Chien-Chang Chen

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.

Rebound burst firing in the reticular thalamus is not essential for pharmacological absence seizures in mice

Significance Intrinsic bursts and rhythmic burst discharges are elicited by activation of T-type Ca2+ channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to spike-and-wave discharges (SWDs) on the cortical electroencephalogram, which are the hallmarks of absence seizures. Using knockout mice for T-type Ca2+ channels that completely lack TRN bursts, however, we show that increased tonic firing in the TRN seems sufficient for drug-induced SWD generation. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction. Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca2+ channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca2+ channel, CaV3.3, was deleted (CaV3.3−/−). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3−/− mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3−/− mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

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.

Functional Evolution of Cardiac MicroRNAs in Heart Development and Functions

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression either by degrading target mRNAs or by suppressing protein translation. miRNAs have been found to be involved in many biological processes, such as development, differentiation, and growth. However, the evolution of miRNA regulatory functions and networks has not been well studied. In this study, we conducted a cross-species analysis to study the evolution of cardiac miRNAs and their regulatory functions and networks. We found that conserved cardiac miRNA target genes have maintained highly conserved cardiac functions. Additionally, most of cardiac miRNA target genes in human with annotations of cardiac functions evolved from the corresponding homologous targets, which are also involved in heart development-related functions. On the basis of these results, we investigated the functional evolution of cardiac miRNAs and presented a functional evolutionary map. From this map, we identified the evolutionary time at which the cardiac miRNAs became involved in heart development or function and found that the biological processes of heart development evolved earlier than those of heart functions, for example, heart contraction/relaxation or cardiac hypertrophy. Our study of the evolution of the cardiac miRNA regulatory networks revealed the emergence of new regulatory functional branches during evolution. Furthermore, we discovered that early evolved cardiac miRNA target genes tend to participate in the early stages of heart development. This study sheds light on the evolution of developmental features of genes regulated by cardiac miRNAs.

Behavior training reverses asymmetry in hippocampal transcriptome of the cav3.2 knockout mice.

Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.

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.

The type VI adenylyl cyclase protects cardiomyocytes from β-adrenergic stress by a PKA/STAT3-dependent pathway

BackgroundThe type VI adenylyl cyclase (AC6) is a main contributor of cAMP production in the heart. The amino acid (aa) sequence of AC6 is highly homologous to that of another major cardiac adenylyl cyclase, AC5, except for its N-terminus (AC6-N, aa 1–86). Activation of AC6, rather than AC5, produces cardioprotective effects against heart failure, while the underlying mechanism remains to be unveiled. Using an AC6-null (AC6−/−) mouse and a knockin mouse with AC6-N deletion (AC6 ΔN/ΔN), we aimed to investigate the cardioprotective mechanism of AC6 in the heart.MethodsWestern blot analysis and immunofluorescence staining were performed to determine the intracellular distribution of AC6, AC6-ΔN (a truncated AC6 lacking the first 86 amino acids), and STAT3 activation. Activities of AC6 and AC6-ΔN in the heart were assessed by cAMP assay. Apoptosis of cardiomyocytes were evaluated by the TUNEL assay and a propidium iodine-based survival assay. Fibrosis was examined by collagen staining.ResultsImmunofluorescence staining revealed that cardiac AC6 was mainly anchored on the sarcolemmal membranes, while AC6-ΔN was redistributed to the sarcoplasmic reticulum. AC6ΔN/ΔN and AC6−/− mice had more apoptotic myocytes and cardiac remodeling than WT mice in experimental models of isoproterenol (ISO)-induced myocardial injury. Adult cardiomyocytes isolated from AC6ΔN/ΔN or AC6−/− mice survived poorly after exposure to ISO, which produced no effect on WT cardiomyocytes under the condition tested. Importantly, ISO treatment induced cardiac STAT3 phosphorylation/activation in WT mice, but not in AC6ΔN/ΔN and AC6−/− mice. Pharmacological blockage of PKA-, Src-, or STAT3- pathway markedly reduced the survival of WT myocytes in the presence of ISO, but did not affect those of AC6ΔN/ΔN and AC6−/− myocytes, suggesting an important role of AC6 in mediating cardioprotective action through the activation of PKA-Src-STAT3-signaling.ConclusionsCollectively, AC6-N controls the anchorage of cardiac AC6 on the sarcolemmal membrane, which enables the coupling of AC6 with the pro-survival PKA-STAT3 pathway. Our findings may facilitate the development of novel therapies for heart failure.