Download Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation

 Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation
Vahagn Ohanyan1, Liya Yin1, Raffi Bardakjian4, Christopher Kolz1, Molly Enrick1, Tatevik Hakobyan1,
John Kmetz1, Ian Bratz1, Jordan Luli1, Masaki Nagane3, Nadeem Khan3, Huagang Hou3, Periannan
Kuppusamy3, Jacqueline Graham1, Frances Kwan Fu1, Danielle Janota1, Moses O. Oyewumi2, Suzanna
Logan1, Jonathan R. Lindner5, William M. Chilian1
1
Department of Integrative Medical Sciences; 2Department of Pharmaceutical Sciences, Northeast Ohio
Medical University; 3Department of Radiology and Medicine, Geisel School of Medicine at Dartmouth
College; 4Departement Internal Medicine, Canton Medical Education Foundation, and; 5Division of
Cardiovascular Medicine, UHN62, Oregon Health and Science University.
V.O. and L.Y. contributed equally to this study.
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Running title: Kv1.5 Channels in Coronary Metabolic Dilation
Subject codes:
[152] Ion channels/membrane transport
[87] Coronary circulation
[31] Echocardiography
[97] Other vascular biology
[118] Cardiovascular pharmacology
Address correspondence to:
Dr. Vahagn Ohanyan
Department of Integrative Medical Sciences
Northeast Ohio Medical University
Rootstown, OH 44272
Tel: 330-325-6535
Fax: 330-325-5912
[email protected]
In June 2015, the average time from submission to first decision for all original research papers submitted to
Circulation Research was 12.31 days.
This manuscript was sent to Jeanne M. Nerbonne, Consulting Editor, for review by expert referees, editorial
decision, and final disposition.
DOI: 10.1161/CIRCRESAHA.115.306642 1
ABSTRACT
Rationale: In the working heart coronary blood flow is linked to the production of metabolites, which
modulate tone of smooth muscle in a redox-dependent manner. Voltage-gated potassium channels, which
play a role in controlling membrane potential in vascular smooth muscle, have certain members that are
redox sensitive.
Objective: To determine the role of redox-sensitive Kv1.5 channels in coronary metabolic flow regulation.
Methods and Results: In mice (wild type [WT], Kv1.5 null [Kv1.5-/-], and Kv1.5-/- and WT with inducible,
smooth muscle specific expression of Kv1.5 channels) we measured mean arterial pressure (MAP),
myocardial blood flow (MBF), myocardial tissue pO2, and ejection fraction (EF) before and after inducing
cardiac stress with norepinephrine (NE). Cardiac work (CW) was estimated as the product of MAP and
heart rate. Isolated arteries were studied to establish if genetic alterations modified vascular reactivity.
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Despite higher levels of CW in the Kv1.5-/- (versus WT at baseline and all doses of NE), MBF was lower
in Kv1.5-/- than in WT. At high levels of CW, tissue pO2 dropped significantly along with EF. Expression
of Kv1.5 channels in smooth muscle in the null background rescued this phenotype of impaired metabolic
dilation. In isolated vessels from Kv1.5-/- mice, relaxation to H2O2 was impaired, but responses to adenosine
and acetylcholine were normal compared to WT.
Conclusions: Kv1.5 channels in vascular smooth muscle play a critical role in coupling myocardial blood
flow to cardiac metabolism. Absence of these channels disassociates metabolism from flow resulting in
cardiac pump dysfunction and tissue hypoxia.
Keywords:
Coronary microcirculation, coronary blood flow, voltage-gated potassium channels, vasodilation, ion
channel, contrast echocardiography, mouse, cardiac function, transgenic mice, mouse blood pressure
measurement/monitoring, hydrogen peroxide.
DOI: 10.1161/CIRCRESAHA.115.306642 2
Nonstandard Abbreviations and Acronyms:
ATP
CW
DP
EF
EPR
FS
H2O2
HR
KCNA-5
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Kir
Kv channel
LVEDV
LVESV
LVID,dLVID,s
LV
MAP
MBF
MCE
MI
NIH
pO2
RBV
RC
ROI
rtTA
SBP
SV
TEA
TP
VGCC
VSMCs
WT
adenosine triphosphate
cardiac work
double product
ejection fraction
electron paramagnetic resonance
fractional shortening
Hydrogen peroxide
heart rate
Potassium voltage-gated channel subfamily A member 5 (Voltage-gated potassium
channel subunit Kv1.5)
inward rectifier
voltage gated potassium channel
left ventricular volume at end diastole
left ventricular volume at end systole
left ventricular internal diameter at end diastole
left ventricular internal diameter at end systole
left ventricle
mean arterial pressure
myocardial blood flow
myocardial contrast echocardiography
mechanical Index
National Institute of Health
oxygen tension
relative blood volume
reconstituted
region of interest
reverse tetracycline transactivator gene
systolic blood pressure
stroke volume
tetraethylammonium
triple product
voltage-gated calcium channels
vascular smooth muscle cells
wild type
DOI: 10.1161/CIRCRESAHA.115.306642 3
INTRODUCTION
The principal function of the coronary circulation is to deliver oxygen and energetic substrates to
the myocardium to match myocardial demand for oxygen and energy with the proper supply under various
physiological conditions. Oxygen extraction in the coronary circulation is 75-80% under baseline
physiological conditions, leaving very little oxygen extraction reserve. Because this extraction is near
maximum to increase oxygen delivery, further extraction is not a viable option,1, 2 which necessitates that
an increase of myocardial work must be met, nearly instantaneously, by an increase of coronary flow to
maintain an adequate oxygen supply. Imbalance of myocardial oxygen supply-to demand ratio results in a
deterioration of myocardial function within a few seconds.3 The tight matching of oxygen supply and
demand must be guaranteed by local flow regulatory mechanism in any condition to avoid pump failure.
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Coronary blood flow is dependent on multiple physiological factors that affect force generation by
coronary vascular smooth muscle cells (VSMCs). These factors include intrinsic response of vascular
smooth muscle cells to intravascular pressure (the vascular myogenic response) and release of vasoactive
metabolites from cell types including endothelium, nerves, and cardiomyocytes.4-7 The tone of vascular
smooth muscle is largely regulated by the membrane potential, which controls the amount of calcium in the
sarcoplasm via the voltage-gated calcium channels (VGCC). Membrane hyperpolarization through the
opening of potassium channels in VSMCs reduces activation of VGCC, leading to reduction of Ca2+ entry
and vasodilatation. In contrast, closure of K+ channels leads to membrane depolarization and causes
vasoconstriction.8-10
Four major classes of potassium channels have been identified in VSMCs: ATP sensitive (KATP),
inward rectifier (Kir), large conductance Ca2+-activated potassium channels (BK), and voltage-dependent
potassium (Kv) channels.8, 9, 11-16 Of these channel families, our previous results have suggested that the Kv
family is involved in coronary metabolic flow regulation,17 in a scheme where production of H2O2 from
mitochondria produces opening of the channels, and thus dilation in feed-forward manner.18 We also found
that H2O2-induced redox-sensitive coronary vasodilatation19 is mediated by 4-aminopyridine-sensitive K+
channels.17 However, it is important to recognize that the use of a pharmacological antagonist does not
irrefutably test for a specific ion channel as drugs, such as 4-aminopyridine, can antagonize other classes
of ion channels, e.g., KATP.20 Moreover, the Kv channel family is large, with 12 families of channels and
multiple channels in each family. Thus the precise ion channel(s) linking the products of metabolism to
coronary blood flow is (are) unknown.
Although several types of Kv family channels are expressed in various cells in the heart, we focused
on Kv1.5 channels, which are reported to be oxygen and redox sensitive and expressed in vascular smooth
muscle cells.21-23 These results, when taken together with our previous observations,17-19 provoked us to
hypothesize that Kv1.5 channels play a critical role in the coupling of myocardial blood flow to cardiac
work. Accordingly, we studied coronary metabolic dilation (changes in myocardial blood flow [MBF] in
response to increases in cardiac work, i.e., the connection of coronary blood flow to myocardial
metabolism) in wild-type mice, mice null for Kv1.5 channels (Kv1.5-/-), and mice with inducible, smooth
muscle-specific expression of Kv1.5 channels (on Kv1.5-/- and wild type backgrounds). We also measured
tissue oxygenation to understand if the balance between oxygen supply (product of blood flow and oxygen
content) and cardiac work, which is a surrogate for myocardial oxygen consumption, was altered in the
genetically modified animals. Our results support a new concept, vis-à-vis, that Kv1.5 channels play a
critical role in connecting blood flow to metabolism in the myocardium.
DOI: 10.1161/CIRCRESAHA.115.306642 4
METHODS
A detailed description of the methodologies, protocols and statistical analyses are presented in the Online
Data Supplement. The murine models are described below.
All procedures were conducted with the approval of the Institutional Animal Care and Use Committee of
the Northeastern Ohio Medical University and 3Department of Radiology and Medicine, Geisel School of
Medicine at Dartmouth College and in accordance with National Institutes of Health Guidelines for the
Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996). Mice were housed in a
temperature-controlled room with a 12:12-h light-dark cycle and maintained with access to food and water
ad libitum.
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Murine models.
Wild type mice (C57Bl/6N and S129) and mice of both backgrounds that were null for the KCNA5 gene,
which encodes Kv1.5 channels (Kv1.5-/-) were used in this study.24 Kv1.5-/- mice on a S129 background
were a gift of Helmut Kettenmann (Max Delbrueck Center for Molecular Medicine, Berlin). These mice
were backcrossed into a C57Bl/6N background (more than 6 generations) to obtain C57Bl/6N null for
KCNA5.
Generation of inducible double transgenic mouse with smooth muscle specific expression of Kv1.5
Channels (Figure 1).
Transgenic mice with smooth muscle–specific expression (SM22-α promoter) of the reverse tetracycline
transactivator gene (rtTA), SM22-rtTA, were purchased from Jackson Lab. Transgenic mice expressing
Td-Tomato and Kv1.5 channel with tet-on expression were made by pronuclear injection of DNA construct
of Tet-On 3G tetracycline inducible system (Clontech) with insertion of Td-Tomato and mouse Kv1.5
cDNA in an IRES construct. After screening, the founders expressing Kv1.5 channels and Td-tomato were
crossed with the SM22-rtTA mice to produce double transgenic mice (SM-22-tet-Kv1.5). Double
transgenic mice have copies of each transgene in all cells, but without tetracycline, the genes are not
expressed. In the presence of doxycycline, the Kv1.5 and Td-Tomato gene are transcribed in smooth
muscle. In this model, 7-10 days of doxycycline treatment (2 mg/ml in drinking water) will induce
expression of Kv1.5 channels and Td-Tomato only in smooth muscle.
Smooth Muscle-Specific Rescue: expression of Kv1.5 in smooth muscle in KV1.5 -/-.
To determine if expression of Kv1.5 channels in smooth muscle is critical for coronary flow regulation
during changes in cardiac work, we determined if expression of Kv1.5 channels in smooth muscle would
rescue the phenotype observed in the global Kv1.5 knockout mice. To accomplish this, double transgenic
mice (SM-22-tet-Kv1.5) were crossed with Kv1.5 null mice to create a mouse with reconstituted (RC)
doxycycline-inducible expression of Kv1.5 channels in smooth muscle in Kv1.5-/- mice, namely SM-Kv1.5
RC (Figure 1).
We studied 4 groups of 4-5 month old mice: Kv1.5-/- (N=15), WT (N=15), double transgenic (SM-22-tetKv1.5, N=15) and reconstituted smooth muscle-specific Kv1.5 expression on the null background (SMKv1.5 RC, N=15).
DOI: 10.1161/CIRCRESAHA.115.306642 5
RESULTS
Cardiac function and hemodynamics.
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Figure 2A shows a typical M-Mode image, obtained at mid-papillary muscle level from which
ejection fraction was calculated. It is also worth noting that differences in cardiac function (EF and
Fractional shortening, FS) between WT and Kv1.5-/- were apparent at all doses of NE (Supplementary
material, Figure I). During NE infusion, EF was significantly lower at all time points in Kv1.5-/- mice
compared to WT mice (Figure 2B). In the double transgenic mice on the Kv1.5-/- background, with 7-10
days of doxycycline treatment to induce smooth muscle expression of Kv1.5 channels, cardiac function
(EF) was increased by NE to levels comparable to WT mice (P=NS), and significantly greater than Kv1.5/(P<0.05). The time course of changes in systolic arterial pressure in WT (Figure 2C) and Kv1.5-/- (Figure
2D) show a striking difference. In WT, during the highest dose of NE blood pressure increased to a steady
state level, and dropped only after the NE infusion was stopped. In contrast, Kv1.5-/- mice maintained
arterial pressure only transiently, for about 30 sec, during infusion of NE, then pressure dropped. If the NE
infusion was not stopped, mortality ensued in the null mice. Figure 2E illustrates the effects of NE on
arterial systolic pressure in the Kv1.5-/- RC mice treated with doxycycline to induce smooth muscle specific
expression of Kv1.5 channels. These mice, like WT, had a steady-state increase in arterial pressure to NE,
which fell only when infusion was stopped. To obtain measurements of myocardial blood flow, we used
the 30 sec period when pressure was elevated as a quasi-steady state to obtain flow and work measurements
during high dose NE (5 µg/kg.min-1). At baseline mean arterial pressure was significantly higher in Kv1.5/mice. After 7 days of treatment with doxycycline the blood pressure dropped significantly in Kv1.5-/--RC
and WT-RC mice. MAP, HR and cardiac work was not significantly different between WT and Kv1.5-/-RC mice after doxycycline (Supplement, Figure I). Left ventricular mass in Kv1.5-/- mice was significantly
higher compared to WT mice, but in Kv1.5-/--RC mice doxycycline treatment LV mass was not significantly
than that of WT mice (Supplement, Figure II). There were no significant differences in body weight among
all 4 groups of mice.
The relationships between MBF and the double product (DP) for the three groups are shown in
Figure 3. MBF in Kv1.5-/- mice was significantly (P<0.05) lower at any given DP than in either WT or the
transgenic (Kv1.5-/- -RC) mice after doxycycline treatment. In Kv 1.5-/- mice at baseline and any given does
of NE the MAP was higher compared to WT and Kv 1.5-/--RC mice, which shifted the DP line to the right.
In transgenic mice (Kv1.5-/--RC), re-expression of Kv1.5 channels in smooth muscle, by administering
doxycycline for 7 days, re-established the connection between MBF and oxygen demands as indicated by
DP. Treatment of the Kv1.5-/--RC with doxycycline, increased expression of mRNA level of Kv1.5 channel
in the aorta about 8 fold (Supplement, Figure III). Doxycycline alone does not change the Kv1.5 channel
expression in null mice or in WT mice, but in WT mice with the two transgenes, increased expression of
Kv1.5 channels shifted the relationship between DP and MBF to the left (Supplement, Figure IV). This
resulted in the observation that for any work load, MBF in the WT-RC animals was greater than in WT,
(P<0.05). We also analyzed MBF against CW (Supplement, Figure V) to ascertain if this relationship
provided different insights than the plot of MBF vs DP; our conclusions were unchanged in that mice null
for Kv1.5 channels had compromised increases in flow during enhanced metabolic demands depicted by
either DP or CW.
Sometimes the deletion of a specific gene in one genetic background (in mice) leads to a different
result than if the deletion is in another background. To determine if the background of the mice makes a
difference in the relationships between work and flow in the heart we compared these variables in WT and
Kv1.5-/- mice of C57Bl/6N and S129 mice (Supplement, Figure VI. The results indicated that background
did not influence our findings. The responses in the WT mice were comparable, and the deletion of Kv1.5
channels showed similar compromised metabolic dilation in both strains. We also would like to add that in
Kv1.5-/- mice expression of other ion channels was altered. In particular we noted that expression of Kv1.2,
DOI: 10.1161/CIRCRESAHA.115.306642 6
Kir6.1, and Kir6.2 channels was upregulated (mRNA measured by real time PCR), Kv1.3 and Kv7.1
appeared to be downregulated, and Kv2.1 was not altered (Supplement, Figure VII).
To determine if the imbalance between metabolism and flow, i.e., insufficient coronary blood flow
to meet cardiac metabolic demands, resulted in tissue hypoxia, we measured myocardial tissue oxygenation.
Myocardial oxygen tension was significantly (P<0.05) lower in Kv1.5-/- mice compared to WT at baseline
and any time point after high dose of NE injection (Figure 4). We also used Hypoxyprobe-1 to identify
hypoxic regions in the myocardium. As shown in Supplemental Figure VIII, augmented metabolic demands
induced by norepinephrine infusion increased myocardial tissue hypoxia in Kv1.5-/- (as indicated by higher
signal intensities for the fluorescence) more than in WT mice. The average signal intensity from the
ischemic zone was significantly higher in Kv1.5-/- mice compared to WT (P<0.05 compared to WT). In
Kv1.5-/--RC mice after 7 days of doxycycline treatment the hypoxic areas were significantly less intense
compared to Kv1.5-/- mice. It is important to note the re-expression of the Kv1.5 channels in smooth muscle
minimized tissue hypoxia, i.e., the fluorescent signals were comparable to controls.
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Figure 5 illustrates vasodilatory responses of small coronary arteries (internal diameter averaged
100-150 µm) to hydrogen peroxide, adenosine, and acetylcholine. In arterioles isolated from Kv1.5-/- mice,
vasodilation to H2O2 was significantly lower than WT. Re-expression of Kv1.5 channels in smooth muscle
(Kv1.5-/--RC after 7 days of doxycycline) increased the dilation compared to Kv1.5-/- (P<0.05) to
vasodilation equivalent to WT. Dilation to adenosine or to acetylcholine was not different between the WT
and Kv1.5-/- mice. Normal dilation to adenosine and to acetylcholine shows that the impaired vasodilation
in Kv1.5-/- mice is not due to a non-specific alteration of smooth muscle or the endothelium in the null mice.
We also evaluated the vasoactive effects of norepinephrine, which did not produce constriction in these
vessels (data not shown). This observation is similar to what we have observed previously in other species25,
26
where coronary resistance vessels do not respond directly to -adrenergic agonists. This latter observation
is critical because it could be argued that the blunted metabolic dilation in Kv1.5-/- mice during the
norepinephrine stress test is due to augmented adrenergic constriction; however this was not the situation.
DISCUSSION
In this study we observed that Kv1.5 channels in smooth muscle play a key role in connecting
myocardial blood flow to cardiac metabolism. This observation was based on impaired increases in MBF
during increased cardiac work in Kv1.5-/- mice. This inadequate dilation during increased cardiac work was
associated with severe tissue hypoxia and decrements in cardiac function, suggesting that oxygen delivery
was not being correctly matched to oxygen consumption. We also found that the phenotype of impaired
metabolic dilation could be rescued by re-expressing Kv1.5 channels in vascular smooth muscle cells,
supporting the concept that the expression of the potassium channels in smooth muscle is critical for
metabolic flow control. Taken together these results support the conclusion that Kv1.5 channels play a key
role in coronary metabolic dilation, i.e., the connection of myocardial blood flow to cardiac metabolism.
We will discuss this conclusion from the perspective of the importance of the coronary microcirculation in
health and disease, results in the literature that are cogent to our findings, and implications of our findings
in understanding the control of coronary blood flow.
The importance of the coronary microcirculation in health and disease.
The microcirculation of the heart comprises the bulk of vascular resistance,27 and is the segment
most responsive to locally produced vasoactive metabolites.28-30 Because of these attributes, under
physiological conditions, the coronary microcirculation is the element of the coronary circulation most
responsible for the dilation of blood vessels that occurs during increases in cardiac blood flow—the
DOI: 10.1161/CIRCRESAHA.115.306642 7
connection of flow to metabolism. Derangements in this connection can have undesirable consequences on
cardiac function in that the myocardium requires a continual supply of oxygen for energy production.
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Perhaps one of the more important implications of our study is shown in Figure 2C-E. Figure 2D
illustrates the outcome when flow, and thus, oxygen delivery, is uncoupled from cardiac work (oxygen
demands). Cardiac pump function and arterial pressure cannot be sustained during the metabolic stress
because there is insufficient flow to meet the metabolic requirements of the heart. The decrease in pressure
and pump function does not occur instantly when demands are increased by norepinephrine, but happens
about 30 seconds after norepinephrine induced the initial increase in arterial pressure. Note, in wild-type
mice (Figure 2C) or those with smooth muscle specific expression of Kv1.5 channels on the null
background (Kv1.5 RC, Figure 2E), arterial pressure was maintained during the entire period of
norepinephrine infusion indicating the oxygen supply matched oxygen demands. In support of this concept,
that in the Kv1.5 null mice flow was uncoupled from metabolism, tissue hypoxia occurred in the null mice
during the norepinephrine stress test (Figure 4 and Supplement, Figure VIII), but tissue oxygenation was
sustained at basal levels in WT and Kv1.5 RC mice. The maintenance of myocardial oxygen tension
indicates that oxygen consumption was matched to oxygen delivery; whereas a decrease in oxygen tension
suggests that flow and oxygen delivery were inadequate to meet the needs of the working heart. We also
will add that the myocardial PO2’s were higher than what we expected. Perhaps this is due to the placement
of the crystals, which are injected in the LV free wall. We cannot eliminate the possibility that some portion
of the signal arises from the LV lumen (perhaps some crystals are close to the lumen); however, despite
this limitation the Kv1.5 null mice show a very different response to metabolic stress with decreases in
oxygen tension, which would not happen if a large portion of the signal arose from oxygenated blood in the
LV lumen. Another caveat is that we are not claiming that norepinephrine produces tissue hypoxia in a wild
type animal, which could be inferred from the results in Supplemental Figure VIII. The presence of hypoxic
tissue could be an artifact of the tissue harvesting and processing in which there are brief periods of time
when the tissue is not perfused and such a period could result in the appearance of hypoxia, when in fact
during perfusion there was none. Nevertheless, even with this hindrance we would like to emphasize that
this would occur in both samples and would not be an explanation for why myocardial tissue hypoxia was
more severe in the Kv1.5-/- compared to the wild type mice.
We believe our observations facilitate an understanding of “microvascular disease” in the heart.
There have been several clinical indications; for example, the WISE trial (Women's Ischemia Syndrome
Evaluation) has revealed that women, without large vessel disease, show symptoms consistent with
myocardial ischemia when stressed.31-33 Also these women show abnormal coronary vasodilator reserve to
adenosine.34 On the surface, results from the WISE trial would seem inconsistent with our observations that
adenosine-induced vasodilation was comparable in isolated coronary arterioles from wild type and Kv1.5/mice. We believe it is difficult to compare in vivo and in vitro responses to an agonist; considering the
WISE trial studied patients with ischemic heart disease. In the GUSTO IIb (Global Use of Strategies to
Open Occluded Coronary Arteries in Acute Coronary Syndromes IIb) trial, it was reported that in patients
with acute coronary syndromes, 30.5% of women with unstable angina and 10.2% of women with STEMI
had normal coronary angiographies.35 Moreover, a recent analysis has further supported the importance of
microvascular disease in the heart; specifically patients without large-vessel disease but with compromised
coronary vasodilator reserve had mortality rates equivalent to those with large-vessel disease.36 It is worth
emphasizing that we are not concluding that Kv1.5 channels are the basis for microvascular disease in the
human heart (also termed non-obstructive coronary disease); rather, we speculate that they may be. More
likely, there may be several genetic polymorphisms, perhaps in different combinations, involved in nonobstructive coronary disease, which may be analogous to a condition like Long QT Syndrome where many
known polymorphisms of ion channels are known to cause the condition.37 Previously, polymorphisms in
several ion channels and in eNOS, but not in Kv1.5 channels were associated with coronary microvascular
disease.38
DOI: 10.1161/CIRCRESAHA.115.306642 8
We would like to speculate about an implication of our results that may also bear upon clinical
observations in patients with non-obstructive coronary disease. For example in mice null for Kv1.5 channels
cardiac function as defined by measurements of ejection fraction and fractional shortening were less than
wild type mice even during basal conditions (Supplement, Figure IX). Perhaps the disassociation of flow
from metabolism induces mild cardiac dysfunction under basal conditions, which becomes more evident
during a stress test. We speculate that in patients with non-obstructive coronary disease the higher incidence
of cardiovascular complications relates to insufficient blood flow to the myocardium.39
Considerations from the literature.
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The factors and their effectors responsible for coronary metabolic dilation have remained elusive.
Historically adenosine was considered to be the metabolite linking coronary blood flow to oxygen
consumption,40, 41 but this hypothesis was rigorously challenged by estimates of interstitial adenosine
concentrations that are insufficient to produce dilation during increased cardiac work.42, 43 Although some
pharmacological studies show an involvement of adenosine in metabolic dilation when other mechanisms
of dilation are blocked,44 this may be more of the result of ischemic dilation as opposed to an aerobic process
linking work to metabolism. Other efforts have suggested a role for KATP channels and the KCa channels in
coronary metabolic dilation,45, 46 but it is important to point out that a limitation of such work is the exquisite
reliance on pharmacological approaches to draw conclusions about particular ion channels. Moreover, other
work has challenged the role of KATP channels in local coronary metabolic dilation.43, 47, 48 More recently,
the role of ATP released by red blood cells was postulated,49, 50 but this hypothesis requires endothelial
production of nitric oxide, which has been found to be not essential for coronary metabolic hyperemia.43
Indeed our present results bear upon these previous findings in that responses to adenosine and to nitric
oxide were not affected in the Kv1.5 null mice; yet coronary metabolic dilation was severely compromised
in these mice. These observations are in concordance with previous studies concluding that neither
adenosine, or nitric oxide are critically involved in coronary metabolic dilation.
Previously, we proposed that that mitochondrial production of H2O2 is a feed-forward link between
metabolism and flow in the heart.18 We further established that the vasodilatory actions of H2O2 were redoxdependent19 and mediated by 4-aminopyridine sensitive ion channels.17 Although it is not unreasonable to
speculate that 4-AP sensitive ion channels are Kv channels, it is presumptuous to make this conclusion only
on the basis of pharmacological evidence.
The current state of knowledge linking specific ion channels to metabolic dilation in the heart is
primarily based on the use of pharmacological agents, e.g., tetraethylammonium (TEA) to block MaxiK
channels,46 glibenclamide to block KATP channels,45 and 4-aminopyridine to block all Kv channels.51, 52 In
these experiments, the antagonists were used to attenuate coronary metabolic dilation to exercise,
administration of inotropes and/or pacing in anesthetized dogs. Definitive conclusions are limited, however,
due to the relative non-specificity of the ion channel antagonists. For example, TEA blocks virtually any K
channel,20, 53, 54 and glibenclamide antagonizes both the sarcolemmal KATP channel and the mitochondrial
KATP channel,55-57 as well as Kv channels.54, 58 In addition to blocking Kv channels, 4-aminopyridine also
can antagonize KATP channels.20 Accordingly, we opine that conclusions about a specific ion channel
transducing metabolic signals into changes in coronary blood flow are based on experiments using
traditional pharmacological responses are premature. What is clear from the literature is the 4aminopyridine-sensitive ion channels are involved in the coronary metabolic dilation,18, 52 but the identity
of the specific channel is not revealed by these previous studies. Although several types of Kv family
channels are expressed by various cells in the heart, we focused on Kv1.5 channels because their oxygen
and redox sensitivity21-23 makes them likely candidates to mediate metabolic dilation.
DOI: 10.1161/CIRCRESAHA.115.306642 9
Implications of Kv1.5 channels in the control of coronary blood flow and vascular tone.
Our results are consistent with the concept that Kv1.5 channels are directly involved in coronary
metabolic dilation and play a role in the link between myocardial blood flow and cardiac metabolism.
Before we discuss evidence supporting this conclusion, we would like to emphasize that we do not believe
this ion channel is the only effector modulated by metabolites that connects flow to metabolism. If it were,
then we would expect the knockout to be lethal, but this is not the observation. Although there were modest
changes in myocardial blood flow at rest between the Kv1.5-/- mice and wild type, this difference was
insufficient to affect basal cardiac function; however when the null mice were subjected to a metabolic
stress (induced by norepinephrine), the increase in myocardial blood flow (metabolic dilation) was
insufficient to sustain cardiac function as indicated by acute decreases in cardiac function and the
development of profound tissue hypoxia (Figures 2D, 3 and 4, respectively). In contrast, wild type mice
subjected to the same metabolic stress maintained cardiac function and the appropriate metabolic dilation
maintained tissue oxygenation during the duration of the increased metabolic demands.
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An important implication of our results is that in addition to the Kv1.5 channels other mechanisms
of vasodilation must play a role in the metabolic control of the coronary circulation. There are several
observations supporting this statement. First, knockout of Kv1.5 channels was not lethal. If Kv1.5 channels
were the only connection between metabolism and flow in the heart, we would expect lethality in a knockout
as cardiac function cannot be sustained by anaerobic metabolism. Second, we observed a connection, albeit
blunted, between cardiac work and coronary blood flow in the Kv1.5 null mice. Accordingly, there must
be other mechanisms involved in coronary metabolic dilation. Third, responses to the coronary metabolic
dilator, H2O2, were not completely abolished in the Kv1.5 null mice. This implies other mechanisms,
perhaps other redox sensitive ion channels, e.g., Kv1.359, or ion channels modulated directly by oxygen,
e.g., Kv1.260, and/or other redox-dependent signaling processes, e.g., dimerization of protein kinase G61
compensate for the loss of the Kv1.5 channel control mechanism. Finally, the knockout of the Kv1.5
channels was associated with an upregulation of Kv1.2, Kir6.1 and Kir6.2 channels (Supplement Figure
VII), which both may compensate for the loss of this membrane ion channel through their control of smooth
muscle membrane potential and vascular tone.60, 62
A potential criticism of our study design is that we used mice with global knockout of Kv1.5
channels in the study of coronary metabolic dilation. Because these channels are located in many cell types,
e.g., cardiac myocytes,63, 64 endothelial cells,65, 66 and neurons,67 it could be argued that the effects observed
in the global knockout can be attributed to cell types other than vascular myocytes. Although a cell specific
knockout would have provided cogent information, given our results that the re-expression of the channel
in smooth muscle on the null background rescued the phenotype, it is likely that our conclusions would be
the same, i.e., the importance of the Kv1.5 channel in facilitating the connection between coronary blood
flow and myocardial metabolism. It is worth noting that our model of conditional (Tet-On) expression of
the channel would not cause developmental compensations as would permanent knockout of the channel—
even in a cell specific manner. These results build upon our previous results suggesting the H2O2 is a
metabolic dilator in the heart in that small arteries isolated from the null mice showed blunted dilation to
this reactive oxygen species (compared to wild type mice) and doxycycline-inducible expression of Kv1.5
channels in smooth muscle restored this vasodilation. It is also worth noting that vessels from the null mice
did not show any response to norepinephrine, so the blunted metabolic dilation during the norepinephrine
stress test should not be interpreted as possible enhanced constriction to this adrenergic agonist.
Another possible explanation is the deletion of Kv1.5 channels in cardiac myocytes renders them
more responsive to the production of vasoconstrictors when stimulated by adrenergic agonists. This
possibility is founded on previous work from our laboratory showing that stimulation of adrenergic
receptors in cardiac myocytes results in the production of a substance or substances that mediate coronary
arterial vasoconstriction. 25, 26 However, we do not think this explanation is plausible given that re-
DOI: 10.1161/CIRCRESAHA.115.306642 10
expression of the Kv1.5 channels only in smooth muscle restored myocardial blood flow during the
norepinephrine stress test.
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The application of contrast echocardiography to measure myocardial blood flow was described
over two decades ago,68 and since this observation the technique has been used to measure blood flow in a
variety of animal models69, 70 and in the clinical setting.71-73 One advantage of this technique is the ability
to measure blood flow in vivo under minimally invasive conditions (intravenous catheter for contrast
infusion). Previous measurements of myocardial blood flow in the mouse heart were confined to bufferperfused isolated heart models,74-77 which impact a variety of control mechanisms for coronary blood flow
causing concerns about physiological relevance of the observations. In our study we used contrast
echocardiography to measure myocardial blood flow in anesthetized mice, and these values were about
40% higher compared to the flows measured with microspheres. Raher et al78 compared microsphere
measurements of flow to those obtained with contrast echocardiography and similar to our results found a
very strong correlation between the two measurements. Although, the slope of their linear regression was
close to identity, they did not quantify blood flow and left the MCE measurement in units of db/sec.
Therefore it is difficult to relate our comparison of blood flows derived from MCE and microspheres to
theirs. Magnetic resonance imaging (MRI) was used to measure myocardial perfusion in a murine model,
and these investigators reported baseline flows in the range of 7 ml/min per g.79 This range was less than
our MCE measurements of about 12 ml/min per g at baseline, but if we corrected based on the microsphere
measurements our values would be comparable. Our values, especially those at the highest dose of
norepinephrine were higher than what we expected but similar to the highest values in buffer perfused,
isolated hearts (Supplement Figure X). We have not “corrected” the blood flow measurements that we
report, but wish to emphasize that any adjustment in the measurement would not change the most important
aspect of our measurements—the impairment in metabolic dilation in Kv1.5-/- mice and restoration of flows
to values comparable to wild type mice in the Kv1.5-/--RC mice in that values for all mice would be
decreased by a certain factor and the differences would remain.
Conclusions.
In the heart, vascular Kv1.5 channels play a critical role in coupling myocardial blood flow to
cardiac metabolism. This coupling is critical in the maintenance of tissue oxygenation during hemodynamic
challenges through balancing oxygen delivery via metabolic coronary dilation and oxygen consumption via
cardiac work.
SOURCES OF FUNDING
This work was supported by NIH/NHLBI (HL100828Z and HL83366) to W.M.C.; NIH 1R15HL115540
and 14BGIA18770028 to LY, AHA (10POST4360030) to VO, and EB004031 to PK.
DISCLOSURES
On behalf of all authors, the corresponding author states that there is no conflict of interest.
DOI: 10.1161/CIRCRESAHA.115.306642 11
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DOI: 10.1161/CIRCRESAHA.115.306642 15
FIGURE LEGENDS
Figure 1. A) Generation of the SM22-tet-KV1.5-Td-Tomato transgenic mouse. The generation of this
mouse results in doxycycline-inducible, smooth muscle-specific expression of Kv1.5 channels. B) Smooth
Muscle-Specific Expression of Kv1.5 channels in smooth muscle in Kv1.5 null mice. Crossing the SM-tetKv1.5 with Kv1.5-/- followed by subsequent back crossing enables the development of Kv1.5-/- mice with
reconstituted doxycycline-inducible expression of Kv1.5 smooth muscle (SM-Kv1.5 RC).
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Figure 2. Cardiac Function and Hemodynamics. A) Transthoracic echocardiographic M-mode images
at the mid-papillary muscle level. LVID- left ventricular internal diameter at diastole and at systole. LVAWleft ventricular anterior wall, LVPW-left ventricular posterior wall. B) Time course of changes in ejection
fraction during norepinephrine infusion (5 µg/kg min-1) in WT, Kv1.5-/-, and Double Transgenic on the
Kv1.5-/- background (Kv1.5-/--RC) with 7 days’ doxycycline treatment. Note in the Kv1.5-/- EF decreased
after one minute of NE infusion, whereas in the other groups EF rose during the duration of infusion. C)
Systolic blood pressure (SBP) in Wild Type (WT) and D) in Kv 1.5-/- mice under basal conditions and
during infusion of norepinephrine (NE 5 µg/kg/min). Note, in Kv1.5-/- mice SBP started to decrease one
minute after injection (prior to stopping the infusion). In contrast, in WT mice SBP increased during the
infusion, and decreased only after NE infusion was stopped. E) Systolic blood pressure in double transgenic
(Kv 1.5-/--RC) after high dose of NE injection. In Kv 1.5-/--RC mice SBP was similar to WT mice and
dropped only after NE infusion was stopped. Data are presented as Mean±SD, -P<0.05 Kv 1.5-/- mice
compared to WT.
Figure 3. Relationship between Double Product (DP, mean arterial pressure X heart rate) and
myocardial blood flow (MBF). MBF and DP were measured at baseline (5-10 minutes after
Hexamethonium) and during norepinephrine administration (0.5 -5.0 µg/kg/min iv,). In Kv 1.5-/- mice MBF
was significantly lower compared to WT mice at any given dose of NE (-P< 0.05). In Kv1.5-/- double
transgenic mice (Kv1.5-/--RC) treated with doxycycline, MBF was comparable to WT at all levels of DP(P=NS, Kv 1.5-/--RC vs WT). MBF in Kv 1.5-/--RC mice was significantly higher compared to Kv 1.5-/- mice
(- P<0.05).
Figure 4: Myocardial oxygen tension (pO2). A) Time course of pO2 values in WT and Kv1.5-/- mice
during the high dose of norepinephrine (NE, 5 µg/kg min-1) (up to 4 minutes after the start of
infusion). Oxygen tension was significantly lower in Kv1.5-/- mice compared to WT mice and declined in
Kv 1.5-/- during NE infusion indicating that myocardial oxygen demand increased beyond the capacity of
supply, resulting in tissue hypoxia. B) Mean values of pO2 at baseline and during NE; pO2 was significantly
lower at baseline and during NE injection in Kv1.5-/- vs WT (-P<0.05). in Kv 1.5-/--RC pO2 was
significantly higher at baseline and NE infusion (-P<0.05) compared to Kv 1.5-/- mice and was comparable
to WT mice ( -P=NS).
Figure 5. Isolated coronary microvessel responses to H2O2, adenosine and acetylcholine (ACH). A)
Vasodilatation response to hydrogen peroxide in coronary vessels isolated from Kv 1.5-/- mice was
significantly lower (-P<0.05) compared to vessels from WT mice or doxycycline-treated transgenic Kv
1.5-/- mice (Kv 1.5-/--RC, -P<0.05). Vasodilation to H2O2 was not different between Kv 1.5-/-RC and WT
mice (-P=NS). There were no significant differences between WT and Kv 1.5-/- dose-response curves to
adenosine (B) or acetylcholine (C).
DOI: 10.1161/CIRCRESAHA.115.306642 16
Novelty and Significance
What Is Known?

The heart is an organ system requiring a continuous supply of oxygen, via myocardial blood flow,
to maintain normal cardiac pump function.

Myocardial blood flow is coupled to cardiac work through a process known as metabolic dilation.

Metabolic dilation in the heart is mediated, in part, by feed-forward production of H2O2 from
mitochondria during aerobic metabolism.

H2O2-induced vasodilation is, in part, mediated by voltage gated potassium (Kv) channels
What New Information Does This Manuscript Contribute?
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
Kv1.5 channels in smooth muscle are critical for metabolic dilation in the heart.

Kv1.5 channels in smooth muscle are critical for maintaining oxygen balance in the heart.

Absence of Kv1.5 channels uncouples myocardial blood flow from cardiac work resulting in tissue
hypoxia and impaired myocardial function.
This study shows the role of Kv1.5 channels in smooth muscle in the regulation of coronary blood flow.
Absence of Kv1.5 channels uncouples myocardial blood flow from cardiac work resulting in coronary
insufficiency, that is, insufficient blood flow to meet the metabolic needs of the myocardium. Insufficient
blood flow creates an imbalance in tissue oxygenation (consumption of oxygen exceeds delivery) resulting
in tissue hypoxia. Expression of Kv1.5 channels in only smooth muscle in a global knockout completely
rescues the abnormality in coronary regulation and restores the balance of tissue oxygenation. Our findings
may help explain how patients with non-obstructive coronary disease show impairments in flow regulation
that can lead to myocardial ischemia.
DOI: 10.1161/CIRCRESAHA.115.306642 17
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A.
Transgenic #1
SM22 promoter
Transgenic #2
PTRE3G
Double transgenic Reverse tetracycline transactivator gene
Kv1.5
SMC transcription factor
(rtTA)
IRES
rtTA
pA
Td‐Tomato
pA
SM‐tet‐Kv1.5
Dox
PTRE3G
Kv1.5 Td‐Tomato
B.
SM‐tet‐Kv1.5
Kv1.5‐/‐
Figure 1
SM‐Kv1.5 RC Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017
Figure 2
LV AW
LVID, D
A
LVID, S
LV PW
HR
456
RESP 152
T
37
B
D
Kv 1.5-/--RC
Systolic pressure
(mmHg)
Systolic pressure
(mmHg)
C
Systolic pressure
(mmHg)

Kv 1.5-/-
WT
E



Figure 3
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NE



 
 
A
B
Figure 4
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R2=0.98
R2=0.89
P<0.05
P<0.05
B
A
C
Figure 5
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Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation
Vahagn Ohanyan, Liya Yin, Raffi Bardakjian, Christopher Kolz, Molly Enrick, Tatevik Hakobyan,
John G Kmetz, Ian Bratz, Jordan Luli, Masaki Nagane, Nadeem Khan, Huagang Hou, Periannan
Kuppusamy, Jacqueline Graham, Frances Shuk Kwan Fu, Danielle Janota, Moses O Oyewumi,
Suzanna J Logan, Jonathan R Lindner and William M Chilian
Circ Res. published online July 29, 2015;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2015/07/29/CIRCRESAHA.115.306642
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2015/07/29/CIRCRESAHA.115.306642.DC1
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Supplemental Material
Data and Methods
Transthoracic stress echocardiography. Transthoracic echocardiography was used to
evaluate cardiac function. 1, 2 M-mode images were obtained from parasternal short axis
view, mid-papillary muscle level using a VEVO 770 High-Resolution echocardiography
Imaging System (VisualSonics, Toronto, Ontario, Canada) designed for small animal
studies. After induction of anesthesia (3% isoflurane with 100% oxygen, 1 L/min, in small
chamber) mice were placed on controlled heating table designed for small animal
echocardiography. The anesthetic and oxygen were delivered through the nose cone (12% isoflurane, 0.5 L/min with oxygen). After removing hair from the chest, warmed
(37°C) Aquasonic 100 gel (Parker Laboratories, Fairfield, NJ) was placed on the chest to
optimize visibility of cardiac structures. Cardiac function was measured at baseline, after
Hexamethonium (5 mg/kg) and different doses of NE (0.5, 1.0, 2.5 and 5.0 µg/kg.min-1,
intravenous, continues for 3 minutes) infusion. Left ventricular volume at end diastole
(LVEDV) and end systole (LVESV), as well left ventricular internal diameter at end
diastole (LVID,d) and end systole (LVID,s) were measured at steady state after drug
infusion. Left ventricular volume was calculated by modified Teichholz formula:
LVV=((7.0 / (2.4 + LVID)) * LVID.3, 4 Left ventricular ejection fraction (LVEF %) was
calculated by: (LVEDV-LVESV)/LVEDV. Fractional shortening was calculated by:
(LVID,d-LVID,s)/LVID,d (Figure 1A). All echocardiographic calculations and
measurements were carried out offline using the Vevo770/3.0.0 software. All
measurements were averaged over 5 cycles.
Hemodynamic measurements and calculation of cardiac work. After induction of
anesthesia (3% isoflurane with 100% oxygen, 1 L/min, in small chamber) mice were
placed on controlled heating surgical table designed for small-animal surgeries and
echocardiography. Body temperature was maintained at 37°C via rectal probe. Mice
were secured in the supine position and placed under a dissecting microscope. The right
jugular vein was cannulated with PE-50 polyethylene tubing (Becton Dickinson, Oakville,
ON, Canada) containing heparin (50 U/ml in Dulbecco’s PBS) in saline for intravenous
drug infusions. The femoral artery was isolated and cannulated with a 1.2F pressure
catheter (Scisense Inc, Ontario, Canada) connected to a data acquisition system
(PowerLab ML820; ADInstrument, Colorado Springs, CO) through a pressure interface
unit (SP200 Pressure System) designed to measure arterial blood pressure and heart
rate (HR). Pressure catheter was advanced ~10mm into abdominal aorta. All measured
variables were continuously recorded and stored on an iMac computer that used the
PowerLab system (AD Instruments; Castle Hill, Australia). The blood pressure data were
collected and analyzed using AD Instruments Chart 7 software. All mice were
euthanized following the experimental protocol with a lethal dose of pentobarbital sodium
(50 µg/kg).
The work that the heart completes per beat is termed stroke work, which is the
area within the ventricular pressure-volume loop. Mathematically stroke work can be
calculated as the product of stroke volume (SV) and mean arterial pressure (MAP).
Mean arterial pressure was calculated by: MAP=2/3 diastolic pressure + 1/3 systolic
pressure. In the current study we used double product to estimate cardiac work (HR x
MAP), but CW is most accurately represented by the triple product of MAP x HR x SV
(stroke volume). Because of technological constraints we could not measure SV during
acquisition of images to measure MBF. In order to calculate stroke volume changes
during NE infusion, we used sparate group of mice and by using M-mode tracing
pictures we calculated SV changes. We used the same age and body waight mice for
this experiments (WT and Kv 1.5-/-). Accordingly we determined how accurately DP
reflects cardiac work in mice by comparing DP to the triple product (CW) (Supplement
Figure 11). We found a linear relationship between triple and double product (R2=0.951)
suggesting DP was a reasonable surrogate for CW. Cardiac work directly reflects
myocardial oxygen consumption, and we employed this index as a surrogate for oxygen
consumption to decipher the basis of coronary metabolic dilation.
Myocardial perfusion imaging by contrast echocardiography. Lipid-shelled
microbubbles were prepared by sonication of an aqueous lipid dispersion of
polyoxyethylene-40-stearate and distearoyl phosphatidylcholine saturated with
decafluorobutane gas. Contrast agent was prepared fresh before the experiments.
Microbubble concentration and size distribution were determined by using Zetasizer
Nano-ZS90 (Malvern Instruments). Prior to particle measurements, the microbubble
suspension was diluted (1:5 v/v) with filtered water (0.22 µm filter, Nalgene International)
to ensure that light scattering signals are within the sensitivity of the instrument.5
For myocardial contrast echocardiography (MCE), animals were prepared as above for
transthoracic echocardiography. Myocardial perfusion imaging was performed with a
linear-array transducer (15L8) interfaced with an ultrasound system (Sequoia 512,
Siemens Medical Systems, Mountain View, CA). The right jugular vein was cannulated
with PE-50 polyethylene tubing containing heparin (50 U/ml) and was used for drug and
contrast infusion. Long axis images were obtained for perfusion imaging. In all mice, the
contrast agent was infused intravenously (jugular vein) at 20 µl/min (5 × 105 bubbles
min-1,). MCE was performed with a multi-pulse contrast-specific pulse sequence
designed to detect the non-linear microbubble signal at a low mechanical index (MI 0.20.25). Data were acquired after a high-MI (1.9) pulse sequence used to destroy
microbubbles in the acoustic field. All settings for processing were adapted and
optimized for each animal: penetration depth was ~2–2.5 cm, near field was focused on
the middle of the left ventricle (short axis view), and gains were adjusted to obtain
images with no signal from the myocardium and then held constant. Regions of interest
(ROI) were positioned within the anterolateral in the short axis view. A curve of signal
intensity over time was obtained in ROI and fitted to an exponential function: y = A(1 - eβt
), where y is the signal intensity at any given time, A is the signal intensity
corresponding to the microvascular cross sectional volume, and β is the initial slope of
the curve, which corresponds to the blood volume exchange frequency. 6, 7 Relative
blood volume (RBV) was calculated as the ratio of myocardial to cavity signal intensity
(RBV=A/ALV). ALV corresponds to the signal intensity for the LV cavity. Color coded
parametric images were used to outline region of interest (region of the left ventricle).
Myocardial blood flow (MBF) was estimated as the product RBV × β.8 The analysis of
nearby regions within the myocardium and the left ventricle is proposed to compensate
for regional beam inhomogeneities and contrast shadowing.9 MBF was calculated from
the blood volume pool relative to the surrounding myocardial tissue, the exchange initial
slope of curve, and tissue density  ( =1.05).6 MBF was measured in 3-5 different
T
images obtained from the same condition (baseline and treatments). MCE analyses
were performed by readers blinded to the genotype and treatment. Although MCE
has been used in mice previously to measure myocardial blood flow, we felt it was
important to correlate flow using MCE to flow measured using microspheres
(Supplement Figure 10). The correlations between the two flow measurements showed
substantial agreement, although the slope of the line indicated that flows were higher in
MCE vs microspheres. We have reported the calculated flows from MCE in all figures in
this paper.
Isolated microvessel studies. Single arterioles and small coronary arteries were dissected
from the epicardial surface of the left ventricle or from the septum. A portion of the left
ventricular free wall or septum was removed and small arteries (100-200 µm, internal diameter)
were located under a dissecting microscope. The vessel with surrounding ventricular muscle
was excised and transferred to a temperature-controlled dissection dish (4°C) containing PSS
and dissected free of the muscle tissue under epi-illumination. The arteriole was cannulated at
both ends using micropipettes that have been shaped in a microforge. The outside of the
arteriole was securely tied to each pipette using 11-0 suture, and then was transferred to the
stage of an inverted microscope for study. Arteriolar dimensions (internal diameter) were
measured using videomicroscopy during the course of an experiment. Vessels were contracted
with the thromboxane mimetic U46619 (1 μM), and subsequent hydrogen peroxide, adenosine
and acetylcholine relaxation was determined. Some vessels segments were used for isometric
force generation experiments. For these preparations, the vessels were isolated as described,
but two small (30 µm diameter) wires were inserted into the vessel and connected to a force
transducer (Living Instruments, Inc). After optimization of tension, experiments were conducted
to determine constrictor or dilator responses to agonists. For either preparation,
pharmacological agents were administered in the bath.
Determination of tissue oxygenation. We used two complementary methods to
evaluate myocardial tissue oxygen levels during the norepinephrine stress test. First, we
analyzed tissue sections using Hypoxyprobe-1, which measures protein modifications
induced by tissue pO2 of less than 10 mmHg. 10 Hypoxyprobe was dissolved in 0.9%
saline and injected intravenously into mice at a dose of 60 mg/kg body weight. Thirty
minutes after the injection of hypoxyprobe, the mice were sacrificed and the heart
tissues were harvested and frozen in liquid nitrogen. Frozen tissues were cryosectioned
into 10 µm sections and were stored at -80oC. After thawing, the sections were fixed in
cold acetone (4oC) for 10 min. The sections were rinsed and incubated overnight at 4oC
with mouse monoclonal anti-pimonidazole antibody (clone 4.3.11.3)(MAb1) diluted at
1:50 in PBS containing 0.1% bovine serum albumin and 0.1% Tween 20. The sections
were then incubated for 2 hours with Alexa Fluor 594-conjugated anti-mouse antibody at
the dilution of 1:500. The slides were mounted with ProLong® Gold Antifade with DAPI
(Invitrogen). Images were visualized and collected using fluorescent microscope and
analyzed using ImageJ (NIH).
We also determined myocardial tissue pO2 as an index of the balance between
oxygen supply and oxygen consumption using L-Band electron paramagnetic resonance
(EPR) oximetry. 11, 12 The principle of EPR oximetry is based on molecular oxygeninduced line-width changes in the EPR spectrum of a paramagnetic probe. The
technology has been well-established and validated for measurements of pO2 in the
heart. The myocardial pO2 measurements in the present study were performed using a
modified Varian E-15 EPR spectrometer, equipped with a home-made low-frequency
(1.2 GHz, L-band) microwave bridge. Microcrystals of LiNc-BuO were used as oxygensensing probes for EPR oximetry. Mice, under inhalation anesthesia (1-2% isoflurane),
had the probes implanted in the left-ventricular mid-myocardium during a left
thoracotomy. In order to recover from surgical trauma and healing, the animals were
allowed to recover for 2-3 weeks before final study of tissue oxygenation. To measure
oxygenation, the mice were situated in a right-lateral position with the chest closer to the
loop of a surface-coil resonator. EPR spectra were acquired as single 30-second
duration scans using the following settings: microwave frequency, 1.2 GHz (L-band),
incident microwave power, 8 mW; modulation amplitude, one-third of the EPR line width
with scan time of 10 sec., modulation frequency 24 kHz; scan range, 2 Gauss; receiver
time constant, 0.2 second. The peak-to-peak width of the EPR spectrum was used to
calculate pO2 using a standard calibration curve. 13
Statistical analysis. Measurements of left ventricular volumes, diameter , EF and FS in
WT , Kv 1.5-/- and Kv 1.5-/- -RC mice are presented as mean values ± SD. Difference of
LV volumes, diameter, EF and FS were analyzed for statistical significance with one-way
ANOVA using GraphPad Prism version 4.0 for Windows (GraphPrism Software Inc, San
Diego, CA,). The correlation of cardiac works (between DP and TP), myocardial blood
flows (MCE determined and microsphere estimated) where assessed by linier regression
analysis. Vasodilation responses to H2O2, adenosine and ACH, relationship between
MBF and DP, myocardial oxygen partial pressure changes were also assessed by linier
regression analysis. R2 and P values are reported for all regression analysis. A
probability of p<0.05 was accepted as statistically significant.
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Supplement data
Online Figure I . Mean Arterial Pressure (panel A), Heart rate (panel B) and cardiac work
(Double Product, panel C ) at baseline and during different doses of Norepinephrine
injection(0.5-5 ug/kg.min-1). P<0.05 Kv 1.5-/- vs WT mice; P<0.05 WT-RC vs. Kv 1.5-/- mice.
Online Figure II. Left ventricular mass (LV mass, panel A), Body weight (Panel B) and LV
mass/body weight ratio ( Panel C). Lv mass in Kv 1.5-/- was higher compared to WT mice, but
LV BW ratio was not significant between all groups. P<0.05 Kv 1.5-/- vs WT mice
Online Figure III. Doxycycline-induced SM22-promoter-driven KV1.5-Td-Tomato expression in
aorta. A and B illustrate tomato red fluorescence in WT and the mice with doxycycline-inducible,
smooth muscle specific expression of Kv1.5 channels (after 7 days of doxycycline). The lower
panels represent higher magnification views of the circumscribed areas in the boxes of the top
panels. The fluorescent signal in WT is due to autofluorescence of elastic lamina (distinct lines
in WT, lower panel). In contrast, note the entire media is fluorescent in the transgenics
indicating smooth muscle expression. Panel C shows m-RNA relative fold changes after 7 days
of doxycycline treatment. -P<0.05 WT-RC compared to WT mice after DOX treatment.
P<0.05 Kv 1.5-/- -RC mice compared to WT mice. Without DOX treatment there was no mRNA
expression Kv 1.5-/- -RC mice.
Online Figure IV. Myocardial blood flow and double product relationship in WT, and WT
transgenic mice (WT-RC). After 7 days doxycycline treatment to both lines (resulting in
increased smooth muscle expression only in WT-RC), the DP-MBF relationship in the WT-RC
was shifted to the left of WT, indicating that for every level of cardiac work, flow was greater in
the WT-RC compared to WT.
Online Figure V. Relationship between myocardial blood (MBF) flow and triple product (Cardiac
Work (CW). MBF was significantly lower in Kv 1.5-/- at any given level of cardiac work (P<0.05).
Online Figure VI. Relationship between MBF and DP in WT (S129 and C57BL/6N
backgrounds) , Kv1.5-/- (S129 amd C57BL/6N backgrounds). Note, there were no significant
diffrences in the relationship between DP and MBF between the backgrounds in the WT or
Kv1.5-/- mice.
Online Figure VII. Expression of other ion channels in Kv 1.5 -/-mice.
Online Figure VIII. A) Hypoxyprobe fluorescent and secondary antibody control signal intensity
in myocardial tissue from wild type (WT), Kv1.5-/-, and Kv1.5-/- RC on doxycycline (7 days). B)
Fold change of signal intensity in the 3 groups. P<0.05 Kv 1.5-/- vs WT mice; P<0.05 Kv 1.5-/RC vs. Kv 1.5-/- mice.
Online Figure IX. Left ventricular volume (LV Vol) at diastole, systole and stroke volumes in Kv
1.5-/- (panel A) and WT (panel B) mice. Panel C- Ejection fraction (EF) at baseline and after NE
treatment. LV internal diameter (LVID) at diastole(d) and systole(s) in Kv 1.5-/- (panel D) and WT
(panel E) mice. Panel F- Fractional shoretening shortening (FS) at baseline and after NE
treatments. Measurements were done at mid-papillary muscle level, and volumes were
calculated by Teichholz formula. Data are presented as Mean±SD, -P<0.05 WT mice
compared to Kv 1.5-/-.
Online Figure X. MBF measured by myocardial contrast echocardiography (MCE) and by
microsphere (BioPal, Inc.). MBF was measured at baseline and during Norepinephrine (0.5-5.0
µg/kg min-1) injection. MBF estimated by MCE was higher compared to microspheres, but there
was an excellent correlation between the two methods. R2 =0.98 and Y=1.39*X-0.7.
Online Figure XI. Correlation between double and triple product as estimates of cardiac work
(CW). R2=0.95.