Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The ß-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2.

Bipolar disorder (BD) is a chronic mood disorder characterized by manic and depressive episodes. Dysregulation of neuroplasticity and calcium homeostasis are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that miR-499-5p regulates dendritogenesis and cognitive function by downregulating the BD risk gene CACNB2. miR-499-5p expression is increased in peripheral blood of BD patients, as well as in the hippocampus of rats which underwent juvenile social isolation. In rat hippocampal neurons, miR-499-5p impairs dendritogenesis and reduces surface expression and activity of the L-type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory ß-subunit of Cav1.2, as a direct functional target of miR-499-5p in neurons. miR-499-5p overexpression in the hippocampus in vivo induces short-term memory impairments selectively in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. In humans, miR-499-5p expression is negatively associated with gray matter volumes of the left superior temporal gyrus, a region implicated in auditory and emotional processing. We propose that stress-induced miR-499-5p overexpression contributes to dendritic impairments, deregulated calcium homeostasis, and neurocognitive dysfunction in BD.

Sex-dependent effects of Cacna1c haploinsufficiency on juvenile social play behavior and pro-social 50-kHz ultrasonic communication in rats.

As cross-disorder risk gene, CACNA1C is implicated in the etiology of all major neuropsychiatric disorders characterized by deficits in social behavior and communication and there is evidence for sex-dependent influences of single-nucleotide polymorphisms within CACNA1C on diagnosis, course, and recovery in humans. In this study, we aimed, therefore, at further exploring the role of Cacna1c in regulating behavioral phenotypes, focusing on sex-specific differences in social behavior and communication during the critical developmental period of adolescence in rats. Specifically, we compared rough-and-tumble play, concomitant emission of pro-social 50-kHz ultrasonic vocalizations, and social approach behavior in response to playback of 50-kHz ultrasonic vocalizations between constitutive heterozygous Cacna1c +/- females and wildtype Cacna1c +/+ littermate controls, and contrasted present female findings to data previously reported in males. Our results show for the first time that partial depletion of Cacna1c leads to sex-dependent alterations in social behavior and communication in rats. In females, Cacna1c haploinsufficiency led to hypermasculinization, with rough-and-tumble play behavior, in general, and pinning behavior, in particular, being even higher than in males without affecting concomitant 50-kHz ultrasonic vocalizations. In males, in contrast, rough-and-tumble play behavior was not altered, yet emission of 50-kHz ultrasonic vocalizations was diminished following partial Cacna1c depletion. The behavioral responses elicited by playback of 50-kHz ultrasonic vocalizations were reduced upon partial Cacna1c depletion in both sexes. It thus can be concluded that Cacna1c plays a prominent sex-dependent role in regulating juvenile rat social play behavior and pro-social 50-kHz ultrasonic communication with relevance to sex-specific effects seen in neuropsychiatric disorders.

Interaction of the Psychiatric Risk Gene Cacna1c With Post-weaning Social Isolation or Environmental Enrichment Does Not Affect Brain Mitochondrial Bioenergetics in Rats.

The pathophysiology of neuropsychiatric disorders involves complex interactions between genetic and environmental risk factors. Confirmed by several genome-wide association studies, Cacna1c represents one of the most robustly replicated psychiatric risk genes. Besides genetic predispositions, environmental stress such as childhood maltreatment also contributes to enhanced disease vulnerability. Both, Cacna1c gene variants and stressful life events are associated with morphological alterations in the prefrontal cortex and the hippocampus. Emerging evidence suggests impaired mitochondrial bioenergetics as a possible underlying mechanism of these regional brain abnormalities. In the present study, we simulated the interaction of psychiatric disease-relevant genetic and environmental factors in rodents to investigate their potential effect on brain mitochondrial function using a constitutive heterozygous Cacna1c rat model in combination with a four-week exposure to either post-weaning social isolation, standard housing, or social and physical environmental enrichment. Mitochondria were isolated from the prefrontal cortex and the hippocampus to evaluate their bioenergetics, membrane potential, reactive oxygen species production, and respiratory chain complex protein levels. None of these parameters were considerably affected in this particular gene-environment setting. These negative results were very robust in all tested conditions demonstrating that Cacna1c depletion did not significantly translate into altered bioenergetic characteristics. Thus, further investigations are required to determine the disease-related effects on brain mitochondria.

Long-term environmental impact on object recognition, spatial memory and reversal learning capabilities in Cacna1c-haploinsufficient rats.

Genetic and environmental influences are thought to interact in their contribution to the etiology of major neuropsychiatric disorders. One of the best replicated findings obtained in genome-wide association studies are genetic variants in the CACNA1C gene. Here, we used our constitutive heterozygous Cacna1c rat model in combination with a 4-week exposure to either post-weaning social isolation, standard housing or social and physical environmental enrichment during the critical juvenile developmental period to observe their long-term interactive effects with Cacna1c haploinsufficiency. Our study provides evidence for a gene × environment interaction, i.e. an interplay between Cacna1c haploinsufficiency and environment during juvenile development, on object recognition, spatial memory and reversal learning capabilities. Social and physical enrichment had a positive influence on Cacna1c+/- rats and Cacna1c+/+ littermate controls on spatial and reversal learning, while post-weaning social isolation negatively affected novel object recognition in both genotypes. Despite intact spatial learning and re-learning abilities in all groups, slight but consistent deficits were evident in Cacna1c+/- rats previously housed under standard conditions particularly during reversal learning but not Cacna1c+/- rats previously exposed to social and physical enrichment. Together, this supports the notion that Cacna1c interacts with the environment to shape disease vulnerability and associated alterations in cognitive functioning.

Sex-specific effects of Cacna1c haploinsufficiency on object recognition, spatial memory, and reversal learning capabilities in rats.

The CACNA1C gene is strongly implicated in the etiology of multiple major neuropsychiatric disorders, such as bipolar disorder, major depression, and schizophrenia, with cognitive deficits being a common feature. It is unclear, however, by which mechanisms CACNA1C variants advance the risk of developing neuropsychiatric disorders. This study set out to investigate cognitive functioning in a newly developed genetic Cacna1c rat model. Specifically, spatial and reversal learning, as well as object recognition memory were assessed in heterozygous Cacna1c+/- rats and compared to wildtype Cacna1c+/+ littermate controls in both sexes. Our results show that both Cacna1c+/+ and Cacna1c+/- animals were able to learn the rewarded arm configuration of a radial maze over the course of seven days. Both groups also showed reversal learning patterns indicative of intact abilities. In females, genotype differences were evident in the initial spatial learning phase, with Cacna1c+/- females showing hypo-activity and fewer mixed errors. In males, a difference was found during probe trials for both learning phases, with Cacna1c+/- rats displaying better distinction between previously baited and non-baited arms; and regarding cognitive flexibility in favor of the Cacna1c+/+ animals. All experimental groups proved to be sensitive to reward magnitude and fully able to distinguish between novel and familiar objects in the novel object recognition task. Taken together, these results indicate that Cacna1c haploinsufficiency has a minor, but positive impact on (spatial) memory functions in rats.

Cacna1c haploinsufficiency leads to pro-social 50-kHz ultrasonic communication deficits in rats.

The cross-disorder risk gene CACNA1C is strongly implicated in multiple neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder (BPD) and schizophrenia (SCZ), with deficits in social functioning being common for all major neuropsychiatric disorders. In the present study, we explored the role of Cacna1c in regulating disorder-relevant behavioral phenotypes, focusing on socio-affective communication after weaning during the critical developmental period of adolescence in rats. To this aim, we used a newly developed genetic Cacna1c rat model and applied a truly reciprocal approach for studying communication through ultrasonic vocalizations, including both sender and receiver. Our results show that a deletion of Cacna1c leads to deficits in social behavior and pro-social 50-kHz ultrasonic communication in rats. Reduced levels of 50-kHz ultrasonic vocalizations emitted during rough-and-tumble play may suggest that Cacna1c haploinsufficient rats derive less reward from playful social interactions. Besides the emission of fewer 50-kHz ultrasonic vocalizations in the sender, Cacna1c deletion reduced social approach behavior elicited by playback of 50-kHz ultrasonic vocalizations. This indicates that Cacna1c haploinsufficiency has detrimental effects on 50-kHz ultrasonic communication in both sender and receiver. Together, these data suggest that Cacna1c plays a prominent role in regulating socio-affective communication in rats with relevance for ASD, BPD and SCZ.This article has an associated First Person interview with the first author of the paper.