Ex vivo Validation of Noninvasive Epicardial and Endocardial Repolarization Mapping

Background: The detection and localization of electrophysiological substrates currently involve invasive cardiac mapping. Electrocardiographic imaging (ECGI) using the equivalent dipole layer (EDL) method allows the noninvasive estimation of endocardial and epicardial activation and repolarization times (AT and RT), but the RT validation is limited to in silico studies. We aimed to assess the temporal and spatial accuracy of the EDL method in reconstructing the RTs from the surface ECG under physiological circumstances and situations with artificially induced increased repolarization heterogeneity.Methods: In four Langendorff-perfused pig hearts, we simultaneously recorded unipolar electrograms from plunge needles and pseudo-ECGs from a volume-conducting container equipped with 61 electrodes. The RTs were computed from the ECGs during atrial and ventricular pacing and compared with those measured from the local unipolar electrograms. Regional RT prolongation (cooling) or shortening (pinacidil) was achieved by selective perfusion of the left anterior descending artery (LAD) region.Results: The differences between the computed and measured RTs were 19.0 ± 17.8 and 18.6 ± 13.7 ms for atrial and ventricular paced beats, respectively. The region of artificially delayed or shortened repolarization was correctly identified, with minimum/maximum RT roughly in the center of the region in three hearts. In one heart, the reconstructed region was shifted by ~2.5 cm. The total absolute difference between the measured and calculated RTs for all analyzed patterns in selectively perfused hearts (n = 5) was 39.6 ± 27.1 ms.Conclusion: The noninvasive ECG repolarization imaging using the EDL method of atrial and ventricular paced beats allows adequate quantitative reconstruction of regions of altered repolarization.

A small molecule inhibitor of Nox2 and Nox4 improves contractile function after ischemia–reperfusion in the mouse heart

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia–reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.

Influence of Serotonin 5-HT4 Receptors on Responses to Cardiac Stressors in Transgenic Mouse Models

The current study aimed to deepen our knowledge on the role of cardiac 5-HT4 receptors under pathophysiological conditions. To this end, we used transgenic (TG) mice that overexpressed human 5-HT4a receptors solely in cardiac myocytes (5-HT4-TG mice) and their wild-type (WT) littermates that do not have functional cardiac 5-HT4 receptors as controls. We found that an inflammation induced by lipopolysaccharide (LPS) was detrimental to cardiac function in both 5-HT4-TG and WT mice. In a hypoxia model, isolated left atrial preparations from the 5-HT4-TG mice went into contracture faster during hypoxia and recovered slower following hypoxia than the WT mice. Similarly, using isolated perfused hearts, 5-HT4-TG mice hearts were more susceptible to ischemia compared to WT hearts. To study the influence of 5-HT4 receptors on cardiac hypertrophy, 5-HT4-TG mice were crossbred with TG mice overexpressing the catalytic subunit of PP2A in cardiac myocytes (PP2A-TG mice, a model for genetically induced hypertrophy). The cardiac contractility, determined by echocardiography, of the resulting double transgenic mice was attenuated like in the mono-transgenic PP2A-TG and, therefore, largely determined by the overexpression of PP2A. In summary, depending on the kind of stress put upon the animal or isolated tissue, 5-HT4 receptor overexpression could be either neutral (genetically induced hypertrophy, sepsis) or possibly detrimental (hypoxia, ischemia) for mechanical function. We suggest that depending on the underlying pathology, the activation or blockade of 5-HT4 receptors might offer novel drug therapy options in patients.

Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.

Amitriptyline functionally antagonizes cardiac H2 histamine receptors in transgenic mice and human atria

We have previously shown that histamine (2-(1H-imidazol-4-yl)ethanamine) exerted concentration-dependent positive inotropic effects (PIE) or positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic (H2R-TG) mice that overexpress the human H2 histamine receptor (H2R) in the heart; however, the effects were not seen in their wild-type (WT) littermates. Amitriptyline, which is still a highly prescribed antidepressant drug, was reported to act as antagonist on H2Rs. Here, we wanted to determine whether the histamine effects in H2R-TG were antagonized by amitriptyline. Contractile studies were performed on isolated left and right atrial preparations, isolated perfused hearts from H2R-TG and WT mice and human atrial preparations. Amitriptyline shifted the concentration-dependent PIE of histamine (1 nM–10 μM) to higher concentrations (rightward shift) in left atrial preparations from H2R-TG. Similarly, in isolated perfused hearts from H2R-TG and WT mice, histamine increased the contractile parameters and the phosphorylation state of phospholamban (PLB) at serine 16 in the H2R-TG mice, but not in the WT mice. However, the increases in contractility and PLB phosphorylation were attenuated by the addition of amitriptyline in perfused hearts from H2R-TG. In isolated electrically stimulated human atria, the PIE of histamine that was applied in increasing concentrations from 1 nM to 10 μM was reduced by 10-μM amitriptyline. In summary, we present functional evidence that amitriptyline also acts as an antagonist of contractility at H2Rs in H2R-TG mouse hearts and in the human heart which might in part explain the side effects of amitriptyline.

Effects of genetic background, sex, and age on murine atrial electrophysiology

Aims Genetically altered mice are powerful models to investigate mechanisms of atrial arrhythmias, but normal ranges for murine atrial electrophysiology have not been robustly characterized. Methods and results We analyzed results from 221 electrophysiological (EP) studies in isolated, Langendorff-perfused hearts of wildtype mice (114 female, 107 male) from 2.5 to 17.7 months (mean 7 months) with different genetic backgrounds (C57BL/6, FVB/N, MF1, 129/Sv, Swiss agouti). Left atrial monophasic action potential duration (LA-APD), interatrial activation time (IA-AT), and atrial effective refractory period (ERP) were summarized at different pacing cycle lengths (PCLs). Factors influencing atrial electrophysiology including genetic background, sex, and age were determined. LA-APD70 was 18 ± 0.5 ms, atrial ERP was 27 ± 0.8 ms, and IA-AT was 17 ± 0.5 ms at 100 ms PCL. LA-APD was longer with longer PCL (+17% from 80 to 120 ms PCL for APD70), while IA-AT decreased (−7% from 80 to 120 ms PCL). Female sex was associated with longer ERP (+14% vs. males). Genetic background influenced atrial electrophysiology: LA-APD70 (−20% vs. average) and atrial ERP (−25% vs. average) were shorter in Swiss agouti background compared to others. LA-APD70 (+25% vs. average) and IA-AT (+44% vs. average) were longer in 129/Sv mice. Atrial ERP was longer in FVB/N (+34% vs. average) and in younger experimental groups below 6 months of age. Conclusion This work defines normal ranges for murine atrial EP parameters. Genetic background has a profound effect on these parameters, at least of the magnitude as those of sex and age. These results can inform the experimental design and interpretation of murine atrial electrophysiology.

Adrenergic supersensitivity and impaired neural control of cardiac electrophysiology following regional cardiac sympathetic nerve loss

Myocardial infarction (MI) can result in sympathetic nerve loss in the infarct region. However, the contribution of hypo-innervation to electrophysiological remodeling, independent from MI-induced ischemia and fibrosis, has not been comprehensively investigated. We present a novel mouse model of regional cardiac sympathetic hypo-innervation utilizing a targeted-toxin (dopamine beta-hydroxylase antibody conjugated to saporin, DBH-Sap), and measure resulting electrophysiological and Ca2+ handling dynamics. Five days post-surgery, sympathetic nerve density was reduced in the anterior left ventricular epicardium of DBH-Sap hearts compared to control. In Langendorff-perfused hearts, there were no differences in mean action potential duration (APD80) between groups; however, isoproterenol (ISO) significantly shortened APD80 in DBH-Sap but not control hearts, resulting in a significant increase in APD80 dispersion in the DBH-Sap group. ISO also produced spontaneous diastolic Ca2+ elevation in DBH-Sap but not control hearts. In innervated hearts, sympathetic nerve stimulation (SNS) increased heart rate to a lesser degree in DBH-Sap hearts compared to control. Additionally, SNS produced APD80 prolongation in the apex of control but not DBH-Sap hearts. These results suggest that hypo-innervated hearts have regional super-sensitivity to circulating adrenergic stimulation (ISO), while having blunted responses to SNS, providing important insight into the mechanisms of arrhythmogenesis following sympathetic nerve loss.

Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of H 2 O 2 activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor–activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of Nppa/Nppb mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.

Cardiac Ischemic Preconditioning Promotes MG53 Secretion Through H 2 O 2 -Activated Protein Kinase C-δ Signaling

Background: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (mitsugumin 53; also called TRIM72), not only plays an essential role in IPC-mediated cardioprotection against ischemia/reperfusion injury but also ameliorates mechanical damage. In addition to its intracellular actions, as a myokine/cardiokine, MG53 can be secreted from the heart and skeletal muscle in response to metabolic stress. However, it is unknown whether IPC-mediated cardioprotection is causally related to MG53 secretion and, if so, what the underlying mechanism is. Methods: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo, isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. Moreover, using recombinant MG53 proteins, we investigated the potential biological function of secreted MG53 in the context of IPC and ischemia/reperfusion injury. Results: We found that IPC triggered robust MG53 secretion in rodents in vivo, perfused hearts, and cultured cardiac myocytes without causing cell membrane leakage. Mechanistically, IPC promoted MG53 secretion through H 2 O 2 -evoked activation of protein kinase-C-δ. Specifically, IPC-induced myocardial MG53 secretion was mediated by H 2 O 2 -triggered phosphorylation of protein kinase-C-δ at Y311, which is necessary and sufficient to facilitate MG53 secretion. Functionally, systemic delivery of recombinant MG53 proteins to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice but also protected rodent hearts from ischemia/reperfusion injury even in the absence of IPC. Moreover, oxidative stress by H 2 O 2 augmented MG53 secretion, and MG53 knockdown exacerbated H 2 O 2 -induced cell injury in human embryonic stem cell–derived cardiomyocytes, despite relatively low basal expression of MG53 in human heart. Conclusions: We conclude that IPC and oxidative stress can trigger MG53 secretion from the heart via an H 2 O 2 –protein kinase-C-δ–dependent mechanism and that extracellular MG53 can participate in IPC protection against cardiac ischemia/reperfusion injury.