The Builders of the Junction: Roles of Junctophilin1 and Junctophilin2 in the Assembly of the Sarcoplasmic Reticulum–Plasma Membrane Junctions in Striated Muscle

Contraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR. This short review focuses on the roles of junctophilins1 and 2 in the formation and organization of SR-PM junctions in skeletal and cardiac muscle and on the functional consequences of the absence or malfunction of these proteins in striated muscle in light of recently published data and recent advancements in protein structure prediction.

MicroRNA-489 Promotes the Apoptosis of Cardiac Muscle Cells in Myocardial Ischemia-Reperfusion Based on Smart Healthcare

With the development of information technology, the concept of smart healthcare has gradually come to the fore. Smart healthcare uses a new generation of information technologies, such as the Internet of Things (loT), big data, cloud computing, and artificial intelligence, to transform the traditional medical system in an all-around way, making healthcare more efficient, more convenient, and more personalized. miRNAs can regulate the proliferation, differentiation, and apoptosis of human cells. Relevant studies have also shown that miRNAs may play a key role in the occurrence and development of myocardial ischemia-reperfusion injury (MIRI). This study aims to explore the effects of miR-489 in MIRI. In this study, miR-489 expression in a myocardial ischemia-reperfusion animal model and H9C2 cells induced by H/R was detected by qRT-PCR. The release of lactate dehydrogenase (LDH) and the activity of creatine kinase (CK) was detected after miR-489 knockdown in H9C2 cells induced by H/R. The apoptosis of H9C2 cells and animal models were determined by ELISA. The relationship between miR-489 and SPIN1 was verified by a double fluorescence reporter enzyme assay. The expression of the PI3K/AKT pathway-related proteins was detected by Western blot. Experimental results showed that miR-489 was highly expressed in cardiac muscle cells of the animal model and in H9C2 cells induced by H/R of the myocardial infarction group, which was positively associated with the apoptosis of cardiac muscle cells with ischemia-reperfusion. miR-489 knockdown can reduce the apoptosis of cardiac muscle cells caused by ischemia-reperfusion. In downstream targeting studies, it was found that miR-489 promotes the apoptosis of cardiac muscle cells after ischemia-reperfusion by targeting the inhibition of the SPIN1-mediated PI3K/AKT pathway. In conclusion, high expression of miR-489 is associated with increased apoptosis of cardiac muscle cells after ischemia-reperfusion, which can promote the apoptosis after ischemia-reperfusion by targeting the inhibition of the SPIN1-mediated PI3K/AKT pathway. Therefore, miR-489 can be one of the potential therapeutic targets for reducing the apoptosis of cardiac muscle cells after ischemia-reperfusion.

Layer-By-Layer Fabrication of Thicker and Larger Human Cardiac Muscle Patches for Cardiac Repair in Mice

The engineered myocardial tissues produced via most manufacturing techniques are typically just a few dozen micrometers thick, which is too thin for therapeutic applications in patients. Here, we used a modified layer-by-layer (LBL) fabrication protocol to generate thick human cardiac muscle patches (hCMPs) with thicknesses of ~3.75 mm. The LBL-hCMPs were composed of a layer of endothelial cells (ECs) sandwiched between two layers of cardiomyocytes (CMs): both cell populations were differentiated from the same human induced pluripotent stem cell line (hiPSCs) and suspended in a fibrin matrix, and the individual layers were sutured together, leaving channels that allowed the culture medium to access the internal cell layer. The LBL-hCMPs were cultured on a dynamic culture platform with electrical stimulation, and when compared to Control-hCMPs consisting of the same total number of hiPSC-ECs and -CMs suspended in a single layer of fibrin, hiPSC-CMs in the LBL-hCMPs were qualitatively more mature with significantly longer sarcomeres and expressed significantly higher levels of mRNA transcripts for proteins that participate in cardiomyocyte contractile activity and calcium handing. Apoptotic cells were also less common in LBL- than in Control-hCMPs. The thickness of fabricated LBL-hCMP gradually decreased to 0.8 mm by day 28 in dynamic culture. When the hCMP constructs were compared in a mouse model of myocardial infarction, the LBL-hCMPs were associated with significantly better measurements of engraftment, cardiac function, infarct size, hypertrophy, and vascularity. Collectively these observations indicate that our modified LBL fabrication protocol produced thicker hCMPs with no decline in cell viability, and that LBL-hCMPs were more potent than Control-hCMPs for promoting myocardial repair in mice.

Plasma and tissue enzyme activities of banded water snakes (Nerodia fasciata) and diamondback water snakes (Nerodia rhombifer)

OBJECTIVE To measure plasma and tissue activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase (AST), creatine kinase, and γ-glutamyltransferase in 2 snake species. ANIMALS 6 banded water snakes (Nerodia fasciata) and 6 diamondback water snakes (Nerodia rhombifer). PROCEDURES Blood was collected via the ventral tail vein to measure plasma enzyme activities. Animals were then euthanized, and samples of 9 tissues were collected from each snake: skeletal muscle, cardiac muscle, liver, spleen, lung, kidney, testicle, pancreas, and gallbladder. Tissues were frozen for 30 days, then homogenized and processed. Supernatants were collected and analyzed within 24 hours of processing. A linear mixed model was used to determine differences in enzyme activity between tissues and species and assess interactions between tissues and species. RESULTS Activities of all enzymes were found to differ significantly among tissues. There were also significant differences between species for all enzyme activities, except AST activity. The kidney had the highest alanine aminotransferase and γ-glutamyltransferase activities. Alkaline phosphatase activity was significantly highest in liver and kidney tissues than in other tissue. Creatine kinase activity was highest in skeletal muscle, followed by cardiac muscle and kidney. AST activity was present in all tissues evaluated, but was highest in liver, kidney, and cardiac muscle in both species. CLINICAL RELEVANCE Results reinforced the importance of characterizing the origin of tissue enzymes in reptiles to improve our understanding of biochemistry results and highlighted the differences that can exist in tissue enzyme activities between closely related species.

X-ray structure of a human cardiac muscle troponin C/troponin I chimera in two crystal forms

The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1–80) fused to the switch region of troponin I (cTnI; residues 138–162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC–cTnI interface.

Automatic Activity Arising in Cardiac Muscle Sleeves of the Pulmonary Vein

Ectopic activity in the pulmonary vein cardiac muscle sleeves can both induce and maintain human atrial fibrillation. A central issue in any study of the pulmonary veins is their difference from the left atrial cardiac muscle. Here, we attempt to summarize the physiological phenomena underlying the occurrence of ectopic electrical activity in animal pulmonary veins. We emphasize that the activation of multiple signaling pathways influencing not only myocyte electrophysiology but also the means of excitation–contraction coupling may be required for the initiation of triggered or automatic activity. We also gather information regarding not only the large-scale structure of cardiac muscle sleeves but also recent studies suggesting that cellular heterogeneity may contribute to the generation of arrythmogenic phenomena and to the distinction between pulmonary vein and left atrial heart muscle.

Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function

Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca2+ sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins’ (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.

Isolation of nuclei from frozen human skeletal and cardiac muscle for single nucleus RNA and chromatin assays v2

This protocol was adapted for the isolation of single nuclei from frozen skeletal and cardiac muscle tissues for molecular characterization with the SNARE-seq2, sci-ATAC-seq, and snRNA-seq assays. References: (1) Preissl et al (2015). Circulation Research. Doi: 10.1161/CIRCRESAHA.115.306337 (2) Hocker et al (2021). Science Advances. Doi: 10.1126/sciadv.abf1444

Effect of Actin Alpha Cardiac Muscle 1 on the Proliferation and Differentiation of Bovine Myoblasts and Preadipocytes

Actin Alpha Cardiac Muscle 1 (ACTC1) gene is a differentially expressed gene screened through the co-culture system of myoblasts-preadipocytes. In order to study the role of this gene in the process of proliferation and differentiation of bovine myoblasts and preadipocytes, the methods of the knockdown, overexpression, and ectopic expression of ACTC1 were used in this study. After ACTC1 knockdown in bovine myoblasts and inducing differentiation, the sizes and numbers of myotube formation were significantly reduced compared to the control group, and myogenic marker genes—MYOD1, MYOG, MYH3, MRF4, MYF5, CKM and MEF2A—were significantly decreased (p < 0.05, p < 0.01) at both the mRNA and protein levels of myoblasts at different differentiation stages (D0, D2, D4, D6 and D8). Conversely, ACTC1 overexpression induced the inverse result. After ectopic expression of ACTC1 in bovine preadipocytes and induced differentiation, the number and size of lipid droplets were significantly higher than those of the control group, and the expression of adipogenic marker genes—FABP4, SCD1, PPARγ and FASN—were significantly increased (p < 0.05, p < 0.01) at the mRNA and protein levels of preadipocytes at different differentiation stages. Flow cytometry results showed that both the knockdown and overexpression of ACTC1 inhibited the normal cell cycle of myoblasts; however, ectopic expression of ACTC1 in adipocytes induced no significant cell cycle changes. This study is the first to explore the role of ACTC1 in bovine myogenesis and lipogenesis and demonstrates that ACTC1 promotes the differentiation of bovine myoblasts and preadipocytes, affecting the proliferation of myoblasts.

Variability of Time and Amplitude ECG Indicators in Older Adults

The purpose of this article was to determine the effect of the clinostatic test on the electrical properties of the myocardium in older adults leading an active lifestyle. Materials and methods. The study involved 38 people aged between 70 and 90 years living in a northern city (Syktyvkar, 61°N) and leading an active lifestyle. The indicators of the electrical properties of the cardiac muscle were determined before and after the clinostatic test. For this purpose, an ECG was recorded in the standard lead II in the standing and lying positions. Results. Using the method of paired comparisons, we showed a decrease in heart rate under the influence of the clinostatic test from 76 ± 10 beats/min to 64 ± 10 beats/min. The variability of ECG components according to pNN50 (the percentage of successive NN intervals differing from each other by more than 50 ms) in older adults in the standing position was, on average, two times lower than in the lying position. The PP interval increased from 0.81 ± 0.12 s (in the standing position) to 0.90 ± 0.10 s (in the lying position). The PT interval remained practically unchanged, staying at the level of (0.46 ± 0.07) – (0.49 ± 0.07) s. The TP segment grew from 0.35 to 0.42 ± 0.10 s. The RD amplitude was 1.04 ± 0.43 mV in the standing and 0.88 ± 0.30 mV in the lying position; the propagation rate of depolarization in the cardiac muscle was 245 ± 137 and 205 ± 106 mm/s, respectively. In addition, the paper discussed the mechanism of opposite reactions to the clinostatic test, i.e. increasing duration of cardiointervals and decreasing amplitude indicators. For citation: Irzhak L.I., Russkikh N.G., Parshukova A.N. Variability of Time and Amplitude ECG Indicators in Older Adults. Journal of Medical and Biological Research, 2021, vol. 9, no. 4, pp. 355–365. DOI: 10.37482/2687-1491-Z073