Fares A. Karamat

Resistance Artery Creatine Kinase mRNA and Blood Pressure in Humans

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6–1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature’s pressor responses.

The Effect of Creatine Kinase Inhibition on Contractile Properties of Human Resistance Arteries.

BACKGROUND Creatine kinase (CK) is a main predictor of blood pressure, and this is thought to largely depend on high resistance artery contractility. We previously reported an association between vascular contractility and CK in normotensive pregnancy, but pregnancy is a strong CK inducer, and data on human hypertension are lacking. Therefore, we further explored CK-dependency of vascular contractility outside the context of pregnancy in normotensive and hypertensive women. METHODS AND RESULTS Nineteen consecutive women, mean age 42 years (SE 1.3), mean systolic/diastolic blood pressure respectively 142.6 (SE 5.9)/85.6 (3.4) mm Hg (9 hypertensive), donated an omental fat sample during abdominal surgery. We compared vasodilation after the specific CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB; 10(-6) mol/l) to sodium nitroprusside (10(-6) mol/l) in isolated resistance arteries using a wire myograph. Additionally, we assessed predictors of vasoconstrictive force. DNFB reduced vascular contractility to 24.3% (SE 4.4), P < 0.001, compared to baseline. Sodium nitroprusside reduced contractility to 89.8% (SE 2.3). Maximum contractile force correlated with DNFB effect as a measure of CK (r = 0.8), and with vessel diameter (r = 0.7). The increase in contractile force was 16.5 mN [9.1-23.9] per unit DNFB effect in univariable and 10.35 mN [2.10-18.60] in multivariable regression analysis. CONCLUSION This study extends on our previous findings in pregnant normotensive women of CK-dependent microvascular contractility, indicating that CK contributes significantly to resistance artery contractility across human normotension and primary hypertension outside the context of pregnancy. Further studies should explore the effect of CK inhibitors on clinical blood pressure.

Creatine kinase inhibition lowers systemic arterial blood pressure in spontaneously hypertensive rats: a randomized controlled trial

Objective: Creatine kinase is reported to be a main predictor of blood pressure (BP) in the general population, with a strong correlation between resistance artery creatine kinase expression and clinical BP in humans. The enzyme rapidly regenerates ATP near cytoplasmic ATPases involved in pressor responses, including resistance artery contractility and renal sodium retention. Therefore, we assessed whether creatine kinase inhibition reduces BP. Methods: We implemented the ‘Animal Research: Reporting of In Vivo Experiments’ guideline. In a 4-week randomized controlled trial, male 16-week-old spontaneously hypertensive rats (N = 16) were randomly assigned to the specific competitive creatine kinase inhibitor beta-guanidinopropionic acid (3%)-supplemented chow vs. standard chow. BP measured by the tail-cuff method was the main outcome. Other outcomes included vasodilation in isolated arteries and renal renin expression. Results: Creatine kinase inhibition reduced BP safely and reversibly. Mean baseline BP of, respectively, 191.5 (standard error 4.3) mmHg SBP and 143.1 (4.1) mmHg DBP was reduced by, respectively, 42.7 (5.5) mmHg SBP and 35.6 (5.0) mmHg DBP (P < 0.001) compared with controls, with evidence of enhanced vasodilation and a diuretic effect. Conclusion: To our knowledge, this is the first report on the BP-lowering effect of creatine kinase inhibition. Our data indicate that modulation of the creatine kinase system is a potential novel treatment target for hypertension.

The acute effect of beta‐guanidinopropionic acid versus creatine or placebo in healthy men (ABC‐Trial): A randomized controlled first‐in‐human trial

Aims Increasing evidence indicates that the ATP‐generating enzyme creatine kinase (CK) is involved in hypertension. CK rapidly regenerates ATP from creatine phosphate and ADP. Recently, it has been shown that beta‐guanidinopropionic acid (GPA), a kidney‐synthesized creatine analogue and competitive CK inhibitor, reduced blood pressure in spontaneously hypertensive rats. To further develop the substance as a potential blood pressure‐lowering agent, we assessed the tolerability of a sub‐therapeutic GPA dose in healthy men. Methods In this active and placebo‐controlled, triple‐blind, single‐centre trial, we recruited 24 healthy men (18–50 years old, BMI 18.5–29.9 kg m−2) in the Netherlands. Participants were randomized (1:1:1) to one week daily oral administration of GPA 100 mg, creatine 5 g, or matching placebo. The primary outcome was the tolerability of GPA, in an intent‐to‐treat analysis. Results Twenty‐four randomized participants received the allocated intervention and 23 completed the study. One participant in the placebo arm dropped out for personal reasons. GPA was well tolerated, without serious or severe adverse events. No abnormalities were reported with GPA use in clinical safety parameters, including physical examination, laboratory studies, or 12‐Lead ECG. At day 8, mean plasma GPA was 213.88 (SE 0.07) in the GPA arm vs. 32.75 (0.00) nmol l−1 in the placebo arm, a mean difference of 181.13 (95% CI 26.53–335.72). Conclusion In this first‐in‐human trial, low‐dose GPA was safe and well‐tolerated when used during 1 week in healthy men. Subsequent studies should focus on human pharmacokinetic and pharmacodynamic assessments with different doses.

Creatine synthesis demands the majority of the bioavailable L-arginine

I n a recent article, Li et al. [1] reviewed the potential mechanisms of reduced nitric oxide bioavailability in hypertension, including a defective L-arginine/nitric oxide pathway. The authors correctly cite earlier work indicating that a reduced bioavailability of L-arginine or circulating asymmetric dimethylarginine acting as a competitive inhibitor of endothelial nitric oxide synthase (eNOS) may attenuate nitric oxide synthesis. However, in their review, the authors decided not to discuss the creatine kinase system, an important enzyme system that may reduce L-arginine bioavailability for the synthesis of nitric oxide [2–5]. We would like to take this opportunity to submit some basic facts concerning the relation between creatine kinase and nitric oxide. Creatine kinase is the main regulatory enzyme of energy metabolism. The enzyme catalyzes the rapid and reversible conversion of ADP into ATP, using creatine and creatine phosphate. At the mitochondrial site, the enzyme facilitates transport of ATP generated by oxidative phosphorylation into the cytoplasm as creatine phosphate. At cytoplasmic subcellular sites of high-energy demands, creatine kinase is bound near ATPases including Naþ/Kþ-ATPase, Ca2þATPase, and myosin ATPase, to rapidly provide ATP for energy-demanding cellular functions including ion transport and contractile responses [2,4,5]. Thus, the enzyme is reported to be involved in energy-demanding conditions including hypertension [4,5]. Creatine synthesis utilizes the major part of available L-arginine [2–5]. L-arginine:glycine amidino transferase in the kidney catalyzes the transfer of the amino group from L-arginine to glycine to yield ornithine and guanidinoacetate [2,3]. Subsequently, liver S-adenosyl-L-methionine: N-guanidinoacetate methyltransferase converts guanidinoacetate into creatine [2,3]. Hence, creatine and nitric oxide are both synthesized from L-arginine, but creatine synthesis demands nearly 10 times the flux of plasma L-arginine represented by nitric oxide synthesis [3–5]. Moreover, L-arginine is reported to induce L-arginine:glycine amidino transferase and increase the flux through the creatine pathway [3]. Therefore, creatine synthesis is thought to reduce the bioavailability of L-arginine for nitric oxide synthesis [2–5]. This might be more apparent in hypertensive patients, obese patients, or patients of African ancestry, as these population subgroups display a greater activity of the creatine kinase system, which induces creatine synthesis [2,4,5]. Despite intracellular L-arginine concentrations that should saturate eNOS, the rate of nitric oxide synthesis is limited by the rate of endothelial L-arginine uptake [1,3–5]. In addition, in the presence of asymmetric dimethylarginine, higher L-arginine

Hypertension and Cardiovascular Risk Profile in a Middle-Income Setting: The HELISUR Study

BACKGROUND Hypertension is the leading risk factor responsible for premature death worldwide, but its burden has shifted to low- and middle-income countries. Therefore, we studied hypertension and cardiovascular risk in the population of Suriname, a middle-income country with a predominantly urban population of African and Asian ancestry. METHODS A random sample of 1,800 noninstitutionalized men and women aged 18-70 years was selected to be interviewed at home and examined at the local hospital for cardiovascular risk factors, asymptomatic organ damage, and cardiovascular disease. RESULTS The 1,157 participants examined (37% men) were mainly of self-defined Asian (43%) or African (39%) ancestry, mean age 43 years (SD 14). The majority of the population (71%) had hypertension or prehypertension, respectively, 40% and 31%. Furthermore, 72% was obese or overweight, while 63% had diabetes or prediabetes. Only 1% of the adult population had an optimal cardiovascular risk profile. Hypertension awareness, treatment, and control were respectively 68%, 56%, and 20%. In line with this, 22% of the adult population had asymptomatic organ damage, including increased arterial stiffness, left ventricular hypertrophy, microalbuminuria, or asymptomatic chronic kidney disease. CONCLUSIONS In this first extensive cardiovascular assessment in the general population of this middle-income Caribbean country, high prevalence of hypertension with inadequate levels of treatment and control was predominant. The findings emphasize the need for collaborative effort from national and international bodies to prioritize the implementation of affordable and sustainable public health programs that combat the escalating hypertension and cardiovascular risk factor burden.

Creatine kinase and renal sodium excretion in African and European men on a high sodium diet

Creatine kinase (CK) rapidly regenerates ATP for Na+/K+‐ATPase driven sodium retention throughout the kidney. Therefore, we assessed whether resting plasma CK is associated with sodium retention after a high sodium diet. Sixty healthy men (29 European and 31 African ancestry) with a mean age of 37.2 years (SE 1.2) were assigned to low sodium intake (< 50 mmol/d) during 7 days, followed by 3 days of high sodium intake (> 200 mmol/d). Sodium excretion (mmol/24‐h) after high sodium was 260.4 (28.3) in the high CK tertile versus 415.2 (26.3) mmol/24‐h in the low CK tertile (P < .001), with a decrease in urinary sodium excretion of 98.4 mmol/24‐h for each increase in log CK, adjusted for age and African ancestry. These preliminary results are in line with the energy buffering function of the CK system, but more direct assessments of kidney CK will be needed to further establish whether this enzyme enhances sodium sensitivity.

Is Creatine Kinase the Intrinsic Factor of Smooth Muscle Enhancing Vascular Contractility in Subjects of African Ancestry

To the Editor: In a recent article, Adefurin et al1 presented interesting results on ethnic differences in venous smooth muscle contractility in response to α-receptor agonists. Healthy subjects of African ancestry showed greater vasoconstriction to phenylephrine than subjects of European ancestry, with a geometric mean ED50 that was 45% lower in the African group, 172 versus 310 ng/min. Notably, the effect was more pronounced in men than in women. The authors concluded that their study did not shed light on …

[OP.LB03.09] THE ACUTE EFFECT OF THE SPECIFIC CREATINE KINASE INHIBITOR BETA- GPA IN HEALTHY MAN (ABC-TRIAL): A RANDOMIZED PLACEBO AND ACTIVE CONTROLLED FIRST-IN-HUMAN TRIAL

Objective: There is increasing evidence that the ATP generating enzyme creatine kinase (CK) is involved in hypertension. The enzyme is central to the regeneration of ATP by using creatine phosphate and ADP, thereby forming creatine and ATP. We recently showed that beta-guanidinopropionic acid (GPA), a creatine analogue and competitive CK inhibitor, effectively and safely reduced blood pressure in the spontaneously hypertensive rat. This renders GPA to be potentially beneficial for blood pressure lowering. However, no human data are available. Therefore, according to FDA and EMEA guidelines, we assessed the tolerability of a subtherapeutic GPA dose in man. Figure. No caption available. Design and method: In this randomized, placebo and active controlled, triple blind, single center trial (randomization 1:1:1), we included healthy male volunteers, 18 to 50 years old, BMI 18.5–29.9 kg/m2, recruited in the Netherlands. The interventions consisted of one week daily oral administration of GPA in a subtherapeutic dose of 100 mg, creatine 5 gram, or placebo. The primary outcome was the tolerability of GPA as a descriptive measure, in an intent-to-treat analysis. Results: Twenty four randomized participants received the allocated intervention (8 in each treatment arm) and 23 completed the study. One participant in the placebo arm dropped out because of an external event in his family. He experienced no side effects, including not during a re-challenge with the assigned drug. GPA was well tolerated. There were no serious or severe adverse events with GPA, creatine and placebo after 1 week of active treatment, and no significant differences in safety measures including self-reported data obtained with structured and unstructured questionnaires. No abnormalities were reported from physical examination, laboratory determined toxicity including kidney and liver parameters, or cardiovascular safety including QT interval between treatment arms (Table 1). Conclusions: To our knowledge, these are the first human data of the specific CK inhibitor and potential new blood pressure lowering agent GPA. We found no evidence of toxicity with subtherapeutic doses, and will proceed to assess safety and tolerance in a dose-escalation trial in humans. Clinical trial registration number The Netherlands National Trial Register (NTR) number 4444, registered March 9, 2014.

LBOS 03-02 PREHYPERTENSION AND HYPERTENSION IN URBAN SURINAME: THE HELISUR STUDY.

Objective: The global burden of disease attributable to high blood pressure showed that 54% of stroke and 47% of ishaemic heart disease worldwide were due to hypertension. Hypertension is still the main risk factor for premature death worldwide and, in particular, in low- and middle-income countries. We aimed to assess prehypertension and hypertension prevalence among a predominantly Asian and African population living in urban Suriname, a middle-income country in South America. Design and Method: We used data from the Healthy Life in Suriname study, in which 1,152 men and women aged 18–70 y living in the capital were randomly selected. Demographic factors, disease history, and use of medication were recorded. Ehtnicity was self-defined. Sitting blood pressure was measured in duplo after a 5-minute rest. The prevalence of prehypertension and hypertension were determined according to international guidelines (JNC 7). Differences between sex and ethnic groups were tested using &khgr;2 tests and logistic regression analyses. Results: Participants with missing blood pressures (n = 4) and of other/mixed ethnicity (n = 199) were excluded. Of the remaining 949 participants (64% women; mean age 43 ± 13 y), 494 subjects were Asian-Surinamese (52%) and 455 were African-Surinamese (48%). The overall prevalence of prehypertension and hypertension was, respectively, 31 and 41% (Figure 1A). Regarding ethnic subgroups analysis, the crude prevalence of prehypertension and hypertension did not differ between Asian and African-Surinamese (p = 0.79 and p = 0.13, respectively). In logistic regression analysis, African-Surinamese showed higher sex-age odds ratios of having hypertension. However, after including BMI in the model, this difference was not statistically significant (Figure 1B). Conclusions: We found a high prevalence of prehypertension (31%) and hypertension (41%) in this urban middle-income population, with no differences between Asian and African-Surinamese. With only 29% having optimal blood pressure levels.Therefore, drastic preventive measures prioritizing screening and control of hypertension need to be taken for cardiovascular health in Surinamese population.