Aims Conflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganized multiple-wavelet activation to organized rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism. Methods and results Optical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact fibrosis (CF), diffuse fibrosis (DiF), and patchy fibrosis (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum. Enhanced GJ coupling with rotigaptide (n = 10) progressively organized fibrillation in a concentration-dependent manner; increasing FDI (0 nM: 0.53 ± 0.04, 80 nM: 0.78 ± 0.03, P < 0.001), increasing RA-sustained VF time (0 nM: 44 ± 6%, 80 nM: 94 ± 2%, P < 0.001), and stabilized RAs (maximum rotations for an RA; 0 nM: 5.4 ± 0.5, 80 nM: 48.2 ± 12.3, P < 0.001). GJ uncoupling with carbenoxolone progressively disorganized VF; the FDI decreased (0 µM: 0.60 ± 0.05, 50 µM: 0.17 ± 0.03, P < 0.001) and RA-sustained VF time decreased (0 µM: 61 ± 9%, 50 µM: 3 ± 2%, P < 0.001). In CF, VF activity was disorganized and the RA-sustained VF time was the lowest (CF: 27 ± 7% vs. PF: 75 ± 5%, P < 0.001). Global fibrillatory organization measured by FDI was highest in PF (PF: 0.67 ± 0.05 vs. CF: 0.33 ± 0.03, P < 0.001). PF harboured the longest duration and most spatially stable RAs (patchy: 1411 ± 266 ms vs. compact: 354 ± 38 ms, P < 0.001). DiF (n = 11) exhibited an intermediately organized VF pattern, sustained by a combination of multiple-wavelets and short-lived RAs. Conclusion The degree of GJ coupling and pattern of fibrosis influences the mechanism sustaining VF. There is a continuous spectrum of organization in VF, ranging between globally organized fibrillation sustained by stable RAs and disorganized, possibly multiple-wavelet driven fibrillation with no RAs.

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H 2 O 2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H 2 O 2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H 2 O 2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H 2 O 2 levels are cytoprotective. We identified p21 Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a “redox rheostat” in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.

Background: The mechanisms sustaining myocardial fibrillation remain disputed, partly due to a lack of mapping tools that can accurately identify the mechanism with low spatial resolution clinical recordings. Granger causality (GC) analysis, an econometric tool for quantifying causal relationships between complex time-series, was developed as a novel fibrillation mapping tool and adapted to low spatial resolution sequentially acquired data. Methods: Ventricular fibrillation (VF) optical mapping was performed in Langendorff-perfused Sprague-Dawley rat hearts (n=18), where novel algorithms were developed using GC-based analysis to (1) quantify causal dependence of neighboring signals and plot GC vectors, (2) quantify global organization with the causality pairing index, a measure of neighboring causal signal pairs, and (3) localize rotational drivers (RDs) by quantifying the circular interdependence of neighboring signals with the circular interdependence value. GC-based mapping tools were optimized for low spatial resolution from downsampled optical mapping data, validated against high-resolution phase analysis and further tested in previous VF optical mapping recordings of coronary perfused donor heart left ventricular wedge preparations (n=12), and adapted for sequentially acquired intracardiac electrograms during human persistent atrial fibrillation mapping (n=16). Results: Global VF organization quantified by causality pairing index showed a negative correlation at progressively lower resolutions (50% resolution: P =0.006, R 2 =0.38, 12.5% resolution, P =0.004, R 2 =0.41) with a phase analysis derived measure of disorganization, locations occupied by phase singularities. In organized VF with high causality pairing index values, GC vector mapping characterized dominant propagating patterns and localized stable RDs, with the circular interdependence value showing a significant difference in driver versus nondriver regions (0.91±0.05 versus 0.35±0.06, P =0.0002). These findings were further confirmed in human VF. In persistent atrial fibrillation, a positive correlation was found between the causality pairing index and presence of stable RDs ( P =0.0005, R 2 =0.56). Fifty percent of patients had RDs, with a low incidence of 0.9±0.3 RDs per patient. Conclusions: GC-based fibrillation analysis can measure global fibrillation organization, characterize dominant propagating patterns, and map RDs using low spatial resolution sequentially acquired data.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mM H 2 O 2 ) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5 to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H 2 O 2 increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. Exposure of cardiac myocytes to sufficiently high levels of reactive oxygen species (ROS) 6 such as H 2 O 2 leads to their death (1-3), and this probably involves a continuum from apoptosis to necrosis, depending on the severity of the oxidative stress (4). In aerobic tissues such as the heart, the mitochondria probably represent a significant source of ROS, and increased ROS production by these organelles during hypoxia and ischemia-reperfusion injury may be particularly important in myocardial injury (5, 6). However, at lower concentrations, ROS have been reported to promote either growth of the cardiac myocyte (7) or to induce "preconditioning" (8), either of which potentially increases the ability of the cardiac myocyte to survive cytotoxic stresses. H 2 O 2 -induced oxidative stress simultaneously stimulates a number of potentially pro-apoptotic and cytoprotective signaling pathways in the whole heart or cardiac myocytes (9), and the final outcome (cell death or survival) could depend on which signaling pathway(s) predominates and endures. As shown by our microarray studies, H 2 O 2 can positively and negatively regulate global gene expression in cardiac myocytes (3, 10). One gene consistently up-regulated by H 2 O 2 in rat cardiac myocytes at toxic and nontoxic concentrations is the orthologue of transformed mouse 3T3 cell double minute 2 (Mdm2) (3, 10), a proto-oncogene (11, 12), to which we will refer as Rdm2. 7 The human orthologue will be abbreviated as HDM2. The Mdm2 protein binds to the pro-apoptotic p53 tumor suppressor transcription factor to inhibit its transactivating activity (13). Perhaps more importantly, Mdm2 is a ubiquitin-protein isopeptide ligase that ubiquitinates p53 and other proteins (13), thus promoting their proteasomal degradation. Indeed, the stability of p53 protein appears to be of major importance in controlling its abundance (13). In addition, Mdm2 may autoubiquitinate to promote its own degradation (14, 15).Regulation of Mdm2 expression is complex and involves two alternative promoters. The P1 promoter lies 5Ј to exon 1 and the P2 promoter lies within intron 1 (16). P1 primarily regulates Th...

Calcium transients. GCaMP6f EHTs were analyzed using standard methods (see Supplemental Methods), and parameters were calculated in pClamp. GCaMP6f cells were obtained courtesy of the Conklin laboratory (Gladstone Institutes,