Heart Failure Remodeling and Ventricular Arrhythmia: The Role of Altered L-type Calcium Channel Function in the Development of Lethal Arrhythmias

Heart failure (HF) is one of the most common causes of morbidity and mortality worldwide. Although many patients suffering from HF die from sudden cardiac death caused by arrhythmias, the mechanism linking HF remodeling to an increased arrhythmogenic propensity remains incomplete. Independently of the etiology of the disease, HF is typically characterized by a progressive loss of transverse tubule (T-tubule) domains, which leads to an altered distribution of L-type Calcium channels (LTCCs). Ischemic cardiomyopathy (ICM) is usually accompanied by an increase in LTCC open probability (Po) in the T-tubules which depends on the activity of protein kinase A (PKA). In dilated cardiomyopathy (DCM) on the other hand, the increased LTCC Po on the non-T-tubule sarcolemma results from enhanced calcium-calmodulin kinase II (CaMKII) modulation. Microdomain degradation also causes the disruption of the β2 adrenergic receptor (β2AR) and phosphodiesterase (PDE) signaling localizations, normally confined to the dyadic space. The goal of this study was to analyze how these subcellular changes affect the function of LTCCs and lead to the emergence of ventricular cell-level triggers of arrhythmias. Furthermore, we aimed to compare how the two different pathways lead to different phenotypes in ICM vs. DCM. Using computational modeling, we analyzed the behavior of the LTCC current (ICaL) under basal and sympathetic stimulation and its effect on cellular action potentials (APs). Our results showed that channels redistributed from the T-tubular membrane to the bulk of the sarcolemma displayed an altered function in their new, non-native signaling domain. The changes in LTCC current led to the development of early afterdepolarizations (EADs) in both types of HF, and triggered reentrant arrhythmias only in the DCM model. Thus, our work shows that altered LTCC function is a potential cause for the emergence of cell-level triggers of arrhythmia, and that CaMKII, β2ARs and PDEs present useful therapeutic targets for treatment of HF and prevention of sudden cardiac death

Long-range orbitofrontal and amygdala axons show divergent patterns of maturation in the frontal cortex across adolescence.

The adolescent transition from juvenile to adult is marked by anatomical and functional remodeling of brain networks. Currently, the cellular and synaptic level changes underlying the adolescent transition are only coarsely understood. Here, we use two-photon imaging to make time-lapse observations of long-range axons that innervate the frontal cortex in the living brain. We labeled cells in the orbitofrontal cortex (OFC) and basolateral amygdala (BLA) and imaged their axonal afferents to the dorsomedial prefrontal cortex (dmPFC). We also imaged the apical dendrites of dmPFC pyramidal neurons. Images were taken daily in separate cohorts of juvenile (P24-P28) and young adult mice (P64-P68), ages where we have previously discovered differences in dmPFC dependent decision-making. Dendritic spines were pruned across this peri-adolescent period, while BLA and OFC afferents followed alternate developmental trajectories. OFC boutons showed no decrease in density, but did show a decrease in daily bouton gain and loss with age. BLA axons showed an increase in both bouton density and daily bouton gain at the later age, suggesting a delayed window of enhanced plasticity. Our findings reveal projection specific maturation of synaptic structures within a single frontal region and suggest that stabilization is a more general characteristic of maturation than pruning

Degradation of T-Tubular Microdomains and Altered cAMP Compartmentation Lead to Emergence of Arrhythmogenic Triggers in Heart Failure Myocytes: An in silico Study

Heart failure (HF) is one of the most common causes of morbidity and mortality worldwide. Although many patients suffering from HF die from sudden cardiac death caused by arrhythmias, the mechanism linking HF remodeling to an increased arrhythmogenic propensity remains incomplete. HF is typically characterized by a progressive loss of transverse tubule (T-tubule) domains, which leads to an altered distribution of L-type calcium channels (LTCCs). Microdomain degradation also causes the disruption of the β2 adrenergic receptor (β2AR) and phosphodiesterase (PDE) signaling localization, normally confined to the dyadic space. The goal of this study was to analyze how these subcellular changes affect the function of LTCCs and lead to the emergence of ventricular cell-level triggers of arrhythmias. To accomplish this, we developed a novel computational model of a human ventricular HF myocyte in which LTCCs were divided into six different populations, based on their location and signaling environment they experience. To do so, we included T-tubular microdomain remodeling which led to a subset of LTCCs to be redistributed from the T-tubular to the surface membrane and allowed for different levels of phosphorylation of LTCCs by PKA, based on the presence of β2ARs and PDEs. The model was used to study the behavior of the LTCC current (ICaL) under basal and sympathetic stimulation and its effect on cellular action potential. Our results showed that channels redistributed from the T-tubular membrane to the bulk of the sarcolemma displayed an altered function in their new, non-native signaling domain. Incomplete calcium dependent inactivation, which resulted in a longer-lasting and larger-in-magnitude LTCC current, was observed when we decoupled LTCCs from ryanodine receptors and removed them from the dyadic space. The magnitude of the LTCC current, especially in the surface sarcolemma, was also increased via phosphorylation by the redistributed β2ARs and PDEs. These changes in LTCC current led to the development of early afterdepolarizations. Thus, our study shows that altered LTCC function is a potential cause for the emergence of cell-level triggers of arrhythmia, and that β2ARs and PDEs present useful therapeutic targets for treatment of HF and prevention of sudden cardiac death

Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase

Energy Restriction during Childhood and Early Adulthood and Ovarian Cancer Risk

Dietary energy restriction may protect against cancer. In parts of the Netherlands, mostly in larger cities, periods of chronically impaired nutrition and even severe famine (Hunger Winter 1944–1945) existed during the 1930s and World War II (1940–1945). We studied the association between energy restriction during childhood and early adulthood on the risk of ovarian cancer later in life. In 1986, the Netherlands Cohort Study was initiated. A self-administered questionnaire on dietary habits and other cancer risk factors was completed by 62,573 women aged 55–69 years at baseline. Follow-up for cancer was established by record linkage to the Netherlands Cancer Registry. After 16.3 years of follow-up, 364 invasive epithelial ovarian cancer cases and 2220 subcohort members (sampled from the total cohort directly after baseline) with complete information confounders were available for case-cohort analyses. In multivariable analysis, ovarian cancer risk was lower for participants with an unemployed father during the 1930s (Hazard Ratio (HR), 0.70; 95% Confidence Interval (CI), 0.47–1.06) compared to participants with an employed father as well as for participants living in a city during World War II (HR, 0.69; 95% CI, 0.54–0.90) compared to participants living in the country-side. Residence in a Western City during the famine (Hunger Winter) was not associated with a decreased risk. Our results show a relation between proxy variables for modest energy restriction over a longer period of time during childhood or early adulthood and a reduced ovarian cancer risk

The role of the basolateral amygdala in flexible behavior

Flexible behavior in response to a changing environment is required for survival, and is the physical manifestation of communication among the prefrontal cortex and different subcortical systems. The basolateral amygdala (BLA), a crucial component of the reward circuitry mediating behavioral choices, is thought to encode the value of discrete cues in the environment. In particular, the BLA is thought to be required in updating the current cue-reward association. Previous studies have shown that the BLA is involved in flexible behavior, demonstrated using reversal learning tasks, where the subject has to inhibit responding to a previously rewarded cue and start responding to a previously unrewarded cue. However, the precise role of the BLA's contribution to inhibiting responding to a previously rewarded cue or learning to respond to a previously unrewarded cue remains unclear. Furthermore, it is not known if the BLA contributes to higher order rule learning that would occur over several reversals. Therefore, we examined the role of the BLA in a 4-choice odor discrimination reversal task across several reversal phases. We found that excitoxic lesions of the BLA impaired reversal learning compared to sham-lesioned mice. BLA-lesioned mice returned more frequently to the previously rewarded odor, indicating that the BLA primarily plays a role in inhibiting response to a previously rewarded cue instead of learning a new cue-reward association. Furthermore, since the BLA acts in concert with the prefrontal cortex to produce flexible behavior, understanding the dynamics of structural connectivity from the BLA to higher-order cortical regions involved in reversal learning is the first step to elucidate how amygdalocortical networks function. To develop our understanding of the development of these critical reward circuit connections, we used two-photon in vivo imaging to compare structural dynamics of axonal boutons of cortical-projecting BLA neurons in adult mice. We found that amygdala axons terminating in the dorsomedial prefrontal cortex are cortical-like in terms of their structural plasticity, and they are highly similar to orbitofrontal axons terminating in the same region. The work in this thesis elucidates a more precise role of the BLA in guiding flexible behavior, while also for the first time defining the baseline structural plasticity of axons from a limbic region projecting to a fronto-cortical region

Assessment of tumor hypoxia in spontaneous canine tumors after treatment with OMX, a novel H-NOX oxygen carrier, with [18F]FMISO PET/CT

BackgroundHypoxia is a detrimental factor in solid tumors, leading to aggressiveness and therapy resistance. OMX, a tunable oxygen carrier from the heme nitric oxide/oxygen-binding (H-NOX) protein family, has the potential to reduce tumor hypoxia. [18F]Fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) is the most widely used and investigated method for non-invasive imaging of tumor hypoxia. In this study, we used [18F]FMISO PET/CT (computed tomography) to assess the effect of OMX on tumor hypoxia in spontaneous canine tumors.ResultsThirteen canine patients with various tumors (n = 14) were randomly divided into blocks of two, with the treatment groups alternating between receiving intratumoral (IT) OMX injection (OMX IT group) and intravenous (IV) OMX injection (OMX IV group). Tumors were regarded as hypoxic if maximum tumor-to-muscle ratio (TMRmax) was greater than 1.4. In addition, hypoxic volume (HV) was defined as the region with tumor-to-muscle ratio greater than 1.4 on [18F]FMISO PET images. Hypoxia was detected in 6/7 tumors in the OMX IT group and 5/7 tumors in the OMX IV injection group. Although there was no significant difference in baseline hypoxia between the OMX IT and IV groups, the two groups showed different responses to OMX. In the OMX IV group, hypoxic tumors (n = 5) exhibited significant reductions in tumor hypoxia, as indicated by decreased TMRmax and HV in [18F]FMISO PET imaging after treatment. In contrast, hypoxic tumors in the OMX IT group (n = 6) displayed a significant increase in [18F]FMISO uptake and variable changes in TMRmax and HV.Conclusions[18F]FMISO PET/CT imaging presents a promising non-invasive procedure for monitoring tumor hypoxia and assessing the efficacy of hypoxia-modulating therapies in canine patients. OMX has shown promising outcomes in reducing tumor hypoxia, especially when administered intravenously, as evident from reductions in both TMRmax and HV in [18F]FMISO PET imaging