Caffeine Cardiovascular Toxicity: Too Much of a Good Thing

Coffee in Health and Disease Preventionhttp://dx.doi.org/10.1016/B978-0-12-409517-5.00078-4 © 2015 Elsevier Inc. All rights reserved.

699

C H A P T E R

78Caffeine Cardiovascular Toxicity:

Too Much of a Good ThingCláudia Deus, Ana F. Branco, Paulo J. Oliveira, Vilma Sardão

CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

List of AbbreviationsAC Adenylate cyclaseACE Angiotensin converting enzymeADP Adenosine diphosphateAMP Adenosine monophosphateAr Adenosine receptorsATP Adenosine triphosphatecAMP Cyclic adenosine monophosphatecAMP PDE 3,5-Cyclic nucleotide phosphodiesteraseCICR Calcium-induced calcium releaseFCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazoneGMP Guanosine monophosphateIMP Inosine monophosphateLDL Low-density lipoproteinMPT Mitochondrial permeability transitionPKA Protein kinase ARAS Renin-angiotensin systemRCR Respiratory control ratioSR Sarcoplasmic reticulumTPP+ Tetraphenylphosphonium cation

78.1 INTRODUCTION

The cardiovascular toxicity of caffeine is still con-troversial and continues to be under active research. Although several studies reported that moderate doses of caffeine are not harmful to the heart,1 improv-ing physical and cognitive performance2 and prevent-ing stroke episodes,3 other reports show that excessive caffeine consumption is harmful.4,5 However, despite the controversy, it is concluded that, for each individ-ual, caffeine toxicity depends on pharmacokinetic and pharmacodynamic variations, clinical condition, and, of course, on the dose consumed.

78.2 CAFFEINE AND THE HEART

78.2.1 Caffeine during Pregnancy: Any Concern for Fetal Heart Development?

Human exposure to caffeine can start in the womb of the progenitor. During pregnancy, the half-life of caf-feine increases from 3–6 h to 10–20 h,6 increasing conse-quently the exposure of the organism to that molecule. Since caffeine is a hydrophobic compound, it crosses the placenta and reaches fetal bloodstream.7 However, neonatal and fetal hepatic detoxification systems are inadequately developed, which increases the half-life of caffeine to 80 h.7 As a consequence, toxic caffeine concen-trations can be quickly achieved and cause perturbations in the fetal heart rate.8 Cases of fetal arrhythmias result-ing from excessive caffeine intake by pregnant women have been reported. In one of those cases, a mother gave birth to a boy with irregular heart rhythm, after drinking 10 cups of coffee during the last hour before initiating spontaneous labor. The effect was reversible since three days later the heart rhythm returned to nor-mal, although traces of caffeine were still detected in the baby’s urine.8 Although the effects observed in the baby’s heart rate induced by excessive consumption of caffeine dissipated several days after birth, the impact on the cardiac tissue can persist and reverberate later in the offspring’s life. In fact, a study performed with C57BL/6 mice revealed that chronic administration of caffeine to pregnant females (20 mg/kg, corresponding to two cups of coffee in humans) promoted a persistent activation of local renin-angiotensin system (RAS) in the heart of the adult offspring, altering their blood pressure and cardiac

78. CAFFEINE CARDIOVASCULAR TOXICITY700

III. EFFECTS OF SPECIFIC COMPOUNDS FOUND IN COFFEE

remodeling.9 RAS is a group of enzymes/substrates that was first identified in the systemic circulation. Thus, once released by juxtaglomerular cells in the kidney, renin converts the substrate angiotensinogen, released by the liver, to angiotensin I. This peptide is in turn con-verted to angiotensin II by the membrane-bound metal-loproteinase enzyme angiotensin converting enzyme (ACE), found in the surface of the pulmonary endothe-lium. Then, angiotensin II, acting on specific receptors on vascular smooth muscle cells, induces vasoconstric-tion and increases blood pressure. In cardiac cells, the local RAS (Figure 78.1) maintains an appropriate cellu-lar remodeling as an adaptive response to myocardial stress. An increase in angiotensin II and angiotensin receptors 1 (AT1) in cardiac cells promotes hypertrophy, which may lead to congestive heart failure and unfore-seen cardiac death. In fact, augmented ACE and AT1 in the cardiac tissue as well as increased cardiomyocyte size were observed in the adult offspring of mice treated

with caffeine during pregnancy.9 In another study, also performed with pregnant CD-1 mice, a modest mater-nal caffeine intake (10 mg/kg/day administered daily to the pregnant mother from embryonic days 9.5 to 18.5, via subcutaneous injection) decreased uteroplacental blood flow and consequently reduced blood volume in the fetoplacental circulation. The embryonic cardio-vascular development was thus affected by maternal caffeine intake and blood flow distribution in the devel-oping embryo can be altered. An interesting observation was that maternal cardiovascular function and maternal weight gain was not affected.10 Also, maternal treatment with adenosine A2A receptor inhibitor mimicked the embryonic hemodynamic effects observed with caffeine. Furthermore, the expression of adenosine A2A receptor in the embryo decreased at embryonic day 11.5. Thus, the adverse effect of maternal caffeine exposure on the developing embryonic cardiovascular system is prob-ably mediated by adenosine A2A receptor inhibition.10

FIGURE 78.1 Local renin-angiotensin system (RAS) in cardiac cells. RAS is a group of enzymes/substrates with endocrine characteristics, first identified in the systemic circulation. However, the detection of those enzymes/substrates in different organs led to the concept of local RAS. The basic enzymatic reactions are common to all organs. The enzyme renin converts the substrate angiotensinogen (AOGEN) to the peptide angiotensin I (ANG I), which is then converted to the peptide angiotensin II (ANG II) by the angiotensin converting enzyme (ACE). Chymase is another enzyme identified in the heart, which also catalyzes the conversion of ANG I to ANG II. This enzyme is not inhibited by the ACE inhibi-tors and is highly specific for the substrate, being a relevant alternative pathway for ANG II generation. It is proposed that in cardiac cells, renin is taken up from circulation through its binding to specific receptors. ACE and chymase expressed in cardiac cells, chymase being more active than ACE. Regarding AOGEN, although its corresponding mRNA was detected in cardiac cells, it is supposed that the major source of this peptide is uptake from the plasma. The action of ANG II is mediated by two membrane receptors, the angiotensin receptor 1 and 2 (AT1R and AT2R), and the major function in local RAS in cardiac cells is to maintain an appropriate cellular remodeling as an adaptive response to myocardial stress. Myocyte hypertrophy is the major consequence observed in cardiac cells when AT1R is activated. Initially, hypertrophy works as a compensa-tory mechanism, preserving cardiac function, but when long-lasting, myocyte hypertrophy can lead to congestive heart failure. Maternal caffeine administration induces an increase in the expression of ACE and AT1R, suggesting that caffeine consumption during pregnancy leads to a persis-tent activation of local RAS in the heart, leading to an adverse cardiac remodeling in the adult offspring.

78.2 CAffEinE And THE HEART 701


Concluding, during pregnancy, daily consumption of caffeine should be minimized or eliminated in order to protect the development of the fetal cardiovascular sys-tem (Tables 78.1 and 78.2).

78.2.2 Caffeine Effect on the Cardiovascular System

Cardiovascular diseases are a major cause of death in developed countries. Lifestyle and diet contribute to those statistics. Among dietary factors, the amount of caffeine consumed may increase blood pressure and contribute to cardiovascular mortality or morbidity.11 Over the years, a large number of epidemiologic studies were performed in many countries, focusing on the rela-tionship between dietary coffee and caffeine intake and cardiovascular function.11,28 However, there are many inconsistencies in the results from these studies, con-tributing to the controversial problem of cardiovascular

toxicity of caffeine, as reported above.1,4 Most of these studies regarding coffee intake have not been able to answer whether caffeine alone or any of the other coffee components responsible for alterations associated with cardiovascular alterations. As discussed elsewhere, cof-fee is a mixture of several chemical compounds that can have a beneficial or deleterious impact for the cardio-vascular system.29 A case–control study demonstrated a significant relationship between coffee consumption and cardiovascular disease with an increased risk usu-ally observed for an intake of five or more cups of coffee during the day (more than 5500 mg caffeine/day). Sev-eral studies demonstrated a direct effect of caffeine on the cardiovascular system, translating into an increase in the incidence of cardiac arrhythmias, increased heart rate, serum cholesterol and homocysteine, as well as trig-gered hypertension (Table 78.1).12,24 Caffeine also alters the contractility of the musculature in the heart and blood vessels, interfering also with neurotransmission.13

TABLE 78.1 Summary of Main Effects of the Caffeine Cardiovascular Toxicity

Effect Amount Alterations References

Blood pressure >250 mg of caffeine(2–3 cups of coffee)

Increased systolic and diastolic blood pressure 11–20

Dose of 410 mg/day of caffeine

Tachyarrhythmias 1–5.0 mg caffeine/kg body weight (intravenous administration)

Increase atrial fibrillation, flutter, and arrhythmias 11,13,18,19,21–23

200 mg of caffeine Increased refractory periods of the right and left atrium, atrioventricular node, and right ventricle.

High quantities of caffeine Supraventricular tachycardia, atrial fibrillation, ventricular tachycardia, and ventricular fibrillation

Coronary heart disease and myocardium infarct

Five or more cups of coffee per day

Risk of 40–60% higher than people not consume coffee

12,14,15,17,20,24,25

At least 10 cups daily of coffee Increased risk of sudden cardiac arrest in patients with established coronary artery disease

Stroke At last 24 oz. coffee daily Risk of thromboembolic (ischemic) stroke 12,14,15,20

LDL High intake of boiled coffee and paper-filtered coffee

Increased LDL cholesterol 14,15

Homocysteine One liter of unfiltered coffee daily (for 2 weeks)

Increased plasma total Homocysteine concentrations by 10%

14,20,26

One liter of filtered coffee daily Increased plasma total Homocysteine concentrations by about 18%

870 mg caffeine per day Homocysteine-raising effect of coffee

Hypertension More than four cans of cola/day The risk was 20% higher lead to hypertension at least in women.

15

Aortic shift 200 mg caffeine (2 cups of coffee) Deteriorate aortic stiffness and wave reflections 27

Acute toxicity and overdose Oral doses of 5–50 g caffeine Fatalities in adults 12,20

Increased blood pressure, development of tachyarrhythmias, coronary heart diseases, myocardium infarct, stroke, hypertension, and aortic stiffness are some of the effects observed after high doses of caffeine consumption. Alterations in low-density lipoprotein (LDL) and homocysteine levels in blood serum are also observed after coffee consumption; however, in this situation, the effect may result not only from caffeine alone, but also from other components present in coffee. Acute toxicity and fatal overdose can be achieved after ingestion of 5–50 g of caffeine.



These effects can range from a moderate increase in heart rate to more severe cardiac arrhythmias, such as atrial fibrillation and ventricular tachycardia.13,31 However, we must take into consideration that some of these effects may be transient, because partial tolerance can be devel-oped after many days of caffeine consumption.26

The effects caused by coffee and caffeine consump-tion in the cardiovascular system depend on whether the population is naïve to coffee or it is comprised of regular consumers.32 Furthermore, the effects of caf-feine on the cardiovascular response depend on several other aspects, such as the amount ingested, the time of consumption, the frequency, degree of absorption, and hepatic metabolism, resulting in exclusive and different response in each individual,12,24,28 as discussed above.

78.2.2.1 Moderate and Severe Cardiac Effects of Caffeine

An elevated heart rate and elevated blood pressure are risk factors for cardiovascular diseases.33 The effects of caffeine on blood pressure differ in habitual or non-habitual caffeine drinkers, and this has been intensively investigated using different types of coffee (boiled, fil-tered, espresso, and decaffeinated).12 A positive associa-tion between coronary heart diseases and caffeine intake was found in several age-adjusted studies.14 Acute and repeated-dose clinical trial studies in healthy people or in people suffering from hypertension showed increased blood pressure by caffeine (higher than 250 mg), in the range of 5–15 mmHg systolic and/or 5–10 mmHg dia-stolic blood pressure, both in men and women of all ages, independently of race, blood pressure, or habits of caffeine intake. Repeated administrations of caffeine resulted in a pressor effect, although more intensively

in risk groups.12,13 Higher consumption of caffeine has also been associated with hypertension in women, which could be a risk factor for coronary heart disease, stroke, and congestive heart failure.15 Nurminen et al.16 showed a similar effect in blood pressure alteration. Two or three cups of coffee containing 200–300 mg of caffeine led to an increase of 3–14 mmHg in systolic blood pressure and an increase of 2–13 mmHg in diastolic blood pressure in normotensive subjects. This study also showed that the effect on blood pressure is stronger among people who do not consume coffee on a regular basis and the pres-sor effect of caffeine was smaller if ingested through cof-fee.16 Blood pressure is also increased after intravenous caffeine but alterations are not found after coffee intake in regular coffee drinkers, confirming that caffeine effects also depend on other variables.32 However, in different studies, where various levels of caffeine intake were used, no or little effect on heart rate and electrocardiographic variables were found in healthy people.17 Ingestion of two or three cups of coffee (300 mg caffeine) in non-habitual consumers did not alter the flow-mediated ves-sel dilation but increased endothelial-mediated response, which is related with vasodilation.32 Besides investigat-ing the effects of caffeine consumption, other studies focused on the consumption of decaffeinated coffee, tea, and non-paper-filtered coffee. However, no significant associations between coffee consumption and coronary heart diseases were observed.14 In studies involving the consumption of decaffeinated and regular coffee intakes, the results were similar between individuals who were not usual coffee consumers and individuals that were decaffeinated-coffee consumers. Thereby, this indicated that the blood pressure-raising effects are due to caffeine rather than other components of coffee,15 and involve vasoconstriction mechanisms that result from the adenos-ine antagonism activity, as well as from increased of stress hormones in plasma, such as epinephrine, norepineph-rine, and cortisol.11,15 The sympathetic nervous system is also activated by caffeine intake and can have an impor-tant role in the regulation of the cardiovascular system.15

Caffeine also has a small, but significant arrhyth-mogenic risk, although many studies in individuals who have cardiac disease or were disease-free have not always shown similar results.11 Caffeine overdose has been linked to coronary vasospasm, as well as to a variety of supraventricular and ventricular arrhyth-mias.18 Normally, cardiac tachyarrhythmias can be divided into arrhythmias of the ventricles, of the atria, and of the atrioventricular node.19 Caffeine toxicity by self-intended poisoning produces tachyarrhythmias, including supraventricular tachycardia, atrial fibrilla-tion, ventricular tachycardia, and ventricular fibrillation. The relationship between consumption of caffeine and supraventricular arrhythmias risk is not clear. Although some reports showed that human healthy individuals exposed to 1 mg caffeine/kg body weight (after 72 h of

TABLE 78.2 Safe Limits of Caffeine Consumption during different Periods of Age

Age GroupCaffeine Intake (mg/day)

Espresso Cups (≈44 ml)

Healthy adults 400–500 5–6

Pregnant women 300 3–4

Children (4–6 years) 45 ½

In this table, we show the safe limits of caffeine consumption for healthy adults, pregnant women, and children and the correspondent amount of coffee that can be consumed daily. It is evident that for pregnant women the limit for daily dose of caffeine consumption is reduced when compared with heath adults. This reduction is advised due to the adverse effects that caffeine causes in the fetus. Caffeine can easily cross the placenta and reach the fetal blood-stream. Since the mechanisms for caffeine clearance in the fetus are not yet developed, caffeine concentration in the fetus bloodstream can reach toxic con-centrations and adverse side effects can be observed. The amount of espresso cups was calculated using the standard value of 77 mg of caffeine/44 ml of espresso. Note that this value is only an average, since the amount of caffeine levels in coffee vary, depending on the type of coffee bean and the method of preparation. In fact, darker roasted and strong flavored coffees have lower levels of caffeine, since the roaster process reduces the amount of caffeine content in coffee beans.Adapted from Butt and Sultan.30



caffeine abstinence) did not exhibit supraventricular arrhythmias, other clinical studies showed that in healthy individuals or patients with ischemic heart disease or serious ventricular ectopia, single doses of caffeine (less than 450 mg) did not increase the frequency or severity of cardiac arrhythmia.12 Also, in a study performed with Wistar rats treated with different doses of caffeine (5, 15, and 45 mg/kg body weight) caffeine induced an increase in spontaneous activity, body temperature, heart rate, and systolic and diastolic blood pressure, although no electrocardiographic alterations or arrhythmias were observed.13 Administration of 25 mg/kg body weight of caffeine to Wistar rats did not eliminate the circadian rhythms of heart rate, body temperature and motor activ-ity but instead increased the MESORS (the average value around which the variables oscillates) and decreased the amplitude of all three circadian rhythms. An advance in the time at which the peak of temperature and motor activity occurred (acrophase of temperature and motor activity) was also observed after caffeine treatment.21

Risk of acute myocardial infarction in middle-aged men free from symptomatic coronary heart disease is increased by heavy consumption of caffeine-containing coffee (daily amounts exceeding 800 ml). Elevated con-centrations of caffeine may have adverse local effects in the ischemic myocardium, by blocking adenosine recep-tors (Ar), which will be discussed later in this chapter.25 Also, caffeine (200 mg) appears to have a synergistic det-rimental effect on aortic stiffness and wave reflections, although this can be related with other risk factors, such as acute smoking.27 Other clinical studies evaluating car-diovascular pathology endpoints, including death from myocardial infarction or coronary heart disease, non-fatal myocardial infarction or coronary event, angina pectoris, and hospitalization for coronary heart disease, showed contradictory results.12

High concentrations of homocysteine in blood appear to increase the risk of developing cardiovascular diseases, including coronary heart disease, stroke, and peripheral vascular disease.20 However, some data in the literature show that caffeine per se does not increase homocysteine in circulation, although coffee intake may cause this effect, suggesting that caffeine plus some other coffee compo-nents may be responsible for these effects.26 In fact, unfil-tered coffee intake (1 l/day) during 4 weeks by healthy people led to increase plasma homocysteine between 10% and 20%, and this effect seems to result from the indepen-dent contribution of both caffeine and coffee itself.32

78.2.3 Caffeine Effect on Cardiac Cells and Cardiac Cellular Organelles

Since caffeine is a modified purine, its action also affects enzymes that use adenosine as substrate, such as 3,5-cyclic nucleotide phosphodiesterase (cAMP PDE), inhibiting its activity through competitive antagonist

mechanism. Although believed to be needed in a much higher concentration, the direct inhibition of cellular phosphodiesterases by caffeine enables the cell to pro-ceed with normal degradation of cyclic adenosine mono-phosphate (cAMP) into adenosine monophosphate (AMP). Increased levels of cAMP modulate intracellular calcium levels and may potentiate heart pathologies. cAMP, through the activation of protein kinase A (PKA) signaling, acts on several sites, stimulating calcium release from intracellular stores or leading to calcium entry in the cell through the activation of calcium chan-nels. Calcium signaling in cardiac cells is fundamental in the control of cardiac contractility. Thus, regulation of calcium fluxes is important for the cardiac function. The sarcoplasmic reticulum (SR) is the major intracellular cal-cium reservoir in cardiac cells, sequestering and releasing calcium when cells are stimulated. Ryanodine receptors are the most important calcium release channels in the SR and are the primary pathway for calcium release dur-ing the excitation-contraction coupling phenomenon. The opening of those channels promotes calcium release from intracellular stores to the cytosol and the activa-tion of several calcium-dependent cellular pathways. Those receptors are controlled by several agonists and antagonists and also by calcium-induced calcium release (CICR). This event is a biological process, where small amounts of calcium in the cytosol, resulting from the activation of voltage dependent calcium channels in the sarcolemma (l-type calcium channels), activate ryano-dine receptors in the SR, promoting calcium release to the cytosol. Ryanodine, dantrolene, and ruthenium red are well-known antagonists of ryanodine receptors, inhibiting calcium release through those channels. Caf-feine, on the other hand, is a ryanodine receptor activa-tor, leading to full depletion of calcium stores, which can explain the increase in the strength and frequency of the heartbeat on caffeine consumption.34 Caffeine activa-tion site in ryanodine receptors is localized in the pore region, in the C-terminal of the protein.35 Upon caffeine binding, conformational changes in the clump region of ryanodine receptors are observed. These conformational changes depend on the ligand, suggesting the existence of multiple activation mechanisms associated with dif-ferent conformational changes.36 Although the average of the open time and the probability of channel opening are increased by caffeine, the conductance of the channel is not affected.35 Myocardial calcium overload is also a precipitating factor for cardiac injury.

Mitochondria can also be targets for caffeine. Altera-tions in mitochondrial function were already observed in the presence of caffeine. Our group had previously dem-onstrated that isolated male rat heart mitochondria, when incubated with supraphysiological concentrations of caf-feine, showed a reduced capacity to accumulate calcium due to increased susceptibility to the opening of the cal-cium-dependent mitochondrial permeability transition



(MPT) pore.37 The MPT phenomenon has been exten-sively reviewed elsewhere.38 Briefly, the MPT is a phe-nomenon characterized by a sudden loss of permeability of the inner mitochondrial membrane and is triggered by opening of pores in the inner mitochondrial membrane, the MPT pore. The MPT pore opening occurs under cer-tain conditions, including oxidative stress in the presence of high calcium loads. The amount of calcium necessary to induce the opening of the MPT pore depends on the species and tissues. Yet, the process can be accelerated by the presence of several compounds, such as caffeine. The uncontrolled opening of MPT pore can lead to the disrup-tion of mitochondrial function and in certain conditions to cell death.38 But, in a controlled situation, MPT pore induction is involved in physiological processes such as the removal of excess calcium from the mitochondrial matrix, or transient mitochondrial depolarization. Our group demonstrated a decreased capacity of rat heart mitochondria to buffer external calcium, resulting in structural alterations.37 In fact, as observed in Figure 78.2, the amount of calcium that isolated rat heart mitochon-dria are able to accumulate is reduced in the presence of caffeine, resulting in an early mitochondrial depolariza-tion. Increasing susceptibility to MPT pore opening by caffeine may cause a decrease in mitochondrial adenosine triphosphate (ATP) production, large amplitude swelling with concomitant mitochondrial structural damage, as well as cell death. Those events will ultimately contrib-ute to the heart failure normally associated with severe caffeine intoxication, as the cardiac muscle would suffer from a decreased supply of mitochondrial-derived ATP and a diminished mitochondrial capacity to help main-tain cytosolic calcium homeostasis.

The effects of caffeine on mitochondrial respiration were also investigated in our study (Figure 78.3). In succinate-energized rat heart mitochondria, caffeine increased stage 4 respiration, not only in the absence of oligomycin (V4), but also upon its addition (Voligo), suggesting an uncou-pler or decoupler action of caffeine on mitochondrial oxidative phosphorylation. However, as caffeine did not decrease the mitochondrial membrane potential, we pro-posed that a decoupler mechanism may be involved in the mechanism behind the stimulation of stage 4 respiration. By definition, decoupling in the respiratory chain implies a slippage of the proton pumps, meaning a different stoi-chiometry between protons pumped per electron trans-ferred in the electron transfer chain, resulting in higher oxygen consumption. Furthermore, stage 3 respiration (mitochondria respiration while producing ATP) was also inhibited by caffeine, explaining the significant reduction of the respiratory control ratio (RCR) values, a measure of the coupling between mitochondrial respiration and ATP production. Uncoupled respiration, triggered by the addition of the uncoupler carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP), was also inhibited by

caffeine in the range of tested concentrations (Figure 78.3), demonstrating also an inhibitory effect of caffeine directly in electron transfer in the respiratory chain when this sys-tem is maximally stimulated. Nevertheless, the adenosine diphosphate (ADP)/O (number of nanomoles ADP phos-phorylated by nanoatoms oxygen consumed) value was not significantly affected (Figure 78.4), demonstrating that, in the presence of caffeine, the efficiency of ATP produc-tion was not affected, although the rate of ATP synthesis was slightly decreased (Figure 78.4). A slight increase in the ADP phosphorylative lag phase in the presence of caffeine was also observed (Figure 78.4), indicating a slower rate in the ATP synthesis, confirming the decreased in stage 3 respiration. Thus, due to the importance of mitochondria

FIGURE 78.2 Evaluation of the mitochondrial transmembrane potential (ΔΨ) fluctuations associated with calcium uptake by heart mitochondria in presence and absence of caffeine. Mitochondrial ΔΨ was evaluated with a tetraphenylphosphonium (TPP+)-selective elec-trode, without corrections for TPP+ passive binding to mitochondrial membranes. A matrix volume of 1.1 μl/mg protein was assumed. The reactions were carried out at 25 °C, in 2 ml of standard media composed of 200 mM sucrose, 10 mM Tris-MOPS, 10 μM EGTA, 5 mM KH2PO4, supplemented with 3 μM TPP+ and 0.5 μg oligomycin. Mitochondria (1 mg) were energized with 8 mM of succinate (plus 4 μM rotenone to inhibit mitochondrial complex I and avoid back-flow of electrons in the respiratory chain, which can result in increased production of oxygen free radicals). After reaching the maximal ΔΨ, several calcium pulses of 50 nmol/mg mitochondrial protein were added to the mitochon-drial suspension. A decrease in ΔΨ was immediately observed due to the entry of positively charged calcium to the mitochondrial matrix through the mitochondrial calcium uniporter channel. Following cal-cium accumulation, ΔΨ repolarization was observed due to proton pumping in the mitochondrial respiratory chain, in order to recover the initial ΔΨ. The same response will be observed after each calcium pulse until a certain threshold is met, when the mitochondrial perme-ability transition occurs due to the formation of special pores (MPT pores) in the inner mitochondrial membrane, leading to complete ΔΨ depolarization. Curve B represents a control situation, in the absence of caffeine. Curves C and D represent respectively the experiment per-formed in the presence of 1 and 2 mM of caffeine after a pre-incubation period of 3 min. An experiment in the presence of 2 mM of caffeine and cyclosporin A (1 μM), an MPT pore inhibitor, was also performed in order to prove that the effect observed is due to MPT pore induction by the acute treatment with caffeine (Vilma Sardão, unpublished results).



FIGURE 78.4 Effects of caffeine on mitochondrial phosphorylative efficiency (A), the rate of adenosine triphosphate (ATP) synthesis (B) and time elapsed for complete adenosine diphosphate (ADP) phosphorylation (lag phase) (C). The effects of caffeine on phosphorylative efficiency was determined polarographically with a Clark-type oxygen electrode connected to a suitable recorder in a 1 ml thermostated, water-jacketed, closed chamber with magnetic stirring at 25 °C. Mitochondria (0.5 mg), isolated from hearts of Wistar-Han rats, were suspended in a reaction medium composed of 200 mM sucrose, 10 mM Tris, 10 mM EGTA, and 5 mM KH2PO4 and supplemented with 4 μM rotenone. The respira-tory V4 was calculated in the presence of 8 mM succinate. ATP synthesis was stimulated by the addition of 100 nmol of ADP. The ADP/O values, which is an endpoint for mitochondrial phosphorylative efficiency, was calculated as the number of nmol ADP phosphorylated per atom oxygen consumed during V3 respiration (mitochondrial oxygen consumption during ATP synthesis). During ATP synthesis, a decrease in the pH of the reaction media is observed. Thus, the rate of ATP synthesis was also evaluated by measuring the pH variation in the reaction medium with an ultrasensitive pH electrode. Mitochondria (0.5 mg), isolated from the hearts of Wistar-Han rats, were suspended in a reaction medium comprising 200 mM sucrose, 10 μM EGTA and 5 mM KH2PO4, supplemented with 3 μM rotenone. The reaction was initiated by the addition of 300 nmol of ADP to the mitochondrial suspension, energized with 8 mM succinate. The time elapsed during complete ADP phosphorylation (lag phase) was also evaluated with a tetraphenylphosphonium (TPP+)—selective electrode. The reactions were carried out at 25 °C, in 2 ml of standard media composed by 200 mM sucrose, 10 mM Tris-MOPS, 10 μM EGTA, 5 mM KH2PO4, supplemented 3 μM TPP+, 4 μM of rotenone, and 8 mM succinate. The reaction was initiated with the addition of 100 nmol ADP. Tests carried out in the presence of caffeine (CAF, 2 mM) started after a period of 3 min of pre-incubation with the compound. Values represent the average ±SEM of four different preparations. Statistical analysis was performed using the unpaired Student’s t-test. *p < 0.05 versus control (Vilma Sardão, unpublished results).

FIGURE 78.3 Effect of caffeine on mitochondrial respiratory parameters. Oxygen consumption of isolated heart mitochondria was moni-tored polarographically with an electrode of the Clark-type oxygen electrode connected to a suitable recorder in a 1 ml thermostated, water-jacketed, closed chamber with magnetic stirring at 25 °C. Mitochondria (0.5 mg), isolated from the hearts of Wistar-Han rats, were suspended in a reaction medium composed of 200 mM sucrose, 10 mM Tris, 10 mM EGTA, and 5 mM KH2PO4 and supplemented with 4 μM rotenone. The respira-tory stage 4 was calculated in the presence of 8 mM succinate. Sequential additions of adenosine diphosphate (100 nmol), oligomycin (2 μg), and the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (1 μM) were performed to induce, respectively, stage 3 respiration (V3), stage 4 respiration (V4) without the contribution of passive proton conductance through the adenosine triphosphate synthase (Voligo), and uncoupled respiration (VFCCP). The respiratory control ratio (RCR) was calculated as the ratio between V3 and V4 respiration. The variations on oxygen consumption after oligomycin (Voligo), FCCP addition (VFCCP), and the ratio Voligo/VFCCP are also presented. The calculations were made assuming an oxygen concentration of 258 μM. Caffeine (CAF, 2 mM) was pre-incubated with mitochondria in the reaction medium for 3 min. The values represent the average ±SEM and were calculated as percentage of the control (absence of caffeine). Statistical analysis was performed using the unpaired t-test. *p < 0.05 versus control; **p < 0.01 versus control, ***p < 0.005 versus control. The figure is based on previously published data.37



in cardiomyocytes, disruptive effects of caffeine on mito-chondrial function must be taken into account. Interest-ingly, the activity of cytochrome c oxidase, the final protein in the mitochondrial respiratory chain was already shown to be increased through caffeine effects on cAMP and PKA activity.39 Nevertheless, this study used lower concentra-tions than the ones used in our study.

A number of findings demonstrated that acute admin-istration of caffeine inhibits ischemic preconditioning,40 potentiating deleterious effects in the heart, similarly to the effect of an equivalent dose of 2–4 cups of coffee. Ischemic preconditioning is a physiological protective response of myocardium to ischemic injury, initially suggested by Murry and coworkers in 1986.41 Several in vivo and in vitro studies demonstrated that, during brief periods of sublethal ischemia followed by periods of reperfusion, protective mechanisms are activated in cardiac cells, adapting the myocardium to ischemic epi-sodes and increasing the threshold to ischemic injury.42 The mechanisms involved in this phenomenon have been already reviewed.42 Production of endogenous autacoids, such as adenosine, opioids, or bradykinin, and their binding to respective receptors promote the activation of important pathways involved in cardiac cell protection against ischemic injury. Activation of Ar, with consequent activation of protein kinase C and ATP-sensitive potassium channels (KATP channels) in sarco-lemma and mitochondria are suggested to be involved

in ischemic preconditioning. Thus, the blockage of Ar by caffeine is thought to inhibit this mechanism and the protection afforded by ischemic preconditioning.

The toxicity of caffeine on cardiac cells is a topic that needs to be further explored. Although we reviewed here several potential cellular targets for caffeine action, several others need to be investigated. A summary of the main targets of caffeine on cardiac cells as discussed above is shown in Figure 78.5.

78.3 SUMMARY POINTS

• The response and tolerance to caffeine are individual and depend on pharmacokinetics and pharmacodynamics.

• Genetic variability in caffeine-metabolizing enzymes affects the susceptibility of each individual to caffeine toxicity.

• Higher exposure to caffeine during pregnancy can be harmful for the development of the fetal cardiovascular system. Thus, daily consumption of caffeine should be maintained reduced or avoided.

• Increased blood pressure, atrial and ventricular fibrillation, arrhythmias, tachycardia, coronary heart disease, and risk of thromboembolic (ischemic) stroke are some of the main effects of caffeine cardiovascular toxicity.

FIGURE 78.5 Caffeine main targets on cardiac cells. By blocking G protein-coupled adenosine receptors (Ar), caffeine is able to mediate the opposite effects to the sedative compound adenosine, producing a stimulatory effect. The activation of adenylate cyclase (AC) that converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), a second messenger that has an important role mediating several cellular responses, increases cAMP intracellular levels. Caffeine action on Ar increases cAMP concentration, which activates cAMP-dependent protein kinase A (PKA) signaling. The phosphorylation by PKA of multiple calcium channels in the plasmatic membrane and in the sarcoplasmic reticulum (SR) increases the concentration of cytosolic calcium, thus stimulating cardiac contraction and, at the organ level, increasing heartbeat. Intracellularly, caffeine has also the capacity to bind to ryanodine receptors (RyR) in the SR, inducing the release of calcium. Caffeine also inhib-its the enzyme that degrades cAMP into 5′AMP, cyclic nucleotide phosphodiesterase (PDE). The consequent calcium release to the cytoplasm through RyR and cAMP accumulation promoted by adenosine receptor stimulation and PDE inhibition, are thought to be the major mechanisms responsible for the caffeine sympathomimetic effect. Abbreviations: PLC; Phospholipase C, NCX; sodium/calcium exchanger, SERCA; sarco/endoplasmic reticulum calcium-ATPase, LTCC; low threshold calcium channels.

REfEREnCES 707


• Both SR and mitochondria can be main intracellular targets for caffeine effects on cardiac cells.

• Cardiovascular toxicity of caffeine is still a controversial topic that needs to be further explored in the context of how host specificity alters the responses to caffeine.

References 1. Mostofsky E, Rice MS, Levitan EB, Mittleman MA. Habitual cof-

fee consumption and risk of heart failure: a dose-response meta-analysis. Circ Heart Fail 2012;5(4):401–5.

2. Einother SJ, Giesbrecht T. Caffeine as an attention enhancer: reviewing existing assumptions. Psychopharmacology (Berl) 2013;225(2):251–74.

3. Kim B, Nam Y, Kim J, Choi H, Won C. Coffee consumption and stroke risk: a meta-analysis of epidemiologic studies. Korean J Fam Med 2012;33(6):356–65.

4. Di Rocco JR, During A, Morelli PJ, Heyden M, Biancaniello TA. Atrial fibrillation in healthy adolescents after highly caffein-ated beverage consumption: two case reports. J Med Case Rep 2011;5:18.

5. McGee MB. Caffeine poisoning in a 19-year-old female. J Forensic Sci 1980;25(1):29–32.

6. Golding J. Reproduction and caffeine consumption–a literature re-view. Early Hum Dev 1995;43(1):1–14.

7. Brent RL, Christian MS, Diener RM. Evaluation of the reproductive and developmental risks of caffeine. Birth Defects Res B Dev Reprod Toxicol 2011;92(2):152–87.

8. Oei SG, Vosters RP, van der Hagen NL. Fetal arrhythmia caused by excessive intake of caffeine by pregnant women. BMJ 1989;298(6673):568.

9. Serapiao-Moraes DF, Souza-Mello V, Aguila MB, Mandarim-de-Lacerda CA, Faria TS. Maternal caffeine administration leads to adverse effects on adult mice offspring. Eur J Nutr 2013;52(8): 1891–900.

10. Momoi N, Tinney JP, Liu LJ, Elshershari H, Hoffmann PJ, Ralphe JC, et al. Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth. Am J Physiol Heart Circ Physiol 2008;294(5):H2248–56.

11. James JE. Critical review of dietary caffeine and blood pressure: a relationship that should be taken more seriously. Psychosom Med 2004;66(1):63–71.

12. Nawrot P, Jordan S, Eastwood J, Rotstein J, Hugenholtz A, Feeley M. Effects of caffeine on human health. Food Addit Contam 2003;20(1):1–30.

13. Ilback NG, Siller M, Stalhandske T. Evaluation of cardiovascular effects of caffeine using telemetric monitoring in the conscious rat. Food Chem Toxicol 2007;45(5):834–42.

14. van Dam RM. Coffee consumption and coronary heart disease: paradoxical effects on biological risk factors versus disease inci-dence. Clin Chem 2008;54(9):1418–20.

15. Bonita JS, Mandarano M, Shuta D, Vinson J. Coffee and cardiovas-cular disease: in vitro, cellular, animal, and human studies. Pharma-col Res 2007;55(3):187–98.

16. Nurminen ML, Niittynen L, Korpela R, Vapaatalo H. Coffee, caffeine and blood pressure: a critical review. Eur J Clin Nutr 1999;53(11):831–9.

17. Ammar R, Song JC, Kluger J, White CM. Evaluation of electrocar-diographic and hemodynamic effects of caffeine with acute dosing in healthy volunteers. Pharmacotherapy 2001;21(4):437–42.

18. Pelchovitz DJ, Goldberger JJ. Caffeine and cardiac arrhythmias: a review of the evidence. Am J Med 2011;124(4):284–9.

19. Katan MB, Schouten E. Caffeine and arrhythmia. Am J Clin Nutr 2005;81(3):539–40.

20. Higdon JV, Frei B. Coffee and health: a review of recent human research. Crit Rev Food Sci Nutr 2006;46(2):101–23.

21. Pelissier AL, Gantenbein M, Bruguerolle B. Caffeine-induced mod-ifications of heart rate, temperature, and motor activity circadian rhythms in rats. Physiol Behav 1999;67(1):81–8.

22. Mattioli AV, Farinetti A, Miloro C, Pedrazzi P, Mattioli G. In-fluence of coffee and caffeine consumption on atrial fibrillation in hypertensive patients. Nutr Metab Cardiovasc Dis 2011;21(6): 412–7.

23. Frost L, Vestergaard P. Caffeine and risk of atrial fibrillation or flutter: the Danish Diet, Cancer, and Health Study. Am J Clin Nutr 2005;81(3):578–82.

24. Baylin A, Hernandez-Diaz S, Kabagambe EK, Siles X, Campos H. Transient exposure to coffee as a trigger of a first nonfatal myocar-dial infarction. Epidemiology 2006;17(5):506–11.

25. Happonen P, Voutilainen S, Salonen JT. Coffee drinking is dose-dependently related to the risk of acute coronary events in middle-aged men. J Nutr 2004;134(9):2381–6.

26. Verhoef P, Pasman WJ, Van Vliet T, Urgert R, Katan MB. Con-tribution of caffeine to the homocysteine-raising effect of cof-fee: a randomized controlled trial in humans. Am J Clin Nutr 2002;76(6):1244–8.

27. Vlachopoulos C, Kosmopoulou F, Panagiotakos D, Ioakeimidis N, Alexopoulos N, Pitsavos C, et al. Smoking and caffeine have a syn-ergistic detrimental effect on aortic stiffness and wave reflections. J Am Coll Cardiol 2004;44(9):1911–7.

28. Echeverri D, Montes FR, Cabrera M, Galan A, Prieto A. Caffeine’s vascular mechanisms of action. Int J Vasc Med 2010;2010:834060.

29. Cornelis MC, El-Sohemy A. Coffee, caffeine, and coronary heart disease. Curr Opin Clin Nutr Metab Care 2007;10(6):745–51.

30. Butt MS, Sultan MT. Coffee and its consumption: benefits and risks. Crit Rev Food Sci Nutr 2011;51(4):363–73.

31. Donnerstein RL, Zhu D, Samson R, Bender AM, Goldberg SJ. Acute effects of caffeine ingestion on signal-averaged electrocardiograms. Am Heart J 1998;136(4 Pt 1):643–6.

32. Cano-Marquina A, Tarin JJ, Cano A. The impact of coffee on health. Maturitas 2013;75(1):7–21.

33. Parati G, Ochoa JE, Lombardi C, Salvi P, Bilo G. Assessment and interpretation of blood pressure variability in a clinical setting. Blood Press; 2013.

34. Kong H, Jones PP, Koop A, Zhang L, Duff HJ, Chen SR. Caf-feine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor. Biochem J 2008;414(3): 441–52.

35. Guerreiro S, Marien M, Michel PP. Methylxanthines and ryano-dine receptor channels. Handb Exp Pharmacol 2011;200:135–50.

36. Tian X, Liu Y, Wang R, Wagenknecht T, Liu Z, Chen SR. Ligand-dependent conformational changes in the clamp region of the car-diac ryanodine receptor. J Biol Chem 2012;288(6):4066–75.

37. Sardao VA, Oliveira PJ, Moreno AJ. Caffeine enhances the calcium-dependent cardiac mitochondrial permeability tran-sition: relevance for caffeine toxicity. Toxicol Appl Pharmacol 2002;179(1):50–6.

38. Siemen D, Ziemer M. What is the nature of the mitochondrial permeability transition pore and what is it not? IUBMB Life 2013;65(3):255–62.

39. Verma R, Huang Z, Deutschman CS, Levy RJ. Caffeine restores myocardial cytochrome oxidase activity and improves cardiac function during sepsis. Crit Care Med 2009;37(4):1397–402.

40. Riksen NP, Zhou Z, Oyen WJ, Jaspers R, Ramakers BP, Brouwer RM, et al. Caffeine prevents protection in two human models of ischemic preconditioning. J Am Coll Cardiol 2006;48(4):700–7.

41. Murry CE, Jennings RB, Reimer KA. Preconditioning with isch-emia: a delay of lethal cell injury in ischemic myocardium. Circula-tion 1986;74(5):1124–36.

42. Minamino T. Cardioprotection from ischemia/reperfusion injury: basic and translational research. Circ J 2012;76(5):1074–82.

http://refhub.elsevier.com/B978-0-12-409517-5.00078-4/ref0010



























































































































Documents

Caffeine Cardiovascular Toxicity: Too Much of a Good Thing