Disruption of a GATA4/Ankrd1 Signaling Axis in Cardiomyocytes Leads to Sarcomere Disarray: Implications for Anthracycline Cardiomyopathy

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy

The Interpersonal Needs Questionnaire: Statistical Considerations for Improved Clinical Application

The interpersonal theory of suicide (ITS) has accumulated empirical support; however, less research has investigated the clinical utility of ITS constructs in suicide risk assessment. The current study sought to increase the clinical utility of the Interpersonal Needs Questionnaire (INQ), an assessment of thwarted belongingness (TB) and perceived burdensomeness (PB), among 318 adult psychiatric outpatients while considering statistical methodology (i.e., multicollinearity and partialling). Results emphasized PB in the prediction of concurrent desire for death/suicide when TB was simultaneously considered. The interaction between TB and PB did not enhance prediction of concurrent desire for death/suicide. Independently, PB was a stronger predictor than TB of concurrent desire for death/suicide in the total sample and gender subsamples. Estimated probabilities of concurrent desire for death/suicide across INQ scores and preliminary INQ clinical cutoff scores are provided to enhance clinical application. These findings suggest the INQ could provide valuable information for suicide risk assessment and conceptualization

Interpersonal Risk Factors, Sexual and Gender Minority Status, and Suicidal Ideation: Is BDSM Disclosure Protective?

Suicidal ideation is elevated among individuals who engage in BDSM practices and those with sexual and gender minority (SGM) identities. There is limited research on the intersectionality of these identities and how they relate to suicidal ideation, especially within a theoretical framework of suicide risk, such as the interpersonal theory of suicide. Thus, we tested the indirect relation between BDSM disclosure and suicidal ideation through thwarted belongingness and perceived burdensomeness, as well as the moderating role of SGM identity on these indirect associations. Participants were 125 (M = 28.27 years; 64% cisgender men) individuals recruited via online BDSM-related forums who endorsed BDSM involvement and recent suicidal ideation. Results indicated significant moderated mediation, such that BDSM disclosure was indirectly negatively related to suicidal ideation through lower thwarted belongingness, but not perceived burdensomeness, among SGM individuals. This was due to the significant relation between BDSM disclosure and thwarted belongingness. There were no significant moderated mediation or indirect effects related to perceived burdensomeness. We also provide supplemental analyses with positive ideation (i.e., positive thoughts toward life) as the criterion variable. In conclusion, BDSM disclosure appears to be protective against suicidal ideation through thwarted belongingness but only for SGM individuals. This work furthers our understanding of the impact of intersecting marginalized identities on suicide risk and resilience. Implications, limitations, and future directions are further discussed

CARP knockdown in ARVMs induces sarcomere disarray.

<p><b>A:</b> ARVMs were transfected with 50 nM nonsilencing siRNA (top panel) or 50 nM CARP-siRNA (bottom panel and fixed at 48 h for immunofluorescence imaging. Cells were stained for CARP (green) myomesin (blue), and filamentous actin (red).</p

Calpain inhibition preserves titin but not CARP levels.

<p>ARVMs were treated for 24 h with 1 µM doxorubicin and cell lysates analyzed for <b>A:</b> titin in Coomassie stained agarose gels (representative of 4 independent experiments, arrowheads indicate titin degradation product), and <b>B:</b> CARP and tubulin by immunoblot. <b>C:</b> Bar graph shows corresponding quantification of CARP immunoblot analysis (% of control, n = 3) and myofibrillar disarray (% of cells scored for >50% disruption of myomesin striations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035743#s2" target="_blank">Methods</a>, n = 4–5, ∼150 cells counted per experiment). Shown are mean±SD, <i>P</i><0.05 relative to control (<b>*</b>) and Doxo (<b>†</b>).</p

CARP siRNA knockdown in ARVMs.

<p><b>A:</b> ARVMs were transfected for 24 h with increasing concentrations of CARP targeted siRNA and lysates analyzed by immunoblot for CARP and tubulin. Also shown is a time course of CARP and tubulin levels in ARVMs transfected with 50 nM CARP- siRNA. <b>B:</b> Cells were harvested at various time-points and fractionated into nuclear and cytoplasmic extracts and analyzed for CARP expression by immunoblot. CARP densitometry values were normalized to tubulin or topoisomerase and expressed as a percentage of the 0 time-point.</p

Doxorubicin inhibits CARP expression at the transcriptional level.

<p><b>A:</b> ARVMs were pretreated with 10 µg/ml cycloheximide (Cyclo), a protein synthesis inhibitor, in the presence or absence of 1 µM doxorubicin (Doxo) and cell lysates analyzed by immunoblot for CARP and actin and corresponding densitometry analysis is shown below. <b>B:</b> Comparison of CARP mRNA decay (quantified by RT-PCR) in ARVMs pretreated with 5 µg/ml actinomycin D (act D) in the presence or absence of 1 µM doxorubicin. <b>C:</b> NRVMs were transfected with a CARP promoter luciferase reporter (CARP-pGL3) and treated with increasing concentrations of doxorubicin. Cell lysates were assayed for luciferase activity and values were normalized to a promoterless control (pGL3 basic). Shown are mean±SD from 4 independent experiments. The qRT-PCR and luciferase-reporter experiments were performed in triplicate. * <i>P</i><0.05, ANOVA.</p