Scleraxis and fibrosis in the pressure-overloaded heart.

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Regulation of Cardiac Fibroblast GLS1 Expression by Scleraxis.

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFß and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFß-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFß induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFß was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

Sirtuin 3 overexpression preserves maximal sarco(endo)plasmic reticulum calcium ATPase activity in the skeletal muscle of mice subjected to high fat - high sucrose feeding.

Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) transports Ca2+ in muscle. Impaired SERCA activity may contribute to diabetic myopathy. Sirtuin (SIRT) 3 regulates muscle metabolism and function; however, it is unknown if SIRT3 regulates muscle SERCA activity or acetylation. We determined if SIRT3 overexpression enhances SERCA activity in mouse gastrocnemius muscle and if SIRT3 overexpression preserves gastrocnemius SERCA activity in a model of type 2 diabetes, induced by high fat - high sucrose (HFHS) feeding. We also determined if the acetylation status of SERCA proteins in mouse gastrocnemius is altered by SIRT3 overexpression or HFHS feeding. Wild-type (WT) and SIRT3 transgenic (SIRT3TG) mice, overexpressing SIRT3 in skeletal muscle, were fed a standard or HFHS diet for 4 months. SIRT3TG and WT mice developed obesity and glucose intolerance after 4 months of HFHS feeding. SERCA Vmax was higher in gastrocnemius of SIRT3TG mice compared with WT mice. HFHS-fed mice had lower SERCA1a protein levels and lower SERCA Vmax in their gastrocnemius than control-fed mice. The decrease in SERCA Vmax in gastrocnemius muscle due to HFHS feeding was attenuated by SIRT3 overexpression in HFHS-fed SIRT3TG mice. SERCA1a and SERCA2a acetylation in mouse gastrocnemius was not altered by genotype or diet. These findings suggest SIRT3 overexpression improves SERCA function in mouse skeletal muscle.

Muscle-specific sirtuin 3 overexpression does not attenuate the pathological effects of high-fat/high-sucrose feeding but does enhance cardiac SERCA2a activity.

Obesity, type 2 diabetes, and heart disease are linked to an unhealthy diet. Sarco(endo)plasmic reticulum calcium (Ca2+ ) ATPase 2a (SERCA2a) controls cardiac function by transporting Ca2+ in cardiomyocytes. SERCA2a is altered by diet and acetylation, independently; however, it is unknown if diet alters cardiac SERCA2a acetylation. Sirtuin (SIRT) 3 is an enzyme that might preserve health under conditions of macronutrient excess by modulating metabolism via regulating deacetylation of target proteins. Our objectives were to determine if muscle-specific SIRT3 overexpression attenuates the pathological effects of high fat-high sucrose (HFHS) feeding and if HFHS feeding alters cardiac SERCA2a acetylation. We also determined if SIRT3 alters cardiac SERCA2a acetylation and regulates cardiac SERCA2a activity. C57BL/6J wild-type (WT) mice and MCK-mSIRT3-M1-Flag transgenic (SIRT3TG ) mice, overexpressing SIRT3 in cardiac and skeletal muscle, were fed a standard-diet or a HFHS-diet for 4 months. SIRT3TG and WT mice developed obesity, glucose intolerance, cardiac dysfunction, and pathological cardiac remodeling after 4 months of HFHS feeding, indicating muscle-specific SIRT3 overexpression does not attenuate the pathological effects of HFHS-feeding. Overall cardiac lysine acetylation was increased by 63% in HFHS-fed mice (p = 0.022), though HFHS feeding did not alter cardiac SERCA2a acetylation. Cardiac SERCA2a acetylation was not altered by SIRT3 overexpression, whereas SERCA2a Vmax was 21% higher in SIRT3TG (p = 0.039) than WT mice. This suggests that SIRT3 overexpression enhanced cardiac SERCA2a activity without direct SERCA2a deacetylation. Muscle-specific SIRT3 overexpression may not prevent the complications associated with an unhealthy diet in mice, but it appears to enhance SERCA2a activity in the mouse heart.

Frailty status and cardiovascular disease risk profile in middle-aged and older females.

OBJECTIVE: Frailty and pre-frailty are known to increase the risk of developing cardiovascular disease (CVD). However, the risk profiles of females are not well characterized. The aim of this study is to characterize the CVD risk profiles of robust, pre-frail and frail females. METHODS: Cross-sectional analysis of 985 females ≥55 years with no self-reported history of CVD were recruited. Frailty was assessed using the Fried Criteria with the cut-points standardized to the cohort. Framingham risk scores (FRS), the 4-test Rasmussen Disease Score (RDS), and the CANHEART health index were used to characterize composite CVD risk. Individual measures of CVD risk included blood lipids, artery elasticity assessments, exercise blood pressure response, 6-min walk test (6MWT), sedentary time and PHQ-9 score. RESULTS: The cohort comprised of 458 (46.4%) robust, 464 (47.1%) pre-frail and 63 (6.4%) frail females with a mean age of 66 ± 6 (SD) years. Pre-frail females were at increased odds of taking diabetes medications (OR 3.04; 95% CI 1.27-7.27), hypertension medications (OR 2.02; 95% CI 1.44-2.82), having an exaggerated blood pressure response to exercise (OR 1.878; 95% CI 1.39-2.50), mild depression symptoms (OR 2.38; 95% CI 1.68-338), and lower fitness as assessed by 6MWT (OR 5.74; 95% CI 3.18-10.37), even after controlling for age and relevant medications. Pre-frail females were also at increased odds for having CVD risk scores indicating higher risk with the FRS (OR 1.52; 95% CI 1.12-2.05), the RDS (OR 1.60; 95% CI 1.21-2.10) and the CANHEART risk score (OR 3.07; 95% CI 2.04-4.62). These odds were higher when frail females were compared to their robust peers. CONCLUSION: Frailty and pre-frailty were associated with higher odds of presenting with CVD risk factors as compared to robust females, even after controlling for age.

Myokines as biomarkers of frailty and cardiovascular disease risk in females.

Frailty is a risk factor for cardiovascular disease (CVD). Biomarkers have the potential to detect the early stages of frailty, such as pre-frailty. Myokines may act as biomarkers of frailty-related disease progression, as a decline in muscle health is a hallmark of the frailty phenotype. This study is a secondary analysis of 104 females 55 years of age or older with no previous history of CVD. Differences in systemic myokine concentrations based on frailty status and CVD risk profile were examined using a case-control design. Propensity matching identified two sets of 26 pairs with pre-frailty as the exposure variable in low or elevated CVD risk groups for a total 104 female participants. Frailty was assessed using the Fried Criteria (FC) and CVD risk was assessed using the Framingham Risk Score (FRS). Factorial ANOVA compared the main effects of frailty, CVD risk, and their interaction on the concentrations of 15 myokines. Differences were found when comparing elevated CVD risk status with low for the concentrations of EPO (384.76 ± 1046.07 vs. 206.63 ± 284.61 pg/mL, p = .001), FABP3 (2772.61 ± 3297.86 vs. 1693.31 ± 1019.34 pg/mL, p = .017), FGF21 (193.17 ± 521.09 vs. 70.18 ± 139.51 pg/mL, p = .010), IL-6 (1.73 ± 4.97 vs. 0.52 ± 0.89 pg/mL, p = .023), and IL-15 (2.62 ± 10.56 vs. 0.92 ± 1.25 pg/mL, p = .022). Pre-frail females had lower concentrations of fractalkine compared to robust (27.04 ± 20.60 vs. 103.62 ± 315.45 pg/mL, p = .004). Interaction effects between frailty status and CVD risk for FGF21 and OSM were identified. In elevated CVD risk, pre-frail females, concentrations of FGF21 and OSM were lower than that of elevated CVD risk, robust females (69.10 ± 62.86 vs. 317.24 ± 719.69, p = .011; 1.73 ± 2.32 vs. 24.43 ± 69.21, p = .018, respectively). These data identified specific biomarkers of CVD risk and biomarkers of frailty that are exacerbated with CVD risk.

Exercise-induced increases in the expression and activity of cardiac sarcoplasmic reticulum calcium ATPase 2 is attenuated in AMPKα2 kinase-dead mice.

Exercise enhances cardiac sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) function through unknown mechanisms. The present study tested the hypothesis that the positive effects of exercise on SERCA2a expression and function in the left ventricle is dependent on adenosine-monophosphate-activated protein kinase (AMPK) α2 function. AMPKα2 kinase-dead (KD) transgenic mice, which overexpress inactivated AMPKα2 subunit, and wild-type C57Bl/6 (WT) mice were randomized into sedentary groups or groups with access to running wheels. After 5 months, exercised KD mice exhibited shortened deceleration time compared with sedentary KD mice. In left ventricular tissue, the ratio of phosphorylated AMPKαThr172:total AMPKα was 65% lower (P < 0.05) in KD mice compared with WT mice. The left ventricle of KD mice had 37% lower levels of SERCA2a compared with WT mice. Although exercise increased SERCA2a protein levels in WT mice by 53%, this response of exercise was abolished in exercised KD mice. Exercise training reduced total phospholamban protein content by 23% in both the WT and KD mice but remained 20% higher overall in KD mice. Collectively, these data suggest that AMPKα influences SERCA2a and phospholamban protein content in the sedentary and exercised heart, and that exercise-induced changes in SERCA2a protein are dependent on AMPKα function.

Standardization of the Fried frailty phenotype improves cardiovascular disease risk discrimination.

BACKGROUND: Standardizing the Fried criteria (S-FC) using cutoffs specific to the patient population improves adverse outcome prediction. However, there is limited evidence to determine if a S-FC assessment can improve discrimination of cardiovascular disease (CVD) risk in middle-aged and older women. DESIGN: The objective of this cross-sectional analysis was to compare the ability of the Fried frailty phenotype criteria (FC) to discriminate between individuals at higher risk for CVD according to the Framingham Risk Score and Rasmussen Disease Score in comparison to the S-FC. SETTING: Asper Clinical Research Institute, St. Boniface Hospital Research Centre. PARTICIPANTS: 985 women 55 years of age or older with no previous history of CVD. MEASUREMENTS: Discrimination of individuals with high CVD risk according to the Framingham and Rasmussen Disease scores was assessed using receiver operating characteristic (ROC) curves, integrated discrimination index (IDI) and net reclassification index (NRI). RESULTS: The S-FC showed superior ability to discriminate CVD risk as assessed by area under the ROC curve (AUROC) based on the Framingham (0.728 vs 0.634, p < 0.001), but not for the Rasmussen (0.594 vs 0.552, p = 0.079) risk score. Net reclassification index identified improved discrimination for both the Framingham (67.9%, p < 0.001) and Rasmussen Disease scores (26.0%, p = 0.003). Integrated discrimination index also identified improved CVD risk discrimination with the Framingham (3.0%, p < 0.001) and Rasmussen Disease scores (1.5%, p < 0.001). CONCLUSION: In this study, the Fried frailty phenotype better discriminated cardiovascular disease risk when standardized to the study population.

Exercise-induced irisin release as a determinant of the metabolic response to exercise training in obese youth: the EXIT trial.

The mechanisms underlying the metabolic improvements following aerobic exercise training remain poorly understood. The primary aim of this study was to determine if an adipomyokine, irisin, responded to acute exercise was associated with the metabolic adaptations to chronic aerobic exercise in obese youth. The acute response to exercise was assessed in 11 obese youth following 45-min acute bouts of aerobic (AE) and resistance exercise (RE). The irisin area under the curve (pre-exercise, 15, 30, and 45 min) during these AE sessions were the main exposure variables. The primary outcome measure was the change in insulin sensitivity using the Matsuda index, following 6 weeks of RE training, delivered for 45 min, three times per week at 60-65% 1RM. Participants were also categorized as either responders (above) or nonresponders (below) based on the percentage change in the Matsuda index following the 6-week intervention. Irisin increased significantly during the acute bout of AE from 29.23 ± 6.96 to 39.30 ± 7.05 ng/mL; P = 0.028, but not significantly during the RE session (P = 0.182). Absolute and relative change in irisin during the acute bout of AE was associated with absolute and relative change in Matsuda index (r = 0.68; P = 0.022 and r = 0.63; P = 0.037) following the 6-week RE intervention. No such association was observed with the irisin response to acute RE (all P > 0.05). Responders to the 6-week RE intervention displayed a fourfold greater irisin response to acute AE (90.0 ± 28.0% vs. 22.8 ± 18.7%; P = 0.024) compared to nonresponders. Irisin increases significantly following an acute bout of AE, but not RE, and this response is associated with a greater improvement in insulin sensitivity in response to chronic resistance training.

Protocol for the HAPPY Hearts study: cardiovascular screening for the early detection of future adverse cardiovascular outcomes in middle-aged and older women: a prospective, observational cohort study.

INTRODUCTION: Efforts to identify individuals at a higher risk for adverse cardiovascular outcomes focus on traditional risk factors, such as age, sex, smoking status, blood pressure and and cholesterol; however, this approach does not directly assess cardiovascular function and may underestimate the risk of experiencing adverse cardiovascular outcomes in women. This prospective, observational cohort study will examine the ability of the Heart Attack Prevention Program for You (HAPPY) Hearts screening protocol, a series of non-invasive procedures to identify middle-aged and older women who are at an elevated risk for experiencing an adverse cardiovascular event in the 5-year period after screening. The predictive value of the HAPPY Hearts protocol will also be compared with the Framingham Risk Score to determine the sensitivity for estimating risk for an adverse cardiovascular outcome. METHODS AND ANALYSIS: One thousand women 55 years of age or older will be recruited to be screened by the HAPPY Hearts protocol. This involves the cardiovascular assessment of resting blood pressure, blood pressure response to 3 min of moderate intensity exercise and large and small arterial elasticity. The participants will be classified into risk categories based on these measures. The incidence of the following adverse cardiovascular outcomes will be assessed in the 5-year period after screening in both groups: ischaemic heart disease, acute myocardial infarction, stroke, percutaneous coronary intervention, coronary bypass surgery, congestive heart failure and new hypertension. ETHICS AND DISSEMINATION: Information gathered in this research will be published in peer-reviewed journals and presented in a programme evaluation report to inform Manitoba Health and key stakeholders about the outcomes of the study. The University of Manitoba Health Research Ethics Board has approved the study protocol V.2.0, dated 29 September 2014 (H2014:224). TRIAL REGISTRATION NUMBER: NCT02863211.

Differential regulation of the fiber type-specific gene expression of the sarcoplasmic reticulum calcium-ATPase isoforms induced by exercise training.

The regulatory role of adenosine monophosphate-activated protein kinase (AMPK)-α2 on sarcoplasmic reticulum calcium-ATPase (SERCA) 1a and SERCA2a in different skeletal muscle fiber types has yet to be elucidated. Sedentary (Sed) or exercise-trained (Ex) wild-type (WT) and AMPKα2-kinase dead (KD) transgenic mice, which overexpress a mutated and inactivated AMPKα2 subunit, were utilized to characterize how genotype or exercise training influenced the regulation of SERCA isoforms in gastrocnemius. As expected, both Sed and Ex KD mice had >40% lower AMPK phosphorylation and 30% lower SERCA1a protein than WT mice (P < 0.05). In contrast, SERCA2a protein was not different among KD and WT mice. Exercise increased SERCA1a and SERCA2a protein content among WT and KD mice, compared with their Sed counterparts. Maximal SERCA activity was lower in KD mice, compared with WT. Total phospholamban protein was higher in KD mice than in WT and lower in Ex compared with Sed mice. Exercise training increased phospholamban Ser(16) phosphorylation in WT mice. Laser capture microdissection and quantitative PCR indicated that SERCA1a mRNA expression among type I fibers was not altered by genotype or exercise, but SERCA2a mRNA was increased 30-fold in WT+Ex, compared with WT+Sed. In contrast, the exercise-stimulated increase for SERCA2a mRNA was blunted in KD mice. Exercise upregulated SERCA1a and SERCA2a mRNA among type II fibers, but was not altered by genotype. Collectively, these data suggest that exercise differentially influences SERCA isoform expression in type I and type II fibers. Additionally, AMPKα2 influences the regulation of SERCA2a mRNA in type I skeletal muscle fibers following exercise training.