Role of dystrophin in acute Trypanosoma cruzi infection

Previous studies have demonstrated loss/reduction of dystrophin in cardiomyocytes in both acute and chronic stages of experimental Trypanosoma cruzi (T. cruzi) infection in mice. The mechanisms responsible for dystrophin disruption in the hearts of mice acutely infected with T. cruzi are not completely understood. The present in vivo and in vitro studies were undertaken to evaluate the role of inflammation in dystrophin disruption and its correlation with the high mortality rate during acute infection. C57BL/6 mice were infected with T. cruzi and killed 14, 20 and 26 days post infection (dpi). The intensity of inflammation, cardiac expression of dystrophin, calpain-1, NF-κB, TNF-α, and sarcolemmal permeability were evaluated. Cultured neonatal murine cardiomyocytes were incubated with serum, collected at the peak of cytokine production and free of parasites, from T. cruzi-infected mice and dystrophin, calpain-1, and NF-κB expression analyzed. Dystrophin disruption occurs at the peak of mortality and inflammation and is associated with increased expression of calpain-1, TNF-α, NF-κB, and increased sarcolemmal permeability in the heart of T. cruzi-infected mice at 20 dpi confirmed by in vitro studies. The peak of mortality occurred only when significant loss of dystrophin in the hearts of infected animals occurred, highlighting the correlation between inflammation, dystrophin loss and mortality.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.     

Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in septic mice

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice. GP was administered to ICR/HSD mice 1 h before induction of CLP. Sham surgically operated mice served as control. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham control. In contrast, GP administration prevented CLP-induced cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP increased myocardial MIF expression by 88.3% (P < 0.05) and cardiomyocyte apoptosis by 7.8-fold (P < 0.05) compared with sham control. GP administration, however, prevented CLP-increased MIF expression and decreased cardiomyocyte apoptosis by 51.2% (P < 0.05) compared with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-GSK-3beta, and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also activates the phosphoinositide 3-kinase/Akt pathway, decreases myocardial MIF expression, and reduces cardiomyocyte apoptosis.     

Loss of duplexmiR-223 (5p and 3p) aggravates myocardial depression and mortality in polymicrobial sepsis

Sepsis is the leading cause of death in critically ill patients. While myocardial dysfunction has been recognized as a major manifestation in severe sepsis, the underlying molecular mechanisms associated with septic cardiomyopathy remain unclear. In this study, we performed a miRNA array analysis in hearts collected from a severe septic mouse model induced by cecal ligation and puncture (CLP). Among the 19 miRNAs that were dys-regulated in CLP-mouse hearts, miR-223(3p) and miR-223*(5p) were most significantly downregulated, compared with sham-operated mouse hearts. To test whether a drop of miR-223 duplex plays any roles in sepsis-induced cardiac dysfunction and inflammation, a knockout (KO) mouse model with a deletion of the miR-223 gene locus and wild-type (WT) mice were subjected to CLP or sham surgery. We observed that sepsis-induced cardiac dysfunction, inflammatory response and mortality were remarkably aggravated in CLP-treated KO mice, compared with control WTs. Using Western-blotting and luciferase reporter assays, we identified Sema3A, an activator of cytokine storm and a neural chemorepellent for sympathetic axons, as an authentic target of miR-223* in the myocardium. In addition, we validated that miR-223 negatively regulated the expression of STAT-3 and IL-6 in mouse hearts. Furthermore, injection of Sema3A protein into WT mice revealed an exacerbation of sepsis-triggered inflammatory response and myocardial depression, compared with control IgG1 protein-treated WT mice following CLP surgery. Taken together, these data indicate that loss of miR-223/-223* causes an aggravation of sepsis-induced inflammation, myocardial dysfunction and mortality. Our study uncovers a previously unrecognized mechanism underlying septic cardiomyopathy and thereby, may provide a new strategy to treat sepsis.     

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth''s 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47^phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47^phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47^phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47^phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.     

Deficiency of Capn4 Gene Inhibits Nuclear Factor-κB (NF-κB) Protein Signaling/Inflammation and Reduces Remodeling after Myocardial Infarction

Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.     

Role of interleukin-6 in cardiac inflammation and dysfunction after burn complicated by sepsis

To examine the role of myocardial interleukin-6 (IL-6) in myocardial inflammation and dysfunction after burn complicated by sepsis, we performed 40% total body surface area contact burn followed by late (7 days) Streptococcus pneumoniae pneumonia sepsis in wild-type (WT) mice, IL-6 knockout (IL-6 KO) mice, and transgenic mice overexpressing IL-6 in the myocardium (TG). Twenty-four hours after sepsis was induced, isolated cardiomyocytes were harvested and cultured in vitro, and supernatant concentrations of IL-6 and tumor necrosis factor (TNF)-alpha were measured. Cardiomyocyte intracellular calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) concentrations were also determined. Separate mice in each group underwent in vivo global hemodynamic and cardiac function assessment by cannulation of the carotid artery and insertion of a left ventricular pressure volume conductance catheter. Hearts from these mice were collected for histopathological assessment of inflammatory response, fibrosis, and apoptosis. In the WT group, there was an increase in cardiomyocyte TNF-alpha, [Ca(2+)](i), and [Na(+)](i) after burn plus sepsis, along with cardiac contractile dysfunction, inflammation, and apoptosis. These changes were attenuated in the IL-6 KO group but accentuated in the TG group. We conclude myocardial IL-6 mediates cardiac inflammation and contractile dysfunction after burn plus sepsis.     

L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy

Under conditions of iron overload, which are now reaching epidemic proportions worldwide, iron-overload cardiomyopathy is the most important prognostic factor in patient survival. We hypothesize that in iron-overload disorders, iron accumulation in the heart depends on ferrous iron (Fe2+) permeation through the L-type voltage-dependent Ca2+ channel (LVDCC), a promiscuous divalent cation transporter. Iron overload in mice was associated with increased mortality, systolic and diastolic dysfunction, bradycardia, hypotension, increased myocardial fibrosis and elevated oxidative stress. Treatment with LVDCC blockers (CCBs; amlodipine and verapamil) at therapeutic levels inhibited the LVDCC current in cardiomyocytes, attenuated myocardial iron accumulation and oxidative stress, improved survival, prevented hypotension and preserved heart structure and function. Consistent with the role of LVDCCs in myocardial iron uptake, iron-overloaded transgenic mice with cardiac-specific overexpression of the LVDCC alpha1-subunit had twofold higher myocardial iron and oxidative stress levels, as well as greater impairment in cardiac function, compared with littermate controls; LVDCC blockade was again protective. Our results indicate that cardiac LVDCCs are key transporters of iron into cardiomyocytes under iron-overloaded conditions, and potentially represent a new therapeutic target to reduce the cardiovascular burden from iron overload.     

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.     

MiR-146b protect against sepsis induced mice myocardial injury through inhibition of Notch1

Myocardial dysfunction is a major cause of death in sepsis. MicroRNA-146b (miR-146b) has been reported to be related to myocardial disease. However, the role of miR-146b in sepsis as well as myocardial injury is still unclear. Septic cardiac dysfunction in mice was induced by cecal ligation and puncture (CLP) and miR-146b was found increased significantly in the myocardium tissue of CLP mice. It was found that up-regulation of miR-146b by agomiR injection suppressed expression of IL-1β in mice as well as myocardium apoptosis in CLP mice. However, suppression of miR-146b by antagomiR injection had inverse effects. Notch1 was identified as a target gene of miR-146b by bioinformatics analysis. And it was verified that in cardiomyocytes, the decrease of miR146b led to increase of both the mRNA and protein level of Notch1 and vice versa. In septic mice serum stimulated cardiomyocytes, up-regulation of miR-146b decreased the level of Notch1 and Hes1. The knockout of Notch1 in transgenic mice showed that the deficiency of Notch1 improved myocardial injury induced by CLP operation. The apoptosis of cardiomyocytes was relieved and the expression of IL-1β was decreased. In conclusion, miR-146b targets to Notch1 and protected cardiomyocytes against inflammation and apoptosis.     

Calpain-dependent Cleavage of N-cadherin Is Involved in the Progression of Post-myocardial Infarction Remodeling

Enzymatic proteolysis by calpains, Ca²⁺-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast^−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast^+/+ mice. The activity of cardiac calpains in Cast^+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast^−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast^−/− and Cast^+/+ mice, Cast^−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast^+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast^−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast^+/+ mice. In the border zone of Cast^−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.     

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.     

Novel function of Roxadustat (FG-4592) as an anti-shock drug in sepsis by regulating mitochondrial oxidative stress and energy metabolism

BackgroundSeptic shock is a serious clinical syndrome leading to high mortality. A new anti-anemia drug Roxadustat (FG-4592) protected against cardiac injury and hypertension. However, its effect and mechanism on shock and cardiac dysfunction induced by sepsis require to be investigated.MethodsC57BL/6j mice received FG-4592 (10 mg/kg/day) by i.p injection, followed by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) treatment. Mortality and shock status were monitored during the experiment. Cardiac function was assessed using echocardiography and serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) assay. TEM, COX-SDH staining and ATP production were used to evaluate mitochondrial function. A non-targeted metabolomic analysis was performed to evaluate the metabolic disorders.ResultsBoth pre- and post-treatment of FG-4592 could improve the survival rate in LPS- and CLP-induced sepsis mice with a better effect in pre-treated animals. Meanwhile, FG-4592 improved systolic blood pressure and body temperature drop in septic mice along with alleviated cardiac dysfunction (as shown by the restoration of decreased LVEF and LVFS and increased LDH and CK-MB) and inflammation. Interestingly, we observed that FG-4592 improved mitochondrial oxidative stress possibly by upregulating the anti-oxidative enzymes of SOD2 and HO-1. Furthermore, FG-4592 improved the energy supply and glycerophospholipid metabolism in cardiomyocytes, possibly through upregulating the HIF-1α-targeted genes of LDHA and PDK1 in glycolysis and CHK-α, respectively.ConclusionsFG-4592 protected against mortality and shock in septic animals possibly by antagonizing mitochondrial oxidative stress and metabolic disorders.General significanceThis study provides a potential of FG-4592 as a novel drug for treating septic shock.     

Autotaxin/lysophosphatidic acid signaling mediates obesity‐related cardiomyopathy in mice and human subjects

Obesity is associated with increased cardiovascular morbidity and mortality, but the direct signals to initiate or exaggerate cardiomyopathy remain largely unknown. Present study aims to explore the pathophysiological role of autotaxin/lysophosphatidic acid (LPA) in the process of cardiomyopathy during obesity. Through utilizing mouse model and clinical samples, present study investigates the therapeutic benefits of autotaxin inhibitor and clinical correlation to obesity‐related cardiomyopathy. The elevated circulating levels of autotaxin are closely associated with cardiac parameters in mice. Administration with autotaxin inhibitor, PF‐8380 effectively attenuates high fat diet‐induced cardiac hypertrophy, dysfunction and inflammatory response. Consistently, autotaxin inhibition also decreases circulating LPA levels in obese mice. In in vitro study, LPA directly initiates cell size enlargement and inflammation in neonatal cardiomyocytes. More importantly, circulating levels of autotaxin are positively correlated with cardiac dysfunction and hypertrophy in 55 patients. In conclusion, present study uncovers the correlation between circulating autotaxin and cardiac parameters in mice and human patient, and provided solid evidence of the therapeutic application of autotaxin inhibitor in combating obesity‐related cardiomyopathy.     

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM^∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.     

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.     

The down-regulation of cardiac contractile proteins underlies myocardial depression during sepsis and is mitigated by carbon monoxide

The aim of this study is to investigate the mechanism underling cardiac dysfunction during sepsis, as well as the possible amelioration of this dysfunction by exogenous carbon monoxide (CO) administration. For this purpose, rats (six-week-old, male, Sprague-Dawley) were administered LPS (15 mg/kg body weight, i.p. 6 h) and/or CORM (30 mg/kg, i.p.). The decreased left ventricular ejection fraction (EF) observed in LPS group rats was recovered in the LSP + CORM group, confirming the protective role of CO against sepsis-induced myocardial depression. Proteomic as well as immunoblot analysis showed that the levels of myosin heavy and light chains (MHC and MLC) as well as α-cardiac actin (ACTC) were decreased in the LPS group, and these decreases were mitigated in the LSP + CORM group, suggesting that the amounts of major contractile proteins are decreased in depressed myocardium. Not only LPS-induced inflammatory cytokine (TNFα and IL-1β) production but also the decrease in myofilament proteins was mitigated by CORM. These results confirm the protective action of exogenously administered CO against myocardial depression during sepsis, and reveal a novel mechanism underling cardiac dysfunction during sepsis.     

Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.     

Development of dilated cardiomyopathy in Bmal1-deficient mice

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1(-/-) mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1(-/-) mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure.