In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca2+ ...uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective.
Key points
Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein‐coupled receptor (GqPCR) stimulation.
Cardiac signalling mechanisms for ...regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood.
We demonstrate that a downstream kinase of GqPCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin‐like protein 1 (DLP1), a mitochondrial fission protein.
The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation.
This study identifies a novel PKD‐specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent GqPCR stimulation.
These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking GqPCR signalling with the regulation of mitochondrial morphology and function.
Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein‐coupled receptor (GqPCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that GqPCR stimulation induced by α1‐adrenergic stimulation mediates mitochondrial fragmentation in a fission‐ and PKD‐dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon GqPCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin‐like protein 1 (DLP1), at S637. PKD‐dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac‐specific overexpression of constitutively active Gαq protein. In conclusion, GqPCR‐PKD signalling induces mitochondrial fragmentation and dysfunction via PKD‐dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD‐specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD‐dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and GqPCR signalling.
Key points
Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein‐coupled receptor (GqPCR) stimulation.
Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood.
We demonstrate that a downstream kinase of GqPCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin‐like protein 1 (DLP1), a mitochondrial fission protein.
The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation.
This study identifies a novel PKD‐specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent GqPCR stimulation.
These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking GqPCR signalling with the regulation of mitochondrial morphology and function.
Objective: Ischemia–reperfusion (I/R) injury, often encountered clinically, results in myocardial apoptosis and necrosis. Hydrogen sulfide (H2S) is produced endogenously in response to ischemia and ...thought to be cardioprotective, although its mechanism of action is not fully known. This study investigates cardioprotection provided by exogenous H2S, generated as sodium sulfide on apoptosis following myocardial I/R injury. Methods: The mid-LAD coronary artery in Yorkshire swine (n = 12) was occluded for 60 min, followed by reperfusion for 120 min. Controls (n = 6) received placebo, and treatment animals (n = 6) received sulfide 10 min prior to and throughout reperfusion. Hemodynamic, global, and regional functional measurements were obtained. Evans blue/TTC staining identified the area-at-risk (AAR) and infarction. Serum CK-MB, troponin I, and FABP were assayed. Tissue expression of bcl-2, bad, apoptosis-inducing-factor (AIF), total and cleaved caspase-3, and total and cleaved PARP were assessed. PAR and TUNEL staining were performed to assess apoptotic cell counts and poly-ADP ribosylation, respectively. Results: Pre-I/R hemodynamics were similar between groups. Post-I/R, mean arterial pressure (mmHg) was reduced by 30.2 ± 4.3 in controls vs 8.2 ± 6.9 in treatment animals (p = 0.01). +LV dP/dt (mmHg/s) was reduced by 1308 ± 435 in controls vs 403 ± 283 in treatment animals (p = 0.001). Infarct size (% of AAR) in controls was 47.4 ± 6.2% vs 20.1 ± 3.3% in the treated group (p = 0.003). In treated animals, CK-MB and FABP were lower by 47.0% (p = 0.10) and 45.1% (p = 0.01), respectively. AIF, caspase-3, and PARP expression was similar between groups, whereas cleaved caspase-3 and cleaved PARP was lower in treated animals (p = 0.04). PAR staining was significantly reduced in sulfide treated groups (p = 0.04). TUNEL staining demonstrated significantly fewer apoptotic cells in sulfide treated animals (p = 0.02). Conclusions: Sodium sulfide is efficacious in reducing apoptosis in response to I/R injury. Along with its known effects on reducing necrosis, sulfide’s effects on apoptosis may partially contribute to providing myocardial protection. Exogenous sulfide may have therapeutic utility in clinical settings in which I/R injury is encountered.
Background New-onset postoperative atrial fibrillation (PAF) continues to be among the most common complications after cardiac surgery, leading to significant morbidity and cost. We studied the role ...of oxidative stress on patients after cardiopulmonary bypass. Methods Patients undergoing coronary artery bypass grafting or valve procedures who exhibited new-onset PAF (n = 11) and those who remained in sinus rhythm (n = 13) were prospectively matched based on preoperative, intraoperative, and postoperative characteristics. Postoperative atrial fibrillation was assessed by electrocardiogram and must have required initiation of antiarrhythmic therapy or anticoagulation. Right atrial and skeletal muscle samples were harvested before and after cardiopulmonary bypass for oxidative protein immunostaining (Oxyblot assay). Serum samples were collected preoperatively and postoperatively at 6 hours and day 4 for microarray assessment of gene expression and to quantify total peroxide levels. Results Patients with PAF had significantly more elevation in total peroxide levels in serum compared with patients in sinus rhythm at 6 hours (5.83 ± 1.9 versus 2.02 ± 0.2 fold, respectively; p = 0.039) but not at day 4 (3.81 ± 1.2 versus 2.17 ± 0.5 fold, respectively; p = 0.188). Patients with PAF also had significantly more myocardial oxidation compared with patients in sinus rhythm at 6 hours (4.19 ± 1.4 versus 0.94 ± 0.3 fold, respectively; p = 0.021). Increased serum peroxide levels in patients who exhibited PAF correlated with elevated myocardial protein oxidation but not peripheral muscle oxidation. Gene expression analysis revealed a differential genomic response in patients with new-onset PAF (more oxidation) compared with patients in sinus rhythm (more reduction). Conclusions Patients who exhibit PAF after cardiac surgery have significantly increased acute oxidative stress, which translates into increased myocardial oxidation. Also, patients with PAF have a differential oxidative genomic response after cardiopulmonary bypass that may predispose them to oxidative stress.
Many cells in both the central visual system and other sensory systems exhibit a center surround organization in their receptive field, where the response to a centrally placed stimulus is modified ...when a surrounding area is also stimulated. This can follow from laterally directed connections in the local circuit at the level of the cell in question but could also involve more complex interactions. In the lateral geniculate nucleus (LGN), the cells relaying the retinal input display a concentric, center surround organization that in part follows from the similar organization characterizing the retinal cells providing their input. However, local thalamic inhibitory interneurons also play a role, and as we examine here, feedback from the visual cortex too. Here, we show in the primate (macaque) that spatially organized cortical feedback provides a clear and differential influence serving to enhance both responses to stimulation within the center of the receptive field and the ability of the nonclassical surround mechanism to attenuate this. In short, both center and surround mechanisms are influenced by the feedback. This dynamically sharpens the spatial focus of the receptive field and introduces nonlinearities from the cortical mechanism into the LGN.
Introduction Hydrogen sulfide is produced endogenously in response to myocardial ischemia and thought to be cardioprotective. The mechanism underlying this protection has yet to be fully elucidated, ...but it may be related to sulfide's ability to limit inflammation. This study investigates the cardioprotection provided by exogenous hydrogen sulfide and its potential anti-inflammatory mechanism of action. Methods The mid left anterior descending coronary artery in 14 Yorkshire swine was acutely occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls (n = 7) received placebo, and treatment animals (n = 7) received sulfide 10 minutes before and throughout reperfusion. Hemodynamic and functional measurements were obtained. Evans blue and triphenyl tetrazolium chloride staining identified the area at risk and infarction. Coronary microvascular reactivity was assessed. Tissue was assayed for myeloperoxidase activity and proinflammatory cytokines. Results Pre-ischemia/reperfusion hemodynamics were similar between groups, whereas post-ischemia/reperfusion mean arterial pressure was reduced by 28.7 ± 5.0 mm Hg in controls versus 6.7 ± 6.2 mm Hg in treatment animals ( P = .03). Positive first derivative of left ventricular pressure over time was reduced by 1325 ± 455 mm Hg/s in controls versys 416 ± 207 mm Hg/s in treatment animals ( P = .002). Segmental shortening in the area at risk was better in treatment animals. Infarct size (percent of area at risk) in controls was 41.0% ± 7.8% versus 21.2% ± 2.5% in the treated group ( P = .036). Tissue levels of interleukin 6, interleukin 8, tumor necrosis factor-alpha, and myeloperoxidase activity decreased in the treatment group. Treated animals demonstrated improved microvascular reactivity. Conclusions Therapeutic sulfide provides protection in response to ischemia/reperfusion injury, improving myocardial function, reducing infarct size, and improving coronary microvascular reactivity, potentially through its anti-inflammatory properties. Exogenous sulfide may have therapeutic utility in clinical settings in which ischemia/reperfusion injury is encountered.
Hypercholesterolemia is prevalent in patients who experience myocardial ischemia-reperfusion injury (IR). We investigate the impact of dietary-induced hypercholesterolemia on the myocardium in the ...setting of acute IR.
In normocholesterolemic (NC, n=7) and hypercholesterolemic (HC, n=7) Yucatan male pigs, the left anterior descending coronary artery was occluded for 60 minutes, followed by reperfusion for 120 minutes. Hemodynamic values were recorded, and TTC staining was used to assess necrosis. Oxidative stress was measured. Specific cell death and survival signaling pathways were assessed by Western blot and TUNEL staining. Infarct size was 45% greater in HC versus NC (42% versus 61%, P<0.05), whereas the area at risk (AAR) was similar in both groups (P=0.61). Whereas global LV function (+dP/dt, P<0.05) was higher during entire period of IR in HC versus NC, regional function deteriorated more following reperfusion in HC (P<0.05). Ischemia increased indices of myocardial oxidative stress such as protein oxidation (P<0.05), lipid peroxidation (P<0.05), and nitrotyrosylation in HC versus NC, as well as the expression of phospho-eNOS (P<0.05). The expression of myeloperoxidase, p38 MAPK, and phospho-p38 MAPK was higher in HC versus NC (all P<05). Ischemia caused higher expression of the proapoptotic protein PARP (P<0.05), and lower expression of the prosurvival proteins Bcl2 (P<0.05), phospho-Akt, (P<0.05), and phospho-PKCepsilon (P<0.05) in the HC versus NC. TUNEL-positive cell count was 3.8-fold (P<0.05) higher in the AAR of HC versus NC.
This study demonstrates that experimental hypercholesterolemia is associated with increased myocardial oxidative stress and inflammation, attenuation of cell survival pathways, and induction of apoptosis in the ischemic territory, which together may account for the expansion of myocardial necrosis in the setting of acute IR.
Small- and large-conductance Ca2+-activated K+channels (SKCa and BKCa, respectively) may be important targets for therapeutic interventions in a variety of cardiac conditions. In cardiomyocytes, BKCa ...channels are localized to mitochondria where they beneficially modulate reactive oxygen species, mitochondrial Ca2+, and respiration. In vascular smooth muscle cells, BKCa channels regulate vascular tone and promote vasodilation. Activation of BKCa channels has demonstrated significant cardioprotection following ischemic injury, including improved function and reduced infarct size. SKCa channels are expressed in both the membrane and mitochondria of cardiomyocytes. Modulation of cardiomyocyte SKCa channels may be beneficial for arrhythmia, heart failure, and ischemia. Mitochondrial SKCa channels may provide similar benefit to BKCa channels. In addition, activation of SKCa channels on the endothelium promotes vasodilation. This mini-review focuses on the modulation of cardiomyocyte BKCa and SKCa channels for cardioprotection and briefly address associated potential therapeutic benefits in the coronary circulation. (Circ J 2015; 79: 455–462)
Abstract Background Calpain inhibition has an enhancing effect on myocardial perfusion and improves myocardial density by inhibiting glycogen synthase kinase 3β (GSK-3β) and up-regulating downstream ...signaling pathways, including the insulin/PI3K and WNT/β-catenin pathways, in a pig model of chronic myocardial ischemia in the setting of metabolic syndrome. Methods Pigs were fed a high-fat diet for 4 weeks, then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, the animals received no drug (high-cholesterol controls HCC), a high-dose calpain inhibitor (HCI), a low-dose calpain inhibitor (LCI), or a GSK-3β inhibitor (GSK-3βI). The diets and drug regimens were continued for 5 weeks and the myocardial tissue was harvested. Results Calpain and GSK-3β inhibition caused an increase in myocardial perfusion ratios at rest and during pacing compared with controls. Pigs in the LCI and HCI groups had increased vessel density in the ischemic myocardium, and pigs in the GSK-3βI group had increased vessel density in the ischemic and nonischemic myocardium compared with the HCC group. Calpain inhibition modulates proteins involved in the insulin/PI3K and WNT/β-catenin pathways. Quantitative proteomics revealed that calpain and GSK-3β inhibition significantly modulated the expression of proteins enriched in cytoskeletal regulation, metabolism, respiration, and calcium-binding pathways. Conclusions In the setting of metabolic syndrome, calpain or GSK-3β inhibition increases vessel density in both ischemic and nonischemic myocardial tissue. Calpain inhibition may exert these effects through the inhibition of GSK-3β and up-regulation of downstream signaling pathways, including the insulin/PI3K and WNT/β-catenin pathways.
Reduced exercise capacity in pulmonary hypertension (PH) significantly impacts quality of life. However, the cause of reduced exercise capacity in PH remains unclear. The objective of this study was ...to investigate whether intrinsic skeletal muscle changes are causative in reduced exercise capacity in PH using preclinical PH rat models with different PH severity. PH was induced in adult Sprague–Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 0–4 weeks of normoxia exposure. Control rats were injected with vehicle and housed in normoxia. Echocardiography was performed to assess cardiac function. Exercise capacity was assessed by VO2 max. Skeletal muscle structural changes (atrophy, fiber type switching, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7‐week timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4‐week timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Our data suggest that cardiopulmonary impairments in PH are the primary cause of reduced exercise capacity, which occurs before intrinsic skeletal muscle dysfunction.
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