Rationale: Exercise training, in addition to reducing cardiovascular risk factors, confers direct protection against myocardial ischemia/reperfusion injury and has been associated with improved heart attack survival in humans. However, the underlying mechanisms of exercise-afforded cardioprotection are still unclear. Objective: To investigate the role of exercise-derived circulating exosomes in cardioprotection and the molecular mechanisms involved. Methods and Results: Circulating exosomes were isolated from the plasma of volunteers with or without exercise training and rats subjected to 4-week swim exercise or sedentary littermates 24 hours after the last training session. Although the total circulating exosome level did not change significantly in exercised subjects 24 hours post-exercise compared with the sedentary control, the isolated plasma exosomes from exercised rats afforded remarkable protection against myocardial ischemia/reperfusion injury. miRNA sequencing combined with quantitative reverse transcription polymerase chain reaction validation identified 12 differentially expressed miRNAs from the circulating exosomes of exercised rats, among which miR-342-5p stood out as the most potent cardioprotective molecule. Importantly, the cardioprotective effects and the elevation of exosomal miR-342-5p were also observed in exercise-trained human volunteers. Moreover, inhibition of miR-342-5p significantly blunted the protective effects of exercise-derived circulating exosomes in hypoxia/reoxygenation cardiomyocytes; in vivo cardiac-specific inhibition of miR-342-5p through serotype 9 adeno-associated virus–mediated gene delivery attenuated exercise-afforded cardioprotection in myocardial ischemia/reperfusion rats. Mechanistically, miR-342-5p inhibited hypoxia/reoxygenation-induced cardiomyocyte apoptosis via targeting Caspase 9 and Jnk2 ; it also enhanced survival signaling (p-Akt) via targeting phosphatase gene Ppm1f . Of note, exercise training or laminar shear stress directly enhanced the synthesis of miR-342-5p in endothelial cells. Conclusions: Our findings reveal a novel endogenous cardioprotective mechanism that long-term exercise-derived circulating exosomes protect the heart against myocardial ischemia/reperfusion injury via exosomal miR-342-5p.

Many biological processes require the co-operative involvement of both microtubules and microfilaments; however, only a few proteins mediating the interaction between microtubules and microfilaments have been identified from plants. In the present study, a cotton kinesin GhKCH2, which contains a CH (calponin homology) domain at the N-terminus, was analysed in vitro and in vivo in order to understand its interaction with the two cytoskeletal elements. A specific antibody against GhKCH2 was prepared and used for immunolabelling experiments. Some GhKCH2 spots appeared along a few microtubules and microfilaments in developing cotton fibres. The His-tagged N-terminus of GhKCH2 (termed GhKCH2-N) could co-precipitate with microfilaments and strongly bind to actin filaments at a ratio of monomeric actin/GhKCH2-N of 1:0.6. The full-length GhKCH2 recombinant protein was shown to bind to and cross-link microtubules and microfilaments in vitro. A GFP-fusion protein GFP-GhKCH2 transiently overexpressed in Arabidopsis protoplasts decorated both microtubules and microfilaments, confirming the binding ability and specificities of GhKCH2 on microtubules and microfilaments in living plant cells. The results of the present study demonstrate that GhKCH2, a plant-specific microtubule-dependent motor protein, not only interacts with microtubules, but also strongly binds to microfilaments. The cytoskeletal dual-binding and cross-linking ability of GhKCH2 may be involved in the interaction between microtubules and microfilaments and the biological processes they co-ordinate together in cotton cells.

c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 48C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plantspecific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.

A 43-bp distal element, the AtKP1-related element (KPRE), was previously shown to repress the promoter activity of the kinesin gene AtKP1 in Arabidopsis thaliana. In order to identify KPRE-binding factor 1 (KBF1), a combination of ion-exchange chromatography, gel-filtration chromatography and DNA-affinity chromatography was used to purify KBF1 from whole cell extracts of Arabidopsis seedlings. Mass spectrometric identification showed that KBF1 contains two members of the whirly family of transcription factors, AtWHY1 and AtWHY3. KBF1 is a single and double-stranded DNA-binding factor. A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. Over-expression of AtWHY1 and AtWHY3 led to an obvious decrease of AtKP1 transcripts, based on quantitative real-time PCR analysis. Interestingly, salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts, and a decrease of AtKP1 transcripts. Thus, AtWHY1 and AtWHY3, as two components of KBF1, can be recruited at the KPRE site to mediate the transcriptional repression of AtKP1. Our results prove that AtKP1 is a new downstream target of the whirly family of transcription factors.

These findings demonstrate that enhancing FA utilization ameliorates mitochondrial fragmentation and cardiac dysfunction via rebalancing OPA1 processing in pressure overload-induced HF, suggesting a unique metabolic intervention approach to improving cardiac functions in HF.

Cardiac pacing is an effective therapy for treating patients with bradycardia due to sinus node dysfunction or atrioventricular block. However, traditional right ventricular apical pacing (RVAP) causes electric and mechanical dyssynchrony, which is associated with increased risk for atrial arrhythmias and heart failure. Therefore, there is a need to develop a physiological pacing approach that activates the normal cardiac conduction and provides synchronized contraction of ventricles. Although His bundle pacing (HBP) has been widely used as a physiological pacing modality, it is limited by challenging implantation technique, unsatisfactory success rate in patients with wide QRS wave, high pacing capture threshold, and early battery depletion. Recently, the left bundle branch pacing (LBBP), defined as the capture of left bundle branch (LBB) via transventricular septal approach, has emerged as a newly physiological pacing modality. Results from early clinical studies have demonstrated LBBP's feasibility and safety, with rare complications and high success rate. Overall, this approach has been found to provide physiological pacing that guarantees electrical synchrony of the left ventricle with low pacing threshold. This was previously specifically characterized by narrow paced QRS duration, large R waves, fast synchronized left ventricular activation, and correction of left bundle branch block. Therefore, LBBP may be a potential alternative pacing modality for both RVAP and cardiac resynchronization therapy with HBP or biventricular pacing (BVP). However, the technique's widespread adaptation needs further validation to ascertain its safety and efficacy in randomized clinical trials. In this review, we discuss the current knowledge of LBBP.

Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6.