Luigino Nascimben

Creatine Kinase System in Failing and Nonfailing Human Myocardium

BACKGROUND The creatine kinase (CK) reaction is important for rapid resynthesis of ATP when the heart increases its work. Studies defining the CK system in human failing and nonfailing myocardium are limited and in conflict. To resolve this conflict, we measured the activities of CK and its isoenzymes and the contents of creatine and CK-B in homogenates of human myocardium. METHODS AND RESULTS Myocardium was sampled from 23 subjects who underwent heart transplant, 36 subjects maintained in an intensive care unit before heart harvesting, 13 accident victims, and 2 patients undergoing heart surgery. Since the characteristics of myocardium of potential organ donors differed from those of myocardium of accident victims, data are presented for three groups: failing, donor, and control. CK activity was 7.7 +/- 1.9 and 6.0 +/- 1.4 IU/mg protein in left (LV) and right (RV) ventricles of failing, 9.4 +/- 2.5 and 10.7 +/- 2 IU/mg protein in LV and RV of donor, and 11.6 +/- 2.4 IU/mg protein in LV of control hearts. CK-MM and the mitochondrial isoenzyme activities were lower in failing and donor LV, and CK-MB activity and CK-B content were higher in failing and donor hearts. Creatine contents were 64 +/- 25 and 56 +/- 18.6 nmol/mg protein in LV and RV of failing, 96 +/- 30 and 110 +/- 24 nmol/mg protein in LV and RV of donor, and 131 +/- 28 nmol/mg protein in LV of control hearts. CONCLUSIONS In failing and nonfailing donor human myocardium, there is a combined decrease of CK activity and creatine that may impair the ability to deliver ATP to energy-consuming systems.

Decreased Energy Reserve in an Animal Model of Dilated Cardiomyopathy: Relationship to Contractile Performance

An animal model was used to test the hypothesis that in heart failure the decrease in the ability to resynthesize ATP through the creatine kinase (CK) reaction (which we call energy reserve) contributes to the inability of the heart to maintain its normal function and contractile reserve. One-week-old turkey poults were fed furazolidone for 14 days to induce dilated cardiomyopathy. Isolated Langendorff-perfused hearts from these myopathic animals showed a 73% decrease in baseline isovolumic contractile performance. Neither increasing [Ca2+]o nor electrical pacing rate increased isovolumic contractile performance. Measured by 31P nuclear magnetic resonance magnetization transfer and chemical assay, ATP concentration was decreased by 23%, phosphocreatine concentration by 42%, CK enzyme activity by 34%, and the pseudo first-order rate constant for the CK reaction by 50%. Measured CK reaction velocity decreased by 71%. The reduced ability to increase cardiac performance in response to increasing [Ca2+]o in hearts with lower CK reaction velocity was reproduced in part by feeding a separate group of turkey poults beta-guanidino-propionic acid to specifically reduce CK reaction velocity by decreasing guanidino substrate concentration. These hearts had normal baseline performance but blunted contractile reserve. These observations provide further support for the hypothesis that a decrease in energy reserve via the CK system contributes to reduced cardiac function in the failing heart.

Defective DNA Replication Impairs Mitochondrial Biogenesis In Human Failing Hearts

Rationale: Mitochondrial dysfunction plays a pivotal role in the development of heart failure. Animal studies suggest that impaired mitochondrial biogenesis attributable to downregulation of the peroxisome proliferator-activated receptor &ggr; coactivator (PGC)-1 transcriptional pathway is integral of mitochondrial dysfunction in heart failure. Objective: The study sought to define mechanisms underlying the impaired mitochondrial biogenesis and function in human heart failure. Methods and Results: We collected left ventricular tissue from end-stage heart failure patients and from nonfailing hearts (n=23, and 19, respectively). The mitochondrial DNA (mtDNA) content was decreased by >40% in the failing hearts, after normalization for a moderate decrease in citrate synthase activity (P<0.05). This was accompanied by reductions in mtDNA-encoded proteins (by 25% to 80%) at both mRNA and protein level (P<0.05). The mRNA levels of PGC-1&agr;/&bgr; and PRC (PGC-1–related coactivator) were unchanged, whereas PGC-1&agr; protein increased by 58% in the failing hearts. Among the PGC-1 coactivating targets, the expression of estrogen-related receptor &agr; and its downstream genes decreased by up to 50% (P<0.05), whereas peroxisome proliferator-activated receptor &agr; and its downstream gene expression were unchanged in the failing hearts. The formation of D-loop in the mtDNA was normal but D-loop extension, which dictates the replication process of mtDNA, was decreased by 75% in the failing hearts. Furthermore, DNA oxidative damage was increased by 50% in the failing hearts. Conclusions: Mitochondrial biogenesis is severely impaired as evidenced by reduced mtDNA replication and depletion of mtDNA in the human failing heart. These defects are independent of the downregulation of the PGC-1 expression suggesting novel mechanisms for mitochondrial dysfunction in heart failure.

Mechanisms for Increased Glycolysis in the Hypertrophied Rat Heart

Glycolysis increases in hypertrophied hearts but the mechanisms are unknown. We studied the regulation of glycolysis in hearts with pressure-overload LV hypertrophy (LVH), a model that showed marked increases in the rates of glycolysis (by 2-fold) and insulin-independent glucose uptake (by 3-fold). Although the Vmax of the key glycolytic enzymes was unchanged in this model, concentrations of free ADP, free AMP, inorganic phosphate (Pi), and fructose-2,6-bisphosphate (F-2,6-P2), all activators of the rate-limiting enzyme phosphofructokinase (PFK), were increased (up to 10-fold). Concentrations of the inhibitors of PFK, ATP, citrate, and H+ were unaltered in LVH. Thus, our findings show that increased glucose entry and activation of the rate-limiting enzyme PFK both contribute to increased flux through the glycolytic pathway in hypertrophied hearts. Moreover, our results also suggest that these changes can be explained by increased intracellular free [ADP] and [AMP], due to decreased energy reserve in LVH, activating the AMP-activated protein kinase cascade. This, in turn, results in enhanced synthesis of F-2,6-P2 and increased sarcolemma localization of glucose transporters, leading to coordinated increases in glucose transport and activation of PFK.

Failure to Maintain a Low ADP Concentration Impairs Diastolic Function in Hypertrophied Rat Hearts

BACKGROUND Mechanisms in addition to diastolic calcium overload may contribute to diastolic dysfunction in hypertrophied hearts. In this study, we tested the hypothesis that failure to maintain a low ADP concentration in hypertrophied hearts contributes to diastolic dysfunction by inhibiting the rate of cross-bridge cycling. METHODS AND RESULTS By perfusing isolated rat hearts with pyruvate and 2-deoxyglucose (2DG), we were able to perturb [ADP] with minimal changes in [ATP] and [inorganic phosphate] or the contribution of glycolytic ATP to ATP synthesis. The effects of 2DG were compared in aortic-banded (LVH, n=5) and sham-operated (control, n=5) rat hearts. 31P NMR spectroscopy was used to measure the concentrations of phosphorus-containing compounds. We found a threefold increase of left ventricular end-diastolic pressure (LVEDP) in LVH during 2DG perfusion, and this increase was concomitant with a threefold increase in intracellular free [ADP]. The [ADP] in the control hearts was maintained <40 micromol/L, and no change in LVEDP was observed. A linear relationship between increases in [ADP] and LVEDP was found (r2=.66, P=.001). Furthermore, the capacity of the creatine kinase reaction, a major mechanism for maintaining a low [ADP], was decreased in LVH (P=.0001). CONCLUSIONS Increased [ADP] contributes to diastolic dysfunction in LVH, possibly due to slowed cross-bridge cycling. Decreased capacity of the creatine kinase reaction to rephosphorylate ADP is a likely contributing mechanism to the failure to maintain a low [ADP] in LVH.

Enalapril Treatment Increases Cardiac Performance and Energy Reserve Via the Creatine Kinase Reaction in Myocardium of Syrian Myopathic Hamsters With Advanced Heart Failure

BACKGROUND Converting enzyme inhibitor treatment of congestive heart failure slows progression to failure and reduces mortality rate. It is known whether these benefits are due solely to improved hemodynamics or also to improved myocyte energetics. This study examines the effect of enalapril treatment on both isovolumic contractile performance and its biochemical correlate, flux through the creatine kinase (CK) system, in an animal model of severely failing myocardium. METHODS AND RESULTS Seven-month-old Syrian cardiomyopathic (TO-2 strain) and normal golden Syrian (FIB strain) hamsters were each randomly assigned to one of three groups supplied daily with either no, low (25 mg/kg body wt), or high (100 mg/kg body wt) doses of enalapril for 12 to 14 weeks. At 10 months of age, all substrates and products and flux through the CK reaction were measured in isolated perfused hearts by 31P magnetization transfer and chemical assay. Compared with normal hamsters, the myopathic hamsters exhibited significantly lower body weights and higher biventricular heart weights, which were partially reversed by drug treatment. The Langendorff-perfused hearts showed decreased isovolumic contractile performance with identical load conditions. This was partially reversed by drug treatment. In the failing hearts, the following substrate and product concentrations and enzyme activities were decreased compared with nonfailing hearts but were unchanged by drug treatment: ATP (-28%), phosphocreatine (-48%), free creatine (-64%), ADP (-51%), and CK (-34%, primarily MM isoenzyme). Flux through the CK reaction for the untreated cardiomyopathic hamster hearts was decreased by 67%, and this decrease was almost completely reversed by enalapril treatment. The increased CK flux is due to an increase in the rate constant for the reaction, since substrate concentrations are unchanged, and is not predicted by the rate equation. In enalapril-treated failing hearts, phosphoryl transfer via the CK reaction increased with contractile performance. This was not observed in the nonfailing hearts, in which energy reserve is adequate to support changes in contractile performance. CONCLUSIONS Decreased flux through CK reaction leads to decreased capacity for ATP synthesis and may contribute to decreased contractile performance in cardiomyopathic hamster hearts. Enalapril treatment results in increased phosphoryl transfer through the CK reaction in failing myocardium, and this increase is coupled to improved cardiac performance. Decreased CK flux in failing hearts is due to a combination of decreased Vmax and lower guanidino pool; this mechanism fails to explain changes in CK flux in enalapril-treated failing hearts.

Troponin is superior to electrocardiogram and creatinine kinase MB for predicting clinically significant myocardial injury after coronary artery bypass grafting

AIMS Cardiac biomarkers are routinely elevated after uncomplicated cardiac surgery to levels considered diagnostic of myocardial infarction in ambulatory populations. We investigated the diagnostic power of electrocardiogram (ECG) and cardiac biomarker criteria to predict clinically relevant myocardial injury using benchmarks of mortality and increased hospital length of stay (HLOS) in patients undergoing coronary artery bypass graft (CABG) surgery. METHODS AND RESULTS Perioperative ECGs, creatinine kinase MB fraction, and cardiac troponin I (cTnI) were assessed in 545 primary CABG patients. None of the ECG criteria for myocardial injury predicted mortality or HLOS. However, post-operative day (POD) 1 cTnI levels independently predicted 5-year mortality (hazard ratio = 1.42; 95% CI 1.14-1.76 for each 10 microg/L increase; P = 0.009), while adjusting for baseline demographic characteristics and perioperative risk factors. Moreover, cTnI was the only biomarker that significantly improved the prediction of 5-year mortality estimated by the logistic Euroscore (P = 0.02). Furthermore, the predictive value of cTnI for 5-year mortality was replicated in a separately collected cohort of 1031 CABG patients using cardiac troponin T. CONCLUSION Electrocardiogram diagnosis of post-operative myocardial injury after CABG does not independently predict an increased risk of 5-year mortality or HLOS. Conversely, cTnI is independently associated with an increased risk of mortality and prolonged HLOS.

Increased Perioperative B-type Natriuretic Peptide Associates with Heart Failure Hospitalization or Heart Failure Death after Coronary Artery Bypass Graft Surgery

Background:Heart failure (HF) is a leading cause of hospitalization and mortality. Plasma B-type natriuretic peptide (BNP) is an established diagnostic and prognostic ambulatory HF biomarker. We hypothesized that increased perioperative BNP independently associates with HF hospitalization or HF death up to 5 yr after coronary artery bypass graft surgery. Methods:The authors conducted a two-institution, prospective, observational study of 1,025 subjects (mean age = 64 ± 10 yr SD) undergoing isolated primary coronary artery bypass graft surgery with cardiopulmonary bypass. Plasma BNP was measured preoperatively and on postoperative days 1–5. The study outcome was hospitalization or death from HF, with HF events confirmed by reviewing hospital and death records. Cox proportional hazards analyses were performed with multivariable adjustments for clinical risk factors. Preoperative and peak postoperative BNP were added to the multivariable clinical model in order to assess additional predictive benefit. Results:One hundred five subjects experienced an HF event (median time to first event = 1.1 yr). Median follow-up for subjects who did not have an HF event = 4.2 yr. When individually added to the multivariable clinical model, higher preoperative and peak postoperative BNP concentrations each, independently associated with the HF outcome (log10 preoperative BNP hazard ratio = 1.93; 95% CI, 1.30–2.88; P = 0.001; log10 peak postoperative BNP hazard ratio = 3.38; 95% CI, 1.45–7.65; P = 0.003). Conclusions:Increased perioperative BNP concentrations independently associate with HF hospitalization or HF death during the 5 yr after primary coronary artery bypass graft surgery. Clinical trials may be warranted to assess whether medical management focused on reducing preoperative and longitudinal postoperative BNP concentrations associates with decreased HF after coronary artery bypass graft surgery.

Osteonecrosis of the Mandible After Laryngoscopy and Endotracheal Tube Placement

Four patients underwent laryngoscopy and general anesthesia surgery without apparent perioperative complications. Within days of their procedure, throat and mouth pain were reported and intraoral examination in all cases revealed exposed bone in the posterior mandible. The sequestra were easily removed and healing was uneventful. These lesions are likely associated with localized oral trauma during intubation causing periosteal damage, compromised blood supply, and subsequent bone necrosis. Because trauma to the mylohyoid ridge during intubation is likely more common than previously appreciated, anesthesiologists should be aware of this potential complication and refer patients to a specialist for management.

Ventricular myosin and creatine-kinase isoenzymes in hypertensive rats treated with captopril.

In the myocardium, myosin and creatine kinase isoforms possess different capacities for using O2 and energy-rich phosphates. We studied electrophoretically the distribution of these isoforms in 19 hypertensive rats (two-kidney, one clip model of hypertension) and in age-matched controls. After 6 weeks of hypertension, seven rats were treated with captopril (2 mg/kg daily) for 4 weeks, six were left hypertensive for another 4 weeks, and the remaining rats were killed under ether anesthesia. In the latter, ventricular mass was significantly higher than in controls; V3 isomyosin was 32.3 ±6.8% versus 0%, and both creatine kinase-MB and -BB were increased at the expense of creatine kinase-MM (creatine kinase-MB=29±2.8% vs. 14.7±1.8%, p < 0.001; creatine kinase-BB=3.1±0.6% vs. 1.7±0.8%, p < 0.001). After 10 weeks of hypertension, ventricular mass, V3 isomyosin, and both creatine kinase-MB and -BB isoforms were found to be persistently higher than in controls. At the same time, captopril-treated rats showed reduced but not normalized blood pressure levels, normalized ventricular mass, and prevalence of the V1 isomyosin (56.9±22% vs. 47.9±23.8% in normotensive controls, p < =NS). However, higher levels of creatine kinase-MB and -BB were still found in these rats in comparison with the normotensive controls (creatine kinase-MB=22.4±5.4% vs. 15.8±2.8%, p < 0.025; creatine kinase-BB=2.3±0.1% vs. 1.8±0.3%, p < 0.02). Therefore, despite the normalization in ventricular mass and isomyosin pattern, captopril-treated rats partly maintain the adaptive changes in creatine kinase isoenzymes that lead to a better use of energy-rich phosphates.