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J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07. Published in final edited form as: J Am Coll Cardiol. 2015 September 08; 66(10): 1157–1159. doi:10.1016/j.jacc.2015.07.028.
How Does Diazoxide Elicit Arrhythmias in Rats With Type 2 Diabetes?: Is This Effect Clinically Significant?* Blake W. Nelson and David R. Van Wagoner, PhD Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio.
Author Manuscript
Keywords ATP-sensitive potassium channel; diabetes; ischemia-reperfusion arrhythmia; mitochondria Under ischemic conditions, adenosine triphosphate (ATP) production is compromised and cell viability is challenged. A homeostatic pathway present to reduce ATP utilization is the reduction of calcium influx and excitability triggered by opening of ATP-sensitive potassium (KATP) channels in the plasma membrane. These channels are abundant in the heart, brain, pancreas, and other tissues.
Author Manuscript
Plasma membrane KATP (pmKATP) channels are well characterized with respect to subunit composition and pharmacology. Similar to many other ion channels, pmKATP channels are macromolecular complexes composed of pore subunits (Kir6.1 or Kir6.2) through which potassium is selectively permeable, and which senses intracellular ATP concentrations via the associated sulfonylurea receptor subunit (either SUR1A, SUR1B, SUR2A, or SUR2B, depending on the species and tissue). Sulfonylurea drugs (tolbutamide, glibenclamide) that block these KATP channels interact with the SUR subunits. The variations in subunit composition contribute to variations in sensitivity to intracellular ATP/adenosine diphosphate reduction, lipids in the cell membrane, and sensitivity to drugs that modulate these channels. Chamber specific variations in subunit expression have been well documented between the atria and ventricle in the heart, and between different organs (e.g., heart vs. pancreas) (1). Species differences in the distribution of the subunit composition of these channels are also apparent, and are especially evident in comparing mice and rats with larger species (1).
Author Manuscript
While the structure and function of the pmKATP channel has been quite well characterized, another KATP channel present in the mitochondrial membrane (mKATP) has been associated with cardioprotection during ischemia, via a modest increase in reactive oxygen species and activation of protein kinase C epsilon (2). Activation of mKATP has been
*Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. REPRINT REQUESTS AND CORRESPONDENCE: Dr. David R. Van Wagoner, Department of Molecular Cardiology, Cleveland Clinic, 9500 Euclid Avenue, M/S NE-61, Cleveland, Ohio 44195. [email protected]. Both authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Nelson and Van Wagoner
Page 2
Author Manuscript
associated with preservation of mitochondrial structure and function, both during ischemia and during reperfusion. While the structure and function of pmKATP is well characterized, efforts to characterize mKATP lagged due to challenges in determining its molecular composition. For years, mKATP was primarily characterized on the basis of low affinity drugs that increase its activity (diazoxide [DZX]) (3) or suppress its activity (5hydroxydecanoate and tolbutamide). At higher concentrations, both DZX (~100 µmol/l) (1) and tolbutamide (1 µmol/l) (4) also modulate pmKATP channels. This cross-reactivity of drugs has led to confusion about the pathophysiologic roles of pmKATP versus mKATP channels.
Author Manuscript
In this issue of the Journal, Xie et al. (5) present a translational study in which they tested the hypothesis that low-dose DZX treatment (30 µmol/l) would be effective as a cardioprotective agent for preventing ischemia-induced arrhythmias in diabetic adult male rat hearts. The authors used optical imaging of ex vivo Langendorff-perfused hearts to compare the effects of DZX, a putative mKATP channel activator, with those of pinacidil, a direct agonist of the pmKATP channel. Male Zucker obese diabetic rats were compared with male Sprague Dawley and Zucker lean rats as controls. The authors noted baseline differences in the response to ischemia between groups, with profound shortening of action potential duration (APD) in nondiabetic controls versus a biphasic response (transient APD shortening) in the diabetic animals resulting in little change in APD. Ischemia slowed conduction in all animals. Upon reperfusion of the untreated hearts, 4 of 9 controls and 3 of 5 diabetic hearts experienced sustained arrhythmias within 1 min. In all of the control and type 2 diabetes mellitus (T2DM) rats, pinacidil, via its profound shortening of ventricular APD, elicited ventricular arrhythmias subsequent to 12 min of no-flow global ischemia.
Author Manuscript
The authors predicted that, as a result of its impact on mKATP, DZX would prevent ischemia-induced arrhythmias in both T2DM and control hearts. In contrast, they found that, while DZX did not elicit arrhythmias during ischemia in any (0 of 9) of the untreated or DZX-treated (0 of 7) control hearts during ischemia, all (7 of 7) DZX treated T2DM and none (0 of 5) untreated T2DM hearts experienced VT during the late stages (10 to 12 min) of ischemia.
Author Manuscript
What underlies the difference in response between control and T2DM rats? To determine whether T2DM affected the composition of known KATP channel subunits, the authors used quantitative PCR normalized to the expression of β-actin. No differences were detected between the Zucker lean versus diabetic rats. A limitation is the small number of hearts (3 per group) used in this analysis. Small differences that might be significant may not be detectable. The authors suggest that if the differences are not at the transcriptional level, post-translational modifications of channel-related proteins may have a significant impact on their function. Diabetes is associated with increased abundance of advanced glycation end products, oxidant stress, and altered nitric oxide metabolism that might lead to altered protein nitration or s-nitrosylation. DZX is currently being evaluated for use as an antidiabetic drug, and diabetic patients have an increased risk of ischemic events subsequent to coronary artery disease. What is the role
J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
Nelson and Van Wagoner
Page 3
Author Manuscript
of the mKATP channel in this response? Is the observed proarrhythmia during ischemia reported by Xie et al. (5) of clinical relevance or significance in human subjects?
Author Manuscript
DZX is a small molecule drug that was created as an antihypertensive agent, as a congener of hydrochlorothiazide, a common diuretic drug. In a 1964 clinical study of its antihypertensive effects, acute DZX infusion was noted to both lower blood pressure and promote hyperglycemia (6). The salt-sparing effects of DZX distinguish it from related antihypertensive medications that promote diuresis. In 1967, it was noted that tolbutamide, a pmKATP channel antagonist, could block the actions of DZX (7). Thus, it may be relevant to consider whether the effects of DZX in diabetes are related to its interactions with the mKATP channel, or also with the pmKATP channel. A 2012 study provided strong evidence that a potassium channel subunit (ROMK, KCNJ1) first described in the kidney is the pore subunit of the mKATP channel (8), consistent with the antihypertensive effects of DZX being mediated by mKATP channels. However, there is also compelling evidence that DZX can activate the pmKATP channel (1).
Author Manuscript
The simplest conclusion is that DZX likely affects both channels, favoring modulation of the mKATP at low concentrations, while both channels are affected at higher concentrations. The authors have not fully eliminated the possibility that both channel types contribute to the pro-arrhythmic response to ischemia in the T2DM rats. Whether or not humans with T2DM have a similar sensitization to DZX cannot easily be predicted, in the absence of direct studies. Some studies with DZX have already been performed in adult human hearts (9). Using similar optical mapping techniques, DZX (300 µmol/l, 10 times higher than in the current study [5]) shortened APD by approximately 20% to 30% in diseased (heart failure or myocardial infarction) atria and ventricles, but not in nonfailing atria, suggesting an important role for the pmKATP channel in the response (9). The drug sensitivity of the pmKATP channel varies in response to the levels of intracellular ATP and as a function of channel subunit composition. That the effects of DZX are evident in T2DM rats may be due to a reduced capacity to generate and maintain ATP via glycolysis (the primary supply available during ischemia). Chamber differences in human and rodent studies suggest that subunit differences can impact the sensitivity to DZX. Additional studies that evaluate the integrated cardiac response to DZX in diabetic humans seem warranted. Studies using longterm exposure of cardiac myocytes derived from inducible pluripotent stem cells to hyperglycemia may supplement efforts to collect primary data from the explanted hearts of subjects with T2DM.
Author Manuscript
Should development of DZX as a treatment for T2DM be halted? The authors suggest that this may be warranted. It would clearly be wise to retrospectively examine the medical records of subjects treated with DZX, and to prospectively closely monitor outcomes. In addition, it would be logical to prospectively assess the electrocardiographic changes in subjects in response to a stress test, in which ischemia can be induced in a controlled setting, with ready access to defibrillation. At all times, it is essential to consider the risk to the patient as the top priority, recalling the guidance of the Hippocratic Oath: First, do no harm.
J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
Nelson and Van Wagoner
Page 4
Author Manuscript
REFERENCES
Author Manuscript
1. Zhang HX, Silva JR, Lin YW, et al. Heterogeneity and function of K(ATP) channels in canine hearts. Heart Rhythm. 2013; 10:1576–1583. [PubMed: 23871704] 2. Garlid AO, Jaburek M, Jacobs JP, Garlid KD. Mitochondrial reactive oxygen species: which ROS signals cardioprotection? Am J Physiol Heart Circ Physiol. 2013; 305:H960–H968. [PubMed: 23913710] 3. Garlid KD, Paucek P, Yarov-Yarovoy VM, et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection. Circ Res. 1997; 81:1072–1082. [PubMed: 9400389] 4. Van Wagoner DR. Mechanosensitive gating of atrial ATP-sensitive potassium channels. Circ Res. 1993; 72:973–983. [PubMed: 8477531] 5. Xie C, Hu J, Motloch LJ, Karam BS, Akar FG. The classically cardioprotective agent diazoxide elicits arrhythmias in type 2 diabetes mellitus. J Am Coll Cardiol. 2015; 66:1144–1156. [PubMed: 26337994] 6. Okun R, Wilson WR, Gelfand MD. The hyperglycemic effect of hypotensive drugs. J Chronic Dis. 1964; 17:31–39. [PubMed: 14107168] 7. Wales JK, Grant AM, Wolff FW. Reversal of diazoxide effects by tolbutamide. Lancet. 1967; 1:1137–1138. [PubMed: 4164793] 8. Foster DB, Ho AS, Rucker J, et al. Mitochondrial ROMK channel is a molecular component of mitoK(ATP). Circ Res. 2012; 111:446–454. [PubMed: 22811560] 9. Fedorov VV, Glukhov AV, Ambrosi CM, et al. Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts. J Mol Cell Cardiol. 2011; 51:215–225. [PubMed: 21586291]
Author Manuscript Author Manuscript J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07. Published in final edited form as: J Am Coll Cardiol. 2015 September 08; 66(10): 1157–1159. doi:10.1016/j.jacc.2015.07.028.
How Does Diazoxide Elicit Arrhythmias in Rats With Type 2 Diabetes?: Is This Effect Clinically Significant?* Blake W. Nelson and David R. Van Wagoner, PhD Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio.
Author Manuscript
Keywords ATP-sensitive potassium channel; diabetes; ischemia-reperfusion arrhythmia; mitochondria Under ischemic conditions, adenosine triphosphate (ATP) production is compromised and cell viability is challenged. A homeostatic pathway present to reduce ATP utilization is the reduction of calcium influx and excitability triggered by opening of ATP-sensitive potassium (KATP) channels in the plasma membrane. These channels are abundant in the heart, brain, pancreas, and other tissues.
Author Manuscript
Plasma membrane KATP (pmKATP) channels are well characterized with respect to subunit composition and pharmacology. Similar to many other ion channels, pmKATP channels are macromolecular complexes composed of pore subunits (Kir6.1 or Kir6.2) through which potassium is selectively permeable, and which senses intracellular ATP concentrations via the associated sulfonylurea receptor subunit (either SUR1A, SUR1B, SUR2A, or SUR2B, depending on the species and tissue). Sulfonylurea drugs (tolbutamide, glibenclamide) that block these KATP channels interact with the SUR subunits. The variations in subunit composition contribute to variations in sensitivity to intracellular ATP/adenosine diphosphate reduction, lipids in the cell membrane, and sensitivity to drugs that modulate these channels. Chamber specific variations in subunit expression have been well documented between the atria and ventricle in the heart, and between different organs (e.g., heart vs. pancreas) (1). Species differences in the distribution of the subunit composition of these channels are also apparent, and are especially evident in comparing mice and rats with larger species (1).
Author Manuscript
While the structure and function of the pmKATP channel has been quite well characterized, another KATP channel present in the mitochondrial membrane (mKATP) has been associated with cardioprotection during ischemia, via a modest increase in reactive oxygen species and activation of protein kinase C epsilon (2). Activation of mKATP has been
*Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. REPRINT REQUESTS AND CORRESPONDENCE: Dr. David R. Van Wagoner, Department of Molecular Cardiology, Cleveland Clinic, 9500 Euclid Avenue, M/S NE-61, Cleveland, Ohio 44195. [email protected]. Both authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Nelson and Van Wagoner
Page 2
Author Manuscript
associated with preservation of mitochondrial structure and function, both during ischemia and during reperfusion. While the structure and function of pmKATP is well characterized, efforts to characterize mKATP lagged due to challenges in determining its molecular composition. For years, mKATP was primarily characterized on the basis of low affinity drugs that increase its activity (diazoxide [DZX]) (3) or suppress its activity (5hydroxydecanoate and tolbutamide). At higher concentrations, both DZX (~100 µmol/l) (1) and tolbutamide (1 µmol/l) (4) also modulate pmKATP channels. This cross-reactivity of drugs has led to confusion about the pathophysiologic roles of pmKATP versus mKATP channels.
Author Manuscript
In this issue of the Journal, Xie et al. (5) present a translational study in which they tested the hypothesis that low-dose DZX treatment (30 µmol/l) would be effective as a cardioprotective agent for preventing ischemia-induced arrhythmias in diabetic adult male rat hearts. The authors used optical imaging of ex vivo Langendorff-perfused hearts to compare the effects of DZX, a putative mKATP channel activator, with those of pinacidil, a direct agonist of the pmKATP channel. Male Zucker obese diabetic rats were compared with male Sprague Dawley and Zucker lean rats as controls. The authors noted baseline differences in the response to ischemia between groups, with profound shortening of action potential duration (APD) in nondiabetic controls versus a biphasic response (transient APD shortening) in the diabetic animals resulting in little change in APD. Ischemia slowed conduction in all animals. Upon reperfusion of the untreated hearts, 4 of 9 controls and 3 of 5 diabetic hearts experienced sustained arrhythmias within 1 min. In all of the control and type 2 diabetes mellitus (T2DM) rats, pinacidil, via its profound shortening of ventricular APD, elicited ventricular arrhythmias subsequent to 12 min of no-flow global ischemia.
Author Manuscript
The authors predicted that, as a result of its impact on mKATP, DZX would prevent ischemia-induced arrhythmias in both T2DM and control hearts. In contrast, they found that, while DZX did not elicit arrhythmias during ischemia in any (0 of 9) of the untreated or DZX-treated (0 of 7) control hearts during ischemia, all (7 of 7) DZX treated T2DM and none (0 of 5) untreated T2DM hearts experienced VT during the late stages (10 to 12 min) of ischemia.
Author Manuscript
What underlies the difference in response between control and T2DM rats? To determine whether T2DM affected the composition of known KATP channel subunits, the authors used quantitative PCR normalized to the expression of β-actin. No differences were detected between the Zucker lean versus diabetic rats. A limitation is the small number of hearts (3 per group) used in this analysis. Small differences that might be significant may not be detectable. The authors suggest that if the differences are not at the transcriptional level, post-translational modifications of channel-related proteins may have a significant impact on their function. Diabetes is associated with increased abundance of advanced glycation end products, oxidant stress, and altered nitric oxide metabolism that might lead to altered protein nitration or s-nitrosylation. DZX is currently being evaluated for use as an antidiabetic drug, and diabetic patients have an increased risk of ischemic events subsequent to coronary artery disease. What is the role
J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
Nelson and Van Wagoner
Page 3
Author Manuscript
of the mKATP channel in this response? Is the observed proarrhythmia during ischemia reported by Xie et al. (5) of clinical relevance or significance in human subjects?
Author Manuscript
DZX is a small molecule drug that was created as an antihypertensive agent, as a congener of hydrochlorothiazide, a common diuretic drug. In a 1964 clinical study of its antihypertensive effects, acute DZX infusion was noted to both lower blood pressure and promote hyperglycemia (6). The salt-sparing effects of DZX distinguish it from related antihypertensive medications that promote diuresis. In 1967, it was noted that tolbutamide, a pmKATP channel antagonist, could block the actions of DZX (7). Thus, it may be relevant to consider whether the effects of DZX in diabetes are related to its interactions with the mKATP channel, or also with the pmKATP channel. A 2012 study provided strong evidence that a potassium channel subunit (ROMK, KCNJ1) first described in the kidney is the pore subunit of the mKATP channel (8), consistent with the antihypertensive effects of DZX being mediated by mKATP channels. However, there is also compelling evidence that DZX can activate the pmKATP channel (1).
Author Manuscript
The simplest conclusion is that DZX likely affects both channels, favoring modulation of the mKATP at low concentrations, while both channels are affected at higher concentrations. The authors have not fully eliminated the possibility that both channel types contribute to the pro-arrhythmic response to ischemia in the T2DM rats. Whether or not humans with T2DM have a similar sensitization to DZX cannot easily be predicted, in the absence of direct studies. Some studies with DZX have already been performed in adult human hearts (9). Using similar optical mapping techniques, DZX (300 µmol/l, 10 times higher than in the current study [5]) shortened APD by approximately 20% to 30% in diseased (heart failure or myocardial infarction) atria and ventricles, but not in nonfailing atria, suggesting an important role for the pmKATP channel in the response (9). The drug sensitivity of the pmKATP channel varies in response to the levels of intracellular ATP and as a function of channel subunit composition. That the effects of DZX are evident in T2DM rats may be due to a reduced capacity to generate and maintain ATP via glycolysis (the primary supply available during ischemia). Chamber differences in human and rodent studies suggest that subunit differences can impact the sensitivity to DZX. Additional studies that evaluate the integrated cardiac response to DZX in diabetic humans seem warranted. Studies using longterm exposure of cardiac myocytes derived from inducible pluripotent stem cells to hyperglycemia may supplement efforts to collect primary data from the explanted hearts of subjects with T2DM.
Author Manuscript
Should development of DZX as a treatment for T2DM be halted? The authors suggest that this may be warranted. It would clearly be wise to retrospectively examine the medical records of subjects treated with DZX, and to prospectively closely monitor outcomes. In addition, it would be logical to prospectively assess the electrocardiographic changes in subjects in response to a stress test, in which ischemia can be induced in a controlled setting, with ready access to defibrillation. At all times, it is essential to consider the risk to the patient as the top priority, recalling the guidance of the Hippocratic Oath: First, do no harm.
J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
Nelson and Van Wagoner
Page 4
Author Manuscript
REFERENCES
Author Manuscript
1. Zhang HX, Silva JR, Lin YW, et al. Heterogeneity and function of K(ATP) channels in canine hearts. Heart Rhythm. 2013; 10:1576–1583. [PubMed: 23871704] 2. Garlid AO, Jaburek M, Jacobs JP, Garlid KD. Mitochondrial reactive oxygen species: which ROS signals cardioprotection? Am J Physiol Heart Circ Physiol. 2013; 305:H960–H968. [PubMed: 23913710] 3. Garlid KD, Paucek P, Yarov-Yarovoy VM, et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection. Circ Res. 1997; 81:1072–1082. [PubMed: 9400389] 4. Van Wagoner DR. Mechanosensitive gating of atrial ATP-sensitive potassium channels. Circ Res. 1993; 72:973–983. [PubMed: 8477531] 5. Xie C, Hu J, Motloch LJ, Karam BS, Akar FG. The classically cardioprotective agent diazoxide elicits arrhythmias in type 2 diabetes mellitus. J Am Coll Cardiol. 2015; 66:1144–1156. [PubMed: 26337994] 6. Okun R, Wilson WR, Gelfand MD. The hyperglycemic effect of hypotensive drugs. J Chronic Dis. 1964; 17:31–39. [PubMed: 14107168] 7. Wales JK, Grant AM, Wolff FW. Reversal of diazoxide effects by tolbutamide. Lancet. 1967; 1:1137–1138. [PubMed: 4164793] 8. Foster DB, Ho AS, Rucker J, et al. Mitochondrial ROMK channel is a molecular component of mitoK(ATP). Circ Res. 2012; 111:446–454. [PubMed: 22811560] 9. Fedorov VV, Glukhov AV, Ambrosi CM, et al. Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts. J Mol Cell Cardiol. 2011; 51:215–225. [PubMed: 21586291]
Author Manuscript Author Manuscript J Am Coll Cardiol. Author manuscript; available in PMC 2017 March 07.
