Richard J. Gumina

Kir6.2 is required for adaptation to stress

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (KATP) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted KATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for KATP channels in the heart.

Inhibition of the Na+/H+ Exchanger Confers Greater Cardioprotection Against 90 Minutes of Myocardial Ischemia Than Ischemic Preconditioning in Dogs

BACKGROUND This study compared the efficacy of ischemic preconditioning (IPC) and sodium-hydrogen exchanger (NHE)-1 inhibition to reduce infarct size (IS) induced by a 90-minute ischemic insult and examined the interaction between NHE-1 inhibition and IPC. METHODS AND RESULTS In a canine infarct model, either IPC, produced by 1 or four 5-minute coronary artery occlusions, or the specific NHE-1 inhibitor BIIB 513, 0.75 or 3.0 mg/kg, was administered 15 minutes before either a 60- or 90-minute coronary artery occlusion followed by 3 hours of reperfusion. IS was determined by TTC staining and expressed as a percentage of the area at risk (IS/AAR). Although both IPC and BIIB 513 at 0.75 mg/kg produced comparable and significant reductions in IS/AAR in the 60-minute occlusion model, insignificant reductions in IS/AAR were observed in the 90-minute occlusion model. However, BIIB 513 at 3.0 mg/kg markedly reduced IS in both models (P<0.05). Next, to examine the interaction between NHE-1 blockade and IPC, BIIB 0.75 mg/kg was administered either before IPC or during the washout phase of IPC before 90 minutes of coronary artery occlusion. Both combinations resulted in a greater-than-additive reduction in IS/AAR (P<0.05). CONCLUSIONS These data demonstrate that although IPC and NHE-1 inhibition provide comparable protection against 60 minutes of myocardial ischemia, NHE-1 inhibition is more efficacious than IPC at protecting against a 90-minute ischemic insult. Furthermore, the combination of NHE-1 inhibition and IPC produces a greater-than-additive reduction in IS/AAR, suggesting either that NHE activity limits the efficacy of IPC or that different mechanisms are involved in the cardioprotective effect of IPC and NHE-1 inhibition.

Cellular remodeling in heart failure disrupts KATP channel‐dependent stress tolerance

ATP‐sensitive potassium (KATP) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on KATP channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor α induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, KATP channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, KATP channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by KATP channel openers. Thus, disease‐induced KATP channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.

Defects in ankyrin-based membrane protein targeting pathways underlie atrial fibrillation

Background— Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting >2 million patients in the United States alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells. Methods and Results— In the present study, we report early-onset AF in patients harboring loss-of-function mutations in ANK2. In mice, we show that ankyrin-B deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B+/− atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca2+ channels (Cav1.3) responsible for low voltage-activated L-type Ca2+ current. Ankyrin-B is associated directly with Cav1.3, and this interaction is regulated by a short, highly conserved motif specific to Cav1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Cav1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF. Conclusions— These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with AF. Additionally, our data demonstrate a novel pathway for ankyrin-B–dependent regulation of Cav1.3 channel membrane targeting and regulation in atrial myocytes.

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, βADP and βATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring β- and γ-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by α-glycerophosphate dehydrogenase (αGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of α-glycerophosphate and nucleoside 5′-monophosphates, through αGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.

Inhibition of the Na(+)/H(+) exchanger attenuates phase 1b ischemic arrhythmias and reperfusion-induced ventricular fibrillation.

The sodium-hydrogen exchanger-isotype 1 (NHE-1) plays a critical role in myocardial ischemia-reperfusion injury. While studies employing less selective sodium-hydrogen inhibitors have demonstrated antiarrhythmic activity, only one study has examined the in vivo efficacy of selective NHE-1 inhibition in a canine model of ischemia-reperfusion-induced arrhythmia. In the present study, the antiarrhythmic activity of Benzamide, N-(aminoiminomethyl)-4-¿4-(2-furanylcarbonyl)-1-piperazinyl -3-(methy lsulfonyl), methanesulfonate (BIIB 513), a novel NHE-1 inhibitor, was examined. An in vivo canine model of myocardial ischemia-reperfusion injury in which 60 min of left anterior descending coronary artery (LAD) occlusion followed by 3 h of reperfusion was employed. BIIB 513 was infused either prior to ischemia or prior to reperfusion. Arrhythmias were quantified by single lead electrocardiogram. Infarct size, determined by triphenyltetrazolium staining, was expressed as a percent of the area-at-risk. In vivo, NHE-1 inhibition did not affect phase 1a arrhythmias, which occur within the first 10 min of occlusion, however, BIIB 513 significantly reduced the incidence of ischemia-induced phase 1b arrhythmias which occur between 10 and 30 min following occlusion and the incidence of reperfusion-induced ventricular fibrillation. Furthermore, NHE-1 inhibition significantly reduced infarct size, when the drug was administered either prior to ischemia or prior to reperfusion. NHE-1 inhibition selectively reduces both ischemia-induced phase 1b arrhythmias and reperfusion-induced ventricular fibrillation, and also markedly reduces myocardial infarct size when the drug is administered prior to ischemia or prior to reperfusion.

Transgenic over expression of ectonucleotide triphosphate diphosphohydrolase-1 protects against murine myocardial ischemic injury.

Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore.

Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation

Myocardial infarction is a leading cause of mortality and morbidity worldwide. Occlusion of a coronary artery produces ischemia and myocardial necrosis that leads to left ventricular (LV) remodeling, dysfunction, and heart failure. Stem cell therapy may decrease infarct size and improve LV function; the hypoxic environment, however, following a myocardial infarction may result in apoptosis, which in turn decreases survival of transplanted stem cells. Therefore, the effects of preconditioned mesenchymal stem cells (MSC) with hyperoxia (100% oxygen), Z‐VAD‐FMK pan‐caspase inhibitor (CI), or both in a hypoxic environment in order to mimic conditions seen in cardiac tissue post‐myocardial infarction were studied in vitro. MSCs preconditioned with hyperoxia or CI significantly decreased apoptosis as suggested by TUNEL assay and Annexin V analysis using fluorescence assisted cell sorting. These effects were more profound when both, hyperoxia and CI, were used. Additionally, gene and protein expression of caspases 1, 3, 6, 7, and 9 were down‐regulated significantly in MSCs preconditioned with hyperoxia, CI, or both, while the survival markers Akt1, NF‐κB, and Bcl‐2 were significantly increased in preconditioned MSCs. These changes ultimately resulted in a significant increase in MSC proliferation in hypoxic environment as determined by BrdU assays compared to MSCs without preconditioning. These effects may prove to be of great clinical significance when transplanting stem cells into the hypoxic myocardium of post‐myocardial infarction patients in order to attenuate LV remodeling and improve LV function. J. Cell. Biochem. 114: 2612–2623, 2013.

Inhibitors of ischemic preconditioning do not attenuate Na+/H+ exchange inhibitor mediated cardioprotection.

Pharmacologic inhibition of the K(ATP) channel with sulfonylureas or the adenosine receptor with methylxanthines has been shown to attenuate ischemic preconditioning (IPC). Both classes of compounds are widely used clinically, and several reports have demonstrated adverse outcomes in patients taking sulfonylureas. Recently inhibition of the sodium/hydrogen exchanger isozyme-1 (NHE-1) has been shown to be equal to IPC at providing myocardial protection in dogs and may be an alternative to IPC in patients taking sulfonylureas or methylxanthines. However, no experiments have examined the pharmacologic overlap between IPC and NHE-1 inhibitor-mediated cardioprotection in dogs. With an in vivo canine infarct model in which the left anterior descending coronary artery was occluded for 60 min and reperfused for 3 h, neither the K(ATP) channel antagonist glibenclamide nor the adenosine-receptor antagonist PD 115199 attenuated NHE-1 inhibitor-mediated reduction in infarct size expressed as a percentage of the area at risk produced by EMD 85131 (Control, 24.2 +/- 3.6%; EMD 85131, 6.4 +/- 2.3%; PD 115199 + EMD 85131, 6.6 +/- 2.4%; glibenclamide + EMD 85131, 3.5 +/- 1.2%). NHE-1 inhibition and IPC do not overlap pharmacologically, and NHE-1 inhibition may be an alternative for cardioprotection in patients taking sulfonylureas or methylxanthines.

If ischemic preconditioning is the gold standard, has a platinum standard of cardioprotection arrived? Comparison with NHE inhibition.

Since ischemic preconditioning (IPC) was ~rst described [1], the reproducibility of this biological phenomenon has been demonstrated across species [2]. Numerous studies have examined the factors that contribute to the cardioprotective effect of IPC, such as the KATP channel [3], adenosine [4], protein kinase C [5], and tyrosine kinases [6,7]. IPC has become the experimental “gold standard” for reduction of myocardial damage and infarct size. However, over the last 10 years a new class of agents that inhibit the sodium-hydrogen exchanger (NHE) has demonstrated marked cardioprotective ef~cacy in a variety of in vitro and in vivo models [8]. Recently, more selective agents that inhibit the NHE isotype 1, the predominant form in the myocardium [9], have been shown to be cardioprotective. Unfortunately, to date the comparison of ischemic preconditioning and NHE inhibition has been limited to a small number of reports (Table 1). Thus, in this article we review the data comparing IPC and NHE inhibition, and speci~cally address three key questions: Do ischemic preconditioning and NHE inhibition work via similar mechanisms? Do ischemic preconditioning and NHE inhibition antagonize one another? Which method of cardioprotection is most ef~cacious, ischemic preconditioning or NHE inhibition?