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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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 REFERENCES 1. Grover RF. Mechanisms augmenting coronary arterial oxygen extraction. Advances in cardiology. 1973;9:89-98. 2. Wolff CB. Normal cardiac output, oxygen delivery and oxygen extraction. Advances in experimental medicine and biology. 2007;599:169-82. 3. Tennant R and Wiggers CJ. THE EFFECT OF CORONARY OCCLUSION ON MYOCARDIAL CONTRACTION; 1935. 4. Jones CJ, Kuo L, Davis MJ and Chilian WM. Myogenic and flow-dependent control mechanisms in the coronary microcirculation. Basic Res Cardiol. 1993;88:2-10. 5. Jones CJ, Kuo L, Davis MJ and Chilian WM. Regulation of coronary blood flow: coordination of heterogeneous control mechanisms in vascular microdomains. Cardiovasc Res. 1995;29:585-96. 6. Jones CJ, Kuo L, Davis MJ and Chilian WM. Distribution and control of coronary microvascular resistance. Adv Exp Med Biol. 1993;346:181-8. 7. Feigl EO. Coronary Physiology. PhysiolRev. 1983;63:1-205. 8. Wu GB, Zhou EX, Qing DX and Li J. Role of potassium channels in regulation of rat coronary arteriole tone. European journal of pharmacology. 2009;620:57-62. 9. Chen TT, Luykenaar KD, Walsh EJ, Walsh MP and Cole WC. Key role of Kv1 channels in vasoregulation. Circulation research. 2006;99:53-60. 10. Knot HJ and Nelson MT. Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries. The American journal of physiology. 1995;269:H348-55. 11. Lu T, Ye D, Wang X, Seubert JM, Graves JP, Bradbury JA, Zeldin DC and Lee HC. Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms. The Journal of physiology. 2006;575:627-44. 12. Zatta AJ and Headrick JP. Mediators of coronary reactive hyperaemia in isolated mouse heart. British journal of pharmacology. 2005;144:576-87. 13. Dick GM and Tune JD. Role of potassium channels in coronary vasodilation. Experimental biology and medicine. 2010;235:10-22. 14. Aziz Q, Thomas AM, Gomes J, Ang R, Sones WR, Li Y, Ng KE, Gee L and Tinker A. The ATPsensitive potassium channel subunit, Kir6.1, in vascular smooth muscle plays a major role in blood pressure control. Hypertension. 2014;64:523-9. 15. Knot HJ, Zimmermann PA and Nelson MT. Extracellular K(+)-induced hyperpolarizations and dilatations of rat coronary and cerebral arteries involve inward rectifier K(+) channels. The Journal of physiology. 1996;492 ( Pt 2):419-30. 16. Jackson WF. Potassium channels in the peripheral microcirculation. Microcirculation. 2005;12:113-27. 17. Rogers PA, Dick GM, Knudson JD, Focardi M, Bratz IN, Swafford AN, Jr., Saitoh S, Tune JD and Chilian WM. H2O2-induced redox-sensitive coronary vasodilation is mediated by 4-aminopyridinesensitive K+ channels. Am J Physiol Heart Circ Physiol. 2006;291:H2473-82. 18. Saitoh S, Zhang C, Tune JD, Potter B, Kiyooka T, Rogers PA, Knudson JD, Dick GM, Swafford A and Chilian WM. Hydrogen peroxide: a feed-forward dilator that couples myocardial metabolism to coronary blood flow. Arterioscler Thromb Vasc Biol. 2006;26:2614-21. 19. Saitoh S, Kiyooka T, Rocic P, Rogers PA, Zhang C, Swafford A, Dick GM, Viswanathan C, Park Y and Chilian WM. Redox-dependent coronary metabolic dilation. Am J Physiol Heart Circ Physiol. 2007;293:H3720-5. Kleppisch T and Nelson MT. ATP-sensitive K+ currents in cerebral arterial smooth muscle: 20. pharmacological and hormonal modulation. Am J Physiol. 1995;269:H1634-40. 21. Archer SL, Weir EK, Reeve HL and Michelakis E. Molecular identification of O2 sensors and O2sensitive potassium channels in the pulmonary circulation. Adv Exp Med Biol. 2000;475:219-40. 22. Archer SL, Wu XC, Thebaud B, Nsair A, Bonnet S, Tyrrell B, McMurtry MS, Hashimoto K, Harry G and Michelakis ED. Preferential expression and function of voltage-gated, O2-sensitive K+ channels in DOI: 10.1161/CIRCRESAHA.115.306642 12 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 resistance pulmonary arteries explains regional heterogeneity in hypoxic pulmonary vasoconstriction: ionic diversity in smooth muscle cells. Circ Res. 2004;95:308-18. 23. Patel AJ and Honore E. Molecular physiology of oxygen-sensitive potassium channels. Eur Respir J. 2001;18:221-7. 24. Pannasch U, Farber K, Nolte C, Blonski M, Yan Chiu S, Messing A and Kettenmann H. The potassium channels Kv1.5 and Kv1.3 modulate distinct functions of microglia. Molecular and cellular neurosciences. 2006;33:401-11. 25. Tiefenbacher CP, DeFily DV and Chilian WM. Requisite role of cardiac myocytes in coronary alpha1-adrenergic constriction. Circulation. 1998;98:9-12. 26. Merkus D, Brzezinska AK, Zhang C, Saito S and Chilian WM. Cardiac myocytes control release of endothelin-1 in coronary vasculature. Am J Physiol Heart Circ Physiol. 2005;288:H2088-92. 27. Chilian WM, Layne SM, Klausner EC, Eastham CL and Marcus ML. Redistribution of coronary microvascular resistance produced by dipyridamole. Am J Physiol. 1989;256:H383-90. 28. Kuo L, Davis MJ and Chilian WM. Longitudinal gradients for endothelium-dependent and independent vascular responses in the coronary microcirculation. Circulation. 1995;92:518-25. 29. Kanatsuka H, Lamping KG, Eastham CL, Dellsperger KC and Marcus ML. Comparison of the effects of increased myocardial oxygen consumption and adenosine on the coronary microvascular resistance. Circulation Research. 1989;65:1296-1305. 30. Marcus ML, Chilian WM, Kanatsuka H, Dellsperger KC, Eastham CL and Lamping KG. Understanding the coronary circulation through studies at the microvascular level. Circulation. 1990;82:17. 31. Johnson BD, Shaw LJ, Buchthal SD, Bairey Merz CN, Kim HW, Scott KN, Doyle M, Olson MB, Pepine CJ, den Hollander J, Sharaf B, Rogers WJ, Mankad S, Forder JR, Kelsey SF and Pohost GM. Prognosis in women with myocardial ischemia in the absence of obstructive coronary disease: results from the National Institutes of Health-National Heart, Lung, and Blood Institute-Sponsored Women's Ischemia Syndrome Evaluation (WISE). Circulation. 2004;109:2993-9. 32. Marroquin OC, Kip KE, Kelley DE, Johnson BD, Shaw LJ, Bairey Merz CN, Sharaf BL, Pepine CJ, Sopko G and Reis SE. Metabolic syndrome modifies the cardiovascular risk associated with angiographic coronary artery disease in women: a report from the Women's Ischemia Syndrome Evaluation. Circulation. 2004;109:714-21. 33. von Mering GO, Arant CB, Wessel TR, McGorray SP, Bairey Merz CN, Sharaf BL, Smith KM, Olson MB, Johnson BD, Sopko G, Handberg E, Pepine CJ and Kerensky RA. Abnormal coronary vasomotion as a prognostic indicator of cardiovascular events in women: results from the National Heart, Lung, and Blood Institute-Sponsored Women's Ischemia Syndrome Evaluation (WISE). Circulation. 2004;109:722-5. 34. Pepine CJ, Anderson RD, Sharaf BL, Reis SE, Smith KM, Handberg EM, Johnson BD, Sopko G and Bairey Merz CN. Coronary microvascular reactivity to adenosine predicts adverse outcome in women evaluated for suspected ischemia results from the National Heart, Lung and Blood Institute WISE (Women's Ischemia Syndrome Evaluation) study. J Am Coll Cardiol. 55:2825-32. 35. Armstrong PW, Fu Y, Chang WC, Topol EJ, Granger CB, Betriu A, Van de Werf F, Lee KL and Califf RM. Acute coronary syndromes in the GUSTO-IIb trial: prognostic insights and impact of recurrent ischemia. The GUSTO-IIb Investigators. Circulation. 1998;98:1860-8. 36. Murthy VL, Naya M, Foster CR, Gaber M, Hainer J, Klein J, Dorbala S, Blankstein R and Di Carli MF. Association between coronary vascular dysfunction and cardiac mortality in patients with and without diabetes mellitus. Circulation. 2012;126:1858-68. 37. Modell SM and Lehmann MH. The long QT syndrome family of cardiac ion channelopathies: a HuGE review. Genetics in medicine : official journal of the American College of Medical Genetics. 2006;8:143-55. 38. Fedele F, Mancone M, Chilian WM, Severino P, Canali E, Logan S, De Marchis ML, Volterrani M, Palmirotta R and Guadagni F. Role of genetic polymorphisms of ion channels in the pathophysiology of coronary microvascular dysfunction and ischemic heart disease. Basic Res Cardiol. 2013;108:387. DOI: 10.1161/CIRCRESAHA.115.306642 13 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 39. Petersen JW and Pepine CJ. Microvascular coronary dysfunction and ischemic heart disease: where are we in 2014? Trends in cardiovascular medicine. 2015;25:98-103. 40. Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res. 1980;47:807813. 41. Berne RM, Rubio R, Dobson JG, Jr. and Curnish RR. Adenosine and adenine nucleotides as possible mediators of cardiac and skeletal muscle blood flow regulation. Circ Res. 1971;28:Suppl 1:115+. 42. Stepp DW, Van Bibber R, Kroll K and Feigl EO. Quantitative relation between interstitial adenosine concentration and coronary blood flow. Circulation Research. 1996;79:601-10. 43. Tune JD, Richmond KN, Gorman MW and Feigl EO. K(ATP)(+) channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation. Am J Physiol Heart Circ Physiol. 2001;280:H868-75. 44. Ishibashi Y, Duncker DJ, Zhang J and Bache RJ. ATP-sensitive K+ channels, adenosine, and nitric oxide-mediated mechanisms account for coronary vasodilation during exercise. Circulation Research. 1998;82:346-359. 45. Duncker DJ, Van Zon NS, Altman JD, Pavek TJ and Bache RJ. Role of K+ATP channels in coronary vasodilation during exercise. Circulation. 1993;88:1245-53. 46. Merkus D, Sorop O, Houweling B, Hoogteijling BA and Duncker DJ. K+Ca-channels contribute to exercise-induced coronary vasodilation in swine. Am J Physiol Heart Circ Physiol. 2006. 47. Richmond KN, Tune JD, Gorman MW and Feigl EO. Role of K+ATP channels in local metabolic coronary vasodilation. Am J Physiol. 1999;277:H2115-23. 48. Stepp DW, Kroll K and Feigl EO. K+ATP channels and adenosine are not necessary for coronary autoregulation. Am J Physiol. 1997;273:H1299-308. 49. Farias M, 3rd, Gorman MW, Savage MV and Feigl EO. Plasma ATP during exercise: possible role in regulation of coronary blood flow. Am J Physiol Heart Circ Physiol. 2005;288:H1586-90. 50. Gorman MW, Rooke GA, Savage MV, Jayasekara MP, Jacobson KA and Feigl EO. Adenine nucleotide control of coronary blood flow during exercise. Am J Physiol Heart Circ Physiol. 2010;299:H1981-9. 51. Saitoh S-i, Zhang C, Tune JD, Potter B, Kiyooka T, Rogers PA, Knudson JD, Dick G, Swafford A and Chilian WM. Hydrogen Peroxide. A Feed-Forward Dilator That Couples Myocardial Metabolism to Coronary Blood Flow. Arterioscler Thromb Vasc Biol. 2006:01.ATV.0000249408.55796.da. 52. Berwick ZC, Dick GM, Moberly SP, Kohr MC, Sturek M and Tune JD. Contribution of voltagedependent K(+) channels to metabolic control of coronary blood flow. J Mol Cell Cardiol. 2012;52:912-9. 53. MacDonald PE, Ha XF, Wang J, Smukler SR, Sun AM, Gaisano HY, Salapatek AM, Backx PH and Wheeler MB. Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion. Mol Endocrinol. 2001;15:1423-35. 54. Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD and Chandy KG. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 1994;45:1227-34. 55. Mironova GD, Negoda AE, Marinov BS, Paucek P, Costa AD, Grigoriev SM, Skarga YY and Garlid KD. Functional distinctions between the mitochondrial ATP-dependent K+ channel (mitoKATP) and its inward rectifier subunit (mitoKIR). J Biol Chem. 2004;279:32562-8. 56. Jaburek M, Yarov-Yarovoy V, Paucek P and Garlid KD. State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate. J Biol Chem. 1998;273:13578-82. 57. Szewczyk A, Czyz A and Nalecz MJ. ATP-regulated potassium channel blocker, glibenclamide, uncouples mitochondria. Pol J Pharmacol. 1997;49:49-52. 58. Yao X, Chang AY, Boulpaep EL, Segal AS and Desir GV. Molecular cloning of a glibenclamidesensitive, voltage-gated potassium channel expressed in rabbit kidney. J Clin Invest. 1996;97:2525-33. 59. Duprat F, Guillemare E, Romey G, Fink M, Lesage F, Lazdunski M and Honore E. Susceptibility of cloned K+ channels to reactive oxygen species. Proc Natl Acad Sci U S A. 1995;92:11796-800. 60. Conforti L, Bodi I, Nisbet JW and Millhorn DE. O2-sensitive K+ channels: role of the Kv1.2 subunit in mediating the hypoxic response. The Journal of physiology. 2000;524 Pt 3:783-93. DOI: 10.1161/CIRCRESAHA.115.306642 14 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 61. Zhang DX, Borbouse L, Gebremedhin D, Mendoza SA, Zinkevich NS, Li R and Gutterman DD. H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and largeconductance Ca2+-activated K+ channel activation. Circ Res. 2012;110:471-80. 62. Standen NB and Quayle JM. K+ channel modulation in arterial smooth muscle. Acta physiologica Scandinavica. 1998;164:549-57. 63. Honore E, Barhanin J, Attali B, Lesage F and Lazdunski M. External blockade of the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:1937-41. 64. Yang T, Wathen MS, Felipe A, Tamkun MM, Snyders DJ and Roden DM. K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells). Circ Res. 1994;75:870-8. 65. Sadanaga T, Ohya Y, Ohtsubo T, Goto K, Fujii K and Abe I. Decreased 4-aminopyridine sensitive K+ currents in endothelial cells from hypertensive rats. Hypertension research : official journal of the Japanese Society of Hypertension. 2002;25:589-96. 66. Chen WL, Huang XQ, Zhao LY, Li J, Chen JW, Xiao Y, Huang YY, Liu J, Wang GL and Guan YY. Involvement of Kv1.5 protein in oxidative vascular endothelial cell injury. PloS one. 2012;7:e49758. 67. Mi H, Deerinck TJ, Ellisman MH and Schwarz TL. Differential distribution of closely related potassium channels in rat Schwann cells. J Neurosci. 1995;15:3761-74. 68. Vandenberg BF, Kieso R, Fox-Eastham K, Chilian W and Kerber RE. Quantitation of myocardial perfusion by contrast echocardiography: analysis of contrast gray level appearance variables and intracyclic variability. J Am Coll Cardiol. 1989;13:200-6. 69. Davidson BP, Chadderdon SM, Belcik JT, Gupta S and Lindner JR. Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression. J Am Soc Echocardiogr. 2014;27:786-793 e2. 70. Kaufmann BA, Lankford M, Behm CZ, French BA, Klibanov AL, Xu Y and Lindner JR. Highresolution myocardial perfusion imaging in mice with high-frequency echocardiographic detection of a depot contrast agent. J Am Soc Echocardiogr. 2007;20:136-43. 71. Lepper W, Belcik T, Wei K, Lindner JR, Sklenar J and Kaul S. Myocardial contrast echocardiography. Circulation. 2004;109:3132-5. 72. Kaufmann BA, Wei K and Lindner JR. Contrast echocardiography. Curr Probl Cardiol. 2007;32:51-96. 73. Gurudevan SV, Nelson MD, Rader F, Tang X, Lewis J, Johannes J, Belcik JT, Elashoff RM, Lindner JR and Victor RG. Cocaine-induced vasoconstriction in the human coronary microcirculation: new evidence from myocardial contrast echocardiography. Circulation. 2013;128:598-604. 74. Bratkovsky S, Aasum E, Birkeland CH, Riemersma RA, Myhre ES and Larsen TS. Measurement of coronary flow reserve in isolated hearts from mice. Acta physiologica Scandinavica. 2004;181:167-72. 75. Reichelt ME, Willems L, Hack BA, Peart JN and Headrick JP. Cardiac and coronary function in the Langendorff-perfused mouse heart model. Experimental physiology. 2009;94:54-70. 76. Zhou X, Teng B, Tilley S, Ledent C and Mustafa SJ. Metabolic hyperemia requires ATP-sensitive K+ channels and H2O2 but not adenosine in isolated mouse hearts. Am J Physiol Heart Circ Physiol. 2014;307:H1046-55. 77. Zhou Z, Rajamani U, Labazi H, Tilley SL, Ledent C, Teng B and Mustafa SJ. Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts. Purinergic Signal. 2015;11:263-73. 78. Raher MJ, Thibault H, Poh KK, Liu R, Halpern EF, Derumeaux G, Ichinose F, Zapol WM, Bloch KD, Picard MH and Scherrer-Crosbie M. In vivo characterization of murine myocardial perfusion with myocardial contrast echocardiography: validation and application in nitric oxide synthase 3 deficient mice. Circulation. 2007;116:1250-7. 79. van Nierop BJ, Coolen BF, Bax NA, Dijk WJ, van Deel ED, Duncker DJ, Nicolay K and Strijkers GJ. Myocardial perfusion MRI shows impaired perfusion of the mouse hypertrophic left ventricle. The international journal of cardiovascular imaging. 2014;30:619-28. 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). Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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? Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 NE A B Figure 4 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 R2=0.98 R2=0.89 P<0.05 P<0.05 B A C Figure 5 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 Downloaded from http://circres.ahajournals.org/ by guest on May 5, 2017 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 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ 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. References 1. 2. 3. 4. Chaves AA, Weinstein DM, Bauer JA. Non-invasive echocardiographic studies in mice: Influence of anesthetic regimen. Life sciences. 2001;69:213-222 Stypmann J, Engelen MA, Troatz C, Rothenburger M, Eckardt L, Tiemann K. Echocardiographic assessment of global left ventricular function in mice. Laboratory animals. 2009;43:127-137 Teichholz LE, Kreulen T, Herman MV, Gorlin R. Problems in echocardiographic volume determinations: Echocardiographic-angiographic correlations in the presence of absence of asynergy. The American journal of cardiology. 1976;37:7-11 van de Weijer T, van Ewijk PA, Zandbergen HR, Slenter JM, Kessels AG, Wildberger JE, Hesselink MK, Schrauwen P, Schrauwen-Hinderling VB, Kooi ME. Geometrical 5. 6. 7. 8. 9. 10. 11. 12. 13. models for cardiac mri in rodents: Comparison of quantification of left ventricular volumes and function by various geometrical models with a full-volume mri data set in rodents. American journal of physiology. Heart and circulatory physiology. 2012;302:H709-715 Kaufmann BA, Lankford M, Behm CZ, French BA, Klibanov AL, Xu Y, Lindner JR. Highresolution myocardial perfusion imaging in mice with high-frequency echocardiographic detection of a depot contrast agent. Journal of the American Society of Echocardiography : official publication of the American Society of Echocardiography. 2007;20:136-143 Vogel R, Indermuhle A, Reinhardt J, Meier P, Siegrist PT, Namdar M, Kaufmann PA, Seiler C. The quantification of absolute myocardial perfusion in humans by contrast echocardiography: Algorithm and validation. Journal of the American College of Cardiology. 2005;45:754-762 Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, Kaul S. Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion. Circulation. 1998;97:473-483 Coggins MP, Sklenar J, Le DE, Wei K, Lindner JR, Kaul S. Noninvasive prediction of ultimate infarct size at the time of acute coronary occlusion based on the extent and magnitude of collateral-derived myocardial blood flow. Circulation. 2001;104:2471-2477 Raher MJ, Thibault H, Poh KK, Liu R, Halpern EF, Derumeaux G, Ichinose F, Zapol WM, Bloch KD, Picard MH, Scherrer-Crosbie M. In vivo characterization of murine myocardial perfusion with myocardial contrast echocardiography: Validation and application in nitric oxide synthase 3 deficient mice. Circulation. 2007;116:1250-1257 Hofer SO, Mitchell GM, Penington AJ, Morrison WA, RomeoMeeuw R, Keramidaris E, Palmer J, Knight KR. The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber. British journal of plastic surgery. 2005;58:1104-1114 Ahmad R, Kuppusamy P. Theory, instrumentation, and applications of electron paramagnetic resonance oximetry. Chemical reviews. 2010;110:3212-3236 Liu KJ, Gast P, Moussavi M, Norby SW, Vahidi N, Walczak T, Wu M, Swartz HM. Lithium phthalocyanine: A probe for electron paramagnetic resonance oximetry in viable biological systems. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:5438-5442 Pandian RP, Kutala VK, Parinandi NL, Zweier JL, Kuppusamy P. Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe. Archives of biochemistry and biophysics. 2003;420:169-175 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.