Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.

Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18 ± 0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 µN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 µM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.

Pathophysiological consequences of TAT-HKII peptide administration are independent of impaired vascular function and ensuing ischemia.

RATIONALE: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 µmol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. OBJECTIVE: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. METHODS AND RESULTS: Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. CONCLUSIONS: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure.

Cardiac gene therapy has emerged as a promising option to treat advanced heart failure (HF). Advances in molecular biology and gene targeting approaches are offering further novel options for genetic manipulation of the cardiovascular system. The aim of this study was to improve cardiac function in chronic HF by overexpressing constitutively active inhibitor-1 (I-1c) using a novel cardiotropic vector generated by capsid reengineering of adeno-associated virus (BNP116). One month after a large anterior myocardial infarction, 20 Yorkshire pigs randomly received intracoronary injection of either high-dose BNP116.I-1c (1.0 × 10(13) vector genomes (vg), n = 7), low-dose BNP116.I-1c (3.0 × 10(12) vg, n = 7), or saline (n = 6). Compared to baseline, mean left ventricular ejection fraction increased by 5.7% in the high-dose group, and by 5.2% in the low-dose group, whereas it decreased by 7% in the saline group. Additionally, preload-recruitable stroke work obtained from pressure-volume analysis demonstrated significantly higher cardiac performance in the high-dose group. Likewise, other hemodynamic parameters, including stroke volume and contractility index indicated improved cardiac function after the I-1c gene transfer. Furthermore, BNP116 showed a favorable gene expression pattern for targeting the heart. In summary, I-1c overexpression using BNP116 improves cardiac function in a clinically relevant model of ischemic HF.

Glutathione oxidation unmasks proarrhythmic vulnerability of chronically hyperglycemic guinea pigs.

Chronic hyperglycemia in type-1 diabetes mellitus is associated with oxidative stress (OS) and sudden death. Mechanistic links remain unclear. We investigated changes in electrophysiological (EP) properties in a model of chronic hyperglycemia before and after challenge with OS by GSH oxidation and tested reversibility of EP remodeling by insulin. Guinea pigs survived for 1 mo following streptozotocin (STZ) or saline (sham) injection. A treatment group received daily insulin for 2 wk to reverse STZ-induced hyperglycemia (STZ + Ins). EP properties were measured using high-resolution optical action potential mapping before and after challenge of hearts with diamide. Despite elevation of glucose levels in STZ compared with sham-operated (P = 0.004) and STZ + Ins (P = 0.002) animals, average action potential duration (APD) and arrhythmia propensity were not altered at baseline. Diamide promoted early (<10 min) formation of arrhythmic triggers reflected by a higher arrhythmia scoring index in STZ (P = 0.045) and STZ + Ins (P = 0.033) hearts compared with sham-operated hearts. APD heterogeneity underwent a more pronounced increase in response to diamide in STZ and STZ + Ins hearts compared with sham-operated hearts. Within 30 min, diamide resulted in spontaneous incidence of ventricular tachycardia and ventricular fibrillation (VT/VF) in 3/6, 2/5, 1/5, and 0/4 STZ, STZ + Ins, sham-operated, and normal hearts, respectively. Hearts prone to VT/VF exhibited greater APD heterogeneity (P = 0.010) compared with their VT/VF-free counterparts. Finally, altered EP properties in STZ were not rescued by insulin. In conclusion, GSH oxidation enhances APD heterogeneity and increases arrhythmia scoring index in a guinea pig model of chronic hyperglycemia. Despite normalization of glycemic levels by insulin, these proarrhythmic properties are not reversed, suggesting the importance of targeting antioxidant defenses for arrhythmia suppression.

Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis.

RATIONALE: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. OBJECTIVE: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. METHODS AND RESULTS: Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 µmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. CONCLUSIONS: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Biophysical properties and functional consequences of reactive oxygen species (ROS)-induced ROS release in intact myocardium.

Reactive oxygen species (ROS)-induced ROS release (RIRR) is a fundamental mechanism by which cardiac mitochondria respond to elevated ROS levels by stimulating endogenous ROS production in a regenerative, autocatalytic process that ultimately results in global oxidative stress (OS), cellular dysfunction and death. Despite elegant studies describing the phenomenon of RIRR under artificial conditions such as photo-induced oxidation of discrete regions within cardiomyocytes, the existence, biophysical properties and functional consequences of RIRR in intact myocardium remain unclear. Here, we used a semi-quantitative approach of optical superoxide (O(2)(-)) mapping using dihydroethidium (DHE) fluorescence to explore RIRR, its arrhythmic consequences and underlying mechanisms in intact myocardium. Initially, perfusion of rat hearts with 200 µM H(2)O(2) for 40 min (n = 4) elicited two distinct O(2)(-) peaks that were readily distinguished by their timing and amplitude. The first peak (P1), which was generated rapidly (within 5-8 min of H(2)O(2) perfusion) was associated with a relatively limited (10 ± 2%) rise in normalized O(2)(-) levels relative to baseline. In contrast, the second peak (P2) occurred 19-26 min following onset of H(2)O(2) perfusion and was associated with a significantly greater amplitude compared to P1. Spatio-temporal ROS mapping during P2 revealed active O(2)(-) propagation across the myocardium at a velocity of ~20 µm s(-1). Exposure of hearts (n = 18) to a short (10 min) episode of H(2)O(2) perfusion revealed consistent generation of P2 by high (≥200 µM, 8/8) but not lower (≤100 µM, 3/8) H(2)O(2) concentrations (P < 0.03). In these hearts, onset of P2 occurred following, not during, the 10 min OS protocol, consistent with RIRR. Importantly, P2 (+) hearts exhibited a markedly greater (by 3.8-fold, P < 0.001) arrhythmia score compared to P2 (-) hearts. To explore the mechanism underlying RIRR in intact myocardium, hearts were perfused with either cyclosporin A (CsA) or 4-chlorodiazepam (4-Cl-DZP) to inhibit the mitochondrial permeability transition pore (mPTP) or the inner membrane anion channel (IMAC), respectively. Surprisingly, perfusion with CsA failed to suppress (P = 0.75, n.s.) or even delay H(2)O(2)-induced P2 or the incidence of arrhythmias compared to untreated hearts. In sharp contrast, perfusion with 4-Cl-DZP markedly blunted O(2)(-) levels during P2, and suppressed the incidence of sustained ventricular tachycardia or ventricular fibrillation (VT/VF). Finally, perfusion of hearts with the synthetic superoxide dismutase/catalase mimetic EUK-134 completely abolished the H(2)O(2)-mediated RIRR response as well as the incidence of arrhythmias. These findings extend the concept of RIRR to the level of the intact heart, establish regenerative O(2)(-) production as the mediator of RIRR-related arrhythmias and reveal their strong dependence on IMAC and not the mPTP in this acute model of OS.

Effects of a high-salt diet on TRPV-1-dependent renal nerve activity in Dahl salt-sensitive rats.

OBJECTIVE: To test the hypothesis that transient receptor potential vanilloid type 1 channel (TRPV1)-mediated increases in afferent renal nerve activity (ARNA) and release of substance P (SP) and calcitonin gene-related peptide (CGRP) from the renal pelvis are suppressed in Dahl salt-sensitive (DS), but not -resistant (DR), rats fed a high-salt (HS) diet. METHODS AND RESULTS: Male DS and DR rats were given a HS or low-salt (LS) diet for 3 weeks. Perfusion of capsaicin (CAP, 10(-6)M), a selective TRPV1 agonist, into the left renal pelvis increased ipsilateral ARNA in all groups, but with a smaller magnitude in DS-HS compared to other groups. CAP increased contralateral urine flow in all groups except DS-HS rats. CAP-induced release of SP and CGRP from the renal pelvis was less in DS-HS compared to other groups. Western blot showed that TRPV1 expression in the kidney decreased while expression of neurokinin 1 receptors increased in DS-HS compared to other groups. CONCLUSION: TRPV1-mediated increases in ARNA and release of SP and CGRP in the renal pelvis are impaired in DS rats fed a HS diet, which can likely be attributed to suppressed TRPV1 expression in the kidney and contributes to increased salt sensitivity.

Interdependent regulation of afferent renal nerve activity and renal function: role of transient receptor potential vanilloid type 1, neurokinin 1, and calcitonin gene-related peptide receptors.

Our previous studies have shown that the activation of the transient receptor potential vanilloid type 1 (TRPV1) expressed in the renal pelvis leads to an increase in ipsilateral afferent renal nerve activity (ARNA) and contralateral renal excretory function, but the molecular mechanisms of TRPV1 action are largely unknown. This study tests the hypothesis that activation of receptors of neurokinin 1 (NK1) or calcitonin gene-related peptide (CGRP) by endogenously released substance P (SP) or CGRP following TRPV1 activation, respectively, governs TRPV1-induced increases in ARNA and renal excretory function. Capsaicin (CAP; 0.04, 0.4, and 4 nM), a selective TRPV1 agonist, administered into the renal pelvis dose-dependently increased ARNA. CAP (4 nM)-induced increases in ipsilateral ARNA or contralateral urine flow rate (Uflow) and urinary sodium excretion (UNa) were abolished by capsazepine (CAPZ), a selective TRPV1 antagonist, or 2-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4-perhydroisoindolone (3aR,7aR) (RP67580) or cis-2-(diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1 azabicyclo[2.2.2]octan-3-amine (L703,606), selective NK1 antagonists, but not by CGRP8-37, a selective CGRP receptor antagonist. Both SP (7.4 nM) and CGRP (0.13 muM) increased ARNA, Uflow, or UNa, and increases in these parameters induced by CGRP but not SP were abolished by CAPZ. CAP at 4 nM perfused into the renal pelvis caused the release of SP and CGRP, which was blocked by CAPZ but not by RP67580, L703,606, or CGRP8-37. Immunofluorescence results showed that NK1 receptors were expressed in sensory neurons in dorsal root ganglion and sensory nerve fibers innervating the renal pelvis. Taken together, our data indicate that NK1 activation induced by SP release upon TRPV1 activation governs TRPV1 function and that a TRPV1-dependent mechanism is operant in CGRP action.

Primary Effect of SERCA 2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction.

Background SERCA 2a gene transfer ( GT ) improves mechano-electrical function in animal models of nonischemic heart failure Whether SERCA 2a GT reverses pre-established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno-associated virus serotype 1. SERCA 2a GT following myocardial infarction ( MI ). Methods and Results Pigs developed mechanical dysfunction 1 month after anterior MI , at which point they received intracoronary adeno-associated virus serotype 1. SERCA 2a ( MI + SERCA 2a) or saline ( MI ) and were maintained for 2 months. Age-matched naive pigs served as controls (Control). In vivo ECG -and-hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential ( AP ) mapping in controls, MI , and MI + SERCA 2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R' waves were present in MI but not MI + SERCA 2a pigs relative to controls. SERCA 2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI + SERCA 2a or control pigs were prone to pacing-induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI + SERCA 2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI + SERCA 2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA 2a GT decreased phosphoactive CAMKII -delta levels by >50%, leading to improved excitability at fast rates. Conclusions SERCA 2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA 2a GT on myocardial excitability, independent of altered mechanical function.

TRPV1-mediated diuresis and natriuresis induced by hypertonic saline perfusion of the renal pelvis.

BACKGROUND: The transient receptor potential vanilloid type 1 (TRPV1) channel is known to be activated by multiple stimuli, albeit its role in mediating renal function is largely unknown. This study was designed to test the hypothesis that TRPV1 mediates diuresis and natriuresis induced by hypertonic saline perfusion into the pelvis. METHODS: NaCl or KCl was perfused into the left renal pelvis of rats at a rate without changing renal pelvic pressure. Afferent renal nerve activity (ARNA), urine flow rate (V) and urinary sodium excretion (UNaV) in the presence or absence of selective antagonists of TRPV1, capsazepine (CAPZ), or neurokinin-1 (NK1) receptors, RP67580, were examined. RESULTS: Unilateral renal pelvis perfusion of NaCl at 600 mM, but not 150 or 300 mM, increased ipsilateral ARNA and contralateral V and UNaV, which were blocked by ipsilateral administration of CAPZ or RP67580. In contrast, KCl perfused at 150 or 300 mM, but not 600 mM, increased ipsilateral ARNA and contralateral V and UNaV, which were insensitive to CAPZ. CONCLUSION: Unilateral hypertonic saline perfusion causes contralateral diuresis and natriuresis via TRPV1 or NK1 activation, indicating that these receptors may play a critical role in sensing microenvironmental changes in the renal pelvis to modulate renal function in health and disease.

Increased afterload following myocardial infarction promotes conduction-dependent arrhythmias that are unmasked by hypokalemia.

Although the pathophysiological significance of resistant hypertension in post-myocardial infarction (MI) patients is established, mechanisms by which increased afterload in that setting worsens outcome are unclear. With regards to sudden cardiac death, whether increased afterload alters the electrophysiological substrate following MI is unknown. We established a new large animal model of chronic post-MI remodeling with increased afterload which exhibits widespread deposition of fibrosis in remote areas from the anterior MI, mimicking the disease phenotype of patients with advanced ischemic heart disease. We identified the mode-of-initiation and mechanism of arrhythmias which were consistently unmasked by hypokalemia in this clinically-relevant model.

Protein Phosphatase Inhibitor-1 Gene Therapy in a Swine Model of Nonischemic Heart Failure.

BACKGROUND: Increased protein phosphatase-1 in heart failure (HF) induces molecular changes deleterious to the cardiac cell. Inhibiting protein phosphatase-1 through the overexpression of a constitutively active inhibitor-1 (I-1c) has been shown to reverse cardiac dysfunction in a model of ischemic HF. OBJECTIVES: This study sought to determine the therapeutic efficacy of a re-engineered adenoassociated viral vector carrying I-1c (BNP116.I-1c) in a preclinical model of nonischemic HF, and to assess thoroughly the safety of BNP116.I-1c gene therapy. METHODS: Volume-overload HF was created in Yorkshire swine by inducing severe mitral regurgitation. One month after mitral regurgitation induction, pigs were randomized to intracoronary delivery of either BNP116.I-1c (n = 6) or saline (n = 7). Therapeutic efficacy and safety were evaluated 2 months after gene delivery. Additionally, 24 naive pigs received different doses of BNP116.I-1c for safety evaluation. RESULTS: At 1 month after mitral regurgitation induction, pigs developed HF as evidenced by increased left ventricular end-diastolic pressure and left ventricular volume indexes. Treatment with BNP116.I-1c resulted in improved left ventricular ejection fraction (-5.9 ± 4.2% vs. 5.5 ± 4.0%; p < 0.001) and adjusted dP/dt maximum (-3.39 ± 2.44 s-1 vs. 1.30 ± 2.39 s-1; p = 0.007). Moreover, BNP116.I-1c-treated pigs also exhibited a significant increase in left atrial ejection fraction at 2 months after gene delivery (-4.3 ± 3.1% vs. 7.5 ± 3.1%; p = 0.02). In vitro I-1c gene transfer in isolated left atrial myocytes from both pigs and rats increased calcium transient amplitude, consistent with its positive impact on left atrial contraction. We found no evidence of adverse electrical remodeling, arrhythmogenicity, activation of a cellular immune response, or off-target organ damage by BNP116.I-1c gene therapy in pigs. CONCLUSIONS: Intracoronary delivery of BNP116.I-1c was safe and improved contractility of the left ventricle and atrium in a large animal model of nonischemic HF.

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart.

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆GATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs.

The Classically Cardioprotective Agent Diazoxide Elicits Arrhythmias in Type 2 Diabetes Mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with an enhanced propensity for ventricular tachyarrhythmias (VTs) under conditions of metabolic demand. Activation of mitochondrial adenosine triphosphate-sensitive potassium (KATP) channels by low-dose diazoxide (DZX) improves hypoglycemia-related complications, metabolic function, and triglyceride and free fatty acid levels and reverses weight gain in T2DM. OBJECTIVES: In this study, we hypothesized that DZX prevents ischemia-mediated arrhythmias in T2DM via its putative cardioprotective and antidiabetic property. METHODS: Zucker obese diabetic fatty (ZO) rats (n = 43) with T2DM were studied. Controls consisted of Zucker lean (ZL; n = 13) and normal Sprague-Dawley (SprD; n = 30) rats. High-resolution optical action potential mapping was performed before and during challenge with no-flow ischemia for 12 min. RESULTS: Electrophysiological properties were relatively stable in T2DM hearts at baseline. In contrast, ischemia uncovered major differences between groups, because action potential duration (APD) in T2DM failed to undergo progressive adaptation to ischemic challenge. DZX promoted the incidence of arrhythmias, because all DZX-treated T2DM hearts exhibited ischemia-induced VTs that persisted on reperfusion. In contrast, untreated T2DM and controls did not exhibit VT during ischemia. Unlike DZX, pinacidil promoted ischemia-mediated arrhythmias in both control and T2DM hearts. Rapid and spatially heterogeneous shortening of APD preceded the onset of arrhythmias in T2DM. DZX-mediated proarrhythmia in T2DM was not related to changes in the messenger ribonucleic acid expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, SUR2B, or ROMK (renal outer medullary potassium channel). CONCLUSIONS: Ischemia uncovers a paradoxical resistance of T2DM hearts to APD adaptation. DZX reverses this property, resulting in rapid and heterogeneous APD shortening. This promotes reentrant VT during ischemia. DZX should be avoided in diabetic patients at risk of ischemic events.

LKB1 deletion causes early changes in atrial channel expression and electrophysiology prior to atrial fibrillation.

AIMS: Liver kinase B1 (LKB1) is a protein kinase that activates the metabolic regulator AMP-activated protein kinase (AMPK) and other related kinases. Deletion of LKB1 in mice leads to cardiomyopathy and atrial fibrillation (AF). However, the specific role of the LKB1 pathway in early atrial biology remains unknown. Thus, we investigated whether LKB1 deletion altered atrial channel expression and electrophysiological function in a cardiomyocyte-specific knockout mouse model. METHODS AND RESULTS: We performed a systematic comparison of αMHC-Cre LKB1(fl/fl) and littermate LKB1(fl/fl) male mice. This included analysis of gene expression, histology, and echocardiography, as well as cellular and tissue-level electrophysiology using patch-clamp recordings in vitro, optical mapping ex vivo, and ECG recordings in vivo. At postnatal day 1, atrial depolarization was prolonged, and Nav1.5 and Cx40 expression were markedly down-regulated in MHC-Cre LKB1(fl/fl) mice. Inward sodium current density was significantly decreased in MHC-Cre LKB1(fl/fl) neonatal atrial myocytes. Subsequently, additional alterations in atrial channel expression, atrial fibrosis, and spontaneous onset of AF developed by 2 weeks of age. In adult mice, abnormalities of interatrial conduction and bi-atrial electrical coupling were observed, likely promoting the perpetuation of AF. Mice with AMPK-inactivated hearts demonstrated modest overlap in channel expression with MHC-Cre LKB1(fl/fl) hearts, but retained normal structure, electrophysiological function and contractility. CONCLUSIONS: Deletion of LKB1 causes early defects in atrial channel expression, action potential generation and conduction, which precede widespread atrial remodelling, fibrosis and AF. LKB1 is critical for normal atrial growth and electrophysiological function.

Functional crosstalk between the mitochondrial PTP and KATP channels determine arrhythmic vulnerability to oxidative stress.

BACKGROUND: Mitochondrial permeability transition pore (mPTP) opening is a terminal event leading to mitochondrial dysfunction and cell death under conditions of oxidative stress (OS). However, mPTP blockade with cyclosporine A (CsA) has shown variable efficacy in limiting post-ischemic dysfunction and arrhythmias. We hypothesized that strong feedback between energy dissipating (mPTP) and cardioprotective (mKATP) channels determine vulnerability to OS. METHODS AND RESULTS: Guinea pig hearts (N = 61) were challenged with H2O2 (200 µM) to elicit mitochondrial membrane potential (ΔΨm) depolarization. High-resolution optical mapping was used to measure ΔΨm or action potentials (AP) across the intact heart. Hearts were treated with CsA (0.1 µM) under conditions that altered the activity of mKATP channels either directly or indirectly via its regulation by protein kinase C. mPTP blockade with CsA markedly blunted (P < 0.01) OS-induced ΔΨm depolarization and delayed loss of LV pressure (LVP), but did not affect arrhythmia propensity. Surprisingly, prevention of mKATP activation with the chemical phosphatase BDM reversed the protective effect of CsA, paradoxically exacerbating OS-induced ΔΨm depolarization and accelerating arrhythmia onset in CsA treated compared to untreated hearts (P < 0.05). To elucidate the putative molecular mechanisms, mPTP inhibition by CsA was tested during conditions of selective PKC inhibition or direct mKATP channel activation or blockade. Similar to BDM, the specific PKC inhibitor, CHE (10 µM) did not alter OS-induced ΔΨm depolarization directly. However, it completely abrogated CsA-mediated protection against OS. Direct pharmacological blockade of mKATP, a mitochondrial target of PKC signaling, equally abolished the protective effect of CsA on ΔΨm depolarization, whereas channel activation with 30 µM Diazoxide protected against ΔΨm depolarization (P < 0.0001). Conditions that prevented mKATP activation either directly or indirectly via PKC inhibition led to accelerated ΔΨm depolarization and early onset of VF in response to OS. Investigation of the electrophysiological substrate revealed accelerated APD shortening in response to OS in arrhythmia-prone hearts. CONCLUSIONS: Cardioprotection by CsA requires mKATP channel activation through a PKC-dependent pathway. Increasing mKATP activity during CsA administration is required for limiting OS-induced electrical dysfunction.

GSH or palmitate preserves mitochondrial energetic/redox balance, preventing mechanical dysfunction in metabolically challenged myocytes/hearts from type 2 diabetic mice.

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the ß-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and ß-stimulated function in db/db myocyte/heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient's inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction.

Inhibition of renin release by arachidonic acid metabolites, 12(s)-HPETE and 12-HETE: role of TRPV1 channels.

We test the hypothesis that 12-hydroperoxyeicosatetraenoic acid (12(s)-HPETE) and 12-hydroxyeicosatetraenoic acid (12-HETE) perfused into the renal pelvis increase afferent renal nerve activity (ARNA) and suppress renin release in rats fed a low-salt (LS) diet via activation of the transient receptor potential vanilloid type 1 (TRPV1) expressed in renal sensory nerves. 12(s)-HPETE or 12-HETE given into the left renal pelvis dose-dependently increased ARNA, which was abolished by AMG9810, a selective TRPV1 antagonist, or by RP67580, a selective neurokinin 1 receptor antagonist, in normal salt or LS-treated rats. 12(s)-HPETE, 12-HETE, or substance P perfused into the left renal pelvis suppressed plasma angiotensin I (Ang I) levels in LS rats, which was abolished by AMG9810 or attenuated by ipsilateral renal denervation (RD). 12(s)-HPETE or 12-HETE increased release of substance P and calcitonin gene-related peptide from the ipsilateral kidney, which was abolished by AMG9810 but not RP67580, RD, or RP67580 plus RD. Immunofluorescence staining showed that TRPV1-positive nerve fibers located in the renal cortex, medulla, and pelvis, and that the sympathetic nerve marker, neuropeptide Y, but not neurokinin 1 receptors expressed in the juxtaglomerular region colocalized with renin. Thus, our data show that 12(s)-HPETE and 12-HETE enhance ARNA and substance P/calcitonin gene-related peptide release but suppress renin activity in LS rats, and these effects are abolished when TRPV1 is blocked. These results indicate that TRPV1 mediates 12(s)-HPETE and 12-HETE action in the kidney in such a way that dysfunction in TRPV1 may lead to disintegrated regulation of renin and renal function.

Ablation of transient receptor potential vanilloid 1 abolishes endothelin-induced increases in afferent renal nerve activity: mechanisms and functional significance.

Endothelin 1 (ET-1) and its receptors, ETA and ETB, play important roles in regulating renal function and blood pressure, and these components are expressed in sensory nerves. Activation of transient receptor potential vanilloid (TRPV) 1 channels expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity (ARNA), diuresis, and natriuresis. We tested the hypothesis that ET-1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in wild-type (WT) but not TRPV1-null mutant mice. ET-1 alone or with BQ123, an ETA antagonist, perfused into the left renal pelvis increased ipsilateral ARNA in WT but not in TRPV1-null mutant mice, and ARNA increases were greater in the latter. [Ala1, 3,11,15]-endothelin 1, an ETB agonist, increased ARNA that was greater than that induced by ET-1 in WT mice only. [Ala1, 3,11,15]-endothelin 1-induced increases in ARNA were abolished by chelerythrine, a protein kinase C inhibitor, but not by H89, a protein kinase A inhibitor. Chelerythrine, H89, and BQ788, an ETB antagonist, did not affect ARNA triggered by capsaicin in WT mice. Substance P release from the renal pelvis was increased by [Ala1, 3,11,15]-endothelin 1 in WT mice only, and the increase was abolished by chelerythrine but not by H89. Chelerythrine, H89, and BQ788 did not affect capsaicin-induced substance P release. Our data show that ET1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in WT but not in TRPV1-null mutant mice, suggesting that TRPV1 mediates ETB-dependent increases in ARNA, diuresis, and natriuresis possibly via the protein kinase C pathway.