Excitation-contraction Coupling Research Articles

Arrhythmogenic calmodulin variants D131E and Q135P disrupt interaction with the L-type voltage-gated Ca2+ channel (Cav1.2) and reduce Ca2+-dependent inactivation.

Long QT syndrome (LQTS) and catecholaminergic polymorphism ventricular tachycardia (CPVT) are inherited cardiac disorders often caused by mutations in ion channels. These arrhythmia syndromes have recently been associated with calmodulin (CaM) variants. Here, we investigate the impact of the arrhythmogenic variants D131E and Q135P on CaM's structure-function relationship. Our study focuses on the L-type calcium channel Cav1.2, a crucial component of the ventricular action potential and excitation-contraction coupling. We used circular dichroism (CD), 1H-15N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca2+ and interaction of Ca2+/CaM with Cav1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Cav1.2 activity was determined using HEK293-Cav1.2 cells (B'SYS) and whole-cell patch-clamp electrophysiology. Using a combination of protein biophysics and structural biology, we show that the disease-associated mutations D131E and Q135P mutations alter apo/CaM structure and stability. In the Ca2+-bound state, D131E and Q135P exhibited reduced Ca2+ binding affinity, significant structural changes, and altered interaction with Cav1.2 domains (increased affinity for Cav1.2-IQ and decreased affinity for Cav1.2-NSCaTE). We show that the mutations dramatically impair Ca2+-dependent inactivation (CDI) of Cav1.2, which would contribute to abnormal Ca2+ influx, leading to disrupted Ca2+ handling, characteristic of cardiac arrhythmia syndromes. These findings provide insights into the molecular mechanisms behind arrhythmia caused by calmodulin mutations, contributing to our understanding of cardiac syndromes at a molecular and cellular level.

Regulation of calcium homeostasis in endoplasmic reticulum–mitochondria crosstalk: implications for skeletal muscle atrophy

This review comprehensively explores the critical role of calcium as an essential small-molecule biomessenger in skeletal muscle function. Calcium is vital for both regulating muscle excitation–contraction coupling and for the development, maintenance, and regeneration of muscle cells. The orchestrated release of calcium from the endoplasmic reticulum (ER) is mediated by receptors such as the ryanodine receptor (RYR) and inositol 1,4,5-trisphosphate receptor (IP3R), which is crucial for skeletal muscle contraction. The sarcoendoplasmic reticulum calcium ATPase (SERCA) pump plays a key role in recapturing calcium, enabling the muscle to return to a relaxed state. A pivotal aspect of calcium homeostasis involves the dynamic interaction between mitochondria and the ER. This interaction includes local calcium signaling facilitated by RYRs and a “quasi-synaptic” mechanism formed by the IP3R-Grp75-VDAC/MCU axis, allowing rapid calcium uptake by mitochondria with minimal interference at the cytoplasmic level. Disruption of calcium transport can lead to mitochondrial calcium overload, triggering the opening of the mitochondrial permeability transition pore and subsequent release of reactive oxygen species and cytochrome C, ultimately resulting in muscle damage and atrophy. This review explores the complex relationship between the ER and mitochondria and how these organelles regulate calcium levels in skeletal muscle, aiming to provide valuable perspectives for future research on the pathogenesis of muscle diseases and the development of prevention strategies.

A new method to evaluate staircase phenomenon in skeletal muscle using piezoelectric sensor.

The staircase phenomenon, which refers to the increases in the force of contraction with repetitive stimulation of the muscle, has been studied for many years, but the method is difficult and not widely used. Our objective was to evaluate the staircase phenomenon in skeletal muscle using a piezoelectric sensor. Thirty-five subjects without neuromuscular diseases (normal controls), 11 patients with Becker muscular dystrophy (BMD), and 19 patients with myotonic dystrophy type 1 (MyD) were studied. Compound muscle action potential (CMAP) and movement-related potential (MRP) waveforms were recorded using piezoelectric sensors during repetitive stimulation of the median nerve, and the amplitudes and durations were measured. Excitation-contraction (E-C) coupling time (ECCT) was calculated from the difference between onset latencies of CMAP and MRP. In normal controls, MRP amplitude ratio (relative to baseline) increased significantly with increase in stimulation duration and with increase in stimulation frequency. In BMD and MyD, however, MRP amplitude ratio did not change significantly with increase in stimulation duration. Especially, in MyD, there was no change in MRP amplitude ratio with increase in frequency. Staircase phenomenon abnormalities can be evaluated easily using piezoelectric sensors, indicating their potential utility for evaluating E-C coupling impairment in myopathies. Piezoelectric sensors may be a useful tool to evaluate staircase phenomenon in skeletal muscle.

Role of the CaV1.2 distal carboxy terminus in the regulation of L-type current

ABSTRACT L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVβ2b. The CaV1.2 channel’s C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels’ basal activity in cardiomyocytes.

Reduced voltage-activated Ca2+ release flux in muscle fibers from a rat model of Duchenne dystrophy

The potential pathogenic role of disturbed Ca2+ homeostasis in Duchenne muscular dystrophy (DMD) remains a complex, unsettled issue. We used muscle fibers isolated from 3-mo-old DMDmdx rats to further investigate the case. Most DMDmdx fibers exhibited no sign of trophic or morphology distinction as compared with WT fibers and mitochondria and t-tubule membrane networks also showed no stringent discrepancy. Under voltage clamp, values for holding current were similar in the two groups, whereas values for capacitance were larger in DMDmdx fibers, suggestive of enhanced amount of t-tubule membrane. The Ca2+ current density across the channel carried by the EC coupling voltage sensor (CaV1.1) was unchanged. The maximum rate of voltage-activated sarcoplasmic reticulum (SR) Ca2+ release was reduced by 25% in the DMDmdx fibers, with no change in voltage dependency. Imaging resting Ca2+ revealed rare spontaneous local SR Ca2+ release events with no sign of elevated activity in DMDmdx fibers. Under current clamp, DMDmdx fibers generated similar trains of action potentials as WT fibers. Results suggest that reduced peak amplitude of SR Ca2+ release is an inherent feature of this DMD model, likely contributing to muscle weakness. This occurs despite a preserved amount of releasable Ca2+ and with no change in excitability, CaV1.1 channel activity, and SR Ca2+ release at rest. Although we cannot exclude that fibers from the 3-mo-old animals do not yet display a fully developed disease phenotype, results provide limited support for pathomechanistic concepts frequently associated with DMD such as membrane fragility, excessive Ca2+ entry, or enhanced SR Ca2+ leak.

Micellized Naringenin Augments Hemodynamics After Myocardial Infarction by Suppressing Tubulin Detyrosination

Impaired contractility after myocardial infarction (MI) causes cardiogenic shock. MARK4 activity impairs contractility post-MI by increasing α-tubulin detyrosination. We assessed the impact of naringenin, a small-molecule MARK4 inhibitor, on contractility post-MI. Naringenin (Nar) was encapsulated in PEG-PCL to augment bioavailability. Wistar rats were randomized to receive either MI + micellized naringenin (0.3 mg/kg) [MI-NarMic], MI + naringenin (0.3 mg/kg) in 1% DMSO [MI-NarDMSO], MI + empty micelle [MI-Mic], MI alone [MI-Untreated], or no MI [Sham]. MI was induced via left anterior descending artery ligation. Invasive hemodynamics with pressure–volume catheterization, cardiomyocyte contractility, and ventricular protein abundance were assessed one day post-MI. A total of 45 rats underwent hemodynamic assessment. MI-NarMic rats demonstrated decreased α-tubulin detyrosination relative to MI-Untreated rats (p < 0.05). Myocytes isolated from peri-infarct tissue had increased contraction and relaxation velocities in MI-NarMic versus MI-Untreated rats (both p < 0.0001). MI-NarMic rats had higher ejection fractions than MI-Mic and MI-Untreated rats (63 ± 3% v. 48 ± 5% vs. 39 ± 4%, p < 0.05) and similar levels to Sham (61 ± 1%, p = 0.97) and MI-NarDMSO (54 ± 5%) rats (p > 0.05). MI-Nar rats had greater stroke work and lower end-diastolic pressure and tau than MI-Untreated rats (all p < 0.05). Micellized naringenin is a translatable agent with the potential to rescue hemodynamics post-MI by inhibiting MARK4 and mitigating myocardial α-tubulin detyrosination.

Proteomic and metabolomic analyses of the human adult myocardium reveal ventricle-specific regulation in end-stage cardiomyopathies

The left and right ventricles of the human heart are functionally and developmentally distinct such that genetic or acquired insults can cause dysfunction in one or both ventricles resulting in heart failure. To better understand ventricle-specific molecular changes influencing heart failure development, we first performed unbiased quantitative mass spectrometry on pre-mortem non-diseased human myocardium to compare the metabolome and proteome between the normal left and right ventricles. Constituents of gluconeogenesis, glycolysis, lipogenesis, lipolysis, fatty acid catabolism, the citrate cycle and oxidative phosphorylation were down-regulated in the left ventricle, while glycogenesis, pyruvate and ketone metabolism were up-regulated. Inter-ventricular significance of these metabolic pathways was then found to be diminished within end-stage dilated cardiomyopathy and ischaemic cardiomyopathy, while heart failure-associated pathways were increased in the left ventricle relative to the right within ischaemic cardiomyopathy, such as fluid sheer-stress, increased glutamine-glutamate ratio, and down-regulation of contractile proteins, indicating a left ventricular pathological bias.

Comparative analysis of biomechanical characteristics of knee flexion and extension muscles in volleyball physical training

This study aimed to analyze the biomechanical characteristics of knee flexion and extension muscles in volleyball physical training from the cellular and molecular biomechanics aspect. Multiple training modes like strength, elasticity, and comprehensive training were chosen to systematically evaluate relevant characteristics of these muscles among volleyball players. Advanced devices such as 3D motion capture systems, ground reaction force platforms, and electromyography equipment were used to gather precise biomechanical data. At the cellular and molecular level, different trainings impact muscle cells differently. For example, strength training might enhance the synthesis of contractile proteins within cells, while elasticity training could influence the elasticity-related molecular structures. With multidimensional data analysis, the effects of various training modes were compared. The comprehensive training group had a kinematic flexion extension angle of 528.27º ± 11.49º, an angular velocity of 135.52º ± 5.97º, and an angular acceleration of 3177.02º ± 116.88º, performing best. This could be due to its comprehensive influence on cellular and molecular processes in muscles, promoting better coordination and force generation. This article offers a theoretical basis for volleyball players to create scientific training plans and gives practical tips for coaches and athletes to optimize programs and prevent injuries. By focusing on cellular and molecular biomechanics, it fills research gaps and helps boost the development of biomechanics in volleyball physical training.

Neddylation drives myofibrillogenesis in the developing heart.

Neddylation is a highly conserved post-translational modification that plays critical roles in various cellular processes through the modulation of cullins and non-cullin substrates. While neddylation is known to be essential for embryonic development, tumor growth, and organogenesis of different tissues, its role in cardiogenesis remains unexplored. Here, we investigated the role of neddylation in early cardiac development by deleting the gene encoding a regulatory subunit of the NEDD8-specific E1 activating enzyme, Nae1, globally and in a heart-specific fashion via Nkx2-5Cre. Global deletion of Nae1 in mice led to embryonic lethality before embryonic day (E) 8.5, whereas cardiac-specific NAE1 knockout mice died at around E12.5 with pronounced cardiac effusion and peripheral hemorrhage, characteristic of cardiac failure. Histological analysis revealed significant thinning of the compact myocardium and reduced trabeculae in mutant hearts, which were accompanied by reduced cardiomyocyte proliferation. Unbiased transcriptomic analysis identified perturbations in cardiomyocyte proliferation and myofibril architecture in mutant hearts. Subsequent analysis showed that loss of NAE1 disrupted sarcomere assembly dysregulated the expression of several important contractile proteins, and impaired mitochondrial function in the developing heart, which was accompanied by downregulation of key cardiac transcription factors including NKX2-5 and SRF. Collectively, our findings demonstrate the essential role of neddylation in cardiogenesis at least in part by driving cardiomyocyte proliferation and myofibrillogenesis.

Inhibition of the upregulated phosphodiesterase 4D isoforms improves SERCA2a function in diabetic cardiomyopathy.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) is impaired in heart failure. Phosphodiesterases (PDEs) are implicated in the modulation of local cAMP signals and protein kinase A (PKA) activity essential for cardiac function. We characterise PDE isoforms that underlie decreased activities of SERCA2a and reduced cardiac contractile function in diabetic cardiomyopathy. Wild type mice were fed with either normal chow or a high-fat diet (HFD). Cardiomyocytes were isolated for excitation-contraction coupling (ECC), fluorescence resonant energy transfer PKA biosensor and proximity ligation assays. The upregulated PDE4D3 and PDE4D9 isoforms in HFD cardiomyocytes specifically bound to SERCA2a but not ryanodine receptor 2 (RyR2) on the sarcoplasmic reticulum (SR). The increased association of PDE4D isoforms with SERCA2a in HFD cardiomyocytes led to reduced local PKA activities and phosphorylation of phospholamban (PLB) but minimally effected the PKA activities and phosphorylation of RyR2. These changes correlate with slower calcium decay tau in the SR and attenuation of ECC in HFD cardiomyocytes. Selective inhibition of PDE4D3 or PDE4D9 restored PKA activities and phosphorylation of PLB at the SERCA2a complex, recovered calcium decay tau, and increased ECC in HFD cardiomyocytes. Therapies with PDE4 inhibitor roflumilast, PDE4D inhibitor BPN14770 or genetical deletion of PDE4D restored PKA phosphorylation of PLB and cardiac contractile function. The current study identifies upregulation of specific PDE4D isoforms that selectively inhibit SERCA2a function in HFD-induced cardiomyopathy, indicating that this remodelling can be targeted to restore cardiac contractility in diabetic cardiomyopathy.

Identification of Novel Nexilin Splice Variants in Mouse and Human Tissues.

There is no doubt that the proper development of the heart is important for its correct function, in addition, maturation processes of the heart are crucial as well. The actin-binding protein nexilin seems to take over central roles in the latter processes, as nexilin-deficient mice are phenotypically inconspicuous at birth but die within short time thereafter. Recently, it has been proposed that nexilin plays a role in the formation and function of transverse tubules (T-tubules), which are essential for excitation-contraction coupling in the hearts of mature animals. Although it has long been known that nexilin is subjected to alternative splicing, a molecular characterization of the respective isoforms is not yet available. Here, we describe novel nexilin splice variants and analyze their expression in tissues of mice and humans. Interestingly, nexilin isoforms segregate to myocyte- and epithelial-specific isoforms. Moreover, heart-specific isoforms of nexilin localize differently between atria and ventricles and are also expressed in the endothelial cells of blood vessels. Further, we narrowed down the critical exons in the actin-binding domains 1 and 2 (ABD1/2), and observed different self-interaction properties by recombinant protein interaction studies. Our results emphasize the diverse tissue and subcellular distribution of the individual nexilin isoforms and point to the importance of taking a closer look at the particular nexilin isoforms investigated.

The sodium/glucose cotransporter 2 inhibitor Empagliflozin inhibits long QT 3 late sodium currents in a mutation specific manner

BackgroundSodium/glucose cotransporter 2 inhibitors (SGLT2is) like empagliflozin have demonstrated cardioprotective effects in patients with or without diabetes. SGLT2is have been shown to selectively inhibit the late component of cardiac sodium current (late INa). Induction of late INa is the primary mechanism in the pathophysiology of congenital long QT syndrome type 3 (LQT3) gain-of-function mutations in the SCN5A gene encoding Nav1.5. We investigated empagliflozin's effect on late INa in thirteen known LQT3 mutations located in distinct regions of the channel. MethodsThe whole-cell patch-clamp technique was used to investigate the effect of empagliflozin on late INa in recombinantly expressed Nav1.5 channels containing different LQT3 mutations. Molecular modeling of human Nav1.5 and simulations in a mathematical model of human ventricular myocytes were used to extrapolate our experimental results to excitation-contraction coupling. ResultsEmpagliflozin selectively inhibited late INa in LQT3 mutations in the inactivation gate region of Nav1.5, without affecting peak current or channel kinetics. In contrast, empagliflozin inhibited both peak and late INa in mutations in the S4 voltage-sensing regions, altered channel gating, and slowed recovery from inactivation. Empagliflozin had no effect on late/peak INa or channel kinetics in channels with mutations in the putative empagliflozin binding region. Simulation results predict that empagliflozin may have a desirable therapeutic effect in LQT3 mutations in the inactivation gate region. ConclusionsEmpagliflozin selectively inhibits late INa, without affecting channel kinetics, in LQT3 mutations in the inactivation gate region. Empagliflozin may thus be a promising precision medicine approach for patients with specific LQT3 mutations.

The foundation of excitation-contraction coupling in skeletal muscle: communication between the transverse tubules and sarcoplasmic reticulum.

The expression excitation-contraction (EC) coupling in skeletal muscle was coined in 1952 (Sandow A. Yale J Biol Med 25: 176-201, 1952). The term evolved narrowly to include only the processes at the triad that intervene between depolarization of the transverse tubular (T-tubular) membrane and Ca2+ release from the sarcoplasmic reticulum (SR). From 1970 to 1988, the foundation of EC coupling was elucidated. The channel through which Ca2+ was released during activation was located in the SR by its specific binding to the plant insecticide ryanodine. This channel was called the ryanodine receptor (RyR). The RyR contained four subunits that together constituted the "SR foot" structure that traversed the gap between the SR and the T-tubular membrane. Ca2+ channels, also called dihydropyridine receptors (DHPRs), were located in the T-tubular membrane at the triadic junction and shown to be essential for EC coupling. There was a precise relationship between the two channels. Four DHPRs, organized as tetrads, were superimposed on alternate RyRs. This structure was consistent with the proposal that EC coupling was mediated via a movement of intramembrane charge in the T-tubular system. The speculation was that the DHPR acted as a voltage sensor transferring information to the RyRs of the SR by protein-protein interaction causing the release of Ca2+ from the SR. A great deal of progress was made by 1988 toward understanding EC coupling. However, the ultimate question of how voltage sensing is coupled to the opening of the SR Ca2+ release channel remains unresolved.NEW & NOTEWORTHY The least understood part of the series of events in excitation-contraction coupling in skeletal muscle was how information was transmitted from the transverse tubules to the sarcoplasmic (SR) and how Ca2+ was released from the SR. Through an explosion of technical approaches including physiological, biochemical, structural, pharmacological, and molecular genetics, much was discovered between 1970 and 1988. By the end of 1988, the foundation of EC coupling in skeletal muscle was established.

Adaptation of Left Ventricular Function and Myocardial Microstructure in Fetuses with Right Ventricular Hypoplasia.

This study aimed to evaluate changes in left ventricular (LV) function and myocardial microstructure in fetuses with right ventricular hypoplasia (RVH) using two-dimensional speckle tracking echocardiography (2D-STE), diffusion tensor cardiovascular magnetic resonance imaging (DT-CMR) and proteomics analysis. 51 singleton fetuses diagnosed with RVH and 51 normal fetuses were retrospectively included. LV global longitudinal strain (GLS) and global circumferential strain (GCS) were acquired by 2D-STE. Fraction anisotropy (FA), mean diffusivity (MD) and helix angle were measured using DT-CMR in four fetal specimens with RVH and three normal fetal specimens. Bioinformatics analysis was performed for differentially expressed proteins between RVH and normal specimens. In RVH fetuses, LVGLS and regional longitudinal strain were significantly lower than in controls (p<0.001), whereas LV sphericity index and LVGCS were increased. In RVH fetuses, FA was higher in middle and apical segments than in normal fetuses (p<0.001). LV MD was reduced in all the segments (p<0.001). Circumferentially oriented myocytes and left-handed oriented myocytes were increased, but right-handed orientated myocytes were decreased (p<0.001). Using proteomics, 95 myocardial proteins differed with upregulation of 66 and downregulation in RVH, hearts including myocardial contractile fibrillar proteins and cell membrane protein complexes. In fetal RVH, the left ventricle demonstrates altered function with reduced longitudinal but augmented circumferential strain which may support its need to augment its preload and consequent cardiac output. Decreased right-handed and increased circumferentially oriented myocytes may contribute to this adaptation. The left ventricle in fetal RVH also demonstrates a differential expression of various myocardial proteins.

IL-1β-induced pericyte dysfunction with a secretory phenotype exacerbates retinal microenvironment inflammation via Hes1/STAT3 signaling pathway

Retinal pericytes are mural cells surrounding capillaries to maintain the integrity of blood-retina barrier and regulate vascular behaviors. Pericyte loss has been considered as the hallmark of diabetic retinopathy (DR), which is a major complication of diabetes and the leading cause of blindness in adults. However, the precise function of pericytes in regulating the retinal microenvironment and the underlying mechanism remains largely unknown. In this study, we observed a secretory phenotype of pericytes with elevated inflammatory cytokines in response to Interleukin-1β (IL-1β), a canonical inflammatory cytokine which significantly increases during the initial stages of diabetic retinopathy. This phenotype is also accompanied by reduced expression of adherent junction proteins and contractile proteins. Paracrine cytokines derived from pericytes further induce the chemotaxis of microglia cells and trigger detrimental changes in endothelial cells, including reduced expression of tight junction protein Occludin and increased apoptosis. Mechanically, the secretion potential in pericytes is partially mediated by Hes1/STAT3 signaling pathway. Moreover, co-injection of stattic, an inhibitor targeting STAT3 activation, could effectively attenuate IL-1β-induced retinal inflammation and microglial activation in retina tissues. Collectively, these findings demonstrate the potential of retinal pericytes as an initial inflammatory sensor prior to their anatomical pathological loss, via undergoing phenotypic changes and secreting paracrine factors to amplify local inflammation and damage endothelial cells in vitro. Furthermore, inhibition of STAT3 activation by inhibitors significantly ameliorates IL-1β-induced retinal inflammation, suggesting STAT3 in retinal pericytes as a promising target for alleviating DR and other IL-1β-induced ocular diseases.

Role of stretch-activated channels in light-generated action potentials mediated by an intramembrane molecular photoswitch

BackgroundThe use of light to control the activity of living cells is a promising approach in cardiac research due to its unparalleled spatio-temporal selectivity and minimal invasiveness. Ziapin2, a newly synthesized azobenzene compound, has recently been reported as an efficient tool for light-driven modulation of excitation-contraction coupling (ECC) in human-induced pluripotent stem cells–derived cardiomyocytes. However, the exact biophysical mechanism of this process remains incompletely understood.MethodsTo address this, we performed a detailed electrophysiological characterization in a more mature cardiac model, specifically adult mouse ventricular myocytes (AMVMs).ResultsOur in vitro results demonstrate that Ziapin2 can photomodulate cardiac ECC in mature AMVMs without affecting the main transporters and receptors located within the sarcolemma. We established a connection between Ziapin2-induced membrane thickness modulation and light-generated action potentials by showcasing the pivotal role of stretch-activated channels (SACs). Notably, our experimental findings, through pharmacological blockade, suggest that non-selective SACs might serve as the biological culprit responsible for the effect.ConclusionsTaken together, these findings elucidate the intricacies of Ziapin2-mediated photostimulation mechanism and open new perspectives for its application in cardiac research.

Loss of Cdc42 causes abnormal optic cup morphogenesis and microphthalmia in mouse.

Congenital ocular malformations originate from defective morphogenesis during early eye development and cause 25% of childhood blindness. Formation of the eye is a multi-step, dynamic process; it involves evagination of the optic vesicle, followed by distal and ventral invagination, leading to the formation of a two-layered optic cup with a transient optic fissure. These tissue folding events require extensive changes in cell shape and tissue growth mediated by cytoskeleton mechanics and intercellular adhesion. We hypothesized that the Rho GTPase Cdc42 may be an essential, convergent effector downstream of key regulatory factors required for ocular morphogenesis. CDC42 controls actin remodeling, apicobasal polarity, and junction assembly. Here we identify a novel essential function for Cdc42 during eye morphogenesis in mouse; in Cdc42 mutant eyes expansion of the ventral optic cup is arrested, resulting in microphthalmia and a wide coloboma. Our analyses show that Cdc42 is required for expression of the polarity effector proteins PRKCZ and PARD6, intercellular junction protein tight junction protein 1, β-catenin, actin cytoskeleton F-actin, and contractile protein phospho myosin light chain 2. Expression of RPE fate determinants OTX2 and MITF, and formation of the RPE layer are severely affected in the temporal domain of the proximal optic cup. EdU incorporation is significantly downregulated. In addition, mitotic retinal progenitor cells mislocalize deeper, basal regions, likely contributing to decreased proliferation. We propose that morphogenesis of the ventral optic cup requires Cdc42 function for coordinated optic cup expansion and establishment of subretinal space, tissue tension, and differentiation of the ventral RPE layer.

Endothelial-derived nitric oxide impacts vascular smooth muscle cell phenotypes under high wall shear stress condition

The Phenotypic states of vascular smooth muscle cells (SMCs) are essential to understanding vascular pathophysiology. SMCs in vessels generally express a specific set of contractile proteins, but decreased contractile protein expression, indicating a phenotypic shift, is a hallmark of vascular diseases. Recent studies have suggested the relation of abnormally high wall shear stress (WSS) of approximately 20 Pa with the aortic disease pathogenesis. However, due to the lack of appropriate experimental models to assess SMC phenotypic states, the details of the phenotypic shift under high WSS conditions remain unclear. In this study, we developed a coculture model where vascular endothelial cells (ECs) were cocultured with SMCs expressing calponin 1, a contractile protein involved in the phenotypic shift of SMCs. We investigated the effects of a pathologically high WSS condition on the phenotypic states of SMCs. Increased calponin 1 expression was found upon exposure to 20 Pa WSS compared with a physiological 2 Pa condition, whereas the expression of another contractile protein, α-smooth muscle actin (αSMA) remained unchanged. Furthermore, the inhibition of EC-derived nitric oxide (NO), which is associated with endothelial dysfunction in vascular diseases, resulted in a trend of decreasing αSMA and Calponin 1 expression under 20 Pa WSS conditions compared with 2 Pa. Our findings suggest that EC-derived NO under pathologically high WSS conditions may impact the expression of contractile proteins implicated in aortic pathophysiology.

Endurance exercise attenuates Gαq-RNAi induced hereditary obesity and skeletal muscle dysfunction via improving skeletal muscle Srl/MRCC-I pathway in Drosophila

G protein alpha q subunit (Gαq) can binds to the G protein-coupled receptor (GPCR) for signaling and is closely related to lipid metabolism. Endurance exercise is an effective means of combating acquired obesity and its complications, but the mechanisms by which endurance exercise modulates hereditary obesity and its complications are unknown. In this study, we achieved knockdown of Gαq in drosophila adipose tissue and skeletal muscle by constructing the Gαq-UAS-RNAi/Ppl-Gal4 and Gαq-UAS-RNAi/Mef2-GAl4 systems. Drosophila were subjected a three-week endurance exercise intervention, and changes in relevant indicators were detected and observed by RT-PCR, ELISA, oil red staining, immunofluorescence staining, and transmission electron microscopy. The results showed that knockdown of Gαq in both adipose tissue and skeletal muscle induced a significant increase in triglycerides accompanied by a decrease in rapid climbing ability, a decrease in Superoxide Dismutase (SOD) activity level, and a decrease in Mitochondrial respiratory chain complexI (MRCC I) content in Drosophila whole body and skeletal muscle, and down-regulated the expression of the G protein alpha q subunit (Gαq), the skeletal muscle myosin heavy chain expression gene (Mhc), mitochondrial biogenesis gene Spargal(the PGC-1alpha homologue in Drosophila). Endurance exercise significantly improved the triglyceride levels in the whole body and skeletal muscle of drosophila with Gαq knockdown in adipose tissue and skeletal muscle, as well as their ability to climb, increased SOD activity level and MRCCI content level, and up-regulated the expression of Gαq, Mhc, and Spargal(Srl). Thus, the present findings suggest that genetic defects in the Gαq gene in adipose and skeletal muscle tissues induce hereditary obesity and skeletal muscle dysfunction, and that endurance exercise attenuates this hereditary obesity and concomitant skeletal muscle dysfunction in drosophila by improving skeletal muscle fiber contractile proteins, mitochondrial function and function, and antioxidant capacity via mediating the Gαq/Mhc, Gαq/Srl/MRCC-I, and Gαq/SOD pathways.

Superacid-Synthesized Fluorinated Diamines Act as Selective hCA IV Inhibitors.

Carbonic anhydrase (CA) IV is a membrane-bound enzyme involved in important physio-pathological processes, such as excitation-contraction coupling in heart muscle, central nervous system (CNS) extracellular buffering, and mediation of inflammatory response after stroke. Known since the mid-1980s, this isoform is still largely unexplored when compared to other isoforms, mostly for the current lack of inhibitors targeting selectively this isoform. The discovery of selective CA IV inhibitors is thus largely awaited. In this work, we report β-(di) fluoropropyl diamines as effective CA IV inhibitors, opening real perspectives for a new mode of selective inhibition of this isoform. Inhibition data reveal that the essential structure core to ensure a potent and selective inhibition of CA IV is the N-propyldiamine. Molecular modeling studies were employed to understand the binding mode of the synthesized amines. Conformational searches within the active site space carried out in an implicit solvent (water) model were also conducted.