MAY/JUNE 2023 VOL 34 NO 2 • Heart fai lure with reduced ejection fraction in a Tunisian hospital • Role of nuclear factor kappa-B in TNF-induced cytoprotection • Cl inical features and outcomes of infective endocarditis • Training rural health workers in acute rheumatic fever in eastern Uganda • CircRNA-mediated pathology: mechanism of type II cardio-renal syndrome • Circulatory soluble LOX-1: a novel predictor for coronary artery disease • Diphtheritic myocarditis: a case report with multi-organ involvement • Conference proceedings of the Nigerian Cardiovascular Symposium CardioVascular Journal of Afr ica (off icial journal for PASCAR) www.cvja.co.za
NEW INTRODUCING OUR TADALAFIL For further product information contact PHARMA DYNAMICS Email info@pharmadynamics.co.za CUSTOMER CARE LINE +27 21 707 7000 TADALAFIL 5, 20 mg DYNA. Each film coated tablet contains 5, 20 mg respectively. S4 A49/7.1.5/0285, 0286. For full prescribing information, refer to the professional information approved by SAHPRA, August 2022. 1) Professional information. TDLA901/04/2023. www.pharmadynamics.co.za 5 mg STRENGTH PACKED IN 30’s Up to minutes 16 An onset of action as early as TADALAFIL HAS 1: HRS duration 36 of action TRAVEL LIGHT IMPROVE QUALITY OF LIFE
ISSN 1995-1892 (print) ISSN 1680-0745 (online) Cardiovascular Journal of Afr ica www.cvja.co.za CONTENTS INDEXED AT SCISEARCH (SCI), PUBMED, PUBMED CENTRAL AND SABINET Vol 34, No 2, MAY/June 2023 EDITORS Editor-in-Chief (South Africa) PROF PAT COMMERFORD Assistant Editor PROF JAMES KER (JUN) Regional Editor DR A DZUDIE Regional Editor (Kenya) DR F BUKACHI Regional Editor (South Africa) PROF R DELPORT EDITORIAL BOARD PROF PA BRINK Experimental & Laboratory Cardiology PROF R DELPORT Chemical Pathology PROF MR ESSOP Haemodynamics, Heart Failure & Valvular Heart Disease DR OB FAMILONI Clinical Cardiology DR V GRIGOROV Invasive Cardiology & Heart Failure PROF J KER (SEN) Hypertension, Cardiomyopathy, Cardiovascular Physiology DR J LAWRENSON Paediatric Heart Disease PROF A LOCHNER Biochemistry/Laboratory Science DR MT MPE Cardiomyopathy PROF DP NAIDOO Echocardiography PROF B RAYNER Hypertension/Society PROF MM SATHEKGE Nuclear Medicine/Society PROF YK SEEDAT Diabetes & Hypertension PROF H DU T THERON Invasive Cardiology INTERNATIONAL ADVISORY BOARD PROF DAVID CELEMAJER Australia (Clinical Cardiology) PROF KEITH COPELIN FERDINAND USA (General Cardiology) DR SAMUEL KINGUE Cameroon (General Cardiology) DR GEORGE A MENSAH USA (General Cardiology) PROF WILLIAM NELSON USA (Electrocardiology) DR ULRICH VON OPPEL Wales (Cardiovascular Surgery) PROF PETER SCHWARTZ Italy (Dysrhythmias) PROF ERNST VON SCHWARZ USA (Interventional Cardiology) SUBJECT EDITORS Nuclear Medicine and Imaging DR MM SATHEKGE Heart Failure DR G VISAGIE Paediatric DR S BROWN Paediatric Surgery DR DARSHAN REDDY Renal Hypertension DR BRIAN RAYNER Surgical DR F AZIZ Adult Surgery DR J ROSSOUW Epidemiology and Preventionist DR AP KENGNE Pregnancy-associated Heart Disease PROF K SLIWA-HAHNLE 67 FROM THE EDITOR’S DESK P Commerford CARDIOVASCULAR TOPICS 68 Epidemiology and management of heart failure with reduced ejection fraction in a Tunisian university hospital H Drissa • M Drissa • S Helali • K Oughlani • A Farah • M Chebbi 74 Role of nuclear factor kappa-B in TNF-induced cytoprotection R Kelly-Laubscher • S Somers • L Lacerda • S Lecour 82 Clinical features and outcomes of infective endocarditis: a single-centre experience H Abdelgawad • S Azab • MA Abdel-Hay • A Almaghraby 89 Positive impact of training rural health workers in identification and prevention of acute rheumatic fever in eastern Uganda J Namuyonga • E Ndagire • D Okumu • O Olugubuyi • S Lubega • J Omagino • P Lwabi • E Okello 93 Association between bifurcation angle of the left main coronary artery and severity of stenosis of the proximal left anterior descending artery M Yahia • W Farid • M Lotfy • M Osama • HA El Deep 98 CircRNA-mediated pathology: a new preliminary insight into the mechanism of type II cardio-renal syndrome H Wang • Y Hu • J Shi • H Wu • Z Qiu • Y Geng 104 Circulatory soluble LOX-1 is a novel predictor for coronary artery disease patients MS Ali Sheikh 109 Assessment of left atrial morphological and functional differences in professional male football players: a prospective, case–control study S Gül • H Güngör
CONTENTS Vol 34, No 2, MAY/June 2023 FINANCIAL & PRODUCTION CO-ORDINATOR ELSABÉ BURMEISTER Tel: 021 976 8129 Fax: 086 664 4202 Cell: 082 775 6808 e-mail: elsabe@clinicscardive.com PRODUCTION EDITOR SHAUNA GERMISHUIZEN Tel: 021 785 7178 Cell: 083 460 8535 e-mail: shauna@clinicscardive.com CONTENT MANAGER MICHAEL MEADON (Design Connection) Tel: 021 976 8129 Fax: 0866 557 149 e-mail: michael@clinicscardive.com The Cardiovascular Journal of Africa, incorporating the Cardiovascular Journal of South Africa, is published 10 times a year, the publication date being the third week of the designated month. COPYRIGHT: Clinics Cardive Publishing (Pty) Ltd. LAYOUT: Jeanine Fourie – TextWrap PRINTER: Tandym Print/Castle Graphics ONLINE PUBLISHING & CODING SERVICES: Design Connection & Active-XML.com All submissions to CVJA are to be made online via www.cvja.co.za Electronic submission by means of an e-mail attachment may be considered under exceptional circumstances. Postal address: PO Box 1013, Durbanville, RSA, 7551 Tel: 021 976 8129 Fax: 0866 644 202 Int.: +27 21 976 8129 e-mail: info@clinicscardive.com Electronic abstracts available on Pubmed Audited circulation Full text articles available on: www.cvja. co.za or via www.sabinet.co.za; for access codes contact elsabe@clinicscardive.com Subscription: To subscribe to the online PDF version of the journal, e-mail elsabe@clinicscardive.com • R500 per issue (excl VAT) • R2 500 for 1-year subscription (excl VAT) The views and opinions expressed in the articles and reviews published are those of the authors and do not necessarily reflect those of the editors of the Journal or its sponsors. In all clinical instances, medical practitioners are referred to the product insert documentation as approved by the relevant control authorities. CASE REPORTS 114 SARS-CoV-2 infection-associated thoraco-abdomino-iliac thrombosis in a patient without cardiac and systemic co-morbidity F Borulu • B Erkut • Y Unlu 117 Diphtheritic myocarditis: a case report, with toxin-mediated complications and multi-organ involvement K Naidoo • M Msimang • M du Plessis • DP Naidoo CONFERENCE PROCEEDINGS 121 Improving cardiovascular outcomes for patients with heart failure in sub-Saharan Africa: conference proceedings of the Nigerian Cardiovascular Symposium OJ Ilonze • A Hicks • B Atanda • MH Abdou • C Onyekwelu • E Chukwu • KM Karaye • I Katibi • OS Ogah • O Emerole • JN Ajuluchukwu • MU Sani • CC Asuzu • MO Ogunniyi NEWS 80 HeFSSA specialist treatment and device therapy course PUBLISHED ONLINE (Available on www.cvja.co.za and in PubMed)
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 67 From the Editor’s Desk I acknowledge it is perhaps unusual to start an editor’s comment with remarks about a case report. I offer no apologies for bringing the case report of Naidoo and colleagues (page 117) to your attention in this issue for two reasons. Firstly, it is a disgrace that diphtheria, a condition that is completely preventable by adequate childhood immunisation, is once again killing young people in South Africa. The authors point out that until the recent outbreaks, diphtheria had been practically eliminated in South Africa, with only three sporadic cases reported between 2008 and 2015. These cases and recently reported outbreaks of measles indicate that the childhood immunisation programme in South Africa is woefully inadequate at present and considerably less effective than in past years. Secondly, it is a reminder that despite the major advances in management of cardiovascular diseases there remain some that were once mainly of historical interest but sadly need to be borne in mind when healthcare systems fail. The authors are to be congratulated for reminding us of this. Namuyonga and co-authors (page 89) address another illness that should have been eliminated by socio-economic intervention rather than immunisation. Recognising that training health workers in high-risk settings to detect acute rheumatic fever/ rheumatic heart disease (ARF/RHD) is a key strategy in preventing ARF, these authors assessed frontline health workers from selected health facilities in Uganda. They were assessed for their knowledge on the clinical features and role of benzathine penicillin G (BPG) in the treatment and prevention of recurrence of ARF. Using the RHD Action Needs assessment tool, they generated and administered a pre-test, then conducted training and re-administered a post-test. Eight months later, health workers were again assessed for knowledge retention and change in practices. During the initial phase, 31% of health workers passed the pre-test, indicating familiarity with clinical features of ARF. The level of knowledge of BPG use in ARF was very poor in all the health units (22.6%), but improved after training to 80%. However, retention of this knowledge waned after eight months and was not significantly different compared to pre-training. This study, which carefully trained, evaluated and, importantly, re-evaluated healthcare workers, identified a critical knowledge gap among health workers, both in awareness and treatment of ARF, and calls for repetitive training as a priority strategy in prevention. Abdelgawad and colleagues’ report (page 82) included 90 consecutive patients admitted to the Cardiology Department in a tertiary-care university hospital in Egypt with a diagnosis of infective endocarditis. In common with the reported experience from other developing countries, the patients were young, with underlying valvular heart disease and intravenous drug abuse being common. Culture-negative endocarditis was common, as was heart failure. Cardiac surgery was considered to be indicated in the majority of patients but was only performed in a minority. Many died before surgery could be performed. The in-hospital mortality rate was high. The above-mentioned articles are a sobering reflection of the state of cardiovascular care in Africa. Success in this area is often measured in the number of high-profile operations or technically complex procedures performed. But the content of these articles indicates that there is still scope for much improvement in very basic levels of care. Pat Commerford Editor-in-Chief Professor PJ Commerford
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 68 AFRICA Cardiovascular Topics Epidemiology and management of heart failure with reduced ejection fraction in a Tunisian university hospital Meriem Drissa, Habiba Drissa, Sana Helali, Khalil Oughlani, Amani Farah, Marwa Chebbi Abstract Introduction: Despite considerable advances in treatment, heart failure (HF) remains a serious public health problem linked to a high rate of mortality. The aim of this work was to describe the epidemiological, clinical and evolutionary features of HF in a Tunisian university hospital. Methods: This was a retrospective study including 350 hospitalised patients diagnosed with HF with reduced ejection fraction (≤ 40%) during the period between 2013 and 2017. Results: The average age was 59 ± 12 years. A male predominance was noted. The main cardiovascular risk factor was the use of tobacco (47%). The electrocardiogram showed atrial fibrillation in 41% of patients and left bundle branch block in 36% of patients. Laboratory results revealed an electrolyte disorder in 30 cases, renal insufficiency in 25% of patients and anaemia in 20%. Echocardiography revealed reduced ejection fraction, with an average of 34 ± 6% (range: 20–40%). The main causes of HF were ischaemic heart disease in 157 patients. The most commonly used medications were diuretics (90% of patients), angiotensin converting enzyme inhibitors (88%), beta-blockers (91%) and mineralocorticoid receptor antagonists (35%). Cardiac resynchronisation therapy was performed on 30 patients and cardioverter defibrillator implantation on 15 patients. The hospital mortality rate was 10% and the average hospital stay was 12 ± 5 days. During six months of follow up, 56 patients died and 126 were re-admitted. Multivariate model predictors of six-month mortality were: age [odds ratio (OR): 8, p = 0.003], ischaemic HF (OR: 1.63, p = 0.01) and diabetes (OR: 21, p = 0.004) Conclusion: This study illustrates the main characteristics of HF in our population. These include relatively young age, a predominance of males, ischaemic heart disease as the main aetiology, insufficient care strategies and a poor prognosis. Keywords: heart failure, epidemiology, aetiology, treatment, Tunisia Submitted 24/5/18, accepted 19/11/18 Published online 3/5/23 Cardiovasc J Afr 2023; 34: 68–72 www.cvja.co.za DOI: 10.5830/CVJA-2018-070 Heart failure (HF) is a serious public health problem leading to increased morbidity and mortality rates worldwide.1,2 Its fast spread over the last few years has been largely linked to aging populations. However, remarkable improvements in HF treatment have increased life expectancy for HF patients and decreased myocardial infarction cases.3 It is important to determine the different diseases leading to HF in order to be able to adjust the diagnosis, and therapeutic and preventative approaches.1 The type of cardiac structural abnormality and triggering factors causing acute decompensation must also be determined. Over the last 20 years, great efforts have been made to improve HF management. These include the use of drugs that interfere with neurohormonal activation, device therapy for selected patients, and multidisciplinary disease management. Despite these advances in therapy and diagnosis, the prognosis of patients with HF remains poor, with increasing mortality rates. There are sufficient data concerning HF characteristics in developed countries while other world regions, including Tunisia, lack sufficient information. The main objective of this work was, therefore, to describe the epidemiological and clinical profile of patients, while providing data about the outcome and management approach in a Tunisian university hospital. The study also aimed to determine the predictors of six-month mortality and re-admission rates. Methods This was a retrospective study including 380 hospitalised chronic HF patients in the adult cardiology department of the Rabta Hospital in Tunis during the period 2013 to 2017. Fig. 1 shows a flow chart of the enrolled patients. Patient consent was obtained and the study was approved by the ethics committee of the Rabta Hospital. We excluded isolated right HF patients (n = 10), those with HF with intermediate ejection fraction (40% < EF < 50%) (n = 15) and patients with HF with preserved ejection fraction (EF ≥ 50%) (n = 5) The final study group encompassed 350 patients with reduced EF (< 40%). Data were extracted from the medical records of the patients. Department of Cardiology, Rabta Hospital, Tunis, Tunisia Meriem Drissa, MD Habiba Drissa, MD, akramhorizon@yahoo.fr Sana Helali, MD Khalil Oughlani, MD Amani Farah, MD Marwa Chebbi, MD
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 69 Analysis of the following information was carried out: age, gender, co-morbidities, electrocardiography and echocardiography assessment, management approach and outcome, which included in-hospital precipitating factors of HF episode, and six-month mortality and re-admission rates. Our main focus was six-month mortality rates and re-admission episodes. Statistical analysis We calculated simple frequencies and relative frequencies (percentages) for qualitative variables, and means, medians and standard deviations for quantitative variables. We also used the chi-squared test and the student or Anova test, respectively, to compare two percentages and two averages. Multivariate analyses were conducted in order to determine the factors predicting mortality. This was performed using a Cox proportional hazard analysis in order to obtain a time-to-event risk ratio after adjustment for confounders. In all the statistical tests, the significance level was fixed at p < 0.05. Results The baseline characteristics of patients are reported in Table 1. The average age was 59 ± 12 years (range: 35–85). A male predominance was noted with a ratio of male:female of 1.2. The main cardiovascular risk factor in 164 patients was the use of tobacco (47%), followed by diabetes in 161 (46%) and hypertension in 150 patients (43%). The clinical presentation was dominated by signs of left HF in 192 patients (55%), global HF in 140 (40%) and cardiogenic shock in 18 patients (5%). The main electrocardiographic abnormalities were atrial fibrillation in 148 patients (41%), ventricular extrasystoles in 28%, non-sustained ventricular tachycardia in 8% and left bundle branch block in 126 patients (36%). Laboratory results showed hyponatraemia in almost 30% of cases (n = 105), renal impairment in 87 patients (25%), anaemia in 70 (20%) and a thyroid disorder was observed in 35 patients (10%). Echocardiography showed that the EF was reduced, with an average of 34 ± 6% (range: 20–40). The distribution of patients according to EF value is shown in Fig. 2. The global longitudinal strain calculated for 277 patients (79%) was altered in all cases, with an average value of –11 (range: –7.3 to –14.2) (26 ± 1.8%). Left ventricular filling pressures were high in 154 patients (44%), functional mitral regurgitation was noted in 214 patients (61%) and the right ventricle was dilated in 84 patients (24%), with impaired function in 11% of cases. Major factors were associated with exacerbation of HF, dominated by bronchopulmonary infection in 143 patients (41%) and non-compliance with medication and/or diet in 105 subjects (30%) (Fig. 3). The main aetiologies of HF were ischaemic heart disease in 157 patients (45%), valvular heart disease in 81 (23%), hypertension in 59 (17%) and cardiomyopathies in 35 patients (10%). The most commonly used medications were diuretics in 315 patients (90%), angiotensin converting enzyme inhibitors Table 1. General characteristics of our population Demographics Patients (n) Age 59 ± 12 Diabetes 161 Gender ratio 1.2 Tobacco use 164 Hypertension 150 Anaemia 70 Atrial fibrillation 148 LBB 126 Ejection fraction (%) 34 ± 6 Aetiology Ischaemic 157 Hypertensive 59 Valvular 81 Cardiomyopathies 35 ACEI use 308 BB use 319 Resynchronisation 30 DAI 16 LBBB: left bundle branch block, ACEI: angiotensin converting enzyme inhibitors, BB: beta-blockers, DAI: defibrillator automatic implantation. Population hospitalised for chronic HF n = 380 Included patients: first episode of HF with EF ≤ 40 % n = 350 Excluded patients Isolated right heart failure n = 10 Excluded patients: mid-range HF 40% < EF < 50% n = 15 Excluded patients: preserved HF EF ≥ 50% n = 5 Fig. 1. Flow chart of the study. HF: heart failure; EF: ejection fraction. EF [20–30%] EF [31–35%] EF [36–40%] 50% 38% 25% 13% 0% 28% 24% 48% Fig. 2. Population distribution according to ejection fraction. EF: ejection fraction.
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 70 AFRICA (ACEI) in 308 (88%), beta-blockers (BB) in 319 (91%) and mineralocorticoid receptor antagonists (MRA) in 123 patients (35%). IschaemicHFwas treatedwith aspirin and statins. Sintrom was prescribed for atrial fibrillation. Electrolyte disturbance was corrected and anaemia was treated with iron therapy in the case of iron deficiency. In our study, the percentage of patients on all threemedications (ACEI, BB, MRA) was 45% (Fig. 4). The recommended optimal dose was reached in only 11% for ACEI, in 15% for BB and in 12% for MRA. None of the patients was put on ivabradine because it was not available in our hospital during the study period. Among the 157 patients with coronary artery disease, revascularisation was done on 125 ischaemic HF patients (80%). This consisted of coronary artery bypass graft in 18% and percutaneous coronary angioplasty in 82%. Fifty-six patients with valvular HF (70%) underwent valve surgery. We reported a low frequency of resynchronisation therapy (15%) and cardioverter defibrillator implantation (4.5%). Heart transplantation was not performed in our study. In-hospital mortality was 10%. The average length of hospital stay was 12 ± 5 days. In the six-month follow up, we reported death in 16% of patients and a 36% re-admission rate. Predictive factors for six-month mortality identified in univariate analysis are shown in Table 2. Table 3 shows the independent factors of six-month mortality rate, which were age [odds ratio (OR): 8, p = 0.003], ischaemic HF (OR: 1.63, p = 0.01) and diabetes (OR: 2, p = 0.004). Discussion HF is a common pathology. Its fast and widespread development makes it a major public health problem. The number of patients with HF is constantly increasing throughout the world due to aging populations and continuous improvement in the management of cardiovascular diseases.1,2 An estimated worldwide prevalence of HF was > 37.7 million in 2011.3 It is expected that by 2030, the number of HF patients would rise by 25%.4 In developed countries, the prevalence of HF is well known while in Tunisia, data are lacking. In the United States, the number of Americans suffering from HF reached five million in 2005 and more than 550 000 new cases are being diagnosed each year.5 Data for developing countries however are scarce in the literature. The prevalence of HF increases with age.6 The average age of our patients in this study was 59 ± 12 years. These data suggest that the Tunisian population with HF is relatively young compared to data from European and American series.7.8 This can be explained by the better quality of management of ischemic heart disease in these countries and by some aetiological characteristics of HF in emerging countries. The majority of our patients were male, which is confirmed by the literature.1,2,9 Left-sided HF was the most frequently observed clinical presentation in several series and also in our study.10 Table 2. Predictive factors of six-month mortality identified in univariate analysis Predictive factors Deaths (n = 56) Survival (n = 294) p-value Age (years) 62 ± 10 54 ± 11 0.04 Diabetes, n (%) 40 (71) 121 (41) < 0.0001 Atrial fibrillation, n (%) 25 (44.5) 132 (44) NS LBBB, n (%) 20 (36) 97 (32) NS Anaemia, n (%) 28 (50) 42 (14) 0.01 Renal impairement, n (%) 30 (53) 55 (18) 0.001 EF (%) 34 ± 4 36 ± 4 0.04 Ischaemic aetiology, n (%) 33 (59) 124 (42) 0.003 Medication with ACEI, n (%) 50 (89) 272 (92) NS Medication with BB, n (%) 29 (52) 299 (98) 0.01 Cardiover defibrillator, n (%) 2 (3.5) 14 (5) NS Resynchronisation therapy, n (%) 5 (5) 29 (10) NS AF: atrial fibrillation, LBBB: left bundle branch block, EF: ejection fraction, BB: beta-blocker. Table 3. Predictors of six-month mortality identified in multivariate analysis Predictors OR 95% CI p-value Diabetes 5 6.5–32 0.004 Age 10 1.3–1.16 0.003 Ischaemic aetiology 1.63 1.21–22 0.01 Medication with beta-blockers 0.4 0.34–0.09 0.01 ACE-I + BB ACE-I + BB + MRA ACE-I or BB 90% 68% 45% 23% 0% 82% 45% 12% Fig. 4. Therapeutic profile in our study. ACEI: angiotensin converting enzyme inhibitors; BB: beta-blockers; MRA: mineralocorticoid receptor antagonists. Ischaemic event Pulmonary embolism Non compliance with medication and/ or diet Arrhthmia Anemia Pulmonary infection 50% 38% 25% 13% 0% 22% 2% 30% 12% 23% 41% Fig. 3. Factors causing exacerbation of HF.
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 71 Dzudie et al. found atrial fibrillation present in HF patients, with a frequency ranging from 23 to 40%.11 It was observed in 41% of patients in our study. Yancy et al. found left bundle branch block in up to 30% of patients with HF in their study,12 and it was found in 40.6% in the study by Bouqat et al.13 We found a comparable rate of left bundle branch block in our study (36%). Determining the decompensation factors of HF should be systematic in order to reduce the risk of rehospitalisation and improve the management of HF.14 Berkovitch,14 in his study including 2 212 patients, found several factors, including infection (21%), non-compliance (17%), renal dysfunction (13%) and other miscellaneous factors (49%). Our results were similar to this study. In our study, the aetiology of HF was mainly ischaemic. In European and American studies,1,2 hypertension represents the leading cause of HF among African adults.15 The increase in incidence of ischaemic heart disease in Tunisia can be explained by a better diagnostic approach and an increase in coronary disease due to changing lifestyles. Valvular disease is a cause of left ventricular (LV) dysfunction in our country. It was observed in 23% of cases and its frequency was 24% in a Moroccan study.1 This was mainly due to frequent rheumatic valvulopathy, in contrast to developed countries where valvulopathies were mainly dystrophic. The relative decline of rheumatic valvulopathies is due to the widespread use of antibiotics and an improvement in general hygiene.13 Over the last 20 years, HF management has been codified thanks to recent updates of European recommendations.16 In our study, the most commonly used drugs were diuretics (mainly furosemide), ACEI, BB and MRA. Data from our study suggest, however, that the prescription of drugs for HF was not optimal. Various factors influencing requirements have been identified, including age, gender and co-morbidities, especially renal insufficiency. Indeed, the triple combination (ACEI, BB and MRA) was prescribed in only 35% of cases in some series17 and the number of patients treated with appropriate doses was low. Our medical prescription included a triple combination in 45% of patients. However, the recommended optimal doses for each class were achieved in only 11% for ACEI, in 15% for BB and in 12% for MRA. Non-pharmacological treatment that can improve the prognosis of HF, such as resynchronisation therapy and defibrillation, remains weak.13,14,16 Indeed, in our study cardiac resynchronisation was performed in only 30 patients (9%) and an implantable cardioverter defibrillator was placed in 15 patients. The explanation for the non-implementation of these devices were: patient’s refusal, problem with logistics and cost, and low percentage of patients eligible for resynchronisation. Cardiac transplantation is not often performed due to its high economic cost and its logistical problem. No case of transplantation is reported in our study. Despite recent advances in treatment, HF remains a serious disease linked to high mortality rates, with a particularly somber, long-term prognosis. Mortality rates ranged across different regions from 6.9 to 15.6% for chronic HF in the study by CrespoLeiro et al.17 Our study revealed that in-hospital mortality was around 10%. This is similar to the report by Savarese and Lars18 and comparatively higher than that reported in other registers,19 which varied from 4 to 7%. Higher rates for six-month mortality from HF were observed in Morocco and ranged from 15 to 35%, depending on the studies.14,20 According to our results, the six-month mortality rate was 16%. It is also noteworthy that the HF mortality rate is higher than that from myocardial infarction and several cancers.19 Hospital re-admissions remain a big challenge for the care of the HF patient. Despite the remarkable progress that has been made over the past five years, more than 20% of patients are re-admitted within 30 days and up to 50% within six months.20 Our study found that the six-month re-admission rate was 36%. Prognostic models provide a means of assessing a patient’s risk of adverse clinical outcomes and may influence clinical management. Several studies used multivariate logistic regression to derive predictive models.21,22 In our study, ischaemic HF was independently predictive of mortality [hazard ratio (HR) = 1.63]. Several studies have corroborated our results.21-23 The independent prognostic value of age of death from any cause was confirmed by the study by Wedel et al.,24 which involved patients from the CORONA trial (HR 1.26 per each 10-year increase; p < 0.0001). The study by Allen et al.25 was conducted on patients from the CHARM trial (HR = 1.32 per each 10 years over the age of 60 years; p = 0.0001) in a follow-up period of 38 months. Wedel et al. found diabetes to be an important precursor to the development of HF and it was associated with an increased mortality rate.24 This confirms our results of a 10% risk of mortality. The use of BB was found to be an independent protective factor in the MAGGIC meta-analysis,26 which included individual data of 3 372 patients with reduced LVEF. Our study showed similar results. As is any research, our study is subject to certain limitations, the most important one being the limited number of patients. This was a major setback while concluding our results. However, it should encourage us to establish a national HF registry that will enable better statistical analysis and lead to more relevant conclusions. Despite these limitations, the study highlighted some limits in the current management system of HF in Tunisia, including non-optimal medication management, poorly supported terminal HF (no transplantation), and very limited recourse to cardiac rehabilitation. Conclusion This study highlighted the epidemiological and clinical features of HF in Tunisia and revealed the deficiencies in patient care. These results should be an incentive to treat HF patients more effectively and to take further preventative measures to improve HF prognosis. The authors thank the managers of the factories, all data collectors and study participants for sharing their precious time and information while collecting data. References 1. Maggioni AD. Epidemiology of heart failure in Europe. Heart Fail Clin 2015; 11: 625–635. 2. Buja A, Solinas G, Visca M, et al. Prevalence of heart failure and adherence to process indicators: which sociodemographic determinants are involved? Int J Environ Res Public Health 2016; 13: 238.
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 72 AFRICA 3. Ziaeian B, Fonarow GC. Epidemiology and etiology of heart failure. Nat Rev Cardiol 2016; 13: 368. 4. Geremy A, Jessup M. Understanding heart failure. Heart Fail Clin 2017; 13: 1–19. 5. Hunt SA, Abraham WT, Chin MH, et al. Focused update incorporated into the ACC/AHA 2005 guidelines for the diagnosis and management of heart failure in adults. Circulation 2009; 119(14): 391–479. 6. Metra M, Carubelli V, Ravera A, et al. Heart failure 2016: still more questions than answers. Int J Cardiol 2017; 227: 766–777. 7. Adams Jr KF, Fonarow GC, Emerman CL, et al. Characteristics and outcomes of patients hospitalized for heart failure in the United States; rational, design and preliminary from the first 100,000 cases in the acute decompensated heart failure national registry (ADHERE). Am Heart J 2005; 149: 209–216. 8. Lappas G, Fu M, Rosengren A. Trends in prevalence from 1990 to 2007 of patients hospitalized with heart failure in Sweden. Eur J Heart Fail 2014; 16: 737–742. 9. Follath F, Yilmaz MB, Delgado JF, et al. Clinical presentation, management and outcomes in the Acute Heart Failure Global Survey of Standard Treatment (ALARM-HF). Intensive Care Med 2011; 37(4): 619–626. 10. Rahman T, Majumder A, Rahman A. Clinical presentation of heart failure patients admitted in National Institute of Cardiovascular Diseases, Dhaka. J Med 2014; 15: 18–22. 11. Dzudie A, Milo O, Edwards CA, et al. Prognostic significance of ECG abnormalities for mortality risk in acute heart failure: insight from the Sub-Saharan Africa Survey of Heart Failure (THESUS-HF). J Card Fail 2014; 20: 45. 12. Yancy CW, Jessup M, Bozkurt B, et al. ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013; 128: 232–240. 13. Epidemiological and evolutionary characteristics of heart failure in patients with left bundle branch block – a Moroccan center-based study. J Saudi Heart Assoc 2015; 27: 1–9. 14. Berkovitch A, Maor A, Sabbag A, et al. Factors for acute heart failure hospitalization and long-term survival. Medicine (Baltimore) 2015; 94(52): e23–30. 15. Dokainish H, Teo K, Zhu J, et al. INTER-CHF investigators. Heart failure in Africa, Asia, the Middle East and South America: The INTERCHF study. Int J Cardiol 2016; 204: 133–141. 16. Ponikowski P, Voors AA, Anker SD. 2016 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). J Heart Fail 2016; 18(8): 891–975. 17. Crespo-Leiro MG, Anker SD, Maggioni AP, et al. Heart Failure Association (HFA) of the European Society of Cardiology (ESC). European Society of Cardiology Heart Failure Long-Term Registry (ESC-HF-LT): 1-year follow-up outcomes and differences across regions. Eur J Heart Fail 2016; 18: 613–625. 18. Savarese G,, Lars H. Global public health burden of heart failure. Card Fail Rev 2017; 3(1): 7–11. 19. Maggioni AP, Dahlström U, Filippatos G, et al. EURO observational research programme: the Heart Failure Pilot Survey (ESC-HF Pilot). Eur J Heart Fail 2010; 12(10): 1076–1084. 20. O’Connor CM. High heart failure readmission rates. Is it the health system’s fault? J Am Coll Cardiol 2017; 5(5): 393. 21. Ponikowski P Anker S, AlHabib KF, et al. Heart failure: preventing disease and death worldwide. ESC Heart Fail 2014; 1: 4–25. 22. Gheorghiade M, Flaherty JD, Fonarow GC, et al. Coronary artery disease, coronary revascularization, and outcomes in chronic advanced systolic heart failure. Int J Cardiol 2011; 151: 69–75. 23. Gajanana D, Shah M, Junpapart P, et al. Mortality in systolic heart failure revisited: ischemic versus non-ischemic cardiomyopathy. Int J Cardiol 2016; 224: 15–17. 24. Wedel H, McMurray JJ, Lindberg M, et al. Predictors of fatal and nonfatal outcomes in the Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA): incremental value of apolipoprotein A-1, high-sensitivity C-reactive peptide and N-terminal pro B-type natriuretic peptide. Eur J Heart Fail 2009; 11: 281–229. 25. Allen LA, Felker GM, Pocock S, et al. Liver function abnormalities and outcome in patients with chronic heart failure: data from the Candesartan in Heart Failure: Assessment of Reduction in Mortality and Morbidity (CHARM) program. Eur J Heart Fail 2009; 11: 170–177. 26. Taneva B, Caparoska D. The impact of treatment with beta-blockers upon mortality in chronic heart failure patients. Maced J Med Sci 2016; 4(1): 94–97.
Pharmaco Distribution (Pty) Ltd. 3 Sandown Valley Crescent, South Tower, 1st Floor Sandton, 2196. PO Box 786522, Sandton, 2146. South Africa Tel: +27 11 784 0077. Fax: +27 11 784 6994. www.pharmaco.co.za ISA _ 21 _ 01 References: 1. Ismo® South African SAHPRA aproved package insert. 2. Ismo 20 Product Monograph(2015). 3. Abshagen,U. 1992. Pharmacokinetics of isosorbide mononitrate. The American Journal of Cradiology, [online] 70 (17),pp.G61-G66 4. Thadani U, Maranda CR, Amsterdam E, et al. Lack of Pharmacological Tolerance and Rebound Angina Pectoris during Twice-daily Therapy with isosorbide-5-mononitrate. Annals of Internal Medicine. 1994.; 120: 353-359. ISMO®-20 R/7.1.4/136. Each Ismo®-20 Tablet contains Isosorbide-5-mononitrate 20mg. S3 For full prescribing information, please refer to the approved package insert R T S Q P R T S Q P P Long term prophylaxis and management of Angina Pectoris1 No first-pass metabolism. 100% bioavalability2,3 Twice-daily dosing regimen shown to avoid withdrawal and tolerance4 Trust the Original!
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 74 AFRICA Role of nuclear factor kappa-B in TNF-induced cytoprotection Roisin Kelly-Laubscher, Sarin Somers, Lydia Lacerda, Sandrine Lecour Abstract Ischaemic heart disease (IHD) is a leading cause of death worldwide. Understanding prosurvival signalling pathways that protect against ischaemia–reperfusion injury (IRI) may assist in the development of novel cardioprotective strategies against IHD. In this regard, the transcription factor, nuclear factor kappa-B (NFκB) is activated by tumour necrosis factor (TNF), but its role in TNF-induced cytoprotection is unknown. Therefore, to investigate the role of NFκB in TNF-induced cytoprotection, C2C12 cells were pretreated with TNF (0.5 ng/ml) in the presence and absence of an NFκB inhibitor, pyrrolidine derivative of dithiocarbamate (PDTC; 100 µM). Cells were subjected to simulated IRI and treated with PDTC, either during TNF exposure or at reperfusion. Phosphorylation of IkB was measured after the TNF stimulus. Cytoprotection by TNF in cells subjected to IRI (cell viability: 43.7 ± 8.1% in control vs 70.6 ± 6.1% with TNF, p < 0.001) was abrogated by co-administration of PDTC (40.6 ± 1.9%, p < 0.001 vs TNF) but not by exposure to PDTC at reperfusion (70.7 ± 1.7%). Cytosolic IkB phosphorylation [1.5 ± 0.2 arbitrary units (AU) for TNF vs 1.0 ± 0.0 for untreated, p < 0.01]) was increased after TNF exposure and this increase was abolished by co-administration with PDTC (0.8 ± 0.3 AU, p < 0 01 vs TNF). Our data suggest that NFκB acts as a key component in TNF-induced cytoprotection. These findings may pave the way for the development of novel therapeutic drugs that target TNF/NFκB signalling to protect against IHD. Keywords: TNF, NFκB, ischaemia–reperfusion, preconditioning, cardioprotection Submitted 18/8/20, accepted 10/5/22 Published online 8/6/22 Cardiovasc J Afr 2023; 34: 74–80 www.cvja.co.za DOI: 10.5830/CVJA-2022-023 Ischaemic heart disease (IHD) is the leading cause of death worldwide and its global prevalence rate is on a constant rise, expected to reach more than 1 845 per 100 000 population by the year 2030.1 Although various possible cardioprotective strategies have been pinpointed as promising therapies to protect against ischaemia–reperfusion injury (IRI) in the preclinical setting, very few have translated into the clinical setting in the past 30 years.2 A better understanding of the signalling pathways that may promote cell survival against IRI are key to the successful development of future therapies that may limit the cellular damage associated with the disease. In this regard, the pro-inflammatory cytokine, tumour necrosis factor (TNF), is implicated as a mediator in cardiovascular disease, including acute myocardial infarction,3,4 atherosclerosis,5 chronic heart failure6,7 and ischaemia–reperfusion.8-10. TNF can induce cardiomyocyte apoptosis via signalling through the TNF receptorassociated death domain (TRADD).11 In contrast, TNF is also a key signalling component whose activation is required to promote cell survival in ischaemic preconditioning and postconditioning.12-14. Most importantly, TNF can mimic the protective effect of ischaemic conditioning in a dose- and time-dependent manner.13,15 TNF is traditionally known as a potent activator of nuclear factor kappa-B (NFκB). This transcription factor mediates the protective effects of various forms of ischaemic and pharmacological preconditioning, including classic ischaemic preconditioning,16,17 late ischaemic preconditioning,18,19, remote ischaemic preconditioning20 and erythropoietininduced cardioprotection.20 Despite a clear role for NFκB in cytoprotection induced by other treatments, the role of NFκB in TNF-induced cytoprotection has not been investigated. Inactive NFκB resides in the cytoplasm bound to inhibitory IkB proteins.21 NFκB activation can be initiated through receptor-mediated events such as ligand binding (e.g. TNF, interleukin-1 or lipopolysaccharide) or non-receptor-mediated pathways such as oxidative stress or ultraviolet radiation.22 This causes phosphorylation and activation of the IkB kinases (IKKs) by upstream protein kinases such as Akt. The IKKs, in turn, phosphorylate IkB at its amino terminus, leading to its disassociation from NFκB and subsequent proteasomal degradation by ubiquination.23 NFκB, now in its active form, translocates to the nucleus where it binds to specific recognition sequences and regulates transcription.24 TNF initiates a prosurvival signalling cascade termed the survivor activating-factor enhancement (SAFE) pathway.25 This pathway includes activation of TNF receptor 2 (TNFR2) by TNF, which leads to activation of the signal transducer and activator of transcription 3 (STAT-3) pathways and ultimately inhibits opening of the mitochondrial permeability transition pore to promote cell survival.13,15 Recently, the mitochondrial effects of TNFR2 activation have been suggested to be mediated by activation of the transcription factor, NFκB,26 but the role of NFκB in TNF-induced cytoprotection against IRI is unknown. Hatter Institute for Cardiovascular Research in Africa, Faculty of Health Sciences, University of Cape Town, South Africa; and Department of Pharmacology and Therapeutics, College of Medicine and Health, University College Cork, Ireland Roisin Kelly-Laubscher, PhD, Roisinkelly@ucc.ie Hatter Institute for Cardiovascular Research in Africa, Faculty of Health Sciences, University of Cape Town, South Africa Sarin Somers, PhD, sarin.somers@gmail.com Lydia Lacerda, PhD Sandrine Lecour, PhD
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 75 Therefore, the current study assessed the role of NFκB and associated respiratory changes in TNF-induced cytoprotection. Methods C2C12 myoblasts (European Collection of Cell Cultures, Centre for Applied Microbiology and Research, UK) were stored in cryovials containing 1 × 106 cells/ml in liquid nitrogen at –196°C. When required, a cryovial was thawed for 30–40 seconds in a 37°C water bath. The contents were sterilely transferred to a 75-cm3 tissue culture flask containing Dulbecco’s modified eagle serum (DMEM) with 4.5 g/l glucose, 0.110 g/l sodium pyruvate and L-glutamine supplemented with 10% foetal calf serum (FCS) and 1% (w/v) penicillin/ streptomycin (PIS) (all purchased from Highveld Biological, RSA). The myoblasts were grown in 5% CO2 trypsinised with 0.25% (w/v) trypsin (Highveld Biological, RSA) supplemented with 0.2% EDTA (w/v) (Sigma, Germany), made up in phosphatebuffered saline (PBS). The trypsinisation process was allowed for three minutes at 37°C before it was neutralised with twice the volume of 10% FCS DMEM. The cells, now in suspension, were transferred to 50 ml polypropylene conical tubes and centrifuged for five minutes at 1 000 rpm. Cells were counted in a Neubauer haemocytometer before centrifugation. Tissue culture flasks (25 cm3) were seeded with enough cells to allow 80% confluency in two to three days. At this point, differentiation was initiated with DMEM supplemented with 1% horse serum (HS) (Sigma, Germany) and 1% PIS. Differentiation was maintained with fresh 1% HS DMEM every two days until day eight. Experiments were performed using differentiated myotubes between days eight and 10. Experimental protocol During physiological ischaemia, the pH becomes mildly acidic, potassium concentration increases and metabolic activity is perturbed. Myocardial ischaemia was simulated in the C2C12 study model under these conditions. The protocol was adapted from Esumi et al.27 The simulated ischaemia (SI) buffer [137 mM NaCl, 12 mM KCI, 0.5 mMMgCI, 0.9 mM CaCl2, 20 mM Hepes and 20 mM 2-deoxy-d-glucose (2-DG)] was adjusted to a pH of 6.4. The inclusion of 2-DG served to inhibit glycolysis and thereby disrupt metabolic activity. To simulate ischaemia, low oxygen consumption was also required. Hence, a multigas incubator (Sanyo Electric Co, Ltd, Japan) supplying 5% CO2, 94% N2 and 1% O2 was employed to house the 25-cm3 tissue culture flasks in a hypoxic environment. The preconditioning protocol involved the following groups, which are schematically represented in Fig. 1. Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Normoxic control Fig. 1. Schematic representation of the protocol. PDTC = ammonium pyrrolidone derivative dithiocarbamate, TNF = tumour necrosis factor, SI = simulated ischaemia on the graph.
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 76 AFRICA Group 1: the normoxic control group represented untreated cells that were maintained in their normal environment in the standard 5% CO2 incubator for the period of the experiment. Group 2: the SI control group for the pre-ischaemia inhibitorexposed groups consisted of cells that were incubated for seven hours in the hypoxic incubator, followed by incubation with 1% HS DMEM for one hour in the normoxic incubator. Group 3: cells were treated with TNF (recombinant murine TNF, Peprotech, USA, 0.5 ng/ml in DMEM with 1% HS) for 30 minutes in the normoxic incubator. Cells were then re-incubated with fresh 1% HS DMEM for 60 minutes in the normoxic incubator before incubation with SI buffer for seven hours in the hypoxic buffer. Finally, cells were incubated with 1% HS DMEM for one hour in the normoxic incubator. Group 4: ammonium pyrrolidone derivative dithiocarbamate (PDTC) (100 µM)28 (Sigma-Aldrich, USA) was added prior to the addition of TNF (0.5 ng/ml) in 1% HS DMEM and incubated in the normoxic incubator. Following re-incubation for 60 minutes with 1% HS DMEM in the normoxic incubator, cells were incubated for seven hours in the hypoxic incubator. Cells were then incubated in 1% HS DMEM for one hour in the normoxic incubator. Group 5: PDTC was added for 30 minutes in 1% HS DMEM followed by two washes, and incubated in 1% HS DMEM for one hour in the normoxic incubator. Following re-incubation for 60 minutes with 1% HS DMEM in the normoxic incubator, cells were incubated for seven hours in the hypoxic incubator. Cells were then incubated in 1% HS DMEM for one hour in the normoxic incubator. Group 6: PDTC (100 µM) was added for one hour after the seven-hour SI with TNF preconditioning. Group 7: PDTC was added for 30 minutes in 1% HS DMEM, followed by two washes, and incubated in 1% HS DMEM for one hour after the seven-hour SI. In all the groups, cells were washed twice with PBS (pH 7.4) prior to the change of medium. Cell viability Cell viability was evaluated using the trypan blue exclusion method.29 Cells were trypsinised and resuspended in PBS. A small volume of cell suspension was mixed in a 1.1 ratio with trypan blue (4 µM) (Sigma, Germany). The trypan blue seeps through the disrupted membrane of dead or dying cells and in the process, stains them blue. The mixture is pipetted onto a Neubauer haemocytometer and the counting is assessed on a light microscope. The ratio of blue-stained cells to grey, unstained live cells is calculated and expressed as a percentage. Respiratory parameters Respiration studies were conducted immediately after the simulated index ischaemia (Fig. 2). State-2 respiration was measured using an Oxytherm respirometer equipped with a Clark-type electrode and a Peltier temperature-control unit (Oxytherm, Hansatech, Norfolk, UK). Calibration was set with PBS at a temperature of 37°C. Calibration was verified by the addition of 1 ml PBS into the chamber followed by a baseline reading. The percentage viability was used to calculate a factor from which the respiration values from viable cells could be determined. Protein isolation To investigate the role of NFκB as a trigger, PDTC was administered during the ischaemic or TNF-preconditioning stimulus. Samples were collected after 15 minutes as this timepoint was observed as the peak of IkB phosphorylation (data not shown). After exposure to the drugs, the cells were washed quickly with PBS (pH 7.4) and then transferred to the respective Eppendorf in the presence of 500 µl solution of lysis buffer containing 10% Nonidet P-40, 4 M NaCl, 1 M Hepes (pH 7.9), 500 mM EDTA and complete EDTA-free protease inhibitor cocktail (Roche Systems, USA). Samples were centrifuged at 3 000 rpm for 30 seconds and the supernatant was transferred to a new tube. These tubes were spun for five minutes at 5 000 rpm. The subsequent supernatant fraction contained the cytosolic proteins. Protein concentrations were determined using Lowry’s protein method.30 Extracts were stored at –80°C. Bio-plex array analysis for phosphoprotein testing A singleplex assay was performed to test for phosphorylated IkB proteins from cell lysates. The Bio-plex phosphorylation assay and testing reagent kits (Bio-Rad Laboratories Inc, USA) were used according to the manufacturer’s instructions. Bio-plex phosphoprotein assays are bead-based singleplex or multiplex assays that detect phosphorylation of proteins in cell and tissue sample lysates. The lysates are coupled to internally dyed beads and incubated with the specific biotinylated detection antibody in a 96-well plate. Streptavidin-phycoerythrin (streptavidin-PE) is then added to bind the detection antibodies. Data are acquired using a dual laser, flow-based microplate reader system and outputted as fluorescence intensity on the Bio-plex Manager™ software. CTL TNF TNF + PDTC Phospho Ikk activity (AU) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 ### Fig. 2. Effect of NFκB inhibitor (PDTC) on the phosphorylation of IkB during the preconditioning stimulus in C2C12 myotubes subjected to TNF-mediated preconditioning. The addition of PDTC (100 µM) reduced the phosphorylation of IkB observed during the TNF preconditioning stimulus. *p < 0.05 vs ischaemic control (CTL), ##p < 0.01 vs TNF; n ≤ 4 per group. AU: arbitrary units.
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 77 Statistical analysis Data are expressed as mean ± standard error of the mean (SEM). Statistical significance between multiple groups was determined by one-way analysis of variance (ANOVA) followed by the Student Newmanm–Keul post hoc test (Graph Pad lnstat). A value of p < 0.05 was considered significant. Results Cell viability was assessed by trypan blue staining as previously described.31 TNF preconditioning improved cell viability (70.6 ± 6.1 vs 43.7 ± 8.1% for ischaemic control group, p < 0.001). The administration of the NFκB inhibitor PDTC, given during the TNF preconditioning stimulus, abolished the cytoprotective effect of TNF (40.9 ± 2.8%, p < 0.001 vs TNF, Fig. 3A). However, PDTC, given at reperfusion, did not decrease the protective effect of TNF (70.7 ± 1.7%, non-significant vs TNF, p < 0.001 vs ischaemic control, Fig. 3B). PDTC alone had no effect on the cell viability. Cellular oxygen consumption (state-2 respiration) in C2C12 myotubes was measured immediately after the index simulated ischaemia (Fig. 4). Normoxic cells presented a state-2 respiration of 6.3 ± 0.4 nM of oxygen per million of viable cells/min. Following seven hours of simulated ischaemia, state-2 respiration was reduced to 2.5 ± 0.1 nM of oxygen per million of viable cells/min. TNF induced preconditioning significantly improved the state-2 respiration (5.7 ± 0.6 nM of oxygen per million of viable cells/min, p < 0.05 vs ischaemic control). Administration of PDTC during the TNF preconditioning stimulus reduced the oxygen consumption (TNF + PDTC: 3.6 ± 0.3, p < 0.05 vs TNF). Phosphorylation of IkB was measured as an indication of NFκB activation. IkB phosphorylation was measured 15 minutes into the 30-minute TNF preconditioning stimulus (Fig. 2). Preconditioning with TNF increased the phosphorylation of IkB (1.5 ± 0.2 arbitrary units for TNF vs 1.0 ± 0.0 for ischaemic control, p < 0.05) and was abolished when TNF was incubated in the presence of PDTC (0.8 ± 0.3 vs TNF, p < 0.01). Discussion The aim of our study was to explore the role of NFκB as a key component to promote cell survival in TNF-induced cytoprotection. Our findings of TNF-induced cytoprotection are in line with previous studies in isolated cells,32 perfused hearts14 and whole animals.12 Furthermore, we showed that inhibition of NFκB abrogates the protection induced by TNF. Although high doses of TNF promoted apoptosis via the activation of the pro-apoptotic factor PHLPP1 via NFκB in neonatal cardiomyocytes,10 our study demonstrated that lower cytoprotective doses of TNF promoted NFκB activation, which is required for TNF-mediated protection against simulated ischaemia–reperfusion. Our study is in agreement with other studies that found that NFκB activation is required for cytoprotection.16,18,19,33,34 While some of these studies coincide with our study in demonstrating that NFκB activation before an ischaemic insult is required CTL TNF TNF + PDTC PDTC % Cell death 80 70 60 50 40 30 20 10 0 *** ### CTL TNF TNF + PDTC PDTC % Cell death 80 70 60 50 40 30 20 10 0 *** *** Fig. 3. Effect of NFκB inhibitor (PDTC) on cell viability in TNF-induced preconditioning. A. Addition of PDTC (100 µM) during the TNF preconditioning stimulus abolished the protective effect of TNF. ***p < 0.001 vs ischaemic control group (CTL); ###p < 0.001 vs TNF; n = 9 per group. B. Addition of PDTC (100 µM) at reperfusion failed to abolish the protective effect of TNF. ***p < 0.001 vs CTL; n ≥ 10 per group. CTL TNF TNF + PDTC Oxygen consumption (mM/oxygen per million cells) 7 6 5 4 3 2 1 0 * # Fig. 4. Effect of NFκB inhibitor (PDTC) given during the TNF preconditioning stimulus on the oxygen consumption in C2C12 myotubes. PDTC (100 µM) abolished the increase of state-2 respiration induced by TNF preconditioning (TNF). *p < 0.05 vs ischaemic control group; #p < 0.05 vs TNF; n ≥ 6. A B
RkJQdWJsaXNoZXIy NDIzNzc=