Ya-Ting Chang

The CaV3.2 T-Type Ca2+ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice

Voltage-gated T-type Ca2+ channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the α1H voltage-gated T-type Ca2+ channel (Cav3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload–induced hypertrophy was severely suppressed in mice deficient for Cav3.2 (Cav3.2−/−) but not in mice deficient for Cav3.1 (Cav3.1−/−). Angiotensin II–induced cardiac hypertrophy was also suppressed in Cav3.2−/− mice. Consistent with these findings, cultured neonatal myocytes isolated from Cav3.2−/− mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Cav3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Cav3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Cav3.2−/−, NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Cav3.2−/−/NFAT-Luc mice. Our results provide strong genetic evidence that Cav3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy.

Cav3.2 T-Type Ca2+ Channel-Dependent Activation of ERK in Paraventricular Thalamus Modulates Acid-Induced Chronic Muscle Pain

Treatments for chronic musculoskeletal pain, such as lower back pain, fibromyalgia, and myofascial pain syndrome, remain inadequate because of our poor understanding of the mechanisms that underlie these conditions. Although T-type Ca2+ channels (T-channels) have been implicated in peripheral and central pain sensory pathways, their role in chronic musculoskeletal pain is still unclear. Here, we show that acid-induced chronic mechanical hyperalgesia develops in Cav3.1-deficient and wild-type but not in Cav3.2-deficient male and female mice. We also show that T-channels are required for the initiation, but not maintenance, of acid-induced chronic muscle pain. Blocking T-channels using ethosuximide prevented chronic mechanical hyperalgesia in wild-type mice when administered intraperitoneally or intracerebroventricularly, but not intramuscularly or intrathecally. Furthermore, we found an acid-induced, Cav3.2 T-channel-dependent activation of ERK (extracellular signal-regulated kinase) in the anterior nucleus of paraventricular thalamus (PVA), and prevention of the ERK activation abolished the chronic mechanical hyperalgesia. Our findings suggest that Cav3.2 T-channel-dependent activation of ERK in PVA is required for the development of acid-induced chronic mechanical hyperalgesia.

Role of Extracellular Signal-Regulated Kinase in Synaptic Transmission and Plasticity of a Nociceptive Input on Capsular Central Amygdaloid Neurons in Normal and Acid-Induced Muscle Pain Mice

Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA–CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC–extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA–CeAC EPSCs, which was also dependent on the PKC–ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA–CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA–CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy

Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

Early growth response 1 is an early signal inducing Cav3.2 T-type calcium channels during cardiac hypertrophy

AIMSnThe Cav3.2 T-channel plays a pivotal role in inducing calcineurin/nuclear factor of activated T cell (NFAT) signalling during cardiac hypertrophy. Because calcineurin/NFAT signalling is induced early after pressure overload, we hypothesized that Cav3.2 is induced by an early signal. Our aim is to investigate when and how Cav3.2 is induced during cardiac hypertrophy.nnnMETHODS AND RESULTSnThe evolutionary conserved promoter Cav3.2-3500 from mouse genome was validated to express the reporter gene as endogenous Cav3.2 in cell lines and transgenic (Tg; Cav3.2-3500-Luc) mice. The early induction of luciferase in Tg mice and Cav3.2 mRNA in wild-type mice after transverse aortic banding (TAB) surgery supported our hypothesis that Cav3.2 is induced early during cardiac hypertrophy. The TAB-responding element [-81 to -41 bp upstream of the transcription start site (TSS) of mouse Cav3.2] was identified by in vivo gene transfer by injecting reporter constructs into the left ventricle followed by TAB surgery. Electrophoresis mobility shift assay and chromatin immunoprecipitation assays revealed that Egr1 bound to the TAB-responding element of Cav3.2. Egr1 level was increased with increased Cav3.2 mRNA level at 3 days after TAB. To demonstrate that Egr1 indeed regulates Cav3.2 expression after hypertrophic stimulation, knockdown of Egr1 with short hairpin RNA prevented the phenylephrine-induced up-regulation of Cav3.2 expression and cellular hypertrophy in neonatal rat ventricular myocytes (NRVMs) and H9c2 cells. Furthermore, overexpression of Cav3.2 in Egr1-knockdown cells restored the phenylephrine-induced hypertrophy.nnnCONCLUSIONnCav3.2 is induced early by Egr1 during cardiac hypertrophy and Cav3.2 is an important mediator of Egr1 in regulating cardiac hypertrophy.

Cardioprotection induced in a mouse model of neuropathic pain via anterior nucleus of paraventricular thalamus

Myocardial infarction is the leading cause of death worldwide. Restoration of blood flow rescues myocardium but also causes ischemia-reperfusion injury. Here, we show that in a mouse model of chronic neuropathic pain, ischemia-reperfusion injury following myocardial infarction is reduced, and this cardioprotection is induced via an anterior nucleus of paraventricular thalamus (PVA)-dependent parasympathetic pathway. Pharmacological inhibition of extracellular signal-regulated kinase activation in the PVA abolishes neuropathic pain-induced cardioprotection, whereas activation of PVA neurons pharmacologically, or optogenetic stimulation, is sufficient to induce cardioprotection. Furthermore, neuropathic injury and optogenetic stimulation of PVA neurons reduce the heart rate. These results suggest that the parasympathetic nerve is responsible for this unexpected cardioprotective effect of chronic neuropathic pain in mice.Various forms of preconditioning can prevent ischemic-reperfusion injury after myocardial infarction. Here, the authors show that in mice, the presence of chronic neuropathic pain can have a cardioprotective effect, and that this is dependent on neural activation in the paraventricular thalamus.

Spinal PKC/ERK signal pathway mediates hyperalgesia priming

Abstract Chronic pain can be initiated by one or more acute stimulations to sensitize neurons into the primed state. In the primed state, the basal nociceptive thresholds of the animal are normal, but, in response to another hyperalgesic stimulus, the animal develops enhanced and prolonged hyperalgesia. The exact mechanism of how primed state is formed is not completely understood. Here, we showed that spinal protein kinase C (PKC)/extracellular signal–regulated kinase (ERK) signal pathway is required for neuronal plasticity change, hyperalgesic priming formation, and the development of chronic hyperalgesia using acid-induced muscle pain model in mice. We discovered that phosphorylated extracellular signal–regulated kinase–positive neurons in the amygdala, spinal cord, and dorsal root ganglion were significantly increased after first acid injection. Inhibition of the phosphorylated extracellular signal–regulated kinase activity intrathecally, but not intracerebroventricularly or intramuscularly before first acid injection, prevented the development of chronic pain induced by second acid injection, which suggests that hyperalgesic priming signal is stored at spinal cord level. Furthermore, intrathecal injection of PKC but not protein kinase A blocker prevented the development of chronic pain, and PKC agonist was sufficient to induce prolonged hyperalgesia response after acid injection. We also found that mammalian target of rapamycin–dependent protein synthesis was required for the priming establishment. To test whether hyperalgesic priming leads to synaptic plasticity change, we recorded field excitatory postsynaptic potentials from spinal cord slices and found enhanced long-term potentiation in mice that received one acid injection. This long-term potentiation enhancement was prevented by inhibition of extracellular signal–regulated kinase. These findings show that the activation of PKC/ERK signal pathway and downstream protein synthesis is required for hyperalgesic priming and the consolidation of pain singling.Chronic pain can be initiated by one or more acute stimulations to sensitize neurons into the primed state. In the primed state, the basal nociceptive thresholds of the animal are normal, but in response to another hyperalgesic stimulus, the animal develops enhanced and prolonged hyperalgesia. The exact mechanism of how primed state is formed is not completely understood. Here we showed that spinal PKC/ERK signal pathway is required for neuronal plasticity change, hyperalgesic priming formation and the development of chronic hyperalgesia using acid-induced muscle pain (AIMP) model in mice. We discovered that pERK-positive neurons in the amygdala, spinal cord and dorsal root ganglion (DRG) were significantly increased after 1st acid injection. Inhibition of the pERK activity intrathecally, but not intracerebroventricularly or intramuscularly before 1st acid injection prevented the development of chronic pain induced by 2nd acid injection which suggests hyperalgesic priming signal is stored at spinal cord level. Furthermore, intrathecal injection of PKC but not PKA blocker prevented the development of chronic pain and PKC agonist was sufficient to induce prolonged hyperalgesia response after acid injection. We also found that mTOR-dependent protein synthesis was required for the priming establishment. To test whether hyperalgesic priming leads to synaptic plasticity change, we recorded fEPSPs from spinal cord slices and found enhanced LTP in mice received one acid injection. This LTP enhancement was prevented by inhibition of ERK. These findings show that the activation of PKC/ERK signal pathway and downstream protein synthesis is required for hyperalgesic priming and the consolidation of pain singling.

Three TF Co-expression Modules Regulate Pressure-Overload Cardiac Hypertrophy in Male Mice

Pathological cardiac hypertrophy, a dynamic remodeling process, is a major risk factor for heart failure. Although a number of key regulators and related genes have been identified, how the transcription factors (TFs) dynamically regulate the associated genes and control the morphological and electrophysiological changes during the hypertrophic process are still largely unknown. In this study, we obtained the time-course transcriptomes at five time points in four weeks from male murine hearts subjected to transverse aorta banding surgery. From a series of computational analyses, we identified three major co-expression modules of TF genes that may regulate the gene expression changes during the development of cardiac hypertrophy in mice. After pressure overload, the TF genes in Module 1 were up-regulated before the occurrence of significant morphological changes and one week later were down-regulated gradually, while those in Modules 2 and 3 took over the regulation as the heart size increased. Our analyses revealed that the TF genes up-regulated at the early stages likely initiated the cascading regulation and most of the well-known cardiac miRNAs were up-regulated at later stages for suppression. In addition, the constructed time-dependent regulatory network reveals some TFs including Egr2 as new candidate key regulators of cardiovascular-associated (CV) genes.

Author Correction: Cardioprotection induced in a mouse model of neuropathic pain via anterior nucleus of paraventricular thalamus

The original version of this Article contained an error in the affiliation of the second author, Ya-Ting Chang. The correct affiliations for Ya-Ting Chang are Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan and International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei 115, Taiwan.