 ± 12	85 ± 14
Systolic Blood Pressure (mmHg)
Constant Pressure	133 ± 11	151 ± 12	156 ± 15*	147 ± 14
Constant Flow	119 ± 11	134 ± 14	137 ± 16	120 ± 21
Diastolic Blood Pressure (mmHg)
Constant Pressure	96 ± 6	104 ± 7	100 ± 10	92 ± 10
Constant Flow	67 ± 14	93 ± 9	90 ± 10	67 ± 11
Heart Rate (beats/min)
Constant Pressure	113 ± 9	116 ± 14	146 ± 17*	188 ± 13*
Constant Flow	122 ± 6	126 ± 8	147 ± 12*	197 ± 13*
Hematocrit (%)
Constant Pressure	33 ± 1	34 ± 2*	36 ± 1*	38 ± 2*
Constant Flow	38 ± 1†	37 ± 1 *	37 ± 1	37 ± 2
O2 Extraction (%)
Constant Pressure	47 ± 7	48 ± 5	44 ± 4	49 ± 4
Constant Flow	46 ± 5	52 ± 6	57 ± 8	62 ± 7
Coronary Venous PO2 (mmHg)
Constant Pressure	38 ± 2	37 ± 4	36 ± 3	34 ± 2
Constant Flow	38 ± 5	40 ± 3	36 ± 3	31 ± 2
Myocardial Oxygen Consumption (µL O2/min/g)
Constant Pressure	65 ± 11	75 ± 10	84 ± 8	126 ± 12*
Constant Flow	68 ± 10	76 ± 11	84 ± 11	93 ± 9
Myocardial Lactate Uptake (µmol/min/g)
Constant Pressure	0.43 ± 0.08	0.48 ± 1.37	0.49 ± 0.21	0.28 ± 0.22
Constant Flow	0.10 ± 0.14†	0.35 ± 0.13	0.65 ± 0.21	-0.82 ± 0.53 †
Stroke Volume (mL)
Constant Pressure	59 ± 1	61 ± 2	56 ± 1	54 ± 2
Constant Flow	55 ± 2	50 ± 3†	45 ± 3†*	43 ± 3†*
Systolic Wall Thickening (%)
Constant Pressure	25.6 ± 1.6	27.3 ± 1.6	27.9 ± 1.7	28.8 ± 1.9
Constant Flow	23.4 ± 1.7	21.7 ± 1.9	18.8 ± 1.2*	15.2 ± 1.1*
Cardiac Output (L/min)
Constant Pressure	6.7 ± 0.6	7.1 ± 0.9	8.1 ± 1.0	10.1 ± 0.8
Constant Flow	6.7 ± 0.4	6.3 ± 0.5	6.7 ± 0.6	8.4 ± 0.5

Values are mean ± SE for n = 6 pigs. * P < 0.05 vs. Baseline, same group; † P < 0.05 vs. Constant Pressure, same dobutamine concentration

Fig. 3.

Interrelationships between coronary blood flow, MVO₂ and regional systolic wall thickening in response to dobutamine in the absence and presence of clamped or restricted coronary flow (n = 6). Group differences in the slopes and resultant intersections between these interrelationships reflect the physiological operating ranges such that progressive impairments of the coronary flow response to dobutamine result in attenuation of increases in MVO₂ (a), reductions in wall thickening (b) and limitations in regional function relative to the level of MVO₂ (c)

Coronary interrelationships in response to elevated MVO₂ with clamped coronary flow

The maintenance of coronary blood flow at baseline levels (0.84 ± 0.07 mL/min/g) during systemic dobutamine administration (1 – 10 μg/kg/min) did not affect the overall increase in heart rate from 122 ± 6 beats/min at rest to 197 ± 13 beats/min at the highest concentration of dobutamine (P = 0.97; Table 3). The limitation of coronary flow (constant flow) significantly diminished systolic (~ 20%; P = 0.01), diastolic (~ 25%; P < 0.01), and mean (~ 25%; P < 0.05) blood pressure relative to 10 μg/kg/min dobutamine administration with normal coronary flow (constant pressure) (Table 3). Reductions in stroke volume (~ 20%; P < 0.001) and a change from myocardial lactate uptake to production (P = 0.05) were also evident during dobutamine infusion (Table 3). Constant flow conditions steepened the relationship between MVO₂ and coronary blood flow (P < 0.05; Fig. 3a) with an ~ 35% increase in myocardial oxygen extraction (Table 3) accounting for a similar ~ 35% increase in MVO₂ in response to dobutamine. The absence of any coronary blood flow response to dobutamine was also associated with a progressive decline in systolic wall thickening from 23 ± 2% at baseline to 15 ± 1% at the highest concentration of dobutamine (P = 0.01; Fig. 3b). Dobutamine-mediated increases in heart rate and myocardial oxygen extraction likely accounted for modest increase in MVO₂, despite reductions in regional wall thickening (Fig. 3c).

Coronary interrelationships in response to reductions in coronary flow with elevated MVO₂

Measurements were also obtained during progressive reductions in coronary blood flow during continuous systemic dobutamine administration (10 μg/kg/min, iv). Coronary flow was decreased in 4 sequential (~ 20%) steps from 1.42 ± 0.15 mL/min/g at highest dobutamine concentration down to a final value of 0.52 ± 0.03 mL/min/g (Table 4). These decreases in coronary flow were accompanied by an ~ 20% reduction in systolic blood pressure (P < 0.01), marked myocardial lactate production (P < 0.001), and an increase in myocardial oxygen extraction from 49 ± 4% to 78 ± 5% (P < 0.05) (Table 4). The relationship between MVO₂ and coronary blood flow was shifted upward (P < 0.01; Fig. 3a) and the relationship between systolic wall thickening and coronary blood flow was shifted downward (P = 0.01; Fig. 3b). Resultant decreases in regional wall thickening from 29 ± 2% at the highest concentration of dobutamine to 16 ± 2% at the lowest coronary flow (P < 0.001) were associated with a progressive (up to ~ 40%) reduction in MVO₂. However, values of systolic wall thickening were diminished relative to respective levels of MVO₂ during flow reductions vs. dobutamine alone (P < 0.05; Fig. 3c).

Table 4.

Hemodynamic and cardiac effects of systemic dobutamine administration with serial reductions in coronary flow

			10 µg/kg/min Dobutamine
	CPP 100	80% Flow	60% Flow	40% Flow	20% Flow
Coronary Perfusion Pressure (mmHg)	100 ± 1	78 ± 8	63 ± 5*	47 ± 3*	39 ± 3*
Coronary Blood Flow (mL/min/g)	1.42 ± 0.15	1.17 ± 0.14*	0.92 ± 0.13*	0.67 ± 0.11*	0.52 ± 0.03*
Mean Blood Pressure (mmHg)	111 ± 11	107 ± 12	94 ± 7	90 ± 7	90 ± 5
Systolic Blood Pressure (mmHg)	147 ± 14	144 ± 15	126 ± 10	122 ± 10*	121 ± 8*
Diastolic Blood Pressure (mmHg)	92 ± 10	88 ± 11	78 ± 7	75 ± 6	75 ± 5
Heart Rate (beats/min)	188 ± 13	189 ± 14	190 ± 14	190 ± 18	198 ± 18
Hematocrit (%)	38 ± 2	38 ± 1	39 ± 1	39 ± 1	38 ± 1
O2 Extraction (%)	49 ± 4	51 ± 5	57 ± 5	64 ± 4	78 ± 5*
Coronary Venous PO2 (mmHg)	34 ± 2	31 ± 2	28 ± 2	26 ± 1*	26 ± 3
Myocardial Oxygen Consumption (µL O2/min/g)	126 ± 12	112 ± 17	101 ± 18	80 ± 14*	75 ± 8*
Myocardial Lactate Uptake (µmol/min/g)	0.28 ± 0.22	-0.13 ± 0.23	-1.22 ± 0.44*	-1.83 ± 0.59*	-2.34 ± 0.42*
Stroke Volume (mL)	54 ± 2	56 ± 2	52 ± 2	50 ± 3	46 ± 3 *
Systolic Wall Thickening (%)	28.8 ± 1.9	24.8 ± 2.2	20.2 ± 1.7	18.6 ± 2.2*	15.7 ± 1.8*
Cardiac Output (L/min)	10.1 ± 0.8	10.6 ± 0.7	9.9 ± 0.7	9.3 ± 0.8	9.0 ± 0.7

Values are mean ± SE for n = 6 pigs. * P < 0.05 vs. Control

Examination of both flow reductions and restrictions (clamp) during dobutamine administration demonstrates the physiological interdependence between coronary blood flow, regional wall thickening, and MVO₂ as these variables are “forced” in different directions. Apparent intersections in key interrelationships illustrate that impairments in coronary blood flow are associated with mounting contractile dysfunction such that wall thickening is diminished to ~ 65 ± 5% of baseline levels when coronary flow responses to increases in MVO₂ are absent (Slope P < 0.01; Fig. 3b). MVO₂ remains elevated (~ 150 ± 20% of baseline values) during limitations when flow is clamped at control levels (Slope P = 0.01; Fig. 3a). Accordingly, heart rates of ~ 200 beats/min (Table 4) and ~ 35% reductions in regional wall thickening correspond with ~ 50% increases in MVO₂ when dobutamine-induced flow responses are abolished (Slope P < 0.001; Fig. 3c).

Myocardial supply/demand imbalance vs. perfusion-contraction matching

Figure 4 provides relationships typically utilized to assess the balance between coronary blood flow and MVO₂ (coronary venous PO₂ vs. MVO₂; Fig. 4a) and overall perfusion-contraction matching (regional wall thickening vs. coronary flow per beat; Fig. 4b) for each experimental condition examined in the present study. Coronary venous PO₂ fell in relation to reductions in coronary blood flow in response to decreases in CPP (Table 1) and to the administration of 4-AP (Table 2). Coronary venous PO₂ also tended to decrease in response to dobutamine-mediated increases in MVO₂ during constant pressure and during constant flow (Table 3). At the highest dobutamine concentration, subsequent reductions in coronary flow also resulted in progressive decreases in coronary venous PO₂ as MVO₂ was reduced (Fig. 4a; Table 4). Multiple linear regression revealed significant differences in slope (P < 0.05) between groups. However, the global fit of all average data points from all experimental groups does not support an association between coronary venous PO₂ and MVO₂ under these conditions (P = 0.38; R² = 0.05; black regression line in Fig. 4a).

Fig. 4.

a) Relationship between coronary venous PO₂ and MVO₂ and between b) regional wall thickening and coronary flow per beat during reductions in coronary perfusion pressure (n = 6), administration of 4-AP (n = 5), and dobutamine infusion in the absence and presence of clamped or restricted coronary flow (n = 6). Multiple linear regression revealed significant differences in the slope of the coronary venous PO₂ vs. MVO₂ relationship (a) between groups (P < 0.05). However, global fit of all average data from all experimental groups (black regression line) showed no association between these variables under these conditions (P = 0.38; R² = 0.05). Alternatively, no differences in the slope of the relationship between wall thickening and coronary flow per beat (b) were noted between treatment groups (P = 0.38) while significant shifts in elevation were noted (P = 0.002). Global fit of all average data from all experimental groups (black regression line) revealed regional wall thickening was highly associated with reductions in the volume of myocardial blood flow normalized per cardiac cycle (P < 0.001; R² = 0.57)

Systemic dobutamine administration resulted in minimal changes in regional wall thickening relative to coronary blood flow normalized per beat (Fig. 4b). In contrast, wall thickening was progressively reduced as coronary flow per beat was diminished below ~ 5.0—7.5 μL/g/beat in response to decreases in resting coronary blood flow (CPP and 4-AP) and when coronary flow was restricted/diminished in the presence of dobutamine-mediated increases in MVO₂. Multiple linear regression demonstrated similar slopes between treatment groups (P = 0.38) while significant shifts in the elevation of wall thickening relative to coronary flow per beat were evident between groups (P = 0.002). Regression analysis of the global fit of all average data points from all experimental groups revealed regional wall thickening was highly associated with reductions in the volume of myocardial blood flow normalized per cardiac cycle (P < 0.001; black regression line in Fig. 4b), regardless of the underlying condition(s) promoting the onset of ischemia.

Discussion

Following over a century of research, understanding of the mechanisms responsible for the coupling of coronary blood flow with myocardial metabolic requirements has remained elusive. Discovery of specific factors/pathways continues to be confounded by continued debate around anticipated experimental outcomes and interpretation of complex circular relationships (Fig. 1) between key variables that are inconsistently measured and reported throughout the literature [14]. To directly address this matter, the present studies were designed to interrogate interrelationships between decreases (CPP and 4-AP) and increases (dobutamine) in regional wall thickening and MVO₂ in the absence and presence of either constant or reduced coronary flow conditions. Our findings support the hypothesis that impairments in the control of coronary blood flow are consistently characterized by progressive decreases in contractile function in response to reductions in volume of myocardial perfusion per beat below ~ 5.0—7.5 μL/g/beat [16]. Results highlight the need for concurrent measurement of contractile function, coronary flow, and MVO₂ and corroborate that antagonist-mediated contractile dysfunction should be corrected by the restoration of coronary flow to normal, pre-treatment levels [14, 27]. Data also provide a definitive framework of anticipated outcomes to guide future investigation of local metabolic mechanisms of coronary blood flow control.

Interrelationships between coronary blood flow, contractile function, and MVO₂

It is well established that increases in cardiac contractile function and MVO₂ are directly linked with proportionate increases in coronary blood flow [17, 21, 28, 29]. Conversely, limitations in coronary flow produce commensurate reductions in MVO₂ and contractile function (Fig. 1). How then would the inhibition of metabolic factors that link perfusion to metabolism influence these variables? Would not primary alterations in flow or MVO₂ simply follow the prescribed relationships outlined in Fig. 1? To what extent would the slopes of these relationships change? How do changes in myocardial oxygen extraction influence these interrelationships? What if the factor/pathway blocked only influenced coronary flow under baseline resting conditions? What would happen to the relationships if local metabolic control was completely abolished? The lack of definitive answers to these, and many other related questions continues to plague the field and thus was the major impetus for this study.

Our results confirm the presence of a threshold of coronary flow, below which MVO₂ (Fig. 2a) and contractile function (Fig. 2b) begin to fall [16, 20]. This threshold was shifted to lower levels of flow as initial reductions in CPP were accompanied by increases in myocardial oxygen extraction that were sufficient to sustain MVO₂ and wall thickening at baseline levels at CPPs ≥ 60 mmHg. Findings also verify that if antagonist-mediated reductions in MVO₂ (Fig. 2d) and function (Fig. 2e) are related to deficits in perfusion, they should be corrected by the restoration of coronary flow to normal, pre-treatment levels [14, 27]. Data from this investigation also established physiological windows that reflect relative operating ranges for changes in regional wall thickening and MVO₂ as coronary blood flow is limited during increases in cardiac workload (Fig. 3). These functional “loops” highlight expected limits of variable degrees of inhibition of any potential local metabolic factor(s)/pathway(s), down to the elimination of changes in blood flow which correspond with an ~ 35% reduction in wall thickening and an ~ 50% increase in MVO₂ relative to normal baseline levels. This increase in MVO₂ is in fact exaggerated in this study by the lower levels of oxygen extraction (baseline = 46 ± 5%; Table 3) observed in our cannulated, extracorporeal perfused coronary preparation. Thus, the working physiological range for changes in the relationship between coronary blood flow and MVO₂ is likely smaller than that depicted in Fig. 3a. The functional coronary “loops” presented in Fig. 3 were generated using coronary flows at or above baseline values and thus do not depict effects of potential antagonist-mediated reductions in baseline coronary blood flow.

Balance between coronary blood flow and MVO₂

Dogma of the coronary field has long held that inhibition of a local metabolic pathway will decrease the balance between coronary flow and MVO₂ [11–14, 18]. Evidence to support the impairment of “supply/demand balance” has largely centered around increases in myocardial oxygen extraction and predicted reductions in coronary venous PO₂ relative to changes in MVO₂ (see Fig. 4a) [11, 13, 30–32]. While augmented oxygen extraction is a logical consequence of reductions in coronary flow, the degree of compensation is quite limited as the left ventricle typically extracts ~ 75–80% of the oxygen delivered at rest [12, 13, 33]. Accordingly, MVO₂ is highly dependent on coronary blood flow, and thus any reduction in flow will lead to a proportional reduction in myocardial oxygen delivery and thus MVO₂ (see Fig. 1a; [13]). Consequently, it is important to recognize that the slope (i.e. “balance”) between coronary blood flow and MVO₂ cannot be appreciably altered within these physiologic constraints. The interdependence of these variables is also evident in calculation of MVO₂ as the product of coronary blood flow and the arterial-coronary venous difference in oxygen content. As such, analysis of the relationship between MVO₂ and coronary blood flow is inherently confounded by the fact that coronary blood flow is represented on both the ordinate and abscissa of the relationship [14]. This point is highlighted in the present study by the relatively modest increase in MVO₂ when coronary flow is clamped at baseline levels during systemic dobutamine administration (Fig. 3a). As outlined above, this increase in MVO₂ is higher than normal as resting oxygen extraction was significantly reduced in our cannulated preparation.

Use of coronary venous PO₂ vs. MVO₂ as an assessment of the balance between myocardial oxygen delivery and metabolism introduces its own set of complications. Current results highlight characteristic decreases in coronary venous PO₂ (Fig. 4a) in response to changes in CPP (~ 30%; Table 1), to increases in MVO₂ (~ 10%; Table 3), and to reductions in coronary flow with elevated MVO₂ (~ 25%; Table 4). However, interpretation of differences in nonlinear responses and/or parallel shifts vs. changes in slope of the coronary venous PO₂ and MVO₂ relationship continue to obscure definitive conclusions. Underlying differences between species [11, 14] and divergent responses to similar patho-physiologic perturbations (e.g. anemia vs. hypoxemia [14]) further confound the utility of this relationship as a reliable means to assess potential mechanisms of coronary flow control.

Perfusion-contraction matching

Our group recently proposed the primary criterion to apply to experimental studies of local metabolic control is that inhibition of the factor/pathway should attenuate the coronary flow response to changes in MVO₂ [14]. Although such an effect cannot markedly diminish the slope of the relationship between coronary blood flow and MVO₂ (i.e. overall supply/demand balance), there is substantial evidence to support that the adequacy of myocardial perfusion is reflected by changes in regional contractile function [16, 17, 19, 20, 22, 23, 28, 29, 34–36]. To examine the perfusion-contraction matching paradigm in the context of local metabolic control, we assessed the relationship between systolic wall thickening and coronary blood flow per beat for all groups in this study. As shown in Fig. 4b, dobutamine-mediated increases in heart rate were matched by increases in coronary blood flow such that the volume of flow per beat remained essentially unchanged. However, the black regression line in Fig. 4b demonstrates wall thickening decreased in direct proportion to reductions in coronary flow per beat in all experimental groups (P < 0.001; R² = 0.57), regardless of whether the decrease was due to a decrease in “supply” (↓ CPP, 4-AP) or a limitation of flow during increases in “demand” (flow reduction vs. flow clamp with dobutamine). These data are consistent with the findings of Gallagher et al. who were the first to observe that the level of contraction is determined by the availability of blood reaching the myocardium on a per beat basis both at rest and during exercise [20]. The progressive decrease in wall thickening as flow per beat falls below an apparent threshold of ~ 5.0—7.5 μL/g/beat is consistent with advancing degrees of myocardial ischemia as proposed by Heusch [16, 23]. More importantly, the escalation of contractile dysfunction in response to antagonist-mediated flow reductions is consistent with the increased contribution of the inhibited pathway to metabolic coronary vasodilation. Accordingly, we submit that progressive decreases in contractile function as a consequence of reductions in coronary flow per beat represents a more reliable means by which to interrogate potential mechanisms of local metabolic control of coronary blood flow.

Limitations of the study

It is important to appreciate that the present results relate to relationships within and below the autoregulatory range, but not above it [37]. Additionally, anesthetized/cannulated preparations typically display poor autoregulation and lower levels of myocardial oxygen extraction in comparison to conscious preparations [28, 38–40]. This augmented oxygen extraction reserve could influence the critical threshold at which regional contractile function begins to decline as coronary flow per beat is reduced. Positioning of the threshold could also be shifted by changes in hematocrit, myocardial compressive forces, or by endogenous factors such as nitric oxide that impact myocardial efficiency by reducing MVO₂ and conserving contractile function [15, 41]. Thus, it is not surprising that the critical flow per beat necessary to induce reductions in regional wall thickening ranged from ~ 5.0 mL/g/beat (↓ CPP) to ~ 7.5 μL/g/beat (Dobutamine ↔ flow) (Fig. 4b). While the experimental preparation and/or condition may influence the value of this threshold, the primary conclusion that impairments in coronary flow control are reliably demonstrated by decreases in contractile function as a consequence of reductions in coronary flow per beat remains evident. In the absence of measures of high energy phosphates, the extent to which this perfusion-contraction matching reflects short term hibernation or overt ischemic contractile dysfunction remains to be determined [42].

We acknowledge that reductions in regional contractile function are largely dictated by decreases in subendocardial blood flow [19, 20, 28] which was not assessed in the present study. However, this limitation is tempered by the paucity of collateral blood flow and the subendocardial dominance of transmural assessments of regional contractile function and perfusion in swine [43]. Transmural flow heterogeneity also confounds interpretation of global assessments of variables such as MVO₂, myocardial oxygen extraction and lactate uptake/release, which not only vary between the subepicardium and subendocardium but also fail to achieve true steady state levels during myocardial ischemia [44]. Prior studies suggest that examination of systemic inotropic stimulation on regionally ischemic myocardium is confounded by effects on nonischemic regions [22]. Accordingly, we acknowledge that percent wall thickening may underestimate the work relevant for MVO₂ as LV pressure is elevated by dobutamine [22]. Return of regional contractile function to normal baseline levels after transient ischemic episodes (↓ CPP or Dobutamine ↓ flow) could also reflect some degree of recruitment of inotropic reserve at the cost of impaired metabolic performance and shifts toward alternative energy sources such as creatine phosphate and lactate production [45].

Implications and conclusions

Data from this investigation argue against the prevailing dogma of myocardial oxygen supply/demand imbalance and in support of the paradigm of myocardial perfusion-metabolism-contraction matching (see schematic in Fig. 5). Our findings highlight that under normal physiological conditions, myocardial metabolism and contractile function dictate the level of myocardial perfusion but if/when myocardial perfusion becomes limited, reductions in perfusion determine the level of myocardial metabolism and contractile function. Importantly, this paradigm applies directly to impairments in the control of coronary blood flow which are consistently characterized by the progressive decline of contractile function as a consequence of reductions in volume of myocardial perfusion per beat below an apparent threshold of ~ 5.0—7.5 μL/g/beat [16]. Results advocate the need for parallel measurements of contractile function, coronary flow, and MVO₂ and validate that experimentally-induced contractile dysfunction should be corrected by the restoration of coronary flow to normal, pre-treatment levels [14, 27]. Our findings provide relative physiological operating ranges for changes in global steady-state relationships if/when local metabolic mechanisms of coronary blood flow control are attenuated by specific antagonists and/or disease states (Fig. 3). The consistency between the present findings and prior studies of reductions in CPP [46, 47] or coronary stenosis [48] establishes an anticipated framework for changes in the relationship between regional contractile function and the volume of myocardial perfusion per beat as a factor/pathway involved in local metabolic control of coronary flow is antagonized.

Fig. 5.

Schematic representation of myocardial perfusion-metabolism-contraction matching as under normal physiological conditions (shown in blue), (1) myocardial metabolism and (2) contractile function drive changes in (3) coronary blood flow. However, when coronary flow becomes limited (shown in orange), decreases in (1) perfusion drive comparable reductions in (2) myocardial metabolism and (3) contractile function

Examination of the influence of specific mechanisms of coronary flow control on steady-state transmural perfusion-contraction matching as well as the potential for the activation of compensatory ischemic vasodilator pathways warrants further investigation. Further resolution of apparent regional and conditional variability in the relationships between myocardial perfusion, contractile function, and metabolism will require additional experimental interventions and measurements as well as theoretical models that account for regional heterogeneity in oxygenation as suggested by prior simulations of myocardial oxygen transport [49–51].

Author contributions

SIE, JDT, and GMD conceived the study design. All authors contributed to the acquisition of presented data and to the analysis and interpretation. SIE, DJD and JDT wrote the manuscript. All authors contributed to critical review and editing of the manuscript and approved the submitted version.

Funding

This work was supported by National Institutes of Health grant R01 HL158723.

Data availability

The data that support the findings of this study are openly available via figshare at: 10.6084/m9.figshare.30066661. Further enquiries can be directed to the corresponding author.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This manuscript does not contain clinical studies or patient data.