Mechanical traumatic injury without circulatory shock causes cardiomyocyte apoptosis: role of reactive nitrogen and reactive oxygen species

Apoptotic cell death plays a critical role in tissue injury and organ dysfunction under a variety of pathological conditions. The present study was designed to determine whether apoptosis may contribute to posttraumatic cardiac dysfunction, and if so, to investigate the mechanisms involved. Male adult mice were subjected to nonlethal traumatic injury, and cardiomyocyte apoptosis, cardiac function, and cardiac production of reactive oxygen/nitrogen species were determined. Modified Noble-Collip drum trauma did not result in circulatory shock, and the 24-h survival rate was 100%. No direct mechanical traumatic injury was observed in the heart immediately after trauma. However, cardiomyocyte apoptosis gradually increased and reached a maximal level 12 h after trauma. Significantly, cardiac dysfunction was observed 24 h after trauma in the isolated perfused heart. This was completely reversed when apoptosis was blocked by administration of a nonselective caspase inhibitor immediately after trauma. In the traumatized hearts, reactive nitrogen species (e.g., nitric oxide) and reactive oxygen species (e.g., superoxide) were both significantly increased, and maximal nitric oxide production preceded maximal apoptosis. Moreover, a highly cytotoxic reactive species, peroxynitrite, was markedly increased in the traumatic heart, and there was a significant positive correlation between cardiac nitrotyrosine content and caspase 3 activity. Our present study demonstrated for the first time that nonlethal traumatic injury caused delayed cell death and that apoptotic cardiomyocyte death contributes to posttrauma organ dysfunction. Antiapoptotic treatments, such as blockade of reactive nitrogen oxygen species generation, may be novel strategies in reducing posttrauma multiple organ failure.     

Myeloperoxidase increased cardiomyocyte protein nitration in mice subjected to nonlethal mechanical trauma

Nonlethal mechanical trauma causes cardiomyocyte apoptosis which contributes to posttraumatic cardiac dysfunction. Apoptosis is positively correlated with protein nitration in the traumatic heart. However, the mechanisms responsible for the cardiomyocyte protein nitration remain unclear. The present study was designed to identify whether myeloperoxidase may contribute to protein nitration in nonlethal mechanical trauma and subsequent cardiomyocyte apoptosis, and, if so, to determine the possible mechanisms responsible. We used Noble-Collip drum to make nonlethal traumatic mice models. Male adult C57B16/J mice were placed in the Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Then myeloperoxidase activity and release, protein nitration, cardiomyocyte apoptosis, endothelial function and intercellular adhesion molecule-1 expression were determined. Nonlethal mechanical trauma was characterized by the 100% survival rate during the first 24 h after trauma, the lack of circulatory shock and without direct heart injury. However, myeloperoxidase activity significantly increased 6 h after trauma, and reached a maximum level 12 h after trauma. Obviously, protein nitration and cardiomyocyte apoptosis increased 12 h after trauma and could be blocked by administration of R15.7, a monoclonal antibody that blocks polymorphonuclear neutrophils adhesion. Moreover, endothelial dysfunction and intercellular adhesion molecule-1 upregulation were observed in traumatic mice. Our present study demonstrated for the first time that myeloperoxidase caused protein nitration and cardiomyocyte apoptosis in nonlethal traumatic mice. Inhibition of polymorphonuclear neutrophils adhesion and antinitration treatments may be novel measures in reducing posttraumatic cardiomyocyte apoptosis and secondary heart injury.     

Tumor necrosis factor-alpha in mechanic trauma plasma mediates cardiomyocyte apoptosis

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation (P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) or TNF-alpha alone, but not with IL-1beta or IFN-gamma alone, caused significant caspase-3 activation (P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-alpha antibody, and TP isolated from TNF-alpha(-/-) mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-alpha (anti-TNF-alpha antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) (P < 0.01). Taken together, our study demonstrated that there exists a TNF-alpha-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.     

Deficiency in Beclin1 attenuates alcohol-induced cardiac dysfunction via inhibition of ferroptosis

BackgroundBinge drinking leads to compromised mitochondrial integrity and contractile function in the heart although little effective remedy is readily available. Given the possible derangement of autophagy in ethanol-induced cardiac anomalies, this study was designed to examine involvement of Beclin1 in acute ethanol-induced cardiac contractile dysfunction, in any, and the impact of Beclin1 haploinsufficiency on ethanol cardiotoxicity with a focus on autophagy-related ferroptosis.MethodsWT and Beclin1 haploinsufficiency (BECN^+/?) mice were challenged with ethanol for one week (2 g/kg, i.p. on day 1, 3 and 7) prior to assessment of cardiac injury markers (LDH, CK-MB), cardiac geometry, contractile and mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis.ResultsEthanol exposure compromised cardiac geometry and contractile function accompanied with upregulated Beclin1 and autophagy, mitochondrial injury, oxidative stress, lipid peroxidation and apoptosis, and ferroptosis (GPx4, SLC7A11, NCOA4). Although Beclin1 deficiency did not affect cardiac function in the absence of ethanol challenge, it alleviated ethanol-induced changes in cardiac injury biomarkers, cardiomyocyte area, interstitial fibrosis, echocardiographic and cardiomyocyte mechanical properties along with mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis. Ethanol challenge evoked pronounced ferroptosis (downregulated GPx4, SLC7A11 and elevated NCOA4, lipid peroxidation), the effect was alleviated by Beclin1 haploinsufficiency. Inhibition of ferroptosis using LIP-1 rescued ethanol-induced cardiac mechanical anomalies. In vitro study noted that ferroptosis induction using erastin abrogated Beclin1 haploinsufficiency-induced response against ethanol.ConclusionsIn sum, our data suggest that Beclin1 haploinsufficiency benefits acute ethanol challenge-induced myocardial remodeling and contractile dysfunction through ferroptosis-mediated manner.     

Nicorandil alleviates myocardial injury and post-infarction cardiac remodeling by inhibiting Mst1

Background

Cardiomyocyte autophagy and apoptosis are crucial events underlying the development of cardiac abnormalities and dysfunction after myocardial infarction (MI). A better understanding of the cell signaling pathways involved in cardiac remodeling may support the development of new therapeutic strategies for the treatment of heart failure (HF) after MI.

Luteolin alleviates post‐infarction cardiac dysfunction by up‐regulating autophagy through Mst1 inhibition

Myocardial infarction (MI), which is characterized by chamber dilation and LV dysfunction, is associated with substantially higher mortality. We investigated the effects and underlying mechanisms of Luteolin on post‐infarction cardiac dysfunction. Myocardial infarction was constructed by left anterior descending coronary artery ligation. In vitro, cultured neonatal cardiomyocytes subjected to simulated MI were used to probe mechanism. Luteolin significantly improved cardiac function, decreased cardiac enzyme and inflammatory cytokines release after MI. Enhanced autophagic flux as indicated by more autophagosomes puncta, less accumulation of aggresomes and P62 in the neonatal cardiomyocytes after hypoxia was observed in the Luteolin pre‐treatment group. Western blot analysis also demonstrated that Luteolin up‐regulated autophagy in the cardiomyocytes subjected to simulated MI injury. Furthermore, Luteolin increased mitochondrial membrane potential, adenosine triphosphate content, citrate synthase activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to simulated MI injury. Interestingly, mammalian sterile 20‐like kinase 1 (Mst1) knockout abolished the protective effects of Luteolin administration. Luteolin enhances cardiac function, reduces cardiac enzyme and inflammatory markers release after MI. The protective effects of Luteolin are associated with up‐regulation of autophagy and improvement of mitochondrial biogenesis through Mst1 inhibition.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

TLR9 deficiency alleviates doxorubicin‐induced cardiotoxicity via the regulation of autophagy

Doxorubicin is a commonly used anthracycline chemotherapeutic drug. Its application for treatment has been impeded by its cardiotoxicity as it is detrimental and fatal. DNA damage, cardiac inflammation, oxidative stress and cell death are the critical links in DOX‐induced myocardial injury. Previous studies found that TLR9‐related signalling pathways are associated with the inflammatory response of cardiac myocytes, mitochondrial dysfunction and cardiomyocyte death, but it remains unclear whether TLR9 could influence DOX‐induced heart injury. Our current data imply that DOX‐induced cardiotoxicity is ameliorated by TLR9 deficiency both in vivo and in vitro, manifested as improved cardiac function and reduced cardiomyocyte apoptosis and oxidative stress. Furthermore, the deletion of TLR9 rescued DOX‐induced abnormal autophagy flux in vivo and in vitro. However, the inhibition of autophagy by 3‐MA abolished the protective effects of TLR9 deletion on DOX‐induced cardiotoxicity. Moreover, TLR9 ablation suppressed the activation of p38 MAPK during DOX administration and may promote autophagy via the TLR9‐p38 MAPK signalling pathway. Our study suggests that the deletion of TLR9 exhibits a protective effect on doxorubicin‐induced cardiotoxicity by enhancing p38‐dependent autophagy. This finding could be used as a basis for the development of a prospective therapy against DOX‐induced cardiotoxicity.     

Decreased Autophagy in Rat Heart Induced by Anti-β1-Adrenergic Receptor Autoantibodies Contributes to the Decline in Mitochondrial Membrane Potential

It has been recognized that changes in mitochondrial structure plays a key role in development of cardiac dysfunction, and autophagy has been shown to exert maintenance of mitochondrial homeostasis effects. Our previous study found that anti-β₁-adrenergic receptor autoantibodies (β₁-AABs) could lead to cardiac dysfunction along with abnormalities in mitochondrial structure. The present study tested the hypothesis that β₁-AABs may induce the decline in mitochondrial membrane potential (ΔΨm) by suppression of cardiac autophagy, which contributed to cardiac dysfunction. Male adult rats were randomized to receive a vehicle or peptide corresponding to the second extracellular loop of the β₁ adrenergic receptor (β₁-AAB group, 0.4 μg/g every two weeks for 12 weeks) and treated with rapamycin (RAPA, an autophagy agonist) at 5 mg/kg/day for two days before detection. At the 4th week, 8th week and 12th week of active immunization, the rats were sacrificed and cardiac function and the levels of cardiac LC3 and Beclin-1 were detected. ΔΨm in cardiac myocytes was determined by myocardial radionuclide imaging technology and JC-1 staining. In the present study, β₁-AABs caused cardiac dysfunction, reduced ΔΨm and decreased cardiac autophagy. Treatment with RAPA markedly attenuated β₁-AABs-induced cardiac injury evidenced by recovered ΔΨm. Taken together, these results suggested that β₁-AABs exerted significant decreased ΔΨm, which may contribute to cardiac dysfunction, most likely by decreasing cardiac autophagy in vivo. Moreover, myocardial radionuclide imaging technology may be needed to assess the risk in developing cardiac dysfunction for the people who have β₁-AABs in their blood.     

Persistent disruption of mitochondrial homeostasis after acute kidney injury

While mitochondrial dysfunction is a pathological process that occurs after acute kidney injury (AKI), the state of mitochondrial homeostasis during the injury and recovery phases of AKI remains unclear. We examined markers of mitochondrial homeostasis in two nonlethal rodent AKI models. Myoglobinuric AKI was induced by glycerol injection into rats, and mice were subjected to ischemic AKI. Animals in both models had elevated serum creatinine, indicative of renal dysfunction, 24 h after injury which partially recovered over 144 h postinjury. Markers of proximal tubule function/injury, including neutrophil gelatinase-associated lipocalin and urine glucose, did not recover during this same period. The persistent pathological state was confirmed by sustained caspase 3 cleavage and evidence of tubule dilation and brush-border damage. Respiratory proteins NDUFB8, ATP synthase β, cytochrome c oxidase subunit I (COX I), and COX IV were decreased in both injury models and did not recover by 144 h. Immunohistochemical analysis confirmed that COX IV protein was progressively lost in proximal tubules of the kidney cortex after ischemia-reperfusion (I/R). Expression of mitochondrial fission protein Drp1 was elevated after injury in both models, whereas the fusion protein Mfn2 was elevated after glycerol injury but decreased after I/R AKI. LC3-I/II expression revealed that autophagy increased in both injury models at the later time points. Markers of mitochondrial biogenesis, such as PGC-1α and PRC, were elevated in both models. These findings reveal that there is persistent disruption of mitochondrial homeostasis and sustained tubular damage after AKI, even in the presence of mitochondrial recovery signals and improved glomerular filtration.     

Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: Role of autophagy

Lipopolysaccharide (LPS) from gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complications in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis, and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity, and carbonyl formation. A Kaplan-Meier curve was constructed for survival after LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice after LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O(2)(-), and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury after LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by the antioxidant N-acetylcysteine and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy.     

Irisin ameliorates angiotensin II-induced cardiomyocyte apoptosis through autophagy

Cardiac hypertrophy is the main cause of heart failure and sudden death in patients. But the pathogenesis is unclear. Angiotensin II may contribute to cardiac hypertrophy in response to pressure overload. In angiotensin II-treated cardiomyocytes, there is a larger cross-sectional area, more apoptosis cells, and a reduction of irisin expression. An increase in P62, an autophagy flux index, as well as LC3II, were observed in cardiomyocytes after angiotensin II-induced injury. Surprisely, irisin supplementation increased LC3II expression and decreased P62 expression, consisted of results of RFP-GFP-LC3B adenovirus transfection, and reduced cardiomyocyte apoptosis, meanwhile, the protection of irisin was reversed by the autophagy inhibitor 3-methyladenine. In animal experiments, overexpression of irisin reduced cardiomyocyte apoptosis and alleviated myocardial hypertrophy caused by pressure overload. The above results indicate that irisin-induced protective autophagy and alleviated the apoptosis signaling pathway in cardiomyocytes, consequently reducing cardiomyocyte apoptosis after angiotensin II-induced injury. Hence, increasing irisin expression may be a new way to improve cardiac function and quality of life in patients with cardiac hypertrophy.     

The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress

Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism in starving cells. Although altered autophagy has been observed in various heart diseases, including cardiac hypertrophy and heart failure, it remains unclear whether autophagy plays a beneficial or detrimental role in the heart. Here, we report that the cardiac-specific loss of autophagy causes cardiomyopathy in mice. In adult mice, temporally controlled cardiac-specific deficiency of Atg5 (autophagy-related 5), a protein required for autophagy, led to cardiac hypertrophy, left ventricular dilatation and contractile dysfunction, accompanied by increased levels of ubiquitination. Furthermore, Atg5-deficient hearts showed disorganized sarcomere structure and mitochondrial misalignment and aggregation. On the other hand, cardiac-specific deficiency of Atg5 early in cardiogenesis showed no such cardiac phenotypes under baseline conditions, but developed cardiac dysfunction and left ventricular dilatation one week after treatment with pressure overload. These results indicate that constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function, and that upregulation of autophagy in failing hearts is an adaptive response for protecting cells from hemodynamic stress.     

Lin28a protects against diabetic cardiomyopathy through Mst1 inhibition

Lin28a has been found to enhance glucose uptake and insulin sensitivity. Lin28a alleviates cardiac dysfunction under various pathological conditions. However, the effects and underlying mechanisms of Lin28a on diabetic cardiomyopathy (DCM) are not well-understood. The aim of this study was to determine whether Lin28a protects against DCM and the potential mechanisms. Two to three days old mouse neonatal primary cardiomyocytes were randomized for treatment with adenoviruses harboring Lin28a and mammalian sterile 20-like kinase 1 (Mst1) short hairpin RNA, 48 hr before culturing in normal or high glucose medium. Cardiomyocyte apoptosis, autophagy, mitochondrial morphology, adenosine triphosphate content, and cytokine levels in the high glucose or normal conditions were observed between all groups. Either Lin28a overexpression or Mst1 knockdown alleviated mitochondrial ultrastructure impairment, decreased cytokine levels, inhibited apoptosis, and enhanced autophagy in primary neonatal mouse cardiomyocytes treated with high glucose. Importantly, the protective effects of Lin28a and Mst1 disappeared after treatment with 3-methyladenine, an autophagy inhibitor. Interestingly, in Mst1 knockdown cardiomyocytes, Lin28a overexpression failed to further enhance autophagy and alleviate high glucose-induced cardiomyocyte injury, which implies the protective roles of Lin28a counteracting high glucose-induced cardiomyocyte injury are dependent on Mst1 inhibition. Furthermore, co-immunoprecipitation and immunofluorescence double staining suggested that there were no direct interactions between Mst1 and Lin28a. Lin28a increased the expression of Akt, which inhibited the activation of Mst1-mediated apoptotic pathways.     

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background

Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury.

Methods and Results

In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress.

Conclusion

Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.     

Burn trauma alters calcium transporter protein expression in the heart.

We have shown previously that burn trauma produces significant cardiac dysfunction, which is first evident 8 h postburn and is maximal 24 h postburn. Because calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism by which burn injury may cause cardiac abnormalities is via calcium dyshomeostasis. We hypothesized that major burn injury alters cardiomyocyte calcium handling through changes in calcium transporter expression. Sprague-Dawley rats were given either burn injury or no burn injury (controls). Cardiomyocyte intracellular calcium and sodium were quantified at various times postburn by fura 2-AM or sodium-binding benzofuran isophthalate fluorescent indicators, respectively. In addition, hearts freeze-clamped at various times postburn (2, 4, 8, and 24 h) were used for Western blot analysis using antibodies against the sarcoplasmic reticulum calcium-ATPase (SERCA), the L-type calcium-channel, the ryanodine receptor, the sodium/calcium exchanger, or the sodium-potassium-ATPase. Intracellular calcium levels were elevated significantly 8-24 h postburn, and intracellular sodium was increased significantly 4 through 24 h postburn. Expression of SERCA was significantly reduced 1-8 h postburn, whereas L-type calcium-channel expression was diminished 1 and 2 h postburn (P < 0.05) but returned toward control levels 4 h postburn. Ryanodine receptor protein was significantly reduced at 1 and 2 h postburn, returning to baseline by 4 h postburn. Sodium/calcium exchanger expression was significantly elevated 2 h postburn but was significantly reduced 24 h postburn. An increase in sodium-potassium-ATPase expression occurred 2-24 h postburn. These data confirm that burn trauma alters calcium transporter expression, likely contributing to cardiomyocyte calcium loading and cardiac contractile dysfunction.     

Role of cytosolic vs. mitochondrial Ca2+ accumulation in burn injury-related myocardial inflammation and function

This study was designed to examine the role of mitochondrial Ca2+ homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial Ca2+ is a primary modulator of cardiomyocyte TNF-alpha, IL-1beta, and IL-6 responses to injury and infection. Ventricular myocytes were prepared by Langendorff perfusion of hearts from adult rats subjected to sham burn or burn injury over 40% of total body surface area to produce enzymatic (collagenase) digestion. Isolated cardiomyocytes were suspended in MEM, cell number was determined, and aliquots of myocytes from each experimental group were loaded with fura 2-AM (2 microg/ml) for 1) 45 min at room temperature to measure total cellular Ca2+, 2) 45 min at 30 degrees C followed by incubation at 37 degrees C for 2 h to eliminate cytosolic fluorescence, and 3) 20 min at 37 degrees C in MnCl2 (200 microM)-containing buffer to quench cytosolic fura 2-AM signal. In vitro studies included preparation of myocytes from control hearts and challenge of myocytes with LPS or burn serum (BS), which have been shown to increase cytosolic Ca2+. Additional aliquots of myocytes were challenged with LPS or BS with or without a selective inhibitor of mitochondrial Ca2+, ruthenium red (RR). All cells were examined on a stage-inverted microscope that was interfaced with the InCyt Im2 fluorescence imaging system. Heat treatment or MnCl2 challenge eliminated myocyte cytosolic fluorescence, whereas cells maintained at room temperature retained 95% of their initial fluorescence. Compared with Ca2+ levels measured in sham myocytes, burn trauma increased cytosolic Ca2+ from 90 +/- 3 to 293 +/- 6 nM (P < 0.05) and mitochondrial Ca2+ from 24 +/- 1 to 75 +/- 2 nM (P < 0.05). LPS (25 microg/5 x 10(4) cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca2+ and promoted myocyte secretion of TNF-alpha, IL-1beta, and IL-6. RR pretreatment decreased LPS- and BS-related rise in mitochondrial Ca2+ and cytokine secretion but had no effect on cytosolic Ca2+. BS challenge in perfused control hearts impaired myocardial contraction/relaxation, and RR pretreatment of hearts prevented BS-related myocardial contractile dysfunction. Our data suggest that a rise in mitochondrial Ca2+ is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis and burn trauma.     

The mechanical effects of CRT promoting autophagy via mitochondrial calcium uniporter down‐regulation and mitochondrial dynamics alteration

The mechanism of cardiac resynchronization therapy (CRT) remains unclear. In this study, mitochondria calcium uniporter (MCU), dynamin‐related protein‐1 (DNM1L/Drp1) and their relationship with autophagy in heart failure (HF) and CRT are investigated. Thirteen male beagle's dogs were divided into three groups (sham, HF, CRT). Animals received left bundle branch (LBB) ablation followed by either 8‐week rapid atrial pacing or 4‐week rapid atrial pacing and 4‐week biventricular pacing. Cardiac function was evaluated by echocardiography. Differentially expressed genes (DEGs) were detected by microarray analysis. General morphological changes, mitochondrial ultrastructure, autophagosomes and mitophagosomes were investigated. The cardiomyocyte stretching was adopted to imitate the mechanical effect of CRT. Cells were divided into three groups (control, angiotensin‐II and angiotensin‐II + stretching). MCU, DNM1L/Drp1 and autophagy markers were detected by western blots or immunofluorescence. In the present study, CRT could correct cardiac dysfunction, decrease cardiomyocyte's size, alleviate cardiac fibrosis, promote the formation of autophagosome and mitigate mitochondrial injury. CRT significantly influenced gene expression profile, especially down‐regulating MCU and up‐regulating DNM1L/Drp1. Cell stretching reversed the angiotensin‐II induced changes of MCU and DNM1L/Drp1 and partly restored autophagy. CRT's mechanical effects down‐regulated MCU, up‐regulated DNM1L/Drp1 and subsequently enhanced autophagy. Besides, the mechanical stretching prevented the angiotensin‐II‐induced cellular enlargement.     

Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.     

Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2^?/? mice were treated with the CYP2E1 inhibitor diallyl sulfide (100?mg/kg/d, i.p.) for 4?weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca²⁺ property, mitochondrial ultrastructural damage, elevated O2^– production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.