Sham-operated Hearts Research Articles

The Structural Basis of Functional Improvement in Response to Human Umbilical Cord Blood Stem Cell Transplantation in Hearts with Postinfarct LV Remodeling

Cellular therapy for myocardial repair has been one of the most intensely investigated interventional strategies for acute myocardial infarction. Although the therapeutic potential of stem cells has been demonstrated in various studies, the underlying mechanisms for such improvements are poorly understood. In the present study, we investigated the long-term effects of stem cell therapy on both myocardial fiber organization and regional contractile function using a rat model of postinfarct remodeling. Human nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs) were administered via tail vein to rats 2 days after infarct surgery. Animals were maintained without immunosuppressive therapy. In vivo and ex vivo MR imaging was performed on infarct hearts 10 months after cell transplantation. Compared to the age-matched rats exposed to the identical surgery, both global and regional cardiac functions of the nh-UCBSC-treated hearts, such as ejection fraction, ventricular strain, and torsion, were significantly improved. More importantly, the treated hearts exhibited preserved fiber orientation and water diffusivities that were similar to those in sham-operated control hearts. These data provide the first evidence that nh-UCBSC treatment may prevent/delay untoward structural remodeling in postinfarct hearts, which supports the improved LV function observed in vivo in the absence of immunosuppression, suggesting a beneficial paracrine effect occurred with the cellular therapy.

Differential cardiac expression of individual lipids in the plasmalogen biosynthetic pathway in response to pressure overload in mice

Introduction: In response to a cardiac insult such as hypertension the heart initially undergoes a compensatory phase followed by a decompensatory phase and failure. Different lipid species are now recognised to be involved in regulating signalling and physiology. Plasmalogens, a class of glycerophospholipids, are enriched in the heart, and previously shown to be protective against reactive oxygen species in mammalian cells. Methods: Mice were subjected to either sham surgery (n=5), moderate [MOD (compensated)], or a severe [SEV (decompensated)] form of transverse aortic constriction (TACpressure overloadmodel, n=6 each) for 4weeks. Lipids were extracted using a single phase chloroform:methanol extraction and run using liquid chromatography electrospray ionisation tandem mass spectrometry. Data was analysed in Multiquant 2.1 software and specific lipid species normalised to total phosphatidylcholine levels. Results: TAC-SEV mice displayed significant cardiac hypertrophy (∼40% increase in heart weight/ tibia length ratio, mean=13.8±1.3mg/mm, p<0.05) as compared to sham (mean=8.3±0.4mg/mm) and TAC-MOD mice (mean=8.9±0.1mg/mm). Individual lipid species involved in the plasmalogen biosynthetic pathway [alkylphosphatidylethanolamine (one ether linked acylchain), PE(O) 40:6, alkyl-phosphatidylcholine, PC(O) 40:6, PE plasmalogen, PE(P) 40:6 and PC plasmalogen, PC(P) 40:6] were all increased (between 15.4%-27.1%, p<0.05) in TAC-MOD hearts vs. sham-operated hearts. In contrast, there was no significant increase in these lipid species in the TAC-SEV hearts. Conclusion: Significant elevation in the lipid species involved in theplasmalogenbiosyntheticpathway ina setting of pressure overload may point to a protective compensatory mechanism of the heart during the onset of hypertrophy.

Regulation of Cardiomyocyte T-Tubule Organization and Density by Ventricular Wall Stress

During heart failure development, t-tubules become disorganized which disrupts Ca2+ homeostasis and weakens contraction of the heart. The mechanisms controlling t-tubular structure in the normal and failing heart remain unknown, but accumulating data suggest that ventricular workload may be an important regulator. In Wistar rats which had developed heart failure 6 weeks following myocardial infarction, we observed that marked t-tubule disruption occurred preferentially in regions of the heart that are proximal to the infarct site, while t-tubule density was normal in distal locations. In vivo imaging by MRI has revealed near-isometric contraction in this proximal zone, with dramatically elevated wall stress due to local thinning of the ventricular wall and elevated diastolic blood pressure. To directly investigate whether elevated wall stress triggers t-tubule disruption, we developed an in vitro model for culturing isolated rat left ventricle papillary muscles in a myobath system (0.5 Hz stimulation, 48 hours). Muscles were subjected to varying amounts of stretch to approximate wall stress values observed in vivo. Muscles exposed to low diastolic wall stress similar to that observed in sham-operated hearts (3.5-4.5 mN/mm2), exhibited well-maintained t-tubule organization during culture (t-tubule fraction of cross-sectional area = 0.174 ± SE 0.007). Exposure to high wall stress (10-15 mN/mm2) triggered marked t-tubule loss during culture (t-tubule fraction = 0.079 ± SE 0.012), and reduction in both peak and between-peak power in Fast-Fourier Transform analyses. In addition, cell size was observed to be markedly increased by elevated wall stress in comparison with muscles that were exposed to low wall stress (cross-sectional area = 1022.71 ± SE 48.00 µm2 vs 564.41 ± SE 25.34 µm2, P < 0.05). Thus, our data indicate that wall stress is an important regulator of both cellular geometry and t-tubular structure.

Integrated Omic Analysis of a Guinea Pig Model of Heart Failure and Sudden Cardiac Death

Mechanistic understanding of heart failure (HF) and sudden cardiac death (SCD) has been hampered by the lack of suitable experimental models with features of human disease. We have developed a guinea pig model of cardiac hypertrophy (HYP) and HF, characterized by predisposition to SCD (Liu et al. Circ. Res. 2014). Our objective was to refine guinea pig models of HF progression by integrating protein, metabolite and transcript levels. Relative protein abundances from sham-operated, HYP and HF hearts were assessed using isobaric tags for relative and absolute quantification, prior to liquid chromatography and tandem mass spectrometry (LC-MS/MS). Metabolites were quantified by LC-MS/MS or gas chromatography coupled to MS. Transcriptome profiles were obtained from Affymetrix microarrays. Theguinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, cellular degeneration, inflammation and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, and other mitochondrial processes, were downregulated. Processes/proteins upregulated in HF include fibrillogenesis, extracellular matrix remodeling, cytoskeletal proteins, the unfolded-protein response, and acute phase inflammation markers. Among metabolites, downregulation of acyl-carnitines was observed in HYP, while fatty acids accumulated in HF. Levels of the TCA cycle metabolite, citrate, and the potent inhibitor, 2-methylcitrate, increased upon transition from HYP to HF. Among transcripts, downregulation of repolarizing K+ channels, SERCA2a, phospholamban and ryanodine receptor was noted, and upregulation of mRNAs for cyclic-nucleotide gated hyperpolarization-activated channels and TRPC6. The biosignature of decompensation in the guinea pig HF/SCD model parallels prior rodent models but spotlights metabolic bottlenecks. Fatty acid use is likely abrogated by impaired mitochondrial import and oxidation, while compensation by glycolytic flux is predicted to be impaired, along with TCA cycle dysfunction and accumulation of inhibitors (e.g. citrate). Pathway analysis suggests avenues for metabolic therapy beyond PPAR activation.

Architectural correlates of myocardial conduction: changes to the topography of cellular coupling, intracellular conductance, and action potential propagation with hypertrophy in Guinea-pig ventricular myocardium.

We tested the hypothesis that alterations to action potential conduction velocity (CV) and conduction anisotropy in left ventricular hypertrophy are associated with topographical changes to gap-junction coupling and intracellular conductance by measuring these variables in the same preparations. Left ventricular papillary muscles were excised from aortic-banded or sham-operated guinea-pig hearts. With intracellular stimulating and recording microelectrodes, CV was measured in 3 dimensions with simultaneous conductance mapping with subthreshold stimuli and correlated with quantitative histomorphometry of myocardial architecture and connexin 43 distribution. In hypertrophied myocardium, CV in the longitudinal axis was smaller and transverse velocity was greater compared with control; associated with similar differences of intracellular conductance, consistent with more cell contacts per cell (5.7 ± 0.2 versus 8.1 ± 0.5; control versus hypertrophy), and more intercalated disks mediating side-to-side coupling (8.2 ± 0.2 versus 10.2 ± 0.4 per cell). Intercalated disk morphology and connexin 43 immunolabelling were not different in hypertrophy. Hypertrophied preparations showed local submillimeter (≈250 μm) regions with slow conduction and low intracellular conductance, which, although not affecting CV on the millimeter scale, were consistent with discontinuities from increased microscopical connective tissue content. With myocardial hypertrophy, altered longitudinal and transverse CV, and greater nonuniformity of CV anisotropy correspond to changes of intracellular conductance. These are associated with alteration of myocardial architecture, specifically the topography of cell-cell coupling and gap-junction connectivity.

Ventricular Hypertrophy Abrogates Intralipid-Induced Cardioprotection by Alteration of Reperfusion Injury Salvage Kinase/Glycogen Synthase Kinase 3β Signal

Recent studies have demonstrated that intralipid (ILP) conferred myocardial protection against ischemia-reperfusion (IR) injury through activation of reperfusion injury salvage kinase (RISK) pathway. As RISK signal has been shown to be impaired in hypertrophied myocardium, we investigated whether ILP-induced cardiac protection was maintained in hypertrophied rat hearts. Transverse aortic constriction was performed on male Sprague-Dawley rats to induce left ventricular hypertrophy, then sham-operated or hypertrophied rat hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The isolated hearts received 15-min episode of 1% ILP separated by 15 min of washout or three episodes of 5-min ischemia followed by 5-min reperfusion before ischemia. The hemodynamics, infarct size, apoptosis, phosphorylated protein kinase B (p-Akt), phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated glycogen synthase kinase 3β (GSK3β), Bcl-2, phosphorylated Bad, and Bax were determined. We found that ILP significantly improved left ventricular hemodynamics and reduced infarct size and the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells in the sham-operated rat hearts exposed to IR. However, such myocardial infarct-sparing effect of ILP was completely blocked by phosphatidylinositol-3-kinase inhibitor wortmannin, but only partially by mitogen-activated protein kinase kinase inhibitor PD98059 in sham-operated hearts. Intralipd upregulated the phosphorylation of Akt, extracellular regulated protein kinase 1/2 (ERK1/2), and their downstream target of GSK3β and antiapoptotic Bcl-2 expression in healthy rat hearts. Nonetheless, ILP failed to improve left ventricular hemodynamics and reduced infarct size and apoptosis and increase the phosphorylated Akt, ERK1/2, GSK3β, and antiapoptotic Bcl-2 in hypertrophied myocardium. In contrast, ischemic preconditioning increased the phosphorylation of Akt, ERK1/2 and GSK3β, improved heart pump function, and reduced myocardial necrosis in sham-operated hearts, a phenomenon partially attenuated by ventricular hypertrophy. Interestingly, GSK inhibitor SB216763 conferred cardioprotection against IR injury in sham-operated hearts, but failed to exert cardioprotection in hypertrophied myocardium. Our results indicated that ventricular hypertrophy abrogated ILP-induced cardioprotection against IR injury by alteration of RISK/GSK3β signal.

Loss of duplexmiR-223 (5p and 3p) aggravates myocardial depression and mortality in polymicrobial sepsis

Sepsis is the leading cause of death in critically ill patients. While myocardial dysfunction has been recognized as a major manifestation in severe sepsis, the underlying molecular mechanisms associated with septic cardiomyopathy remain unclear. In this study, we performed a miRNA array analysis in hearts collected from a severe septic mouse model induced by cecal ligation and puncture (CLP). Among the 19 miRNAs that were dys-regulated in CLP-mouse hearts, miR-223(3p) and miR-223*(5p) were most significantly downregulated, compared with sham-operated mouse hearts. To test whether a drop of miR-223 duplex plays any roles in sepsis-induced cardiac dysfunction and inflammation, a knockout (KO) mouse model with a deletion of the miR-223 gene locus and wild-type (WT) mice were subjected to CLP or sham surgery. We observed that sepsis-induced cardiac dysfunction, inflammatory response and mortality were remarkably aggravated in CLP-treated KO mice, compared with control WTs. Using Western-blotting and luciferase reporter assays, we identified Sema3A, an activator of cytokine storm and a neural chemorepellent for sympathetic axons, as an authentic target of miR-223* in the myocardium. In addition, we validated that miR-223 negatively regulated the expression of STAT-3 and IL-6 in mouse hearts. Furthermore, injection of Sema3A protein into WT mice revealed an exacerbation of sepsis-triggered inflammatory response and myocardial depression, compared with control IgG1 protein-treated WT mice following CLP surgery. Taken together, these data indicate that loss of miR-223/-223* causes an aggravation of sepsis-induced inflammation, myocardial dysfunction and mortality. Our study uncovers a previously unrecognized mechanism underlying septic cardiomyopathy and thereby, may provide a new strategy to treat sepsis.

Hypertrophied Myocardium Is Refractory to Sevoflurane-Induced Protection With Alteration of Reperfusion Injury Salvage Kinase/Glycogen Synthase Kinase 3β Signals

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired by ventricular hypertrophy, we investigate whether anesthetic-induced cardiac protection was maintained in rat hearts with ventricular hypertrophy. Transverse aortic constriction operation was performed on male Sprague-Dawley rats to induce left ventricular (LV) hypertrophy, then sham-operated or hypertrophied rat hearts were subjected to 40 min of global ischemia and 2 h of reperfusion. The isolated hearts received 3% sevoflurane for 10 min or six cycles of 10-s ischemia/10-s reperfusion after reperfusion. The hemodynamics, infarct size, PTEN (phosphatase and tensin homolog deleted on chromosome ten), phosphorylated Akt, phosphorylated extracellular regulated protein kinase (ERK) 1/2, and phosphorylated glycogen synthase kinase 3β (GSK3β) were determined. We found the myocardial expression of PTEN, phosphorylated Akt, ERK1/2, and phosphorylated GSK3β did not significantly differ between sham-operated and transverse aortic constriction-control groups. Both sevoflurane and ischemic postconditioning significantly improved LV hemodynamics, reduced infarct size, and increased the phosphorylation of Akt, ERK1/2, and their downstream target of GSK3β in the sham-operated rat hearts. In contrast, neither sevoflurane nor ischemic postconditioning improved LV hemodynamic, reduced infarct size, and increased the phosphorylated Akt, ERK1/2, and GSK3β in hypertrophied myocardium. All the results above indicate that ventricular hypertrophy abrogated sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of RISK/GSK3β signals.

A novel approach for assessing cardiac fibrosis using label-free second harmonic generation

To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red stainingwas used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p<0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts.

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are re-expressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P<0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development.

Pyruvate restores β-adrenergic sensitivity of L-type Ca2+channels in failing rat heart: role of protein phosphatase

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

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.

Resveratrol Reverses Remodeling in Hearts with Large, Old Myocardial Infarctions through Enhanced Autophagy-Activating AMP Kinase Pathway

We investigated the effect of resveratrol, a popular natural polyphenolic compound with antioxidant and proautophagic actions, on postinfarction heart failure. Myocardial infarction was induced in mice by left coronary artery ligation. Four weeks postinfarction, when heart failure was established, the surviving mice were started on 2-week treatments with one of the following: vehicle, low- or high-dose resveratrol (5 or 50 mg/kg/day, respectively), chloroquine (an autophagy inhibitor), or high-dose resveratrol plus chloroquine. High-dose resveratrol partially reversed left ventricular dilation (reverse remodeling) and significantly improved cardiac function. Autophagy was augmented in those hearts, as indicated by up-regulation of myocardial microtubule-associated protein-1 light chain 3-II, ATP content, and autophagic vacuoles. The activities of AMP-activated protein kinase and silent information regulator-1 were enhanced in hearts treated with resveratrol, whereas Akt activity and manganese superoxide dismutase expression were unchanged, and the activities of mammalian target of rapamycin and p70 S6 kinase were suppressed. Chloroquine elicited opposite results, including exacerbation of cardiac remodeling associated with a reduction in autophagic activity. When resveratrol and chloroquine were administered together, the effects offset one another. In vitro, compound C (AMP-activated protein kinase inhibitor) suppressed resveratrol-induced autophagy in cardiomyocytes, but did not affect the events evoked by chloroquine. In conclusion, resveratrol is a beneficial pharmacological tool that augments autophagy to bring about reverse remodeling in the postinfarction heart.

New Insights into the Beneficial Electrophysiologic Profile of Ranolazine in Heart Failure: Prevention of Ventricular Fibrillation With Increased Postrepolarization Refractoriness and Without Drug-Induced Proarrhythmia

BackgroundRanolazine inhibits late Na+ and K+ currents. Earlier studies have reported an antiarrhythmic effect. The aim of the present study was to understand whether ranolazine could still preserve its antiarrhythmic properties in the settings of chronic heart failure (CHF). Methods and ResultsIn 12 female rabbits, CHF was induced by 4 weeks of rapid ventricular pacing leading to a decrease in ejection fraction. Twelve rabbits underwent sham operation. Isolated hearts were Langendorff perfused and demonstrated a significant QT prolongation after induction of heart failure. Ranolazine caused a concentration-dependent (10 and 30 μmol/L) increase of action potential duration (APD90) in sham-operated and failing hearts. Eight endo- and epicardial monophasic action potentials revealed a nonsignificant increase in spatial and temporal dispersion of repolarization. The increase in APD90 was accompanied by a greater increase in refractory period, resulting in a significant increase in postrepolarization refractoriness in sham-operated (+29 ms and +55 ms; P < .01) and failing (+22 ms and +30 ms; P < .05) hearts. In control conditions, programmed ventricular stimulation and a burst pacing protocol led to ventricular fibrillation (VF) in 5 of the 12 sham-operated (6 episodes) and in 7 of the 12 failing (18 episodes) hearts. In the presence of ranolazine, VF was inducible in only 2 of 12 failing hearts (5 episodes). In the presence of low [K+], only 1 ranolazine-treated sham-operated heart developed early afterdepolarizations and ventricular tachyarrhythmias despite significant QT prolongation. ConclusionsRanolazine decreases inducibility of VF in the presence of a significant increase in postrepolarization refractoriness. This antiarrhythmic effect in the intact heart is preserved in CHF and is not associated with drug-induced proarrhythmia.

Abstract 215: Systemic Inflammation Exacerbates Electrophysiological Remodeling and Arrhythmia Following Myocardial Infarction

Introduction: Inflammatory macrophages infiltrate the heart following myocardial infarction (MI) and secrete proteases and cytokines that may contribute to adverse electrophysiological remodeling. Atherosclerosis, the leading cause of MI, is associated with elevated systemic inflammation. Using a novel combination of optical mapping and molecular imaging, we sought to determine if systemic inflammation exacerbates electrophysiological remodeling and arrhythmogenesis. Methods: A protease activatable fluorescent sensor (Prosense680) was injected I.V. 4 days post MI in mice. Optical mapping of transmembrane potential was performed 5 days post MI on Langendorff-perfused hearts to assess electrophysiology. Fluorescence reflectance imaging of Prosense680 was then performed to visualize and quantify inflammatory protease activity. Lipopolysaccharide (LPS:10 μ g/day, n=6) was injected I.P. to promote systemic inflammation or saline as control (SAL, n=7). Results: Inflammation levels were greater in MI+LPS vs MI+SAL hearts (5.626±1.5 vs 1.73±0.25 A.U., p&lt;0.02) and negligible in sham-operated hearts (SH, n=4). MI+LPS hearts had prolonged action potential (AP) duration vs MI+SAL vs SH (62±4.5 vs 56±1.8 vs 45±3.7 ms, p&lt;0.05) and increased AP rise time (5.5±0.5 vs 4.2±0.2 vs 2.9±0.3 ms, p&lt;0.05). A single premature pacing stimulus induced ventricular tachycardia in 67%(4 of 6) of MI+LPS vs 15%(1 of 7) MI+SAL vs 0% (0 of 4) SH, p&lt;0.02. Conclusions: Elevated post-MI inflammation may contribute to electrophysiological remodeling and arrhythmias. These findings have important implications for the etiology of post-MI arrhythmias in humans with systemic inflammation.

Abstract 221: Chronic Alterations of the Tissue Dendritic Cell and Macrophage Network Underlies the Th1 Response in the Failing Heart

Little is known about changes in the immune cell network that underlie pro-inflammatory cytokine elaboration in chronic heart failure (HF). Dendritic cells (DCs) and macrophages are antigen-presenting cells that play a central role in inflammation and immune tolerance. We investigated the pattern of infiltrating cardiac DCs and macrophages in chronic post-ischemic HF. Myocardial infarction (MI) was induced by left coronary ligation in male C57BL/6 mice. Pathological remodeling and HF were confirmed by hemodynamics, gravimetry, and echocardiography at 8 weeks post-MI. Sham-operated mice served as controls. At 8 weeks, cardiac infiltrating immune cells were isolated by collagenase digestion followed by density gradient centrifugation. Trypan blue-negative viable cells were quantified using flow cytometry. DCs were characterized using specific markers for classical (cDC) (CD11C+B220-), plasmacytoid (pDC) (CD11C+B220+), and immunogenic (CD11c+B220+CD86hi) and tolerogenic (CD11c+B220+CD86low) DCs. Macrophages were classified as activated (CD11b+F480+), pro- or anti-inflammatory (CD11b+F480+Gr1hi or low), and M1 (CD11b+F480+CD206-) or M2 (CD11b+F480+CD206+). Significant increases in cDCs (1.4 fold; p=0.016) and pDCs (4.3 fold; p=0.0016) were observed in failing myocardium. Surface expression of the maturation marker CD86 was increased 8.6 fold (p=0.009), whereas immature DCs exhibited a robust 24.4 fold (p=0.0005) increase. Activated macrophages were also significantly elevated in the failing heart as compared with sham-operated hearts (19.2 ± 1.9 vs 10.9 ± 2.9%; p=0.033), with marked infiltration of pro-inflammatory and M1 macrophages (6.2 ± 0.6 vs 3.8 ± 0.7%; p=0.019). In contrast, levels of anti-inflammatory and M2 macrophages were unchanged. We conclude that in chronic HF, there is marked upregulation of cDC and pDC subsets and pro-inflammatory/M1 macrophages that alter immune homeostasis, contribute to the recruitment and activation of immune cells, and underlie the chronic Th1 response in failing myocardium that promotes pathological remodeling. This suggests that targeting or altering immune cell subsets in the failing heart, rather than specific cytokines, may be a better approach to modulating inflammation in HF.

Blockade of ICa suppresses early afterdepolarizations and reduces transmural dispersion of repolarization in a whole heart model of chronic heart failure

BACKGROUND AND PURPOSE Chronic heart failure (CHF) is associated with action potential prolongation and Ca(2+) overload, increasing risk of ventricular tachyarrhythmias (VT). We therefore investigated whether I(Ca) blockade was anti-arrhythmic in an intact perfused heart model of CHF. EXPERIMENTAL APPROACH CHF was induced in rabbits after 4 weeks of rapid ventricular pacing. Hearts from CHF and sham-operated rabbits were isolated and perfused (Langendorff preparation), with ablation of the AV node. VT was induced by erythromycin and low [K(+) ] (1.5mM). Electrophysiology of cardiac myocytes, with block of cation currents, was simulated by a mathematical model. KEY RESULTS Repolarization was prolonged in CHF hearts compared with sham-operated hearts. Action potential duration (APD) and overall dispersion of repolarization were further increased by erythromycin (300 µM) to block I(Kr) in CHF hearts. After lowering [K(+) ] to 1.5mM, CHF and sham hearts showed spontaneous episodes of polymorphic non-sustained VT. Additional infusion of verapamil (0.75 µM) suppressed early afterdepolarizations (EAD) and VT in 75% of sham and CHF hearts. Verapamil shortened APD and dispersion of repolarization, mainly by reducing transmural dispersion of repolarization via shortening of endocardial action potentials. Mathematical simulations showed that EADs were more effectively reduced by verapamil assuming a state-dependent block than a simple block of I(Ca) . CONCLUSIONS AND IMPLICATIONS Blockade of I(Ca) was highly effective in suppressing VT via reduction of transmural dispersion of repolarization and suppression of EAD. Such blockade might represent a novel therapeutic option to reduce risk of VT in structurally normal hearts and also in heart failure. LINKED ARTICLE This article is commented on by Stams et al., pp. 554-556 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01818.x.

Metabolic Adaptation to a Disruption in Oxygen Supply during Myocardial Ischemia and Reperfusion Is Underpinned by Temporal and Quantitative Changes in the Cardiac Proteome

Despite decades of intensive research, there is still no effective treatment for ischemia/reperfusion (I/R) injury, an important corollary in the treatment of ischemic disease. I/R injury is initiated when the altered biochemistry of cells after ischemia is no longer compatible with oxygenated microenvironment (or reperfusion). To better understand the molecular basis of this alteration and subsequent incompatibility, we assessed the temporal and quantitative alterations in the cardiac proteome of a mouse cardiac I/R model by an iTRAQ approach at 30 min of ischemia, and at 60 or 120 min reperfusion after the ischemia using sham-operated mouse heart as the baseline control. Of the 509 quantified proteins identified, 121 proteins exhibited significant changes (p-value<0.05) over time and were mostly clustered in eight functional groups: Fatty acid oxidation, Glycolysis, TCA cycle, ETC (electron transport chain), Redox Homeostasis, Glutathione S-transferase, Apoptosis related, and Heat Shock proteins. The first four groups are intimately involved in ATP production and the last four groups are known to be important in cellular antioxidant activity. During ischemia and reperfusion, the short supply of oxygen precipitates a pivotal metabolic switch from aerobic metabolism involving fatty acid oxidation, TCA, and phosphorylation to anaerobic metabolism for ATP production and this, in turn, increases reactive oxygen species (ROS) formation. Therefore the implication of these 8 functional groups suggested that ischemia-reperfusion injury is underpinned in part by proteomic alterations. Reversion of these alterations to preischemia levels took at least 60 min, suggesting a refractory period in which the ischemic cells cannot adjust to the presence of oxygen. Therefore, therapeutics that could compensate for these proteomic alterations during this interim refractory period could alleviate ischemia-reperfusion injury to enhance cellular recovery from an ischemic to a normoxic microenvironment. Among the perturbed proteins, Park7 and Ppia were selected for further investigation of their functions under hypoxia. The results show that Park7 plays a key role in regulating antioxidative stress and cell survival, and Ppia may function in coping with the unfolded protein stress in the I/R condition.

Direct Renin Inhibition Exerts an Anti-hypertrophic Effect Associated with Improved Mitochondrial Function in Post-infarction Heart Failure in Diabetic Rats

Background: In addition to hypertension control, direct renin inhibition has been shown to exert direct beneficial effects on the heart in post-infarction cardiac remodeling. This study elucidates the possible contribution of mitochondria to the anti-hypertrophic effects of the direct renin inhibitor aliskiren in post-infarction heart failure complicated with diabetes in rats. Methods: Diabetes was induced in male Sprague-Dawley rats by a single injection of streptozotocin (IP, 65 mg/kg body weight). After 7 days, the animals were randomly assigned to 4 groups: sham, heart failure, sham+aliskiren, and heart failure+aliskiren. Post-infarction HF was induced by coronary artery ligation for 4 weeks. Results: showed that heart failure reduced ejection fraction and cardiac output by 41% (P<0.01) and 42% (P<0.05), respectively, compared to sham-operated hearts. Cardiac dysfunction was associated with suppressed state 3 respiration rates and respiratory control index in mitochondria, and increased mitochondrial permeability transition pore (PTP) opening. In addition, heart failure reduced expression of the major mitochondrial sirtuin, SIRT3 and increased acetylation of cyclophilin D, a regulatory component of the PTP. Aliskiren significantly improved cardiac function and abrogated mitochondrial perturbations. Conclusion: Our results demonstrate that aliskiren attenuates post-infarction remodeling which is associated with its beneficial effects on mitochondria.

Abstract P126: Calpain Protects the Heart from Hemodynamic Stress

Although calpains are well-known Ca 2+ -dependent intracellular cysteine proteases, the pathophysiological function of calpains in the heart remains to be elucidated. Previous reports have suggested that calpains play detrimental roles to induce apoptotic or necrotic cell death in pathological Ca 2+ -overloaded conditions such as ischemia-reperfusion injury or myocardial infarction. Recent loss of function studies have suggested that calpains play important physiological roles in development, cell migration, organization of actin cytoskeleton, and plasma membrane repair. In the present study, we generated and analyzed cardiac-specific calpain 4-deficient mice (CKO) to answer unresolved questions as to in vivo function of calpains in the heart. Ubiquitous calpain is consisted of a common regulatory subunit (calpain 4) and a large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain). In agreement with previous reports, cardiac-specific deletion of calpain 4 markedly resulted in a simultaneous decrease in protein levels of calpain 1 and 2, indicating that calpain activity was almost absent in CKO hearts. CKO showed no cardiac phenotypes under basal conditions. Then, we subjected CKO and control mice (CTL) to pressure overload by means of transverse aortic constriction (TAC). One week after TAC, CKO showed left ventricle dilatation (LVDd, CKO 3.64 ± 0.2 mm versus CTL 2.66 ± 0.05 mm), and contractile dysfunction (FS, CKO 33.2 ± 3.9% versus CTL 47 ± 0.7%). CKO hearts took up Evans blue, a membrane-impermeant dye, within cardiomyocytes after TAC, whereas CTL hearts or sham-operated CKO hearts did not. This indicates plasma membrane was disrupted in CKO hearts in response to pressure overload. We performed membrane repair assays on isolated cardiomyocytes from CKO hearts using a two-photon laser-scanning microscope. CKO cardiomyocytes continued to take up FM1-43FX dye for at least 480 sec after disruption of a plasma membrane by laser irradiation, although CTL cardiomyocytes resealed within 250 sec. These data indicate that plasma membrane of cardiomyocytes disrupted by pressure overload failed to be resealed in CKO hearts. Thus, we conclude that calpains protect the heart from hemodynamic stresses by promoting membrane repair.