Adam J. Chicco

Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure

The mitochondrial phospholipid cardiolipin is required for optimal mitochondrial respiration. In this study, cardiolipin molecular species and cytochrome oxidase (COx) activity were studied in interfibrillar (IF) and subsarcolemmal (SSL) cardiac mitochondria from Spontaneously Hypertensive Heart Failure (SHHF) and Sprague-Dawley (SD) rats throughout their natural life span. Fisher Brown Norway (FBN) and young aortic-constricted SHHF rats were also studied to investigate cardiolipin alterations in aging versus pathology. Additionally, cardiolipin was analyzed in human hearts explanted from patients with dilated cardiomyopathy. A loss of tetralinoleoyl cardiolipin (L4CL), the predominant species in the healthy mammalian heart, occurred during the natural or accelerated development of heart failure in SHHF rats and humans. L4CL decreases correlated with reduced COx activity (no decrease in protein levels) in SHHF cardiac mitochondria, but with no change in citrate synthase (a matrix enzyme) activity. The fraction of cardiac cardiolipin containing L4CL became much lower with age in SHHF than in SD or FBN mitochondria. In summary, a progressive loss of cardiac L4CL, possibly attributable to decreased remodeling, occurs in response to chronic cardiac overload, but not aging alone, in both IF and SSL mitochondria. This may contribute to mitochondrial respiratory dysfunction during the pathogenesis of heart failure.

Exercise training attenuates acute doxorubicin-induced cardiac dysfunction.

The use of doxorubicin, a highly effective antitumor antibiotic, is limited by a dose-dependent cardiotoxicity. The purpose of this study was to determine whether chronic exercise training (ET) prior to doxorubicin treatment would preserve cardiac function and reduce myocardial oxidative stress following treatment. Rats were exercise trained on a motorized treadmill or confined to sedentary cage activity for 12 weeks, then administered an intraperitoneal injection of doxorubicin (15 mg/kg) or 0.9% saline. Five days following the injections, hearts were isolated and Langendorf perfused to assess cardiac function and then processed for biochemical analyses. Doxorubicin treatment induced significant inotropic, lusitropic, and chronotropic cardiac dysfunction, reduced coronary flow, and increased cardiac lipid peroxidation in the sedentary animals. Doxorubicin treatment was also associated with a decrease in cardiac manganese superoxide dismutase protein expression and an increase in heat shock protein-72 (Hsp72) compared with saline-treated animals. Exercise training attenuated doxorubicin-induced cardiac dysfunction, and lipid peroxidation, and led to a greater cardiac expression of Hsp72 compared with the sedentary animals. The results of this study demonstrate for the first time that chronic exercise training before doxorubicin treatment protects against cardiac dysfunction following treatment, and provide evidence for a sustained increase in myocardial Hsp72 following exercise training and doxorubicin treatment in vivo.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Low-Intensity Exercise Training Delays Heart Failure and Improves Survival in Female Hypertensive Heart Failure Rats

Exercise training improves functional capacity and quality of life in patients with heart failure. However, the long-term effects of exercise on mortality associated with hypertensive heart disease have not been well defined. In the present study, we investigated the effect of low-intensity exercise training on disease progression and survival in female spontaneously hypertensive heart failure rats. Animals with severe hypertension (16 months old) were treadmill trained (14.5 m/min, 45 min/d, 3 d/wk) until they developed terminal heart failure or were euthanized because of age-related complications. Exercise delayed mortality resulting from heart failure (P<0.001) and all causes (P<0.05) and transiently attenuated the systolic hypertension and contractile dysfunction observed in the sedentary animals but had no effect on cardiac morphology or contractile function in end-stage heart failure. Training had no effect on terminal myocardial protein expression of antioxidant enzymes, calcium handling proteins, or myosin heavy chain isoforms but was associated with higher cytochrome oxidase activity in cardiac mitochondria (P<0.05) and a greater mitochondrial content of cardiolipin, a phospholipid that is essential for optimal mitochondrial energy metabolism. In conclusion, low-intensity exercise training significantly delays the onset of heart failure and improves survival in female hypertensive heart failure rats without eliciting sustained improvements in blood pressure, cardiac function, or expression of several myocardial proteins associated with the cardiovascular benefits of exercise. The effects of exercise on cytochrome oxidase and cardiolipin provide novel evidence that training may improve prognosis in hypertensive heart disease by preserving mitochondrial energy metabolism.

Apelin administration ameliorates high fat diet-induced cardiac hypertrophy and contractile dysfunction

Apelin has been recognized as an adipokine that plays an important role in regulating energy metabolism and is credited with antiobesity and antidiabetic properties. This study was designed to examine the effect of exogenous apelin on obesity-associated cardiac dysfunction. Oral glucose tolerance test, echocardiography, cardiomyocyte contractile and intracellular Ca(2+) properties were assessed in adult C57BL/6J mice fed - low or a - high-fat diet for 24weeks followed by apelin treatment (100nmol/kg, i.p. for 2weeks). High-fat diet resulted in increased left ventricular diastolic and systolic diameters, and wall thickness, compromised fractional shortening, impaired cardiomyocyte mechanics (peak-shortening, maximal velocity of shortening/relengthening, and duration of shortening and relengthening) and compromised intracellular Ca(2+) handling, all of which were reconciled by apelin. Apelin treatment also reversed high fat diet-induced changes in intracellular Ca(2+) regulatory proteins, ER stress, and autophagy. In addition, microRNAs (miR) -133a, miR-208 and miR-1 which were elevated following high-fat feeding were attenuated by apelin treatment. In cultured cardiomyocytes apelin reconciled palmitic acid-induced cardiomyocyte contractile anomalies. Collectively, these data depict a pivotal role of apelin in obesity-associated cardiac contractile dysfunction, suggesting a therapeutic potential of apelin in the management of cardiac dysfunction associated with obesity.

The role of calcium-independent phospholipase A2 in cardiolipin remodeling in the spontaneously hypertensive heart failure rat heart

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)4CL]. A decrease in (18:2)4CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)4CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A2 (iPLA2) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)4CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA2 inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)4CL in the failing rat heart, and that mitochondrial iPLA2 plays a role in the remodeling of CLs acyl composition.

Linoleate-Rich High-Fat Diet Decreases Mortality in Hypertensive Heart Failure Rats Compared With Lard and Low-Fat Diets

Recent studies indicate that high-fat diets may attenuate cardiac hypertrophy and contractile dysfunction in chronic hypertension. However, it is unclear whether consuming a high-fat diet improves prognosis in aged individuals with advanced hypertensive heart disease or the extent to which differences in its fatty acid composition modulate its effects in this setting. In this study, aged spontaneously hypertensive heart failure rats were administered a standard high-carbohydrate diet or high-fat diet (42% of kilocalories) supplemented with high-linoleate safflower oil or lard until death to determine their effects on disease progression and mortality. Both high-fat diets attenuated cardiac hypertrophy, left ventricular chamber dilation, and systolic dysfunction observed in rats consuming the high-carbohydrate diet. However, the lard diet significantly hastened heart failure mortality compared with the high-carbohydrate diet, whereas the linoleate diet significantly delayed mortality. Both high-fat diets elicited changes in the myocardial fatty acid profile, but neither had any effect on thromboxane excretion or blood pressure. The prosurvival effect of the linoleate diet was associated with a greater myocardial content and linoleate-enrichment of cardiolipin, an essential mitochondrial phospholipid known to be deficient in the failing heart. This study demonstrates that, despite having favorable effects on cardiac morphology and function in hypertension, a high-fat diet may accelerate or attenuate mortality in advanced hypertensive heart disease depending on its fatty acid composition. The precise mechanisms responsible for the divergent effects of the lard and linoleate-enriched diets merit further investigation but may involve diet-induced changes in the content and/or composition of cardiolipin in the heart.

Dietary linoleate preserves cardiolipin and attenuates mitochondrial dysfunction in the failing rat heart

AIMS Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L(4)CL). A selective loss of L(4)CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L(4)CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure. METHODS AND RESULTS Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L(4)CL and total CL to 90% of non-failing levels (vs. 61-75% in control and lard groups), and attenuated 17-22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure. CONCLUSION Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.

Pro-Inflammatory Mediation of Myoblast Proliferation

Skeletal muscle satellite cell function is largely dictated by the surrounding environment following injury. Immune cell infiltration dominates the extracellular space in the injured area, resulting in increased cytokine concentrations. While increased pro-inflammatory cytokine expression has been previously established in the first 3 days following injury, less is known about the time course of cytokine expression and the specific mechanisms of cytokine induced myoblast function. Therefore, the expression of IL-1β and IL-6 at several time points following injury, and their effects on myoblast proliferation, were examined. In order to do this, skeletal muscle was injured using barium chloride in mice and tissue was collected 1, 5, 10, and 28 days following injury. Mechanisms of cytokine induced proliferation were determined in cell culture using both primary and C2C12 myoblasts. It was found that there is a ∼20-fold increase in IL-1β (p≤0.05) and IL-6 (p = 0.06) expression 5 days following injury. IL-1β increased proliferation of both primary and C2C12 cells ∼25%. IL-1β stimulation also resulted in increased NF-κB activity, likely contributing to the increased proliferation. These data demonstrate for the first time that IL-1β alone can increase the mitogenic activity of primary skeletal muscle satellite cells and offer insight into the mechanisms dictating satellite cell function following injury.

Cathepsin K Knockout Alleviates Pressure Overload–Induced Cardiac Hypertrophy

Evidence from human and animal studies has documented elevated levels of lysosomal cysteine protease cathepsin K in failing hearts. Here, we hypothesized that ablation of cathepsin K mitigates pressure overload–induced cardiac hypertrophy. Cathepsin K knockout mice and their wild-type littermates were subjected to abdominal aortic constriction, resulting in cardiac remodeling (heart weight, cardiomyocyte size, left ventricular wall thickness, and end diastolic and end systolic dimensions) and decreased fractional shortening, the effects of which were significantly attenuated or ablated by cathepsin K knockout. Pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance, which were partly resolved by cathepsin K knockout. Cardiac mammalian target of rapamycin and extracellular signal-regulated kinases (ERK) signaling cascades were upregulated by pressure overload, the effects of which were attenuated by cathepsin K knockout. In cultured H9c2 myoblast cells, silencing of cathepsin K blunted, whereas cathepsin K transfection mimicked phenylephrine–induced hypertrophic response, along with elevated phosphorylation of mammalian target of rapamycin and ERK. In addition, cathepsin K protein levels were markedly elevated in human hearts of end-stage dilated cardiomyopathy. Collectively, our data suggest that cathepsin K ablation mitigates pressure overload–induced hypertrophy, possibly via inhibition of the mammalian target of rapamycin and ERK pathways.