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Background Collagen cross‐linking is covalent bonds among collagen fibers from catalysis of lysyl oxidase (LOX) and advanced glycation end products (AGEs). We aimed to evaluate the formation of enzymatic and nonenzymatic collagen cross‐linking and its clinical significance in patients with hypertrophic obstructive cardiomyopathy. Methods and Results Forty‐four patients with hypertrophic obstructive cardiomyopathy who underwent surgical myectomy were consecutively enrolled. Cardiovascular magnetic resonance parameters of left atrial/left ventricular function were measured, including peak filling rate (PFR) and early peak emptying rate (PER‐E). Total collagen was the sum of soluble and insoluble collagen, which were assessed by collagen assay. The myocardial LOX and AGEs expression were measured by molecular and biochemical methods. Compared with patients without atrial fibrillation, insoluble collagen ( P =0.018), insoluble collagen fraction ( P =0.017), and AGEs ( P =0.039) were higher in patients with atrial fibrillation, whereas LOX expression was similar ( P =0.494). The insoluble collagen fraction was correlated with PFR index (PFR normalized by left ventricular filling volume) (r=−0.44, P =0.005), left atrial diameters (r=0.36, P =0.021) and PER‐E index (PER‐E normalized by left ventricular filling volume) (r=−0.49, P =0.001).Myocardial LOX was positively correlated with total collagen (r=0.37, P =0.025) and insoluble collagen fraction (r=0.53, P P =0.006) and PER‐E index (r=−0.35, P =0.027). In multiple regression analysis, myocardial LOX was independently associated with PFR, while insoluble collagen fraction showed independent correlation with PER‐E after adjustment for clinical confounders. Conclusions Collagen cross‐linking plays an important role on heart remodeling in hypertrophic obstructive cardiomyopathy. Myocardial LOX expression is independently correlated with left ventricular stiffness, while accumulation of AGEs cross‐links might be associated with the occurrence of atrial fibrillation in patients with hypertrophic obstructive cardiomyopathy.

Background Approximately 1 in 6 adolescents report regular binge alcohol consumption, and we hypothesize it affects heart growth during this period. Methods and Results Adolescent, genetically diverse, male Wistar rats were gavaged with water or ethanol once per day for 6 days. In vivo structure and function were assessed before and after exposure. Binge alcohol exposure in adolescence significantly impaired normal cardiac growth but did not affect whole‐body growth during adolescence, therefore this pathology was specific to the heart. Binge rats also exhibited signs of accelerated pathological growth (concentric cellular hypertrophy and thickening of the myocardial wall), suggesting a global reorientation from physiologic to pathologic growth. Binge rats compensated for their smaller filling volumes by increasing systolic function and sympathetic stimulation. Consequently, binge alcohol exposure increased PKA (protein kinase A) phosphorylation of troponin I, inducing myofilament calcium desensitization. Binge alcohol also impaired in vivo relaxation and increased titin‐based cellular stiffness due to titin phosphorylation by PKCα (protein kinase C α). Mechanistically, alcohol inhibited extracellular signal‐related kinase activity, a nodal signaling kinase activating physiology hypertrophy. Thus, binge alcohol exposure depressed genes involved in growth. These cardiac structural alterations from binge alcohol exposure persisted through adolescence even after cessation of ethanol exposure. Conclusions Alcohol negatively impacts function in the adult heart, but the adolescent heart is substantially more sensitive to its effects. This difference is likely because adolescent binge alcohol impedes the normal rapid physiological growth and reorients it towards pathological hypertrophy. Many adolescents regularly binge alcohol, and here we report a novel pathological consequence as well as mechanisms involved.

Background Laboratory studies demonstrate glucose-insulin-potassium (GIK) as a potent cardioprotective intervention, but clinical trials have yielded mixed results, likely because of varying formulas and timing of GIK treatment and different clinical settings. This study sought to evaluate the effects of modified GIK regimen given perioperatively with an insulin-glucose ratio of 1:3 in patients undergoing cardiopulmonary bypass surgery. Methods and Results In this prospective, randomized, double-blinded trial with 930 patients referred for cardiac surgery with cardiopulmonary bypass, GIK (200 g/L glucose, 66.7 U/L insulin, and 80 mmol/L KCl) or placebo treatment was administered intravenously at 1 mL/kg per hour 10 minutes before anesthesia and continuously for 12.5 hours. The primary outcome was the incidence of in-hospital major adverse cardiac events including all-cause death, low cardiac output syndrome, acute myocardial infarction, cardiac arrest with successful resuscitation, congestive heart failure, and arrhythmia. GIK therapy reduced the incidence of major adverse cardiac events and enhanced cardiac function recovery without increasing perioperative blood glucose compared with the control group. Mechanistically, this treatment resulted in increased glucose uptake and less lactate excretion calculated by the differences between arterial and coronary sinus, and increased phosphorylation of insulin receptor substrate-1 and protein kinase B in the hearts of GIK-treated patients. Systemic blood lactate was also reduced in GIK-treated patients during cardiopulmonary bypass surgery. Conclusions A modified GIK regimen administered perioperatively reduces the incidence of in-hospital major adverse cardiac events in patients undergoing cardiopulmonary bypass surgery. These benefits are likely a result of enhanced systemic tissue perfusion and improved myocardial metabolism via activation of insulin signaling by GIK. Clinical Trial Registration URL: clinicaltrials.gov. Identifier: NCT01516138.

Background Dilated cardiomyopathy (DCM) is a common cause of heart failure in adult and pediatric patients, but the underlying mechanism may vary in adults and children, with few studies conducted to date. The objective of the present study was to determine whether differential remodeling of the extracellular matrix contributes to the differences between pediatric and adult DCM hearts. Methods and Results Explanted hearts were procured from adult (age, 46–61 years) and pediatric (age, 2–8) patients with DCM‐related heart failure and nonfailing control hearts. Fibrillar and nonfibrillar extracellular matrix (proteoglycans, glycosaminoglycans, glycoprotein), their regulatory enzymes (matrix metalloproteinases, disintegrin and metalloproteinases, and disintegrin and metalloproteinases with a thrombospondin domain), and their inhibitors (tissue inhibitor of metalloproteinases) were assessed. Pediatric DCM hearts exhibited less fibrosis compared with adult DCMs. Total glycosaminoglycans increased similarly in both DCM groups but exhibited a significantly lower affinity for transforming growth factor‐β in adult DCMs versus pediatric DCMs, resulting in increased bioavailability of transforming growth factor‐β1 and a significantly higher activity of the Smad2/3 pathway in adult DCMs. Glycosylated biglycan and versican, and cleaved thrombospondin‐1 increased in both DCMs. Protein expression of disintegrin and metalloproteinases with thrombospondin domains (‐1, ‐2, ‐4, ‐7) and disintegrin and metalloproteinases (‐12, ‐15, ‐17, ‐19) were altered differently in pediatric and adult control and failing hearts. Total matrix metalloproteinase activity increased in both DCMs. Tissue inhibitor of metalloproteinase levels were altered similarly with heart failure in both age groups, and only tissue inhibitor of metalloproteinase 3 decreased in both DCM groups. Conclusions Differential remodeling of glycosaminoglycans in pediatric DCMs versus adult DCMs could underlie the enhanced activation of the transforming growth factor‐β pathway, leading to more fibrosis in adult DCM hearts. The distinct remodeling of the fibrillar and nonfibrillar extracellular matrix between pediatric and adult DCM hearts highlights a distinct pathophysiological basis for these cohorts.

Background Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal‐responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor‐2. β‐Adrenoceptor (β‐AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation‐independent nuclear export and phosphorylation‐dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β‐AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. Methods and Results A novel 3‐dimensional confocal microscopy method that objectively quantifies the whole‐cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β‐AR agonist isoproterenol to induce β1‐AR‐mediated and protein kinase A‐dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor‐2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol‐induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol‐induced HDAC5 dephosphorylation. Co‐immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3‐fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol‐induced HDAC5 dephosphorylation. Conclusions β‐AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A‐dependent but requires B55α‐PP2A‐mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A‐mediated phosphorylation of Ser279.

Background High levels of ketone bodies are associated with improved survival as observed with regular exercise, caloric restriction, and—most recently—treatment with sodium–glucose linked transporter 2 inhibitor antidiabetic drugs. In heart failure, indices of ketone body metabolism are upregulated, which may improve energy efficiency and increase blood flow in skeletal muscle and the kidneys. Nevertheless, it is uncertain how ketone bodies affect myocardial glucose uptake and blood flow in humans. Our study was therefore designed to test whether ketone body administration in humans reduces myocardial glucose uptake ( MGU ) and increases myocardial blood flow. Methods and Results Eight healthy subjects, median aged 60 were randomly studied twice: (1) During 390 minutes infusion of Na‐3‐hydroxybutyrate ( KETONE ) or (2) during 390 minutes infusion of saline ( SALINE ), together with a concomitant low‐dose hyperinsulinemic–euglycemic clamp to inhibit endogenous ketogenesis. Myocardial blood flow was measured by 15 O‐H 2 O positron emission tomography/computed tomography, myocardial fatty acid metabolism by 11 C‐palmitate positron emission tomography/computed tomography and MGU by 18 F‐fluorodeoxyglucose positron emission tomography/computed tomography. Similar euglycemia, hyperinsulinemia, and suppressed free fatty acids levels were recorded on both study days; Na‐3‐hydroxybutyrate infusion increased circulating Na‐3‐hydroxybutyrate levels from zero to 3.8±0.5 mmol/L. MGU was halved by hyperketonemia ( MGU [nmol/g per minute]: 304±97 [ SALINE ] versus 156±62 [ KETONE ], P Conclusions Ketone bodies displace MGU and increase myocardial blood flow in healthy humans; these novel observations suggest that ketone bodies are important cardiac fuels and vasodilators, which may have therapeutic potentials.

Background Chronic cardiotoxicity is less common in male than in female patients receiving doxorubicin and other anthracyclines at puberty and adolescence. We hypothesized that this sex difference might be secondary to distinct activities of sex hormones on cardiomyocyte senescence, which is thought to be central to the development of long‐term anthracycline cardiomyopathy. Methods and Results H9c2 cells and neonatal mouse cardiomyocytes were exposed to doxorubicin with or without prior incubation with testosterone or 17β‐estradiol, the main androgen and estrogen, respectively. Testosterone, but not 17β‐estradiol, counteracted doxorubicin‐elicited senescence. Downregulation of telomere binding factor 2, which has been pinpointed previously as being pivotal to doxorubicin‐induced senescence, was also prevented by testosterone, as were p53 phosphorylation and accumulation. Pretreatment with the androgen receptor antagonist flutamide, the phosphatidylinositol 3 kinase inhibitor LY 294002, and the nitric oxide synthase inhibitor L‐ NG ‐nitroarginine methyl ester abrogated the reduction in senescence and the normalization of telomere binding factor 2 levels attained by testosterone. Consistently, testosterone enhanced the phosphorylation of AKT and nitric oxide synthase 3. In H9c2 cells, doxorubicin‐stimulated senescence was still observed up to 21 days after treatment and increased further when cells were rechallenged with doxorubicin 14 days after the first exposure to mimic the schedule of anthracycline‐containing chemotherapy. Remarkably, these effects were also inhibited by testosterone. Conclusions Testosterone protects cardiomyocytes against senescence caused by doxorubicin at least in part by modulating telomere binding factor 2 via a pathway involving the androgen receptor, phosphatidylinositol 3 kinase, AKT , and nitric oxide synthase 3. This is a potential mechanism by which pubescent and adolescent boys are less prone to chronic anthracycline cardiotoxicity than girls.