Mitochondrial connexin 43 in sex-dependent myocardial responses and estrogen-mediated cardiac protection following acute ischemia/reperfusion injury Abstract Preserving mitochondrial activity is crucial in rescuing cardiac function following acute myocardial ischemia/reperfusion (I/R). The sex difference in myocardial functional recovery has been observed after I/R. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we aimed to determine the implication of mitochondrial Cx43 in sex-related myocardial responses and to examine the effect of estrogen (17β-estradiol, E2) on Cx43, particularly mitochondrial Cx43-involved cardiac protection following I/R. Mouse primary cardiomyocytes and isolated mouse hearts (from males, females, ovariectomized females, and doxycycline-inducible Tnnt2-controlled Cx43 knockout without or with acute post-ischemic E2 treatment) were subjected to simulated I/R in culture or Langendorff I/R (25-min warm ischemia/40-min reperfusion), respectively. Mitochondrial membrane potential and mitochondrial superoxide production were measured in cardiomyocytes. Myocardial function and infarct size were determined. Cx43 and its isoform, Gja1-20k, were assessed in mitochondria. Immunoelectron microscopy and co-immunoprecipitation were also used to examine mitochondrial Cx43 and its interaction with estrogen receptor-α by E2 in mitochondria, respectively. There were sex disparities in stress-induced cardiomyocyte mitochondrial function. E2 partially restored mitochondrial activity in cardiomyocytes following acute injury. Post-ischemia infusion of E2 improved functional recovery and reduced infarct size with increased Cx43 content and phosphorylation in mitochondria. Ablation of cardiac Cx43 aggravated mitochondrial damage and abolished E2-mediated cardiac protection during I/R. Female mice were more resistant to myocardial I/R than age-matched males with greater protective role of mitochondrial Cx43 in female hearts. Post-ischemic E2 usage augmented mitochondrial Cx43 content and phosphorylation, increased mitochondrial Gja1-20k, and showed cardiac protection.

Therapies to limit myocardial injury in animal models of myocarditis: a systematic review and meta-analysis Abstract Current myocarditis guidelines do not advocate treatment to prevent myocardial injury and scar deposition in patients with myocarditis and normal left ventricular ejection fraction. We aimed to ascertain the utility of beta blockers, calcium channel blockers and antagonists of the renin–angiotensin system in ameliorating myocardial injury, scar formation and calcification in animal in vivo models of myocarditis. The project was prospectively registered with the PROSPERO database of systematic reviews (CRD42018089336). Primary outcomes (necrosis, fibrosis and calcification) were meta-analysed with random-effects modelling. 52 studies were systematically reviewed. Meta-analysis was performed compared with untreated controls. In each study, we identified all independent comparisons of treatment versus control groups. The pooled weighted mean difference (WMD) indicated treatment reduced necrosis by 16.9% (71 controlled analyses, 95% CI 13.2–20.7%; P < 0.001), however there was less evidence of an effect after accounting for publication bias. Treatment led to a 12.8% reduction in fibrosis (73 controlled analyses, 95% CI 7.6–18.0%; P < 0.001). After accounting for publication bias this was attenuated to 7.8% but remained significant. Treatment reduced calcification by 4.1% (28 controlled analyses, 95% CI 0.2–8.0%; P < 0.0395). We observed significant heterogeneity in effect size in all primary endpoints, which was predominantly driven by differences between drug categories. Beta blockers and angiotensin-converting enzyme (ACE) inhibitors were the only agents that were effective for both necrosis and fibrosis, while only ACE inhibitors had a significant effect on calcification. This study provides evidence for a role for ACE inhibitors and beta blockers to prevent myocardial injury and scar deposition in in vivo models of myocarditis. There is a need for further well-designed studies to assess the translational application of these treatments.

Glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux Abstract A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte transverse tubular system (t-system), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-system is altered in GRKO hearts and if GR signalling is required for t-system preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-system density and increased distances between ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC). Because t-system remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-system density than cells treated with dexamethasone (EC50 1.1 nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR–LTCC coupling and synchrony of intracellular Ca2+ release, but did not alter expression levels of t-system-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-system preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-system loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-system. These findings suggest that GR signalling and autophagy are critically involved in t-system preservation and remodelling in the heart.

Acute exposure to nocturnal train noise induces endothelial dysfunction and pro-thromboinflammatory changes of the plasma proteome in healthy subjects Abstract Nocturnal train noise exposure has been associated with hypertension and myocardial infarction. It remains unclear whether acute nighttime train exposure may induce subclinical atherosclerosis, such as endothelial dysfunction and other functional and/or biochemical changes. Thus, we aimed to expose healthy subjects to nocturnal train noise and to assess endothelial function, changes in plasma protein levels and clinical parameters. In a randomized crossover study, we exposed 70 healthy volunteers to either background or two different simulated train noise scenarios in their homes during three nights. After each night, participants visited the study center for measurement of vascular function and assessment of other biomedical and biochemical parameters. The three nighttime noise scenarios were exposure to either background noise (control), 30 or 60 train noise events (Noise30 or Noise60), with average sound pressure levels of 33, 52 and 54 dB(A), respectively. Flow-mediated dilation (FMD) of the brachial artery was 11.23 ± 4.68% for control, compared to 8.71 ± 3.83% for Noise30 and 8.47 ± 3.73% for Noise60 (p < 0.001 vs. control). Sleep quality was impaired after both Noise30 and Noise60 nights (p < 0.001 vs. control). Targeted proteomic analysis showed substantial changes of plasma proteins after the Noise60 night, mainly centered on redox, pro-thrombotic and proinflammatory pathways. Exposure to simulated nocturnal train noise impaired endothelial function. The proteomic changes point toward a proinflammatory and pro-thrombotic phenotype in response to nocturnal train noise and provide a molecular basis to explain the increased cardiovascular risk observed in epidemiological noise studies.

Coronary microvascular obstruction: the new frontier in cardioprotection Abstract Cardioprotection aims at infarct size reduction and improvement of clinical outcome from myocardial infarction. The translation of cardioprotection from preclinical and promising proof-of-concept studies to clinical benefit for patients has been largely disappointing. Almost all of these studies which did not translate into clinical benefit had infarct size reduction as the primary endpoint and used protocols selected to achieve infarct size reduction. The present review puts forward the hypothesis that the coronary circulation is a so far neglected target of cardioprotection. Mechanisms of ischemia–reperfusion injury to the coronary circulation are detailed. Clinical methods and measures to assess coronary microvascular impairment in reperfused acute myocardial infarction are presented. A comparison of magnetic resonance imaging data on infarct size vs. coronary microvascular obstruction is made. Finally, the design of studies which are optimized not only for infarct size reduction but also for salvage of the coronary microcirculation is advocated.

Correction to: Exercise-induced circulating extracellular vesicles protect against cardiac ischemia–reperfusion injury The original version of this article unfortunately contained a mistake.

Enhancement of cardiac lymphangiogenesis by transplantation of CD34 + VEGFR-3 + endothelial progenitor cells and sustained release of VEGF-C Abstract Impairment of cardiac lymphatic vessels leads to cardiac lymphedema. Recent studies have suggested that stimulation of lymphangiogenesis may reduce cardiac lymphedema. However, effects of lymphatic endothelial progenitor cells (LEPCs) on cardiac lymphangiogenesis are poorly understood. Therefore, this study investigated effectiveness of LEPC transplantation and VEGF-C release with self-assembling peptide (SAP) on cardiac lymphangiogenesis after myocardial infarction (MI). CD34+VEGFR-3+ EPCs isolated from rat bone marrow differentiated into lymphatic endothelial cells after VEGF-C induction. VEGF-C also stimulated the cells to incorporate into the lymphatic capillary-like structures. The functionalized SAP could adhere with the cells and released VEGF-C sustainedly. In the condition of hypoxia and serum deprivation or abdominal pouch assay, the SAP hydrogel protected the cells from apoptosis and necrosis. At 4 weeks after intramyocardial transplantation of the cells and VEGF-C loaded with SAP hydrogel in rat MI models, cardiac lymphangiogenesis was increased, cardiac edema and reverse remodeling were reduced, and cardiac function was improved significantly. Delivery with SAP hydrogel favored survival of the engrafted cells. VEGF-C released from the hydrogel promoted differentiation and incorporation of the cells as well as growth of pre-existed lymphatic vessels. Cardiac lymphangiogenesis was beneficial for elimination of the inflammatory cells in the infarcted myocardium. Moreover, angiogenesis and myocardial regeneration were enhanced after reduction of lymphedema. These results demonstrate that the combined delivery of LEPCs and VEGF-C with the functionalized SAP promotes cardiac lymphangiogenesis and repair of the infarcted myocardium effectively. This study represents a novel therapy for relieving myocardial edema in cardiovascular diseases.

Junctophilin-2 is a target of matrix metalloproteinase-2 in myocardial ischemia–reperfusion injury Abstract Junctophilin-2 is a structural membrane protein that tethers T-tubules to the sarcoplasmic reticulum to allow for coordinated calcium-induced calcium release in cardiomyocytes. Defective excitation–contraction coupling in myocardial ischemia–reperfusion (IR) injury is associated with junctophilin-2 proteolysis. However, it remains unclear whether preventing junctophilin-2 proteolysis improves the recovery of cardiac contractile dysfunction in IR injury. Matrix metalloproteinase-2 (MMP-2) is a zinc and calcium-dependent protease that is activated by oxidative stress in myocardial IR injury and cleaves both intracellular and extracellular substrates. To determine whether junctophilin-2 is targeted by MMP-2, isolated rat hearts were perfused in working mode aerobically or subjected to IR injury with the selective MMP inhibitor ARP-100. IR injury impaired the recovery of cardiac contractile function which was associated with increased degradation of junctophilin-2 and damaged cardiac dyads. In IR hearts, ARP-100 improved the recovery of cardiac contractile function, attenuated junctophilin-2 proteolysis, and prevented ultrastructural damage to the dyad. MMP-2 was co-localized with junctophilin-2 in aerobic and IR hearts by immunoprecipitation and immunohistochemistry. In situ zymography showed that MMP activity was localized to the Z-disc and sarcomere in aerobic hearts and accumulated at sites where the striated JPH-2 staining was disrupted in IR hearts. In vitro proteolysis assays determined that junctophilin-2 is susceptible to proteolysis by MMP-2 and in silico analysis predicted multiple MMP-2 cleavage sites between the membrane occupation and recognition nexus repeats and within the divergent region of junctophilin-2. Degradation of junctophilin-2 by MMP-2 is an early consequence of myocardial IR injury which may initiate a cascade of sequelae leading to impaired contractile function.

Carotid baroreceptor stimulation suppresses ventricular fibrillation in canines with chronic heart failure Abstract Carotid baroreceptor stimulation (CBS) has been shown to improve cardiac dysfunction and pathological structure remodelling. This study aimed to investigate the effects of CBS on the ventricular electrophysiological properties in canines with chronic heart failure (CHF). Thirty-eight beagles were randomized into control (CON), CHF, low-level CBS (LL-CBS), and moderate-level CBS (ML-CBS) groups. The CHF model was established with 6 weeks of rapid right ventricular pacing (RVP), and concomitant LL-CBS and ML-CBS were applied in the LL-CBS and ML-CBS groups, respectively. After 6 weeks of RVP, ventricular electrophysiological parameters and left stellate ganglion (LSG) neural activity and function were measured. Autonomic neural remodelling in the LSG and left ventricle (LV) and ionic remodelling in the LV were detected. Compared with the CHF group, both LL-CBS and ML-CBS decreased spatial dispersion of action potential duration (APD), suppressed APD alternans, reduced ventricular fibrillation (VF) inducibility, and inhibited enhanced LSG neural discharge and function. Only ML-CBS significantly inhibited ventricular repolarization prolongation and increased the VF threshold. Moreover, ML-CBS inhibited the increase in growth-associated protein-43 and tyrosine hydroxylase-positive nerve fibre densities in LV, increased acetylcholinesterase protein expression in LSG, and decreased nerve growth factor protein expression in LSG and LV. Chronic RVP resulted in a remarkable reduction in protein expression encoding both potassium and L-type calcium currents; these changes were partly amended by ML-CBS and LL-CBS. In conclusion, CBS suppresses VF in CHF canines, potentially by modulating autonomic nerve and ion channels. In addition, the effects of ML-CBS on ventricular electrophysiological properties, autonomic remodelling, and ionic remodelling were superior to those of LL-CBS.

Cardioprotection of post-ischemic moderate ROS against ischemia/reperfusion via STAT3-induced the inhibition of MCU opening Abstract Enhanced reactive oxygen species (ROS) at the beginning of reperfusion activated signal transducer and activator of transcription 3 (STAT3) in intermittent hypobaric hypoxia (IHH)-afforded cardioprotection against ischemia/reperfusion (I/R). However, its mechanism remains largely unknown. This study aimed to investigate the role and the downstream of STAT3 in exogenous enhanced post-ischemic ROS-induced cardioprotection using the model of moderate hydrogen peroxide postconditioning (H2O2PoC) mimicking endogenous ROS in IHH. Moderate H2O2PoC not only improved the post-ischemic myocardial contractile recovery and reduced the infarct size in isolated rat I/R hearts, but also alleviated mitochondrial calcium overload and ameliorated Ca2+ transients, cell contraction, and mitochondrial membrane potential in rat I/R cardiomyocytes. However, the cardioprotective effects of moderate H2O2PoC were abrogated by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490 in rat hearts as well as adenovirus-delivered short hairpin RNA specific for STAT3 and the opener of mitochondrial calcium uniporter (MCU) spermine in rat cardiomyocytes. Notably, the moderate H2O2PoC-afforded cardioprotection abrogated by spermine could be rescued by STAT3 over-expression with adenovirus in rat I/R cardiomyocytes. Besides, moderate H2O2PoC enhanced mitochondrial STAT3 expression during I/R. A co-localization/interaction of STAT3 or phospho-STAT3ser727 and MCU was observed in rat cardiomyocytes with moderate H2O2PoC at 5 and 30 min of reperfusion but not in rat I/R cardiomyocytes. Further, STAT3 interacted with the N-terminal domain (NTD) of MCU in rat cardiomyocytes with moderate H2O2PoC. These findings indicated that post-ischemic moderate ROS activate STAT3 against cardiac I/R by inhibiting MCU opening via its interaction with the NTD of MCU to alleviate mitochondrial calcium overload.