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Background: Elevated myocardial intracellular sodium ([Na + ] i ) was shown to decrease mitochondrial calcium ([Ca 2+ ] MITO ) via mitochondrial sodium/calcium exchanger (NCX MITO ), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na + ] i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na + ] i ., Methods: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31 P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCX MITO inhibitor. Myocardial [Na + ] i was measured by 23 Na NMR spectroscopy., Results: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na + ] i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na + ] i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts., Conclusions: Elevated myocardial [Na + ] i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na + ] i and/or NCX MITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na + ] i ., Competing Interests: Declaration of Competing Interest Dr. Colucci is supported by an investigator‐initiated grant (IISP‐57335) from Merck Pharmaceuticals (Kenilworth, NJ, USA). The remaining authors have no disclosures to report., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background Inhibitors of the sodium-glucose linked transporter 2 improve cardiovascular outcomes in patients with or without type 2 diabetes mellitus, but the effects on cardiac energetics and mitochondrial function are unknown. We assessed the effects of sodium-glucose linked transporter 2 inhibition on mitochondrial function, high-energy phosphates, and genes encoding mitochondrial proteins in hearts of mice with and without diet-induced diabetic cardiomyopathy. Methods and Results Mice fed a control diet or a high-fat, high-sucrose diet received ertugliflozin mixed with the diet (0.5 mg/g of diet) for 4 months. Isolated mitochondria were assessed for functional capacity. High-energy phosphates were assessed by 31 P nuclear magnetic resonance spectroscopy concurrently with contractile performance in isolated beating hearts. The high-fat, high-sucrose diet caused myocardial hypertrophy, diastolic dysfunction, mitochondrial dysfunction, and impaired energetic response, all of which were prevented by ertugliflozin. With both diets, ertugliflozin caused supernormalization of contractile reserve, as measured by rate×pressure product at high work demand. Likewise, the myocardial gene sets most enriched by ertugliflozin were for oxidative phosphorylation and fatty acid metabolism, both of which were enriched independent of diet. Conclusions Ertugliflozin not only prevented high-fat, high-sucrose-induced pathological cardiac remodeling, but improved contractile reserve and induced the expression of oxidative phosphorylation and fatty acid metabolism gene sets independent of diabetic status. These effects of sodium-glucose linked transporter 2 inhibition on cardiac energetics and metabolism may contribute to improved structure and function in cardiac diseases associated with mitochondrial dysfunction, such as heart failure.

Background: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes., Methods: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H 2 O 2 (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction., Results: In adult rat ventricular myocytes expressing wild-type SERCA, H 2 O 2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H 2 O 2 -stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H 2 O 2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice., Conclusions: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Mice with obesity and metabolic heart disease (MHD) due to a high-fat, high-sucrose diet were treated with placebo, a clinically relevant dose of sacubitril (SAC)/valsartan (VAL), or an equivalent dose of VAL for 4 months. There were striking differences between SAC/VAL and VAL with regard to: 1) diastolic dysfunction; 2) interstitial fibrosis; and to a lesser degree; 3) oxidative stress-all of which were more favorably affected by SAC/VAL. SAC/VAL and VAL similarly attenuated myocardial hypertrophy and improved myocardial energetics. In mice with obesity-related MHD, neprilysin inhibition exerts favorable effects on diastolic function., (© 2020 The Authors.)

Multiplexed imaging is essential for the evaluation of substrate utilization in metabolically active organs, such as the heart and brown adipose tissue (BAT), where substrate preference changes in pathophysiologic states. Optical imaging provides a useful platform because of its low cost, high throughput and intrinsic ability to perform composite readouts. However, the paucity of probes available for in vivo use has limited optical methods to image substrate metabolism. Here, we present a novel near-infrared (NIR) free fatty acid (FFA) tracer suitable for in vivo imaging of deep tissues such as the heart. Using click chemistry, Alexa Fluor 647 DIBO Alkyne was conjugated to palmitic acid. Mice injected with 0.05 nmol/g bodyweight of the conjugate (AlexaFFA) were subjected to conditions known to increase FFA uptake in the heart (fasting) and BAT [cold exposure and injection with the β 3 adrenergic agonist CL 316, 243(CL)]. Organs were subsequently imaged both ex vivo and in vivo to quantify AlexaFFA uptake. The blood kinetics of AlexaFFA followed a two-compartment model with an initial fast compartment half-life of 0.14 h and a subsequent slow compartment half-life of 5.2 h, consistent with reversible protein binding. Ex vivo fluorescence imaging after overnight cold exposure and fasting produced a significant increase in AlexaFFA uptake in the heart (58 ± 12%) and BAT (278 ± 19%) compared to warm/fed animals. In vivo imaging of the heart and BAT after exposure to CL and fasting showed a significant increase in AlexaFFA uptake in the heart (48 ± 20%) and BAT (40 ± 10%) compared to saline-injected/fed mice. We present a novel near-infrared FFA tracer, AlexaFFA, that is suitable for in vivo quantification of FFA metabolism and can be applied in the context of a low cost, high throughput, and multiplexed optical imaging platform.

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG ~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG ~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities., (© 2020 John Wiley & Sons, Ltd.)

Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches., (© 2019 American Heart Association, Inc.)

Aims: Metabolic syndrome is associated with metabolic heart disease (MHD) that is characterized by left ventricular (LV) hypertrophy, interstitial fibrosis, contractile dysfunction, and mitochondrial dysfunction. Overexpression of catalase in mitochondria (transgenic expression of catalase targeted to the mitochondria [mCAT]) prevents the structural and functional features of MHD caused by a high-fat, high-sucrose (HFHS) diet for ≥4 months. However, it is unclear whether the effect of mCAT is due to prevention of reactive oxygen species (ROS)-mediated cardiac remodeling, a direct effect on mitochondrial function, or both. To address this question, we measured myocardial function and energetics in mice, with or without mCAT, after 1 month of HFHS, before the development of cardiac structural remodeling. Results: HFHS diet for 1 month had no effect on body weight, heart weight, LV structure, myocyte size, or interstitial fibrosis. Isolated cardiac mitochondria from HFHS-fed mice produced 2.2- to 3.8-fold more H 2 O 2 , and 16%-29% less adenosine triphosphate (ATP). In isolated beating hearts from HFHS-fed mice, [phosphocreatine (PCr)] and the free energy available for ATP hydrolysis (ΔG ∼ATP ) were decreased, and they failed to increase with work demands. Overexpression of mCAT normalized ROS and ATP production in isolated mitochondria, and it corrected myocardial [PCr] and ΔG ∼ATP in the beating heart. Innovation: This is the first demonstration that in MHD, mitochondrial ROS mediate energetic dysfunction that is sufficient to impair contractile function. Conclusion: ROS produced and acting in the mitochondria impair myocardial energetics, leading to slowed relaxation and decreased contractile reserve. These effects precede structural remodeling and are corrected by mCAT, indicating that ROS-mediated energetic impairment, per se , is sufficient to cause contractile dysfunction in MHD.

Calcium (Ca 2+ ), an important second messenger, regulates many cellular activities and varies spatiotemporally within the cell. Conventional methods to monitor Ca 2+ changes, such as synthetic Ca 2+ indicators, are not targetable, while genetically encoded Ca 2+ indicators (GECI) can be precisely directed to cellular compartments. GECIs are chimeric proteins composed of calmodulin (or other proteins that change conformation on Ca 2+ binding) coupled with two fluorescent proteins that come closer together after an increase in [Ca 2+ ], and enhance Förster resonance energy transfer (FRET) that allows for ratiometric [Ca 2+ ] assessment. Here, adult rat ventricular myocytes were transfected with specifically targeted calmodulin-based GECIs and Ca 2+ responses to a physiological stimulus, norepinephrine (NE, 10 μM), were observed in a) sarcoplasmic reticulum (SR), b) mitochondria, c) the space between the mitochondria and SR, termed the Mitochondria Associated Membrane space (MAM) and d) cytosol for 10 min after stimulation. In SR and mitochondria, NE increased the [Ca 2+ ] ratio by 17% and by 8%, respectively. In the MAM the [Ca 2+ ] ratio decreased by 16%, while in cytosol [Ca 2+ ] remained unchanged. In conclusion, adrenergic stimulation causes distinct responses in the cardiomyocyte SR, mitochondria and MAM. Additionally, our work provides a toolkit-update for targeted [Ca 2+ ] measurements in multiple cellular compartments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mice challenged with lipopolysaccharide develop cardiomyopathy in a sex and redox-dependent fashion. Here we extended these studies to the cecal ligation and puncture (CLP) model.We compared male and female FVB mice (wild type, WT) and transgenic littermates overexpressing myocardial catalase (CAT). CLP induced 100% mortality within 4 days, with similar mortality rates in male and female WT and CAT mice. 24 h after CLP, isolated (Langendorff) perfused hearts showed depressed contractility in WT male mice, but not in male CAT or female WT and CAT mice. In WT male mice, CLP induced a depression of cardiomyocyte sarcomere shortening (ΔSS) and calcium transients (ΔCai), and the inhibition of the sarcoplasmic reticulum Ca ATPase (SERCA). These deficits were associated with overexpression of NADPH-dependent oxidase (NOX)-1, NOX-2, and cyclooxygenase 2 (COX-2), and were partially prevented in male CAT mice. Female WT mice showed unchanged ΔSS, ΔCai, and SERCA function after CLP. At baseline, female WT mice showed partially depressed ΔSS, ΔCai, and SERCA function, as compared with male WT mice, which were associated with NOX-1 overexpression and were prevented in CAT female mice.In conclusion, in male WT mice, septic shock induces myocardial NOX-1, NOX-2, and COX-2, and redox-dependent dysregulation of myocardial Ca transporters. Female WT mice are resistant to CLP-induced cardiomyopathy, despite increased NOX-1 and COX-2 expression, suggesting increased antioxidant capacity. Female resistance occurred in association with NOX-1 overexpression and signs of increased oxidative signaling at baseline, indicating the presence of a protective myocardial redox hormesis mechanism.

Background Obesity is a precursor to heart failure with preserved ejection fraction. Biomarkers that identify preclinical metabolic heart disease ( MHD ) in young obese patients would help identify high-risk individuals for heart failure prevention strategies. We assessed the predictive value of GAL3 (galectin-3), FSTL3 (follistatin-like 3 peptide), and NT-proBNP (N-terminal pro-B-type natriuretic peptide) to identify stage B MHD in young obese participants free of clinically evident cardiovascular disease. Methods and Results Asymptomatic obese patients (n=250) and non-obese controls (n=21) underwent echocardiographic cardiac phenotyping. Obese patients were classified as MHD positive ( MHD - POS ; n=94) if they had abnormal diastolic function or left ventricular hypertrophy and had estimated pulmonary artery systolic pressure ≥35 mm Hg. Obese patients without such abnormalities were classified as MHD negative (MHD-NEG; n=52). Serum biomarkers timed with echocardiography. MHD - POS and MHD-NEG individuals were similarly obese, but MHD - POS patients were older, with more diabetes mellitus and metabolic syndrome. Right ventricular coupling was worse in MHD - POS patients ( P<0.001). GAL 3 levels were higher in MHD - POS versus MHD -NEG patients (7.7±2.3 versus 6.3±1.9 ng/mL, respectively; P<0.001). Both GAL 3 and FSTL 3 levels correlated with diastolic dysfunction and increased pulmonary artery systolic pressure but not with left ventricular mass. In multivariate models including all 3 biomarkers, only GAL 3 remained associated with MHD (odds ratio: 1.30; 95% CI , 1.01-1.68; P=0.04). Conclusions In young obese individuals without known cardiovascular disease, GAL 3 is associated with the presence of preclinical MHD . GAL 3 may be useful in screening for preclinical MHD and identifying individuals with increased risk of progression to obesity-related heart failure with preserved ejection fraction.

Obesity, particularly visceral adiposity, has been linked to mitochondrial dysfunction and increased oxidative stress, which have been suggested as mechanisms of insulin resistance. The mechanism(s) behind this remains incompletely understood. In this study, we hypothesized that mitochondrial complex II dysfunction plays a role in impaired insulin sensitivity in visceral adipose tissue of subjects with obesity. We obtained subcutaneous and visceral adipose tissue biopsies from 43 subjects with obesity (body mass index ≥ 30 kg/m 2 ) during planned bariatric surgery. Compared with subcutaneous adipose tissue, visceral adipose tissue exhibited decreased complex II activity, which was restored with the reducing agent dithiothreitol (5 mM) ( P < 0.01). A biotin switch assay identified that cysteine oxidative posttranslational modifications (OPTM) in complex II subunit A (succinate dehydrogenase A) were increased in visceral vs. subcutaneous fat ( P < 0.05). Insulin treatment (100 nM) stimulated complex II activity in subcutaneous fat ( P < 0.05). In contrast, insulin treatment of visceral fat led to a decrease in complex II activity ( P < 0.01), which was restored with addition of the mitochondria-specific oxidant scavenger mito-TEMPO (10 µM). In a cohort of 10 subjects with severe obesity, surgical weight loss decreased OPTM and restored complex II activity, exclusively in the visceral depot. Mitochondrial complex II may be an unrecognized and novel mediator of insulin resistance associated with visceral adiposity. The activity of complex II is improved by weight loss, which may contribute to metabolic improvements associated with bariatric surgery.

Metabolic syndrome is a cluster of obesity-related metabolic abnormalities that lead to metabolic heart disease (MHD) with left ventricular pump dysfunction. Although MHD is thought to be associated with myocardial energetic deficiency, two key questions have not been answered. First, it is not known whether there is a sufficient energy deficit to contribute to pump dysfunction. Second, the basis for the energy deficit is not clear. To address these questions, mice were fed a high fat, high sucrose (HFHS) 'Western' diet to recapitulate the MHD phenotype. In isolated beating hearts, we used 31 P NMR spectroscopy with magnetization transfer to determine a) the concentrations of high energy phosphates ([ATP], [ADP], [PCr]), b) the free energy of ATP hydrolysis (∆G ~ATP ), c) the rate of ATP production and d) flux through the creatine kinase (CK) reaction. At the lowest workload, the diastolic pressure-volume relationship was shifted upward in HFHS hearts, indicative of diastolic dysfunction, whereas systolic function was preserved. At this workload, the rate of ATP synthesis was decreased in HFHS hearts, and was associated with decreases in both [PCr] and ∆G ~ATP . Higher work demands unmasked the inability of HFHS hearts to increase systolic function and led to a further decrease in ∆G ~ATP to a level that is not sufficient to maintain normal function of sarcoplasmic Ca 2+ -ATPase (SERCA). While [ATP] was preserved at all work demands in HFHS hearts, the progressive increase in [ADP] led to a decrease in ∆G ~ATP with increased work demands. Surprisingly, CK flux, CK activity and total creatine were normal in HFHS hearts. These findings differ from dilated cardiomyopathy, in which the energetic deficiency is associated with decreases in CK flux, CK activity and total creatine. Thus, in HFHS-fed mice with MHD there is a distinct metabolic phenotype of the heart characterized by a decrease in ATP production that leads to a functionally-important energetic deficiency and an elevation of [ADP], with preservation of CK flux., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Substrate utilization in tissues with high energetic requirements could play an important role in cardiometabolic disease. Current techniques to assess energetics are limited by high cost, low throughput, and the inability to resolve multiple readouts simultaneously. Consequently, we aimed to develop a multiplexed optical imaging platform to simultaneously assess energetics in multiple organs in a high throughput fashion., Methods and Results: The detection of 18F-Fluordeoxyglucose uptake via Cerenkov luminescence and free fatty acid uptake with a fluorescent C 16 free fatty acid was tested. Simultaneous uptake of these agents was measured in the myocardium, brown/white adipose tissue, and skeletal muscle in mice with/without thoracic aortic banding. Within 5 weeks of thoracic aortic banding, mice developed left ventricular hypertrophy and brown adipose tissue activation with upregulation of β 3 AR (β 3 adrenergic receptors) and increased natriuretic peptide receptor ratio. Imaging of brown adipose tissue 15 weeks post thoracic aortic banding revealed an increase in glucose ( P <0.01) and free fatty acid ( P <0.001) uptake versus controls and an increase in uncoupling protein-1 ( P <0.01). Similar but less robust changes were seen in skeletal muscle, while substrate uptake in white adipose tissue remained unchanged. Myocardial glucose uptake was increased post-thoracic aortic banding but free fatty acid uptake trended to decrease., Conclusions: A multiplexed optical imaging technique is presented that allows substrate uptake to be simultaneously quantified in multiple tissues in a high throughput manner. The activation of brown adipose tissue occurs early in the onset of left ventricular hypertrophy, which produces tissue-specific changes in substrate uptake that may play a role in the systemic response to cardiac pressure overload., (© 2018 American Heart Association, Inc.)

Preparations from Crataegus (hawthorn) have a long history in the treatment of heart failure. WS 1442 is a dry extract from hawthorn leaves with flowers (4-6.6:1), extraction solvent of ethanol 45% (w/w), adjusted to 17.3-20.1% of oligomeric procyanidins. Nonclinical studies show that WS 1442 has positive inotropic and antiarrhythmic properties and protects the myocardium from ischemic damage, reperfusion injury, and hypertension-related hypertrophy, improves endothelial functions such as NO synthesis, and delays endothelial senescence. Randomized, controlled trials in patients with heart failure have demonstrated that the herbal medicinal product increases functional capacity, alleviates disabling symptoms, and improves health-related quality of life, all of which have become important targets of heart failure therapy according to current disease management guidelines. Clinical trials (including a 2-year mortality study with polypharmacy and > 1300 patients exposed) and post-marketing surveillance studies have shown that WS 1442 has a very favorable safety profile both as monotherapy and as add-on therapy, where no drug interactions have been observed. No specific adverse reactions to WS 1442 are known to date. WS 1442 may thus help to close the therapeutic gap between systolic and diastolic heart failure for which evidence of efficacy for other cardioactive drugs is sparse. Scientific evidence shows that WS 1442 is safe and has a beneficial effect in patients with heart failure corresponding to New York Heart Association classes II or III. The benefit-risk assessment for WS 1442 is therefore positive.

Objective: Metabolic syndrome (MetS) can lead to myocardial fibrosis, diastolic dysfunction, and eventual heart failure. This study evaluated alterations in myocardial microstructure in people with MetS by using a novel algorithm to characterize ultrasonic signal intensity variation., Methods: Among 254 participants without existing cardiovascular disease (mean age 42 ± 11 years, 75% women), there were 162 with MetS, 47 with obesity without MetS, and 45 nonobese controls. Standard echocardiography was performed, and a novel validated computational algorithm was used to investigate myocardial microstructure based on sonographic signal intensity and distribution. The signal intensity coefficient (SIC [left ventricular microstructure]) was examined., Results: The SIC was significantly higher in people with MetS compared with people with (P < 0.001) and without obesity (P = 0.04), even after adjustment for age, sex, body mass index, hypertension, diabetes mellitus, and the ratio of triglyceride (TG) to high-density lipoprotein (HDL) cholesterol (P < 0.05 for all). Clinical correlates of SIC included TG concentrations (r = 0.21, P = 0.0007) and the TG/HDL ratio (r = 0.2, P = 0.001)., Conclusions: This study's findings suggest that preclinical MetS and dyslipidemia in particular are associated with altered myocardial signal intensity variation. Future studies are needed to determine whether the SIC may help detect subclinical diseases in people with metabolic disease, with the ultimate goal of targeting preventive efforts., (© 2017 The Obesity Society.)

Background: In surviving patients, sepsis-induced cardiomyopathy is spontaneously reversible. In the absence of any experimental data, it is generally thought that cardiac recovery in sepsis simply follows the remission of systemic inflammation. Here the authors aimed to identify the myocardial mechanisms underlying cardiac recovery in endotoxemic mice., Methods: Male C57BL/6 mice were challenged with lipopolysaccharide (7 μg/g, intraperitoneally) and followed for 12 days. The authors assessed survival, cardiac function by echocardiography, sarcomere shortening, and calcium transients (with fura-2-acetoxymethyl ester) in electrically paced cardiomyocytes (5 Hz, 37°C) and myocardial protein expression by immunoblotting., Results: Left ventricular ejection fraction, cardiomyocyte sarcomere shortening, and calcium transients were depressed 12 h after lipopolysaccharide challenge, started to recover by 24 h (day 1), and were back to baseline at day 3. The recovery of calcium transients at day 3 was associated with the up-regulation of the sarcoplasmic reticulum calcium pump to 139 ± 19% (mean ± SD) of baseline and phospholamban down-regulation to 35 ± 20% of baseline. At day 6, calcium transients were increased to 123 ± 31% of baseline, associated with increased sarcoplasmic reticulum calcium load (to 126 ± 32% of baseline, as measured with caffeine) and inhibition of sodium/calcium exchange (to 48 ± 12% of baseline)., Conclusions: In mice surviving lipopolysaccharide challenge, the natural recovery of cardiac contractility was associated with the up-regulation of cardiomyocyte calcium handling above baseline levels, indicating the presence of an active myocardial recovery process, which included sarcoplasmic reticulum calcium pump activation, the down-regulation of phospholamban, and sodium/calcium exchange inhibition.

Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria.

In male mice, sepsis-induced cardiomyopathy develops as a result of dysregulation of myocardial calcium (Ca) handling, leading to depressed cellular Ca transients (ΔCai). ΔCai depression is partially due to inhibition of sarcoplasmic reticulum Ca ATP-ase (SERCA) via oxidative modifications, which are partially opposed by cGMP generated by the enzyme soluble guanylyl cyclase (sGC). Whether similar mechanisms underlie sepsis-induced cardiomyopathy in female mice is unknown.Male and female C57Bl/6J mice (WT), and mice deficient in the sGC α1 subunit activity (sGCα1), were challenged with lipopolysaccharide (LPS, ip). LPS induced mouse death and cardiomyopathy (manifested as the depression of left ventricular ejection fraction by echocardiography) to a similar degree in WT male, WT female, and sGCα1 male mice, but significantly less in sGCα1 female mice. We measured sarcomere shortening and ΔCai in isolated, externally paced cardiomyocytes, at 37°C. LPS depressed sarcomere shortening in both WT male and female mice. Consistent with previous findings, in male mice, LPS induced a decrease in ΔCai (to 30 ± 2% of baseline) and SERCA inhibition (manifested as the prolongation of the time constant of Ca decay, τCa, to 150 ± 5% of baseline). In contrast, in female mice, the depression of sarcomere shortening induced by LPS occurred in the absence of any change in ΔCai, or SERCA activity. This suggested that, in female mice, the causative mechanism lies downstream of the Ca transients, such as a decrease in myofilament sensitivity for Ca. The depression of sarcomere shortening shortening after LPS was less severe in female sGCα1 mice than in WT female mice, indicating that cGMP partially mediates cardiomyocyte dysfunction.These results suggest, therefore, that LPS-induced cardiomyopathy develops through distinct sex-specific myocardial mechanisms. While in males LPS induces sGC-independent decrease in ΔCai, in female mice LPS acts downstream of ΔCai, possibly via sGC-dependent myofilament dysfunction., Competing Interests: None.

Background: Obesity and metabolic syndrome lead to the development of metabolic heart disease (MHD) that is characterized by left ventricular hypertrophy (LVH), diastolic dysfunction, and increased mitochondrial ROS. Caloric restriction (CR) is a nutritional intervention that protects against obesity, diabetes, and cardiovascular disease. Healthy adipose tissue is cardioprotective via releasing adipokines such as adiponectin. We tested the hypothesis that CR can ameliorate MHD and it is associated with improved adipose tissue function as reflected by increased circulating levels of high molecular weight (HMW) adiponectin and AMP-activated protein kinase (AMPK) in db/db mice., Methods: Genetically obese db/db and lean db/+ male mice were fed either ad libitum or subjected to 30% CR for 5 weeks. At the end of the study period, echocardiography was carried out to assess diastolic function. Blood, heart, and epididymal fat pads were harvested for mitochondrial study, ELISA, and Western blot analyses., Results: CR reversed the development of LVH, prevented diastolic dysfunction, and decreased cardiac mitochondrial H 2 O 2 in db/db ( vs. ad lib ) mice. These beneficial effects on the heart were associated with increased circulating level of HMW adiponectin. Furthermore, CR increased AMPK and eNOS activation in white adipose tissue of db/db mice, but not in the heart., Conclusions: These findings indicate that even short-term CR protects the heart from MHD. Whether the beneficial effects of CR on the heart could be related to the improved adipose tissue function warrants future investigation.

Background: Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD., Methods and Results: Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium., Conclusion: Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Myocardial hypertrophy and dysfunction are key features of metabolic heart disease due to dietary excess. Metabolic heart disease manifests primarily as diastolic dysfunction but may progress to systolic dysfunction, although the mechanism is poorly understood. Liver kinase B1 (LKB1) is a key activator of AMP-activated protein kinase and possibly other signaling pathways that oppose myocardial hypertrophy and failure. We hypothesized that LKB1 is essential to the heart's ability to withstand the metabolic stress of dietary excess., Methods and Results: Mice heterozygous for cardiac LKB1 were fed a control diet or a high-fat, high-sucrose diet for 4 months. On the control diet, cardiac LKB1 hearts had normal structure and function. After 4 months of the high-fat, high-sucrose diet, there was left ventricular hypertrophy and diastolic dysfunction in wild-type mice. In cardiac LKB1 (versus wild-type) mice, high-fat, high-sucrose feeding caused more hypertrophy (619 versus 553 μm(2), P<0.05), the de novo appearance of systolic dysfunction (left ventricular ejection fraction; 41% versus 59%, P<0.01) with left ventricular dilation (3.6 versus 3.2 mm, P<0.05), and more severe diastolic dysfunction with progression to a restrictive filling pattern (E/A ratio; 5.5 versus 1.3, P=0.05). Myocardial dysfunction in hearts of cardiac LKB1 mice fed the high-fat, high-sucrose diet was associated with evidence of increased apoptosis and apoptotic signaling via caspase 3 and p53/PUMA (p53 upregulated modulator of apoptosis) and more severe mitochondrial dysfunction., Conclusions: Partial deficiency of cardiac LKB1 promotes the adverse effects of a high-fat, high-sucrose diet on the myocardium, leading to worsening of diastolic function and the de novo appearance of systolic dysfunction. LKB1 plays a key role in protecting the heart from the consequences of metabolic stress., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

Background: Despite the substantial overlap of obesity and metabolic disease, there is heterogeneity with respect to cardiovascular risk. We sought to investigate preclinical differences in systolic and diastolic function in obesity, and specifically compare obese individuals with and without metabolic syndrome (MS)., Methods and Results: Obese individuals without cardiac disease with (OB/MS+, n=124) and without (OB/MS-, n=37) MS were compared with nonobese controls (n=29). Diastolic function was assessed by transmitral and tissue Doppler. Global longitudinal strain (LS) and time-based dyssynchrony were assessed by speckle tracking. Both OB/MS- and OB/MS+ groups had similar ejection fraction but worse systolic mechanics as assessed by LS and dyssynchrony when compared with nonobese controls. Specifically, OB/MS- had 2.5% lower LS (SE, 0.7%; P=0.001 in multivariable-adjusted analyses) and 10.8 ms greater dyssynchrony (SE, 3.3 ms; P=0.002), and OB/MS+ had 1.0% lower LS (SE, 0.3%; P<0.001) and 7.8 ms greater dyssynchrony (SE, 1.5 ms; P<0.001) when compared with controls. Obesity was associated with impaired diastolic function regardless of MS status, as evidenced by greater left atrial diameter and left ventricular mass although diastolic dysfunction was more pronounced in OB/MS+ than in OB/MS- individuals., Conclusions: Obesity is associated with subclinical differences in both systolic and diastolic function regardless of the presence or absence of MS although MS seems to be associated with worse diastolic dysfunction. When compared with controls, metabolically healthy obese had lower LS, greater dyssynchrony, and early diastolic dysfunction, supporting the notion that obesity per se may have adverse cardiovascular effects regardless of metabolic disease., (© 2015 American Heart Association, Inc.)

Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Metabolic disease can lead to intrinsic pulmonary hypertension in experimental models. The contributions of metabolic syndrome (MetS) and obesity to pulmonary hypertension and right ventricular dysfunction in humans remain unclear. We investigated the association of MetS and obesity with right ventricular structure and function in patients without cardiovascular disease., Methods and Results: A total of 156 patients with MetS (mean age 44 years, 71% women, mean body mass index 40 kg/m(2)), 45 similarly obese persons without MetS, and 45 nonobese controls underwent echocardiography, including pulsed wave Doppler measurement of pulmonary artery acceleration time (PAAT) and ejection time. Pulmonary artery systolic pressure was estimated from PAAT using validated equations. MetS was associated with lower tricuspid valve e' (right ventricular diastolic function parameter), shorter PAAT, shorter ejection time, and larger pulmonary artery diameter compared with controls (P<0.05 for all). Estimated pulmonary artery systolic pressure based on PAAT was 42±12 mm Hg in participants with MetS compared with 32±9 and 32±10 mm Hg in obese and nonobese controls (P for ANOVA <0.0001). After adjustment for age, sex, hypertension, diabetes, body mass index, and triglycerides, MetS remained associated with a 20-ms-shorter PAAT (β=-20.4, SE=6.5, P=0.002 versus obese). This association persisted after accounting for left ventricular structure and function and after exclusion of participants with obstructive sleep apnea., Conclusions: MetS is associated with abnormal right ventricular and pulmonary artery hemodynamics, as shown by shorter PAAT and subclinical right ventricular diastolic dysfunction. Estimated pulmonary artery systolic pressures are higher in MetS and preclinical metabolic heart disease and raise the possibility that pulmonary hypertension contributes to the pathophysiology of metabolic heart disease., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Sepsis-induced cardiomyopathy (SIC) is thought to be the result of detrimental effects of inflammatory mediators on the cardiac muscle. Here we studied the effects of prolonged (24 ± 4 h) exposure of adult rat ventricular myocytes (ARVM) to bacterial lipopolysaccharide (LPS) and inflammatory cytokines tumor necrosis factor (TNF) and interleukins-1 (IL-1) and IL-6., Materials and Methods: We measured sarcomere shortening (SS) and cellular calcium (Ca(2+)) transients (ΔCai, with fura-2 AM) in isolated cardiomyocytes externally paced at 5 Hz at 37°C., Results: SS decreased after incubation with LPS (100 μg/mL), IL-1 (100 ng/mL), and IL-6 (30 ng/mL), but not with lesser doses of these mediators, or TNF (10-100 ng/mL). A combination of LPS (100 μg/mL), TNF, IL-1, and IL-6 (each 100 ng/mL; i.e., "Cytomix-100") induced a maximal decrease in SS and ΔCai. Sarcoplasmic reticulum (SR) Ca(2+) load (CaSR, measured with caffeine) was unchanged by Cytomix-100; however, SR fractional release (ΔCai/CaSR) was decreased. Underlying these effects, Ca(2+) influx into the cell (via L-type Ca(2+) channels, LTCC) and Ca(2+) extrusion via Na(+)/Ca(2+) exchange were decreased by Cytomix-100. SR Ca(2+) pump (SERCA) (SR Ca(2+) ATPase) was not affected., Conclusions: Prolonged exposure of ARVM to a mixture of LPS and inflammatory cytokines inhibits cell contractility. The effect is mediated by the inhibition of Ca(2+) influx via LTCC, and partially opposed by the inhibition of Na(+)/Ca(2+) exchange. Because both mechanisms are commonly seen in animal models of SIC, we conclude that prolonged challenge with Cytomix-100 of ARVM may represent an accurate in vitro model for SIC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Obesity leads to metabolic heart disease (MHD) that is associated with a pathologic increase in myocardial fatty acid (FA) uptake and impairment of mitochondrial function. The mechanism of mitochondrial dysfunction in MHD, which results in oxidant production and decreased energetics, is poorly understood but may be related to excess FAs. Determining the effects of cardiac FA excess on mitochondria can be hindered by the systemic sequelae of obesity. Mice with cardiomyocyte-specific overexpression of the fatty acid transport protein FATP1 have increased cardiomyocyte FA uptake and develop MHD in the absence of systemic lipotoxicity, obesity or diabetes. We utilized this model to assess 1) the effect of cardiomyocyte lipid accumulation on mitochondrial structure and energetic function and 2) the role of lipid-driven transcriptional regulation, signaling, toxic metabolite accumulation, and mitochondrial oxidative stress in lipid-induced MHD., Methods: Cardiac lipid species, lipid-dependent signaling, and mitochondrial structure/function were examined from FATP1 mice. Cardiac structure and function were assessed in mice overexpressing both FATP1 and mitochondrial-targeted catalase., Results: FATP1 hearts exhibited a net increase (+12%) in diacylglycerol, with increases in several very long-chain diacylglycerol species (+160-212%, p<0.001) and no change in ceramide, sphingomyelin, or acylcarnitine content. This was associated with an increase in phosphorylation of PKCα and PKCδ, and a decrease in phosphorylation of AKT and expression of CREB, PGC1α, PPARα and the mitochondrial fusion genes MFN1, MFN2 and OPA1. FATP1 overexpression also led to marked decreases in mitochondrial size (-49%, p<0.01), complex II-driven respiration (-28.6%, p<0.05), activity of isolated complex II (-62%, p=0.05), and expression of complex II subunit B (SDHB) (-60% and -31%, p<0.01) in the absence of change in ATP synthesis. Hydrogen peroxide production was not increased in FATP1 mitochondria, and cardiac hypertrophy and diastolic dysfunction were not attenuated by overexpression of catalase in mitochondria in FATP1 mice., Conclusions: Excessive delivery of FAs to the cardiac myocyte in the absence of systemic disorders leads to activation of lipid-driven signaling and remodeling of mitochondrial structure and function., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sepsis-induced cardiomyopathy (SIC) develops as the result of myocardial calcium (Ca) dysregulation. Here we reviewed all published studies that quantified the dysfunction of intracellular Ca transporters and the myofilaments in animal models of SIC. Cardiomyocytes isolated from septic animals showed, invariably, a decreased twitch amplitude, which is frequently caused by a decrease in the amplitude of cellular Ca transients (ΔCai) and sarcoplasmic reticulum (SR) Ca load (CaSR). Underlying these deficits, the L-type Ca channel is downregulated, through mechanisms that may involve adrenomedullin-mediated redox signaling. The SR Ca pump is also inhibited, through oxidative modifications (sulfonylation) of one reactive thiol group (on Cys) and/or modulation of phospholamban. Diastolic Ca leak of ryanodine receptors is frequently increased. In contrast, Na/Ca exchange inhibition may play a partially compensatory role by increasing CaSR and ΔCai. The action potential is usually shortened. Myofilaments show a bidirectional regulation, with decreased Ca sensitivity in milder forms of disease (due to troponin I hyperphosphorylation) and an increase (redox mediated) in more severe forms. Most deficits occurred similarly in two different disease models, induced by either intraperitoneal administration of bacterial lipopolysaccharide or cecal ligation and puncture. In conclusion, substantial cumulative evidence implicates various Ca transporters and the myofilaments in SIC pathology. What is less clear, however, are the identity and interplay of the signaling pathways that are responsible for Ca transporters dysfunction. With few exceptions, all studies we found used solely male animals. Identifying sex differences in Ca dysregulation in SIC becomes, therefore, another priority.

Background: Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM., Methods and Results: Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB., Conclusions: MHD due to consumption of a HFHS "Western" diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease"., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Metabolic syndrome (MS) is commonly associated with left ventricular (LV) diastolic dysfunction and LV hypertrophy. We sought to examine whether preclinical LV diastolic dysfunction can occur independent of LV hypertrophy in MS. We recruited 90 consecutive participants with MS and without cardiovascular disease (mean age 46 years, 78% women) and 26 controls (no risk factors for MS; mean age 43 years, 65% women). Participants underwent echocardiography with tissue Doppler imaging. In age- and gender-adjusted analyses, MS was associated with higher left atrial (LA) diameter, higher LV mass, lower E/A ratio, and lower mean e' (p <0.001 for all). These associations remained significant after further adjusting for blood pressure, antihypertensive medication use, and body mass index. After adjusting for LV mass, MS remained independently associated with higher LA diameter, lower E/A ratio, and lower mean e' (p ≤0.01 for all). Specifically, subjects with MS had a 1.8 cm/s lower mean e' compared with controls (p = 0.01). Notably, differences in mean e' between those with and without MS were more pronounced at younger ages (p for interaction = 0.003). In conclusion, MS was associated with preclinical LV diastolic dysfunction independent of LV mass, as reflected by higher LA diameter, lower E/A ratio, and lower mean e'. This suggests that MS can lead to the development of diastolic dysfunction through mechanisms independent of hypertrophy. Differences in diastolic function were more pronounced at younger ages, highlighting the potential importance of early risk factor modification and preventive strategies in MS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Oxidative stress in the myocardium plays an important role in the pathophysiology of hemodynamic overload. The mechanism by which reactive oxygen species (ROS) in the cardiac myocyte mediate myocardial failure in hemodynamic overload is not known. Accordingly, our goals were to test whether myocyte-specific overexpression of peroxisomal catalase (pCAT) that localizes in the sarcoplasm protects mice from hemodynamic overload-induced failure and prevents oxidation and inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), an important sarcoplasmic protein. Chronic hemodynamic overload was caused by ascending aortic constriction (AAC) for 12 wk in mice with myocyte-specific transgenic expression of pCAT. AAC caused left ventricular hypertrophy and failure associated with a generalized increase in myocardial oxidative stress and specific oxidative modifications of SERCA at cysteine 674 and tyrosine 294/5. pCAT overexpression ameliorated myocardial hypertrophy and apoptosis, decreased pathological remodeling, and prevented the progression to heart failure. Likewise, pCAT prevented oxidative modifications of SERCA and increased SERCA activity without changing SERCA expression. Thus cardiac myocyte-restricted expression of pCAT effectively ameliorated the structural and functional consequences of chronic hemodynamic overload and increased SERCA activity via a post-translational mechanism, most likely by decreasing inhibitory oxidative modifications. In pressure overload-induced heart failure cardiac myocyte cytosolic ROS play a pivotal role in mediating key pathophysiologic events including hypertrophy, apoptosis, and decreased SERCA activity., (Copyright © 2014 the American Physiological Society.)

Digoxin is the oldest cardiac drug still in contemporary use, yet its role in the management of patients with heart failure (HF) remains controversial. A purified cardiac glycoside derived from the foxglove plant, digoxin increases ejection fraction, augments cardiac output, and reduces pulmonary capillary wedge pressure without causing deleterious increases in heart rate or decreases in blood pressure. Moreover, it is also a neurohormonal modulator at low doses. In the pivotal DIG (Digitalis Investigation Group) trial, digoxin therapy was shown to reduce all-cause and HF-specific hospitalizations but had no effect on survival. With the discovery of neurohormonal blockers capable of reducing mortality in HF with reduced ejection fraction, the results of the DIG trial were viewed as neutral, and the use of digoxin declined precipitously. Although modern drug and device-based therapies have dramatically improved the survival of ambulatory patients with HF, outcomes for patients with worsening chronic HF, defined as deteriorating signs and symptoms on standard therapy often leading to unscheduled clinic or emergency department visits or hospitalization, have largely remained unchanged over the past 2 decades. The available data suggest that a therapeutic trial of digoxin may be appropriate in patients with worsening chronic heart failure who remain symptomatic., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, β-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

Background: Currently available screening tools for left ventricular (LV) hypertrophy (LVH) and systolic dysfunction (LVSD) are either expensive (echocardiography) or perform suboptimally (B-type natriuretic peptide [BNP]). It is unknown whether newer biomarkers are associated with LVH and LVSD and can serve as screening tools., Methods and Results: We studied 2460 Framingham Study participants (mean age 58 years, 57% women) with measurements of biomarkers mirroring cardiac biomechanical stress (soluble ST-2 [ST2], growth differentiation factor-15 [GDF-15] and high-sensitivity troponin I [hsTnI]) and BNP. We defined LVH as LV mass/height(2) ≥the sex-specific 80th percentile and LVSD as mild/greater impairment of LV ejection fraction (LVEF) or a fractional shortening <0.29. Adjusting for standard risk factors in logistic models, BNP, GDF-15, and hsTnI were associated with the composite echocardiographic outcome (LVH or LVSD), odds ratios (OR) per SD increment in log-biomarker 1.29, 1.14, and 1.18 (95% CI: 1.15 to 1.44, 1.004 to 1.28, and 1.06 to 1.31), respectively. The C-statistic for the composite outcome increased from 0.765 with risk factors to 0.770 adding BNP, to 0.774 adding novel biomarkers. The continuous Net Reclassification Improvement was 0.212 (95% CI: 0.119 to 0.305, P<0.0001) after adding the novel biomarkers to risk factors plus BNP. BNP was associated with LVH and LVSD in multivariable models, whereas GDF-15 was associated with LVSD (OR 1.41, 95% CI: 1.16 to 1.70), and hsTnI with LVH (OR 1.22, 95% CI: 1.09 to 1.36). ST2 was not significantly associated with any outcome., Conclusions: Our community-based investigation suggests that cardiac stress biomarkers are associated with LVH and LVSD but may have limited clinical utility as screening tools.

Background: The nitroxyl (HNO) donor, Angeli's salt, exerts positive inotropic, lusitropic, and vasodilator effects in vivo that are cAMP independent. Its clinical usefulness is limited by chemical instability and cogeneration of nitrite which itself has vascular effects. Here, we report on effects of a novel, stable, pure HNO donor (CXL-1020) in isolated myoctyes and intact hearts in experimental models and in patients with heart failure (HF)., Methods and Results: CXL-1020 converts solely to HNO and inactive CXL-1051 with a t1/2 of 2 minutes. In adult mouse ventricular myocytes, it dose dependently increased sarcomere shortening by 75% to 210% (50-500 μmol/L), with a ≈30% rise in the peak Ca(2+) transient only at higher doses. Neither inhibition of protein kinase A nor soluble guanylate cyclase altered this contractile response. Unlike isoproterenol, CXL-1020 was equally effective in myocytes from normal or failing hearts. In anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced left ventricular end-diastolic pressure and myocardial oxygen consumption while increasing ejection fraction from 27% to 40% and maximal ventricular power index by 42% (both P<0.05). In conscious dogs with tachypacing-induced HF, CXL-1020 increased contractility assessed by end-systolic elastance and provided venoarterial dilation. Heart rate was minimally altered. In patients with systolic HF, CXL-1020 reduced both left and right heart filling pressures and systemic vascular resistance, while increasing cardiac and stroke volume index. Heart rate was unchanged, and arterial pressure declined modestly., Conclusions: These data show the functional efficacy of a novel pure HNO donor to enhance myocardial function and present first-in-man evidence for its potential usefulness in HF., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01096043, NCT01092325.

The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 μg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.

Background: A hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum (SR) calcium uptake and relaxation due to decreased SR calcium ATPase (SERCA) activity. We tested the hypothesis that H2O2-mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging., Methods and Results: Young (5-month-old) and senescent (21-month-old) FVB wild-type (WT) or transgenic mice with myocyte-specific overexpression of catalase were studied. In senescent mice, myocyte-specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S-glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H2O2., Conclusion: Oxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.

Objectives: Levosimendan is an inodilator indicated for acute heart failure (AHF). Its vasodilatory and anti-ischemic effects are mediated by the opening of ATP-dependent potassium channels (K(ATP) channels). Diabetes mellitus is common in AHF patients and sulfonylureas are often prescribed. Sulfonylureas act by blocking the K(ATP) channels. An interaction between levosimendan and sulfonylureas has been shown in preclinical models and could be hypothesized in clinical practice., Design: We produced a pooled analysis of six randomized levosimendan trials (in total of 3004 patients of which 1700 were treated with levosimendan and 226 both with levosimendan and sulfonylureas) with the aim to study the influence of concurrent sulfonylurea treatment to the levosimendan effects. Invasive and non-invasive hemodynamics, biomarkers (BNP), adverse events related to myocardial ischemia, and survival were evaluated., Results: In our relatively small data set, we could not detect any clinically relevant interactions between the sulfonylureas and levosimendan. Similar decreases in systolic and diastolic blood pressure, pulmonary capillary wedge pressure and BNP, and similar survival and adverse event profiles were seen in sulfonylurea users and non-users exposed to levosimendan., Conclusions: Concomitant use of sulfonylureas with levosimendan does not attenuate the hemodynamic or other effects of levosimendan.

Significance: Oxidative post-translational modifications (OPTMs) have been demonstrated as contributing to cardiovascular physiology and pathophysiology. These modifications have been identified using antibodies as well as advanced proteomic methods, and the functional importance of each is beginning to be understood using transgenic and gene deletion animal models. Given that OPTMs are involved in cardiovascular pathology, the use of these modifications as biomarkers and predictors of disease has significant therapeutic potential. Adequate understanding of the chemistry of the OPTMs is necessary to determine what may occur in vivo and which modifications would best serve as biomarkers., Recent Advances: By using mass spectrometry, advanced labeling techniques, and antibody identification, OPTMs have become accessible to a larger proportion of the scientific community. Advancements in instrumentation, database search algorithms, and processing speed have allowed MS to fully expand on the proteome of OPTMs. In addition, the role of enzymatically reversible OPTMs has been further clarified in preclinical models., Critical Issues: The identification of OPTMs suffers from limitations in analytic detection based on the methodology, instrumentation, sample complexity, and bioinformatics. Currently, each type of OPTM requires a specific strategy for identification, and generalized approaches result in an incomplete assessment., Future Directions: Novel types of highly sensitive MS instrumentation that allow for improved separation and detection of modified proteins and peptides have been crucial in the discovery of OPTMs and biomarkers. To further advance the identification of relevant OPTMs in advanced search algorithms, standardized methods for sample processing and depository of MS data will be required.

Background: Galectin-3 (GAL-3), a β-galactoside-binding protein, is a new clinical biomarker believed to reflect cardiac remodeling/fibrosis in patients with heart failure (HF). Plasma GAL-3 is inversely related to renal function. It is not known whether the relationship between renal function and GAL-3 is influenced by clinical decompensation, type of HF, or the presence or absence of clinical HF., Methods and Results: Patients were prospectively categorized as having acute decompensated HF or stable HF on the basis of clinical status and as having HF with reduced left ventricular ejection fraction or HF with preserved left ventricular ejection fraction. Plasma GAL-3 was measured by enzyme-linked immunosorbent assay in patients with HF (n=75), control patients without HF (n=32), and control patients without HF with moderate renal insufficiency (n=12). Compared to controls without HF (14±4 ng/mL), GAL-3 was higher in patients with both acute decompensated HF (23±11 ng/mL) and stable HF (22±10 ng/mL) (P<0.001 versus controls for both) but did not differ between acute decompensated HF and stable HF (P=0.75). Likewise, GAL-3 was elevated in both HF with preserved left ventricular ejection fraction (23±9 ng/mL) and HF with reduced left ventricular ejection fraction (22±11 ng/mL) (P<0.001 versus controls for both) but did not differ between HF with preserved ejection fraction and HF with reduced ejection fraction (P=0.37). GAL-3 correlated strongly with estimated glomerular filtration rate, both in patients with HF (r=-0.75, P<0.001) and in patients without HF (r=-0.82, P<0.001), and this relationship was unaffected by the presence or absence of clinical HF., Conclusions: Plasma GAL-3 is inversely related to renal function in patients with and without clinical HF. Concentrations of plasma GAL-3 do not seem to depend on the level of compensation or type of HF. Furthermore, the relationship between GAL-3 and renal function seems to be affected little or not at all by the presence or absence of clinical HF.

Background: Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes mellitus and chronic hemodynamic overload., Methods and Results: We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or resveratrol for up to 8 months. HFHS diet-fed mice developed progressive left ventricular hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS diet-fed mice, there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol adduction. HFHS diet-fed mice also exhibited increases in plasma fasting glucose, insulin, and homeostasis model assessment of insulin resistance indicative of insulin resistance. Treatment with S17834 or resveratrol prevented left ventricular hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyperinsulinemia and increased plasma adiponectin., Conclusions: Resveratrol and S17834 administered concurrently with a HFHS diet prevent the development of left ventricular hypertrophy, interstitial fibrosis, and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols, including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance, and increased plasma adiponectin. The polyphenols resveratrol and S17834 may be of value in the prevention of diet-induced metabolic heart disease.

Background: Patients with heart failure (HF) develop metabolic derangements including increased adipokine levels, insulin resistance, inflammation and progressive catabolism. It is not known whether metabolic dysfunction and adipocyte activation worsen in the setting of acute clinical decompensation, or conversely, improve with clinical recovery., Methods and Results: We assessed insulin resistance using homeostasis model assessment of insulin resistance (HOMA-IR), and measured plasma levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), adiponectin, visfatin, resistin, leptin, and tumor necrosis factor (TNF) α in 44 patients with acute decompensated HF (ADHF) due to left ventricular (LV) systolic dysfunction and again early (<1 wk) and late (> 6 mo) after clinical recovery, in 26 patients with chronic stable HF, and in 21 patients without HF. NT-proBNP was not increased in control subjects, mildly elevated in patients with stable HF, markedly elevated in patients with ADHF, and decreased progressively early and late after treatment. Compared to control subjects, plasma adiponectin, visfatin, leptin, resistin, and TNF-α were elevated in patients with chronic stable HF and increased further in patients with ADHF. Likewise, HOMA-IR was increased in chronic stable HF and increased further during ADHF. Adiponectin, visfatin, and HOMA-IR remained elevated at the time of discharge from the hospital, but returned to chronic stable HF levels. Adipokine levels were not related to body mass index in HF patients. HOMA-IR correlated positively with adipokines and TNF-α in HF patients., Conclusions: ADHF is associated with worsening of insulin resistance and elevations of adipokines and TNF-α, indicative of adipocyte activation. These metabolic abnormalities are reversible, but they temporally lag behind the clinical resolution of decompensated HF., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart., Methods and Results: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion., Conclusions: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Objective: Levosimendan is indicated for acute heart failure. The formation of levosimendan's active metabolite OR-1896 is dependent on the acetylator status. We evaluated whether acetylator status affects the hemodynamic responses after levosimendan infusion., Methods: Forty-one NYHA III to IV heart failure patients were divided into rapid and slow acetylators by population kinetic modeling. Invasive hemodynamics and plasma concentrations of levosimendan and its metabolites were followed serially., Results: Fifty-six percent of the patients were rapid and 44% slow acetylators. Levosimendan induced increases in heart rate and cardiac output, and decreases in pulmonary capillary wedge pressure (PCWP) and blood pressure, which were sustained at 24 hours after stopping the infusion. At this time, levosimendan levels were undetectable, and OR-1896 levels were about two-fold higher in rapid acetylators. However, hemodynamic effects were similar; mean(SEM) change from baseline in cardiac output was +2.0(0.3) vs. +1.6(0.3) l/min (p = 0.309), and in PCWP -8(2) vs. -7(1) mmHg (p = 0.536), in rapid and slow acetylators, respectively., Conclusion: The sustained hemodynamic effects of levosimendan are similar irrespective of acetylator status.

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outer mitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is a mediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance of mitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomorphic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrial dysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeability transition downstream of Ca(2+) stimulation or due to local generation of reactive oxygen species (ROS). Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducing stimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude that in cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells to mitochondrial permeability transition and to trigger cell death.

Objective: To evaluate if biomarkers reflecting left ventricular/vascular extracellular matrix remodeling are associated with cardiovascular disease (CVD) and death in the community., Methods and Results: In 922 Framingham Study participants (mean age, 58 years; 56% women), we related circulating concentrations of matrix metalloproteinase-9 (binary variable: detectable versus undetectable), log of tissue inhibitor of matrix metalloproteinase-1, and log of procollagen type III aminoterminal peptide (PIIINP) to incident CVD and death. On follow-up (mean, 9.9 years), 51 deaths and 81 CVD events occurred. Each SD increment of log of tissue inhibitor of matrix metalloproteinase-1 and log-PIIINP was associated with multivariable-adjusted hazards ratios of 1.72 (95% CI, 1.30 to 2.27) and 1.47 (95% CI, 1.11 to 1.96), respectively, for mortality risk. Log-PIIINP concentrations were also associated with CVD risk (hazard ratio [95% CI] per SD, 1.35 [1.05 to 1.74]). Death and CVD incidence rates were 2-fold higher in participants with both biomarkers higher than the median (corresponding hazard ratio [95% CI], 2.78 [1.43 to 5.40] and 1.77 [1.04 to 3.03], respectively) compared with those with either or both less than the median. The inclusion of both biomarkers improved the C-statistic (for predicting mortality) from 0.78 to 0.82 (P=0.03). Matrix metalloproteinase-9 was unrelated to either outcome., Conclusions: Higher circulating tissue inhibitor of matrix metalloproteinase-1 and PIIINP concentrations are associated with mortality, and higher PIIINP is associated with incident CVD, in the community.

Background: Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte-specific overexpression of Galphaq develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation., Objective: We tested the hypothesis that myocyte contractile dysfunction in the Galphaq mouse heart is mediated by reactive oxygen species, and in particular, oxidative posttranslational modifications, which impair the function of sarcoplasmic reticulum Ca2+-ATPase (SERCA)., Methods and Results: Freshly isolated ventricular myocytes from Galphaq mice had marked abnormalities of myocyte contractile function and calcium transients. In Galphaq myocardium, SERCA protein was not altered in quantity but displayed evidence of oxidative cysteine modifications reflected by decreased biotinylated iodoacetamide labeling and evidence of specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295. Maximal calcium-stimulated SERCA activity was decreased 47% in Galphaq myocardium. Cross-breeding Galphaq mice with transgenic mice that have cardiac myocyte-specific overexpression of catalase (a) decreased SERCA oxidative cysteine modifications, (b) decreased SERCA cysteine 674 sulfonylation and tyrosine 294/295 nitration, (c) restored SERCA activity, and (d) improved myocyte calcium transients and contractile function., Conclusions: In Galphaq-induced cardiomyopathy, myocyte contractile dysfunction is mediated, at least in part, by 1 or more oxidative posttranslational modifications of SERCA. Protein oxidative posttranslational modifications contribute to the pathophysiology of myocardial dysfunction and thus may provide a target for therapeutic intervention.