The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.

Functional defects in hiPSCs-derived cardiomyocytes from patients with a PLEKHM2-mutation associated with dilated cardiomyopathy and left ventricular non-compaction.

Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and ß-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient's iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.

316Engineered extracellular vesicle-mediated delivery of miR-199a-3p increases the viability of 3D-printed cardiac patches.

In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

Defining the timeline of periostin upregulation in cardiac fibrosis following acute myocardial infarction in mice.

After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2-7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.

Dock10 Regulates Cardiac Function under Neurohormonal Stress.

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.

Obstructive Sleep Apnea Syndrome In Vitro Model: Controlled Intermittent Hypoxia Stimulation of Human Stem Cells-Derived Cardiomyocytes.

Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-CMs following IH. IH induces the co-activation of inflammatory features together with cardiomyocyte alterations which are consistent with myocardial damage in OSA. This study provides an innovative approach for in vitro studies of OSA cardiovascular morbidity and supports the search for new pharmacological agents and molecular targets to improve diagnosis and treatment of patients.

The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells.

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell's morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.

Rapid Atrial Pacing Promotes Atrial Fibrillation Substrate in Unanesthetized Instrumented Rats.

AIM: The self-perpetuating nature of atrial fibrillation (AF) has been a subject of intense research in large mammalian models exposed to rapid atrial pacing (RAP). Recently, rodents are increasingly used to gain insight into the pathophysiology of AF. However, little is known regarding the effects of RAP on the atria of rats and mice. Using an implantable device for electrophysiological studies in rodents, we examined on a daily basis, the effects of continuous RAP on the developed AF substrate of unanesthetized rats and mice. METHODS AND RESULTS: Aggressive burst pacing did not induce AF at baseline in the large majority of rodents, but repeatedly induced AF episodes in rats exposed to RAP for more than 2 days. A microarray study of left atrial tissue from rats exposed to RAP for 2 days vs. control pacing identified 304 differentially expressed genes. Enrichment analysis and comparison with a dataset of atrial tissue from AF patients revealed indications of increased carbohydrate metabolism and changes in pathways that are thought to play critical roles in human AF, including TGF-beta and IL-6 signaling. Among 19 commonly affected genes in comparison with human AF, downregulation of FOXP1 and upregulation of the KCNK2 gene encoding the Kir2.1 potassium channel were conspicuous findings, suggesting NFAT activation. Further results included reduced expression of MIR-26 and MIR-101, which is in line with NFAT activation. CONCLUSION: Our results demonstrate electrophysiological evidence for AF promoting effects of RAP in rats and several molecular similarities between the effects of RAP in large and small mammalian models.

MiR-499 Responsive Lethal Construct for Removal of Human Embryonic Stem Cells after Cardiac Differentiation.

Deriving cell populations from human embryonic stem cells (hESCs) for cell-based therapy is considered a promising strategy to achieve functional cells, yet its translation to clinical practice depends on achieving fully defined differentiated cells. In this work, we generated a miRNA-responsive lethal mRNA construct that selectively induces rapid apoptosis in hESCs by expressing a mutant (S184del) Bax variant. Insertion of miR-499 target sites in the construct enabled to enrich hESC-derived cardiomyocytes (CMs) in culture. A deterministic non-linear model was developed and validated with experimental data, to predict the outcome for each treatment cycle and the number of treatment cycle repetitions required to achieve completely purified cTNT-positive cells. The enriched hESC-CMs displayed physiological sarcomere orientation, functional calcium handling and after transplantation into SCID-NOD mice did not form teratomas. The modular miRNA responsive lethal mRNA construct could be employed in additional directed differentiation protocols, by adjusting the miRNA to the specific cells of choice.

CAP2 mutation leads to impaired actin dynamics and associates with supraventricular tachycardia and dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure and excessive risk of sudden cardiac death. Around half of DCM cases are idiopathic, and genetic factors seem to play an important role. AIM: We investigated a possible genetic cause of DCM in two consanguineous children from a Bedouin family. METHODS AND RESULTS: Using exome sequencing and searching for rare homozygous variations, we identified a nucleotide change in the donor splice consensus sequence of exon 7 in CAP2 as the causative mutation. Using patient-derived fibroblasts, we demonstrated that the mutation causes skipping of exons 6 and 7. The resulting protein is missing 64 amino acids in its N-CAP domain that should prevent its correct folding. CAP2 protein level was markedly reduced without notable compensation by the homolog CAP1. However, ß-actin mRNA was elevated as demonstrated by real-time qPCR. In agreement with the essential role of CAP2 in actin filament polymerization, we demonstrate that the mutation affects the kinetics of repolymerization of actin in patient fibroblasts. CONCLUSIONS: This is the first report of a recessive deleterious mutation in CAP2 and its association with DCM in humans. The clinical phenotype recapitulates the damaging effects on the heart observed in Cap2 knockout mice including DCM and cardiac conduction disease, but not the other effects on growth, viability, wound healing and eye development. Our data underscore the importance of the proper kinetics of actin polymerization for normal function of the human heart.

Nanoparticle Delivery of miRNA-21 Mimic to Cardiac Macrophages Improves Myocardial Remodeling after Myocardial Infarction.

MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from pro-inflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA-21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21 with a potential for use to attenuate post-MI remodeling and heart failure.

Prominent differences in left ventricular performance and myocardial properties between right ventricular and left ventricular-based pacing modes in rats.

Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements (pressure-volume analysis) or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms (n = 6, p < 0.05), dispersed refractoriness between the right and left pacing sites and induced important molecular changes mainly in the early-activated septal tissue. These effects were not observed during biventricular tachypacing (n = 6). Our results demonstrate that the rat is an attractive new model to study the biological consequences of LV dyssynchrony and resynchronization.

HPA axis linkage in parent-child dyads: Effects of parent sex, autism spectrum diagnosis, and dyadic relationship behavior.

Families of preschoolers participated in two dyadic home visits, once with mother (56 dyads) and once with father (59 dyads). Each member of the dyad provided three cortisol samples and participated in several interaction tasks that were behaviorally coded. Approximately half of the children had been diagnosed with autism spectrum disorders (ASD), whereas half were typically developing (TD). In a multilevel model, father's cortisol level at each timepoint predicted child cortisol. Father-child linkage was stronger in dyads that showed less reciprocity, in which fathers showed less sensitivity, and in which children showed less self-regulation and more withdrawal. Cortisol levels were not significantly correlated in mother-child dyads, and there was a trend toward moderation by ASD diagnosis, such that linkage was greater in TD children. Mother-child linkage was stronger in dyads that showed less behavioral coordination and less sensitivity. HPA axis linkage may be stronger in less behaviorally attuned dyads.

Self-regulated compliance in preschoolers with autism spectrum disorder: The role of temperament and parental disciplinary style.

Regulatory difficulties are common in children with autism spectrum disorder. This study focused on an important aspect of self-regulation-the ability to willingly comply with frustrating demands of socialization agents, termed "self-regulated compliance." We studied compliance to parental demands in 40 preschoolers with autism spectrum disorder and 40 matched typically developing preschoolers, during separate interactions with mother and father, while engaging in two paradigms: toy pick-up and delayed gratification, which tap the "do" and "don't" aspects of self-regulated socialization at this age. Parents' disciplinary style was micro-coded from the two paradigms and child temperament was parent reported. Compared to their typically developing peers, children with autism spectrum disorder showed more noncompliance and less self-regulated compliance to parental demands and prohibitions and greater temperamental difficulties across several domains. No group differences were found in parental disciplinary style. Child self-regulated compliance was associated with parental supportive disciplinary style and with child attention focusing. Findings highlight the importance of parental supportive presence in structuring the development of socialization in children with autism spectrum disorder. Implications for parent-child emotion regulation interventions are discussed.

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.

Self- and Co-regulation of Anger and Fear in Preschoolers with Autism Spectrum Disorders: The Role of Maternal Parenting Style and Temperament.

Emotion regulation (ER) difficulties are a major concern in children with autism spectrum disorder (ASD). Maternal temperament and parenting style have significant effects on children's ER. However, these effects have not been studied in children with ASD. Forty preschoolers with ASD and their mothers and forty matched controls engaged in fear and anger ER paradigms, micro-coded for child self- and co-regulatory behaviors and parent's regulation-facilitation. Mothers' parenting style and temperament were self-reported. In the ASD group only, maternal authoritarian style predicted higher self-regulation and lower co-regulation of anger and maternal authoritative style predicted higher self-regulation of fear. Maternal temperament did not predict child's ER. Findings emphasize the importance of maternal flexible parenting style in facilitating ER among children with ASD.

Neuroendocrine and behavioral response to social rupture and repair in preschoolers with autism spectrum disorders interacting with mother and father.

BACKGROUND: Preschoolers with autism spectrum disorder (ASD) exhibit difficulties in handling social stress and utilizing efficient emotion regulation (ER) strategies to manage high arousal. While researchers called to assess ER in ASD, few studies utilized direct observations. We tested children's behavioral and cortisol response to maternal and paternal unavailability and hypothesized that children with ASD will employ less complex ER strategies and their parents would show increased regulation facilitation effort to accommodate their child's difficulties. METHODS: Forty preschoolers with ASD were matched with 40 typically developing (TD) preschoolers. Children were seen twice for identical battery with mother or father in the face-to-face-still-face paradigm, a three-episode paradigm where parent-child play (free play (FP)) is interrupted by elimination of communication (still face (SF)) followed by resuming play (reunion (RE)). Micro-coding of parent and child's social behavior and ER strategies was conducted. Parent and child's cortisol was assessed at baseline, following stress, and at recovery. RESULTS: Children with ASD exhibited the typical SF effect, indexed by an increase in negative affect and decrease in positive communications, but employed more simple regulatory behavior (self-soothing, proximity-seeking) and less complex strategies involving attention redirection and substitutive play. Their parents used more regulation-facilitation behavior, both simple and complex. All children showed initial cortisol response to novelty, which declined over time. However, maternal presence suppressed initial cortisol response in children with ASD. CONCLUSIONS: Children with ASD form typical expectations of parental availability and their parents increase effort to help repair social rupture. Among children with ASD, maternal presence and regulation facilitation provide social buffering for the child's HPA stress response in a manner similar to mammalian neonates. Results highlight the importance of assessing ER by combining direct observations and physiological measures and including fathers in empirical studies and intervention efforts for children with ASD during sensitive periods for social growth.

Mothering, fathering, and the regulation of negative and positive emotions in high-functioning preschoolers with autism spectrum disorder.

BACKGROUND: Children with autism spectrum disorder (ASD) exhibit difficulties in regulating emotions and authors have called to study the specific processes underpinning emotion regulation (ER) in ASD. Yet, little observational research examined the strategies preschoolers with ASD use to regulate negative and positive emotions in the presence of their mothers and fathers. METHODS: Forty preschoolers with ASD and 40 matched typically developing children and their mothers and fathers participated. Families were visited twice for identical battery of paradigms with mother or father. Parent-child interactions were coded for parent and child behaviors and children engaged in ER paradigms eliciting negative (fear) and positive (joy) emotions with each parent. ER paradigms were microcoded for negative and positive emotionality, ER strategies, and parent regulation facilitation. RESULTS: During free play, mothers' and fathers' sensitivity and warm discipline were comparable across groups; however, children with ASD displayed lower positive engagement and higher withdrawal. During ER paradigms, children with ASD expressed less positive emotionality overall and more negative emotionality during fear with father. Children with ASD used more simple self-regulatory strategies, particularly during fear, but expressed comparable levels of assistance seeking behavior toward mother and father in negative and positive contexts. Parents of children with ASD used less complex regulation facilitation strategies, including cognitive reappraisal and emotional reframing, and employed simple tactics, such as physical comforting to manage fear and social gaze to maintain joy. CONCLUSION: Findings describe general and parent- and emotion-specific processes of child ER and parent regulation facilitation in preschoolers with ASD. Results underscore the ability of such children to seek parental assistance during moments of high arousal and the parents' sensitive adaptation to their children's needs. Reduced positive emotionality, rather than increased negative reactivity and self-regulatory efforts, emerges as the consistent element associated with ER processes in this group.

Parent-child interaction and oxytocin production in pre-schoolers with autism spectrum disorder.

BACKGROUND: Autism spectrum disorder (ASD) is associated with genetic risk on the oxytocin system, suggesting oxytocin involvement in ASD; yet oxytocin functioning in young children with ASD is unknown. AIMS: To assess baseline oxytocin in pre-schoolers with ASD and test whether oxytocin production may be enhanced by parent-child contact. METHOD: Forty pre-schoolers with high-functioning ASD were matched with 40 typically developing controls. Two home visits included an identical 45-minute social battery once with the mother and once with the father. Four saliva oxytocin samples were collected from each parent and the child during each visit. RESULTS: Children with ASD had lower baseline oxytocin. Following 20 min of parent-child interactions, oxytocin normalised and remained high during social contact. Fifteen minutes after contact, oxytocin fell to baseline. Oxytocin correlated with parent-child social synchrony in both groups. CONCLUSIONS: Oxytocin dysfunction in ASD is observed in early childhood. The quick improvement in oxytocin production following parent-child contact underscores the malleability of the system and charts future directions for attachment-based behavioural and pharmacological interventions.