Therapeutic potential of modulating potassium currents in the diseased myocardium.

Myocardial disease states are characterized by multiple electrophysiologic abnormalities, including alterations in potassium channel activities. During acute myocardial ischemia, activation of ATP-regulated K+ current (IK(ATP)) results in shortening of action potential duration and elevation of extracellular K+ concentration. In hypertrophied myocardium, increases in inward rectifier K+ current (IK1) and decreases in delayed rectifier K+ current (IK) are observed. Alterations in K+ channel activity in myocardial disease states suggest the potential to therapeutically modify cardiac rhythm and function with K+ channel modulators. Class III anti-arrhythmic agents, which prolong myocardial refractoriness predominantly via a blockade of IK, have demonstrated efficacy in suppressing reentrant atrial and ventricular arrhythmias in animal models as well as promising efficacy in initial clinical studies. Potassium channel openers (PCOs), which activate cardiac IK(ATP), have demonstrated both antiarrhythmic and proarrhythmic activities in various experimental settings, and also are being investigated as potential cardioprotective agents. Sulfonylureas, which block cardiac IK(ATP), also have been investigated as potential antiarrhythmic agents with equivocal results, and have displayed a propensity to exacerbate ischemic myocardial dysfunction in experimental studies. A more comprehensive understanding of K+ channel activity in various myocardial disease states, including concomitant disorders such as myocardial ischemia and hypertrophy, will facilitate the development of more useful potassium channel modulators, as well as a clearer recognition of the undesirable effects of such agents.     

S100B: a multifunctional role in cardiovascular pathophysiology

S100B, a calcium-binding protein of the EF-hand type exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures, and high level expression of S100B in transgenic mice hearts and aortic smooth muscle cells inhibit cardiac hypertrophy and the associated phenotype, arterial smooth muscle proliferation, respectively, but demonstrate increased apoptosis following α₁-adrenergic stimulation or myocardial infarction. Knocking out S100B, augmented hypertrophy, decreased apoptosis and preserved cardiac function following myocardial infarction. S100B induces apoptosis by an extracellular mechanism by interacting with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular, and extracellular, roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular disease.     

Ca2+/calmodulin-dependent protein kinase II inhibition reduces myocardial fatty acid uptake and oxidation after myocardial infarction

An AMP-activated kinase (AMPK) signaling pathway is activated during myocardial ischemia and promotes cardiac fatty acid (FA) uptake and oxidation. Similarly, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also triggered by myocardial ischemia, but its function in FA metabolism remains unclear. Here, we explored the role of CaMKII in FA metabolism during myocardial ischemia by investigating the effects of cardiac CaMKII on AMPK-acetyl-CoA carboxylase (ACC), malonyl CoA decarboxylase (MCD), and FA translocase cluster of differentiation 36 (FAT/CD36), as well as cardiac FA uptake and oxidation. Moreover, we tested whether CaMKII and AMPK are binding partners. We demonstrated that diseased hearts from patients with terminal ischemic heart disease displayed increased phosphorylation of CaMKII, AMPK, and ACC and increased expression of MCD and FAT/CD36. AC3-I mice, which have a genetic myocardial inhibition of CaMKII, had reduced gene expression of cardiac AMPK. In post-MI (myocardial infarction) AC3-I hearts, AMPK-ACC phosphorylation, MCD and FAT/CD36 levels, cardiac FA uptake, and FA oxidation were significantly decreased. Notably, we demonstrated that CaMKII interacted with AMPK α1 and α2 subunits in the heart. Additionally, AC3-I mice displayed significantly less cardiac hypertrophy and apoptosis 2 weeks post-MI. Overall, these findings reveal a unique role for CaMKII inhibition in repressing FA metabolism by interacting with AMPK signaling pathways, which may represent a novel mechanism in ischemic heart disease.     

Molecular Mechanisms of Inherited Arrhythmias

Inherited arrhythmias and conduction system diseases are known causes of sudden cardiac death and are responsible for significant mortality and morbidity in patients with congenital heart disease and electrical disorders. Knowledge derived from human genetics and studies in animal models have led to the discovery of multiple molecular defects responsible for arrhythmogenesis. This review summarizes the molecular basis of inherited arrhythmias in structurally normal and altered hearts.On the cellular and molecular levels, minor disturbances can provoke severe arrhythmias. Ion channels are responsible for the initiation and propagation of the action potential within the cardiomyocyte. Structural heart diseases, such as hypertrophic or dilated cardiomyopathies, increase the likelihood of cardiac electrical abnormalities. Ion channels can also be up- or down-regulated in congenital heart disease, altering action potential cellular properties and therefore triggering arrhythmias. Conduction velocities may be inhomogeneously altered if connexin function, density or distribution changes. Another important group of electrophysiologic diseases is the heterogeneous category of inherited arrhythmias in the structurally normal heart, with a propensity to sudden cardiac death. There have been many recent relevant discoveries that help explain the molecular and functional mechanisms of long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and other electrical myopathies. Identification of molecular pathways allows the identification of new therapeutic targets, for both disease palliation and cure. As more disease-causing mutations are identified and genotypic-phenotypic correlation is defined, families can be screened prior to symptom-onset and patients may potentially be treated in a genotype-specific manner, opening the doors of cardiac electrophysiology to the emerging field of pharmacogenomics.     

Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal K(ATP) channels

The functional significance of ATP-sensitive K(+) (K(ATP)) channels is controversial. In the present study, transgenic mice expressing a mutant Kir6.2, with reduced ATP sensitivity, were used to examine the role of sarcolemmal K(ATP) in normal cardiac function and after an ischemic or metabolic challenge. We found left ventricular developed pressure (LVDP) was 15-20% higher in hearts from transgenics in the absence of cardiac hypertrophy. beta-Adrenergic stimulation caused a positive inotropic response from nontransgenic hearts that was not observed in transgenic hearts. Decreasing extracellular Ca(2+) decreased LVDP in hearts from nontransgenics but not in those from transgenics. These data suggest an increase in intracellular [Ca(2+)] in transgenic hearts. Additional studies have demonstrated hearts from nontransgenics and transgenics have a similar postischemic LVDP. However, ischemic preconditioning does not improve postischemic recovery in transgenics. Transgenic hearts also demonstrate a poor recovery after metabolic inhibition. These data are consistent with the hypothesis that sarcolemmal K(ATP) channels are required for development of normal myocardial function, and perturbations of K(ATP) channels lead to hearts that respond poorly to ischemic or metabolic challenges.     

Phospholipid lysophosphatidylcholine as a metabolic trigger and HERG as an ionic pathway for extracellular K accumulation and "short QT syndrome" in acute myocardial ischemia.

The most profound abnormalities during acute myocardial ischemia are extracellular K(+) accumulation ([K(+)](o)- upward arrow) and shortening of action potential duration or QT interval (APD- downward arrow or QT- downward arrow), which are pivotal in the genesis of ischemic arrhythmias and sudden cardiac death. The ionic mechanisms however remained obscured. We performed studies in a rabbit model of acute global myocardial ischemia in order to explore ionic and metabolic mechanisms for ischemic [K(+)](o)- upward arrow and QT- downward arrow. Exogenous 1-palmitoyl-lysophosphatidylcholine (LPC-16) mimicked the low-perfusion ischemia to produce significant [K(+)](o)- upward arrow and QT- downward arrow. The [K(+)](o)- upward arrow and QT- downward arrow induced by either LPC-16 or ischemia were prevented by dofetilide, a blocker of rapid delayed rectifier K(+) current (I(Kr)), but not by blockers for other K(+) channels. Consistently, dofetilide efficiently abolished the ventricular tachy-arrhythmias induced by ischemia or LPC-16. LPC-16 remarkably shortened APD and enhanced the function of I(Kr) and HERG (the pore-forming subunit of I(Kr)). The effects of LPC-16 manifested with shorter APD (faster repolarization rate) and at more negative potential (membrane repolarization). Dofetilide abolished the I(Kr)/HERG enhancing and APD shortening effects of LPC-16. Our results suggest that LPC-16 accumulation/HERG enhancement may be a link between metabolic trigger and ionic pathway for ischemic [K(+)](o)- upward arrow and QTc- downward arrow. This represents the first documentation of I(Kr)/HERG as the ionic mechanism in ischemic [K(+)](o)- upward arrow and QTc- downward arrow. Inhibition of LPC-16 production and accumulation and/or of I(Kr)/HERG may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease.     

心脏疾病中G蛋白的变化

G蛋白是一类重要的信号转导分子,其生理功能是将细胞膜受体所识别的各种细胞外信号同细胞内一系列效应分子偶联起来,引起核基因转录及蛋白质结构和功能的变化。G蛋白在心脏表达的亚型有Gs、Gi/o、Gq／11、G12／13,参与心肌收缩力、心率、心律和心肌细胞生长的调节。本文着重讨论了心脏G蛋白的分类、结构和功能,以及在心肌肥大、心力衰竭、急性心肌缺血和心律失常等心脏疾病中的改变,以加深对这些疾病的发病机制和病理生理过程的认识。     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats.

The subcellular mechanisms underlying intrinsic myocardial depression during sepsis remain poorly defined, in particular the relative roles of altered intracellular Ca2+ transients versus changes in myofilament properties. We studied contractile function of cardiac myocytes isolated 12 h after induction of endotoxemia (5 mg/kg intravenous E. coli lipopolysaccharide [LPS]) in conscious rats. Cardiomyocytes from LPS-injected rats had depressed twitch shortening compared with control cells (4.10.2% versus 7.80.3%; P2+ transients (peak indo-1 ratio 1.130.02 versus 1.120.02; P = NS). Contractile depression was unaffected by inhibitors of nitric oxide synthase. Steady-state myofilament response to Ca2+, assessed by tetanization of intact cells over a range of [Ca2+], was reduced significantly in the LPS group (P2+ was unaffected by isoproterenol (3 nmol/L) in endotoxemic cells, whereas there was a rightward shift in control cells. A reduction in myofilament response to Ca2+ is the major determinant of intrinsic cardiac depression in systemic endotoxemia. This condition appears to be related to an increase in myocardial troponin I phosphorylation.     

Defective calcium inactivation causes long QT in obese insulin-resistant rat

The majority of diabetic patients who are overweight or obese die of heart disease. We suspect that the obesity-induced insulin resistance may lead to abnormal cardiac electrophysiology. We tested this hypothesis by studying an obese insulin-resistant rat model, the obese Zucker rat (OZR). Compared with the age-matched control, lean Zucker rat (LZR), OZR of 16-17 wk old exhibited an increase in QTc interval, action potential duration, and cell capacitance. Furthermore, the L-type calcium current (I(CaL)) in OZR exhibited defective inactivation and lost the complete inactivation back to the closed state, leading to increased Ca(2+) influx. The current density of I(CaL) was reduced in OZR, whereas the threshold activation and the current-voltage relationship of I(CaL) were not significantly altered. L-type Ba(2+) current (I(BaL)) in OZR also exhibited defective inactivation, and steady-state inactivation was not significantly altered. However, the current-voltage relationship and activation threshold of I(BaL) in OZR exhibited a depolarized shift compared with LZR. The total and membrane protein expression levels of Cav1.2 [pore-forming subunit of L-type calcium channels (LTCC)], but not the insulin receptors, were decreased in OZR. The insulin receptor was found to be associated with the Cav1.2, which was weakened in OZR. The total protein expression of calmodulin was reduced, but that of Cavβ2 subunit was not altered in OZR. Together, these results suggested that the 16- to 17-wk-old OZR has 1) developed cardiac hypertrophy, 2) exhibited altered electrophysiology manifested by the prolonged QTc interval, 3) increased duration of action potential in isolated ventricular myocytes, 4) defective inactivation of I(CaL) and I(BaL), 5) weakened the association of LTCC with the insulin receptor, and 6) decreased protein expression of Cav1.2 and calmodulin. These results also provided mechanistic insights into a remodeled cardiac electrophysiology under the condition of insulin resistance, enhancing our understanding of long QT associated with obese type 2 diabetic patients.     

Functions of Autophagy in Pathological Cardiac Hypertrophy

Pathological cardiac hypertrophy is the response of heart to various biomechanical and physiopathological stimuli, such as aging, myocardial ischemia and hypertension. However, a long-term exposure to the stress makes heart progress to heart failure. Autophagy is a dynamic self-degradative process necessary for the maintenance of cellular homeostasis. Accumulating evidence has revealed a tight link between cardiomyocyte autophagy and cardiac hypertrophy. Sophisticatedly regulated autophagy protects heart from various physiological and pathological stimuli by degradating and recycling of protein aggregates, lipid drops, or organelles. Here we review the recent progresses concerning the functions of autophagy in cardiac hypertrophy induced by various hypertrophic stimuli. Moreover, the therapeutic strategies targeting autophagy for cardiac hypertrophy will also be discussed.     

Ratiometric imaging of calcium during ischemiareperfusion injury in isolated mouse hearts using Fura-2

ABSTRACT: BACKGROUND: We present an easily implementable method for measuring Fura-2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of intracellular [Ca2+] with both high spatial and temporal resolutions, permitting study of changes in dispersion of diastolic [Ca2+], Ca2+ transient dynamics, and conduction velocities in mouse hearts. In a proof-of-principle study, we imaged isolated Langendorff-perfused mouse hearts with reversible regional myocardial infarctions. METHODS: Isolated mouse hearts were perfused in the Landendorff-mode and loaded with Fura-2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura-2 fluorescence was recorded at regular intervals using a high-speed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart. RESULTS: Fura-2 produced consistent Ca2+ transients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in diastolic Ca2+ without a significant change in transient amplitude; Ca2+ handling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion. CONCLUSIONS: Our method of imaging Fura-2 from isolated whole hearts is capable of detecting pathological changes in intracellular Ca2+ levels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue connectivity rather than altered intracellular Ca2+ handling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction.     

Scoparone alleviates Ang II-induced pathological myocardial hypertrophy in mice by inhibiting oxidative stress

Long-term poorly controlled myocardial hypertrophy often leads to heart failure and sudden death. Activation of ras-related C3 botulinum toxin substrate 1 (RAC1) by angiotensin II (Ang II) plays a pivotal role in myocardial hypertrophy. Previous studies have demonstrated that scoparone (SCO) has beneficial effects on hypertension and extracellular matrix remodelling. However, the function of SCO on Ang II-mediated myocardial hypertrophy remains unknown. In our study, a mouse model of myocardial hypertrophy was established by Ang II infusion (2 mg/kg/day) for 4 weeks, and SCO (60 mg/kg bodyweight) was administered by gavage daily. In vitro experiments were also performed. Our results showed that SCO could alleviate Ang II infusion-induced cardiac hypertrophy and fibrosis in mice. In vitro, SCO treatment blocks Ang II-induced cardiomyocyte hypertrophy, cardiac fibroblast collagen synthesis and differentiation to myofibroblasts. Meanwhile, we found that SCO treatment blocked Ang II-induced oxidative stress in cardiomyocytes and cardiac fibroblasts by inhibiting RAC1-GTP and total RAC1 in vivo and in vitro. Furthermore, reactive oxygen species (ROS) burst by overexpression of RAC1 completely abolished SCO-mediated protection in cardiomyocytes and cardiac fibroblasts in vitro. In conclusion, SCO, an antioxidant, may attenuate Ang II-induced myocardial hypertrophy by suppressing of RAC1 mediated oxidative stress.     

Regulation of Ion Gradients across Myocardial Ischemic Border Zones: A Biophysical Modelling Analysis

The myocardial ischemic border zone is associated with the initiation and sustenance of arrhythmias. The profile of ionic concentrations across the border zone play a significant role in determining cellular electrophysiology and conductivity, yet their spatial-temporal evolution and regulation are not well understood. To investigate the changes in ion concentrations that regulate cellular electrophysiology, a mathematical model of ion movement in the intra and extracellular space in the presence of ionic, potential and material property heterogeneities was developed. The model simulates the spatial and temporal evolution of concentrations of potassium, sodium, chloride, calcium, hydrogen and bicarbonate ions and carbon dioxide across an ischemic border zone. Ischemia was simulated by sodium-potassium pump inhibition, potassium channel activation and respiratory and metabolic acidosis. The model predicted significant disparities in the width of the border zone for each ionic species, with intracellular sodium and extracellular potassium having discordant gradients, facilitating multiple gradients in cellular properties across the border zone. Extracellular potassium was found to have the largest border zone and this was attributed to the voltage dependence of the potassium channels. The model also predicted the efflux of from the ischemic region due to electrogenic drift and diffusion within the intra and extracellular space, respectively, which contributed to depletion in the ischemic region.     

Effect of Global Cardiac Ischemia on Human Ventricular Fibrillation: Insights from a Multi-scale Mechanistic Model of the Human Heart

Acute regional ischemia in the heart can lead to cardiac arrhythmias such as ventricular fibrillation (VF), which in turn compromise cardiac output and result in secondary global cardiac ischemia. The secondary ischemia may influence the underlying arrhythmia mechanism. A recent clinical study documents the effect of global cardiac ischaemia on the mechanisms of VF. During 150 seconds of global ischemia the dominant frequency of activation decreased, while after reperfusion it increased rapidly. At the same time the complexity of epicardial excitation, measured as the number of epicardical phase singularity points, remained approximately constant during ischemia. Here we perform numerical studies based on these clinical data and propose explanations for the observed dynamics of the period and complexity of activation patterns. In particular, we study the effects on ischemia in pseudo-1D and 2D cardiac tissue models as well as in an anatomically accurate model of human heart ventricles. We demonstrate that the fall of dominant frequency in VF during secondary ischemia can be explained by an increase in extracellular potassium, while the increase during reperfusion is consistent with washout of potassium and continued activation of the ATP-dependent potassium channels. We also suggest that memory effects are responsible for the observed complexity dynamics. In addition, we present unpublished clinical results of individual patient recordings and propose a way of estimating extracellular potassium and activation of ATP-dependent potassium channels from these measurements.