2024 Volume 47 Issue 7 Pages 1360-1367
The current study aimed to investigate the anti-atrial fibrillatory (AF) effects of a combination of valsartan and a calcium channel blocker (cilnidipine or amlodipine) in Dahl salt-sensitive (Dahl S) rats. Seven-week-old male Dahl S rats were fed an 8% salt diet. Six weeks later, valsartan (60 mg/kg, Val group), cilnidipine + valsartan (10 + 60 mg/kg, CV group), amlodipine + valsartan (3 + 60 mg/kg, AV group), or vehicle was orally administered daily for 5 weeks. Echocardiography and atrial electrophysiological evaluations were performed on the last day of treatment. Blood pressure in each drug treatment group was lower than in the Vehicle group. The duration of AF induced by atrial burst stimulation was shorter in the Val group (3.2 ± 1.6 s) than in the Vehicle group (11.2 ± 6.0 s), which was further shortened in the CV and AV groups (1.1 ± 0.3 and 1.3 ± 0.3 s, respectively). Left ventricular ejection fraction and left ventricular fractional shortening were greater in the CV and AV groups than those in the Vehicle group. Urinary albumin excretion in the CV group was the lowest among the drug-treated groups. The results collectively suggest that the combination of a calcium channel blocker with valsartan could be useful in terms of its anti-AF action as well as for improving cardiac and renal functions.
Patients with hypertension have a 1.7-fold higher risk of developing atrial fibrillation (AF) than normotensive individuals,1) and hypertension is common in patients with AF. Risk factors for the occurrence of AF include left ventricular (LV) hypertrophy, kidney dysfunction, and cardiovascular and cerebrovascular disorders, which are common consequences of hypertension.2) Therefore, adequate management of hypertension using angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) is recommended as upstream therapies for preventing AF, per clinical guidelines in Europe, as they might modify the atrial substrate, prevent inflammation, and reduce the risk of AF.3) Based on the J-RHIYTHM II trial, the L-type Ca2+ channel blocker (CCB) amlodipine has been shown to exert similar efficacy in reducing the frequency of paroxysmal AF as candesartan.4) According to the Japanese Society of Hypertension Guidelines for the Management of Hypertension (JSH2019), two or three drugs should be combined when poorly controlled blood pressure as combination therapy with different classes of antihypertensives exhibits effective therapeutic actions to achieve the desired blood pressure level. However, information regarding the effectiveness of combination antihypertensive therapy for AF is still limited.
Our previous study demonstrated that chronic administration of the L/N-type CCB cilnidipine effectively suppressed atrial fibrosis and persistent AF in a Dahl salt-loaded rat model of AF.5) Cilnidipine has been widely used for patients with hypertension in Japan as well as India and is known as a unique antihypertensive drug with an anti-sympathetic action through N-type Ca2+ channel blockade at the sympathetic nerve endings.6–9) Using the same animal model, in this study, we aimed to investigate the effect of combination therapy with the typical ARB valsartan plus cilnidipine or amlodipine on AF occurrence. We further analyzed the relationship between the anti-AF properties and cardiorenal protective effects of the combination drug.
The animal experiment was approved by the Institutional Animal Care and Use Committee of the Pharmaceutical Research Laboratories of Ajinomoto Pharmaceuticals Co., Ltd. Five-week-old male Dahl salt-sensitive (Dahl S) rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). Tap water was provided ad libitum throughout the experiment, and standard laboratory chow (CRF-1, Charles River, Kanagawa, Japan) was fed until 6 weeks of age.
MaterialsCilnidipine (Ajinomoto Co., Inc., Tokyo, Japan), amlodipine (Lek Co., Inc., Kolodvorska, Slovenia), and valsartan (Livzon Group Changzhou Kony Pharmaceutical Co., Inc., Changzhou, China) were obtained and suspended in 0.5% hydroxypropyl methylcellulose (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) immediately before daily oral administration to animals.
Experimental ProcedureThe Dahl S rats (n = 44) were fed standard laboratory chow till seven weeks of age. Afterward, the rats were fed a high-salt diet (F2Dahl-8.0 containing 8% NaCl; Oriental Yeast Co., Ltd., Tokyo, Japan) till the end of the experiment (18 weeks of age). Six weeks after starting the high-salt diet, the animals were divided into four groups by stratified randomization according to their body weight and blood pressure. Every morning from 13 to 18 weeks of age, valsartan (60 mg/kg per day, Val group, n = 11), cilnidipine + valsartan (10 mg/kg per day + 60 mg/kg per day, CV group, n = 11), amlodipine + valsartan (3 mg/kg per day + 60 mg/kg per day, AV group, n = 11), or vehicle containing 0.5% hydroxypropyl methylcellulose (2 mL/kg per day, Vehicle group, n = 11) was orally given to the rats. The dosages used were determined on the basis of our previous study.5) Nine Dahl S rats fed a normal-salt diet (F2Dahl-0.3 containing 0.3% NaCl; Oriental Yeast Co., Ltd.) from 6 to 18 weeks of age served as the normal control group.
Blood Pressure MonitoringBlood pressure monitoring was performed weekly using tail-cuff plethysmography (BP-98A, Softron Co., Ltd., Tokyo, Japan), as described previously.10) Rats were placed in a thermostatically warmed tube maintained at 34–36 °C, and the mean values were recorded from three measurements of systolic blood pressure (SBP) for each animal.
Urine AnalysisOn the last week of drug treatment, urine samples were collected for 24 h from the rats individually placed in metabolic cages to quantify urinary albumin excretion. Albumin concentrations in the urine samples were determined using a commercially manufactured enzyme-linked immunosorbent assay (ELISA) kit (Nephrat II; Exocell Inc., Philadelphia, PA, U.S.A.). Urinary albumin excretion was normalized to 24 h urine volume.
Echocardiographic AnalysisOn the last day of the drug administration, transthoracic echocardiography was performed under pentobarbital anesthesia using a 12.5-MHz transducer (Xario SSA-660A; Toshiba Medical Systems, Tochigi, Japan). Based on the M-mode echocardiography recording, the left ventricular (LV) end-diastolic and end-systolic dimensions (LVDd and LVDs, respectively), interventricular septum thickness (IVST), and LV posterior wall thickness (LVPWT) were measured. LV fractional shortening (FS) was calculated using the following formula:
 |
LV ejection fraction (EF) was calculated using the formula reported by Teichholz.11)
Electrophysiological StudyAfter the echocardiographic assessment, the rats were subjected to electrophysiological study, as described previously.5) The atrial electrogram recording/pacing combination catheter (3 F, Physio-Tech, Tokyo, Japan) was positioned at the atrial septum through the right jugular vein under the surface lead II electrocardiogram (ECG) recordings. The electrograms were amplified with a bioelectric amplifier (AB-621G, Nihon Kohden, Tokyo, Japan) and acquired into the PowerLab system (ADInstruments, Castle Hill, Australia).
The measurement of the atrial effective refractory period (AERP) and induction of AF were done with a cardiac stimulator (SEN-7203, Nihon Kohden), as described previously.5) AF was induced at the atrial septum by burst pacing. The AERP and AF were defined as the shortest coupling interval still producing an electrical response and a period of rapid, irregular atrial rhythm resulting in an irregular baseline of the ECG, respectively.
Organ Weights and Histological StudiesAfter the electrophysiological experiments, the hearts and kidneys were removed following euthanasia under deep anesthesia, and they were weighed and fixed in 10% formalin. Myocardial sections stained with Masson’s trichrome stain were analyzed in a blinded fashion under a light microscope (BX50; Olympus Corporation, Tokyo, Japan) to quantify the myocyte cross-sectional dimensions and interstitial fibrosis. The relative volume occupied by each tissue element of the atrium and ventricle (myocardial fibers and fibrous tissue) was quantified using image processing software (WinROOF version 3.5, Mitani Corporation, Fukui, Japan).
Measurements of Biological SubstancesBefore the electrophysiological study, the blood was collected from the jugular vein under pentobarbital anesthesia. Blood samples were centrifuged (3000 × g at 4 °C) for 15 min, and the supernatants were stored at −80 °C for later analysis. Plasma concentrations of angiotensin I and angiotensin II were determined using HPLC.10) To measure plasma levels of brain natriuretic peptide (BNP), the blood was collected from the postcaval vein after the electrophysiological study, centrifuged, and stored at −80 °C for later analysis. Plasma levels of BNP were determined using commercially manufactured ELISA kits (AssayMax Rat BNP-32, Assaypro, St. Charles, MO, U.S.A.).
Gene Expression AnalysisRNA extraction and quantitative (q)RT-PCR were performed as described previously.12) Total RNA was isolated from renal cortical tissues using the RNeasy Mini kit (QIAGEN GmbH, Hilden, Germany). One microgram of total RNA was reverse-transcribed using SuperScript III (Invitrogen Corporation, Grand Island, NY, U.S.A.). Real-time qRT-PCR was performed with a 7500 Real-Time PCR System using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, U.S.A.) and sequence-specific primers. mRNA expression levels were normalized to β-actin levels.
Statistical AnalysisData are expressed as the means ± standard error of the mean (S.E.M.). Dunnett’s multiple tests were used to assess the differences between the Vehicle group and the Val, CV, or AV group. Parameters in the Vehicle and normal control groups were compared using unpaired t-tests. AF duration was analyzed after converting the numerical value to a logarithmic value. Differences were considered statistically significant at p values less than 0.05.
SBP and urinary albumin excretion were significantly higher in the Vehicle group than those in the normal control group in Dahl S rats (p < 0.001 and p < 0.0001, respectively; Table 1). The SBP decreased after oral administration of valsartan and further decreased with concomitant administration of CCBs, cilnidipine or amlodipine. The extent of the decrease in the CV group was similar to that in the AV group. Among the three groups, only the CV group showed a significant decrease in urinary albumin excretion.
| n | Systolic blood pressure (mmHg) | Urinary albumin excretion (mg/d) | ||||
|---|---|---|---|---|---|---|
| Pre | Four weeksafter administration | Pre | Four weeksafter administration | |||
| Normal diet | Vehicle | 9 | 134.5 ± 3.1 | 129.0 ± 3.1 | 20.3 ± 4.7 | 18.3 ± 5.3 |
| High-Na+ diet | Vehicle | 11 | 207.9 ± 6.7#### | 226.9 ± 5.6#### | 94.0 ± 10.3#### | 164.2 ± 16.8#### |
| Valsartan (Val) | 11 | 206.5 ± 7.5 | 203.8 ± 6.7* | 92.8 ± 9.8 | 133.7 ± 11.2 | |
| Cilnidipine + Valsartan (CV) | 11 | 205.6 ± 7.2 | 156.4 ± 3.4**** | 87.0 ± 9.1 | 94.1 ± 9.0*** | |
| Amlodipine + Valsartan (AV) | 11 | 203.3 ± 6.6 | 159.1 ± 4.7**** | 93.9 ± 9.3 | 130.1 ± 13.0 | |
Data are expressed as means ± S.E.M. Valsartan (60 mg/kg, n = 11), cilnidipine + valsartan (10 +60 mg/kg, n = 11), amlodipine + valsartan (3 +60 mg/kg, n = 11), or vehicle (n = 11) was administered to Dahl S rats fed a high-Na+ diet for 5 weeks. Vehicle was also administered to Dahl S rats fed a normal diet for 5 weeks (n = 9). “Pre” indicates before administration. #### p < 0.0001 vs. the normal diet group using t-test. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. the Vehicle group using Dunnett’s multiple comparisons tests.
Typical tracings of the right atrial (RA) electrogram and surface ECG in Dahl S rats are depicted in Fig. 1A. Longer duration of AF was induced in the Vehicle group by burst pacing at the right atrium (11.2 ± 6.02 s) than that in the normal control group (0.333 ± 0.128 s, p < 0.001, Fig. 1B). The duration of AF in the CV group (1.09 ± 0.26 s) was significantly shorter than that in the Vehicle group (p < 0.01) while those in the Val and AV groups (3.19 ± 1.58 and 1.33 ± 0.286 s) were also shorter than that in the Vehicle group, though statistically insignificant (Fig. 1B).
(A) Representative electrograms of burst pacing-induced AF in Dahl S rats that received the vehicle with a normal or high-salt diet. (B) Effects on burst pacing-induced AF in Dahl S rats fed a high-salt diet. Valsartan (60 mg/kg, Val group, n = 11), cilnidipine + valsartan (10 + 60 mg/kg, CV group, n = 11), amlodipine + valsartan (3 + 60 mg/kg, AV group, n = 11), or their vehicle (n = 11) was administered for 5 weeks. The vehicle was administered for 5 weeks to Dahl S rats fed a normal diet (n = 9). Values are represented as means ± S.E.M.; ### p < 0.001 vs. normal group. ** p < 0.01 vs. Vehicle group.
Table 2 summarizes the electrocardiographic variables of the Dahl S rats. The P duration, PR interval, QRS width, QT interval, R amplitude, AERP, and AF cycle length in the Vehicle group were greater than those in the normal diet group. The PR interval, QT interval, R amplitude, and AF cycle length in the CV group were reduced compared to those in the Vehicle group.
| Normal diet | High-Na+ diet | ||||
|---|---|---|---|---|---|
| Vehicle | Vehicle | Valsartan (Val) | Cilnidipine + valsartan (CV) | Amlodipine + valsartan (AV) | |
| Heart rate (bpm) | 386 ± 13 | 378 ± 12 | 403 ± 13 | 419 ± 12* | 407 ± 9 |
| P duration (ms) | 15 ± 0 | 19 ± 1### | 17 ± 1 | 18 ± 1 | 19 ± 1 |
| PR interval (ms) | 50 ± 1 | 54 ± 1# | 53 ± 1 | 49 ± 1** | 52 ± 1 |
| QRS width (ms) | 17 ± 1 | 30 ± 2### | 31 ± 3 | 30 ± 2 | 25 ± 1 |
| QT interval (ms) | 84 ± 2 | 97 ± 2### | 89 ± 3 | 84 ± 3** | 92 ± 2 |
| R amplitude (mV) | 0.322 ± 0.042 | 0.647 ± 0.064### | 0.514 ± 0.050 | 0.336 ± 0.045*** | 0.542 ± 0.063 |
| AERP (ms) | 42 ± 2 | 37 ± 3 | 37 ± 1 | 38 ± 3 | 37 ± 2 |
| AF cycle length (ms) | 36 ± 2 | 42 ± 1## | 43 ± 1 | 38 ± 1* | 40 ± 1 |
Data are expressed as means ± S.E.M. Valsartan (60 mg/kg, n = 8), cilnidipine + valsartan (10 + 60 mg/kg, n = 11), amlodipine + valsartan (3 + 60 mg/kg, n = 10), or vehicle (n = 9) was administered to Dahl S rats fed a high-Na+ diet for 5 weeks. Vehicle was also administered to Dahl S rats fed a normal diet for 5 weeks (n = 9). An electrophysiological study was performed after the termination of the 5-week-long drug treatment. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the normal diet group using t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the Vehicle group using Dunnett’s multiple comparisons tests.
Figure 2 shows the indices related to cardiac function. Left ventricular EF and left ventricular FS were significantly lower in the Vehicle group than those in the normal control group, whereas EF and FS were greater in the CV and AV groups than those in the Vehicle group.
Valsartan (60 mg/kg, Val group, n = 11), cilnidipine + valsartan (10 + 60 mg/kg, CV group, n = 11), amlodipine + valsartan (3 + 60 mg/kg, AV group, n = 11), or their vehicle (n = 11) was administered for 5 weeks. The vehicle was administered for 5 weeks to Dahl salt-sensitive rats fed a normal diet (n = 9). Values are represented as means ± S.E.M.; ### p < 0.001 vs. normal group. ** p < 0.01, *** p < 0.001 vs. Vehicle group.
The heart and kidney weights in the Vehicle group were higher than those in the normal control group (Table 3). The heart and kidney weights in the Val, CV, and AV groups were significantly lower than those in the Vehicle group. Weights of the atria and ventricles in the CV and AV groups were also lower than those in the Vehicle group. No significant difference was observed in organ weights between the CV and AV groups (Table 3).
| Normal diet | High-Na+ diet | ||||
|---|---|---|---|---|---|
| Vehicle | Vehicle | Valsartan (Val) | Cilnidipine + valsartan (CV) | Amlodipine + valsartan (AV) | |
| Heart (g/100 g BW) | 0.30 ± 0.00 | 0.52 ± 0.02#### | 0.45 ± 0.01*** | 0.39 ± 0.00**** | 0.41 ± 0.00**** |
| Atrium (g/100 g BW) | 0.031 ± 0.001 | 0.061 ± 0.007#### | 0.049 ± 0.004 | 0.039 ± 0.003** | 0.043 ± 0.003* |
| Ventricle (g/100 g BW) | 0.26 ± 0.00 | 0.45 ± 0.02#### | 0.39 ± 0.02** | 0.34 ± 0.00**** | 0.35 ± 0.00**** |
| Kidney (g/100 g BW) | 0.67 ± 0.00 | 1.22 ± 0.04#### | 1.04 ± 0.03*** | 0.91 ± 0.02**** | 0.99 ± 0.03**** |
Data are expressed as means ± S.E.M. Valsartan (60 mg/kg, n = 11), cilnidipine + valsartan (10 + 60 mg/kg, n = 11), amlodipine + valsartan (3 + 60 mg/kg, n = 11), or vehicle (n = 11) was administered to Dahl S rats fed a high-Na+ diet for 5 weeks. Vehicle was also administered to Dahl S rats fed a normal diet for 5 weeks (n = 9). Tissues were obtained after the termination of the 5-week-long drug treatment. ####p < 0.0001 vs. the normal diet group using t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the Vehicle group using Dunnett’s multiple comparisons tests.
Typical photomicrographs of longitudinal sections of atrial tissues obtained from a Dahl S rat in each of the three animals in all the groups are shown in Fig. 3; interstitial fibrosis was observed, and the area of fibrosis in the atria in the Vehicle group was greater than that in the normal control group. The areas of atrial fibrosis in the Val, CV, and AV groups were smaller than those in the Vehicle group.
Atrial tissues were obtained at the termination of drug treatment after 5 weeks. Valsartan (60 mg/kg, Val group, n = 11), cilnidipine + valsartan (10 + 60 mg/kg, CV group, n = 11), amlodipine + valsartan (3 + 60 mg/kg, AV group, n = 11), or their vehicle (n = 11) was administered for 5 weeks. Vehicle was administered for 5 weeks to Dahl S rats fed a normal diet (n = 9). The fibrosis area in atria was measured with three individuals in each treatment group, and the average values are shown in each photograph.
Expression of Col1a1, Ctgf, Cccf2 (MCP-1), Osteopontin, and Caspase-3 genes was analyzed by quantitative PCR (qPCR) using atrial tissue, as shown in Table 4. Expression of the genes related to fibrosis (Col1a1 and Ctgf) in the Vehicle group was higher than that in the normal control group. The expression level of Ctgf was lower in the Val, CV, and AV groups than that in the Vehicle group. The genes related to inflammation (Ccf2 (MCP-1) and Osteopontin) were upregulated in the Vehicle group. The Ccf2 levels in the Val and CV groups and the Osteopontin levels in the CV and AV groups were significantly lower than those in the Vehicle group. The expression of Caspase-3, a gene related to apoptosis, was significantly higher in the AV group than that in the Val group.
| Normal diet | High-Na+ diet | ||||
|---|---|---|---|---|---|
| Vehicle | Vehicle | Valsartan (Val) | Cilnidipine +valsartan (CV) | Amlodipine +valsartan (AV) | |
| Col1a1 (Collagen1) | 1.0 ± 0.1 | 3.8 ± 0.6## | 2.5 ± 0.5 | 2.1 ± 0.3 | 2.7 ± 0.4 |
| Ctgf | 1.0 ± 0.2 | 2.8 ± 0.5## | 1.1 ± 0.2** | 0.8 ± 0.1*** | 0.9 ± 0.1*** |
| Ccl2 (MCP-1) | 1.0 ± 0.4 | 1.9 ± 0.4 | 0.9 ± 0.1* | 0.9 ± 0.1* | 1.1 ± 0.2 |
| Osteopontin | 1.0 ± 0.3 | 50.0 ± 16.9# | 15.0 ± 5.8 | 5.6 ± 0.7** | 9.9 ± 4.3* |
| Caspase-3 | 1.0 ± 0.1 | 1.6 ± 0.1## | 1.1 ± 0.2 | 1.4 ± 0.1 | 1.7 ± 0.1** |
Data are expressed as means ± S.E.M. Valsartan (60 mg/kg, n = 8), cilnidipine + valsartan (10 + 60 mg/kg, n = 8), amlodipine + valsartan (3 + 60 mg/kg, n = 8), or vehicle (n = 8) was administered to Dahl S rats fed a high-Na+ diet for 5 weeks. Vehicle was also administered to Dahl S rats fed a normal diet for 5 weeks (n = 7). Atrial tissues were obtained and subjected to qPCR analysis after the termination of the 5-week-long drug treatment. #p < 0.05, ##p < 0.01 vs. the normal diet group using t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the Vehicle group using Dunnett’s multiple comparisons tests.
As shown in Fig. 4A, plasma BNP levels were higher in the Vehicle group than those in the normal control group. BNP levels in the CV and AV groups decreased compared to those in the Vehicle group, whereas no significant difference was seen between the Val and Vehicle groups. Figures 4B and 4C show the angiotensin levels in the Dahl S rats. Plasma levels of angiotensin I and II in the Vehicle group were higher than those in the normal control group. Among the three groups, the plasma levels of angiotensin I in the CV group were lower than those in the Vehicle group.
Valsartan (60 mg/kg, Val group, n = 11), cilnidipine + valsartan (10 + 60 mg/kg, CV group, n = 11), amlodipine + valsartan (3 + 60 mg/kg, AV group, n = 11), or their vehicle (n = 11) was administered for 5 weeks. The vehicle was administered for 5 weeks to Dahl salt-sensitive rats fed a normal diet (n = 5). Values are means ± S.E.M.; #p < 0.05, ##p < 0.01, ### p < 0.001 vs. normal group. * p < 0.05, *** p < 0.001 vs. Vehicle group.
Feeding Dahl S rats a high-salt diet increased the duration of burst-pacing-induced AF, accompanied by enhanced tissue weight and interstitial fibrosis in the atrium. Combination therapy with the angiotensin II AT1 receptor blocker valsartan and the L/N-type CCB cilnidipine effectively ameliorated these electrical and histological abnormalities, which were additively observed relative to that with monotherapy with valsartan.
Hemodynamic and Renoprotective Effects of Cilnidipine and Valsartan Combined TreatmentFour weeks after the start of drug administration, SBP was lower by 13, 70, and 67 mmHg in the Val, CV, and AV groups, respectively, than that in the Vehicle group of the high-salt diet-fed Dahl S rats. Our previous study using the same animal model demonstrated that cilnidipine (10 mg/kg) and amlodipine (3 mg/kg) decreased SBP by 53 and 51 mmHg, respectively.5) These findings suggest that the reductions in SBP observed with cilnidipine plus valsartan or amlodipine plus valsartan may result from their additive effects.
Urinary albumin excretion was effectively decreased by the combination of cilnidipine and valsartan. Dahl S rats are known to be susceptible to renal complications due to the high-salt diet, showed a more potent improvement in albuminuria with the combination of cilnidipine and valsartan than the combination of amlodipine and valsartan or by valsartan alone.12) Combining the results of our previous study5) and the present study, the suppression of urinary albumin excretion in the cilnidipine group was 57%, while in the CV group, it was 42%, suggesting that the suppression of urinary albumin excretion was mainly due to the effect of cilnidipine. Studies have shown that cilnidipine protects against a wide range of renal injuries caused by hypertension, diabetes, or drugs that impair kidney function, including adriamycin13–15); such effects of cilnidipine could be the basis for the potent renoprotective action of its combination with valsartan.
Atrial Electrophysiological and Anti-AF Actions by Combination of Cilnidipine and ValsartanThe atrium of the high-salt diet-fed Dahl S rats was remodeled electrically due to the shortened AERP and increased P-wave duration in comparison with those of the normal diet-fed Dahl S rats (Table 2). These changes could be aggravating factors for reentry in the atrium, leading to a longer duration of AF. Moreover, burst pacing-induced AF in the high-salt diet-fed Dahl S rats was more stable than that in the normal diet-fed Dahl S rats, as the AF cycle length of the former was greater than AERP. The duration of burst pacing-induced AF was effectively suppressed by the drug treatments (Fig. 1B). Our previous study showed a 60% suppression against AF with cilnidipine alone, while in the current study, the suppression rate escalated to 90% in the CV group, providing substantial evidence for the combined effect of cilnidipine and valsartan. However, despite their remarkable anti-AF effects, the AERP or P-wave duration between the Vehicle group and drug treatment groups was not significantly different, suggesting no amelioration of the atrial refractoriness or transmissibility of atrial pulse after the drug treatments. In the case of the combination of cilnidipine and valsartan, more potent inhibition of the duration of AF was observed; the AF cycle length was significantly decreased and was close to the value of AERP, which possibly made AF more unstable, leading to a shorter duration of AF.
Atrial Anatomical and Histological Amelioration by Combination of Cilnidipine and ValsartanAtrial enlargement is an important clinical predictor of AF maintenance,16) and can occur with heart failure-related remodeling.17) Stretching and dilation of the atrium due to volume and pressure loading cause mechanical stimulation, leading to increased angiotensin II levels triggering atrial fibrosis via ERK1/2 activation.18) Mice overexpressing myocardial-specific angiotensin II-converting enzyme are reported to be prone to AF, showing remarkable enlargement and fibrosis of the atria.19) As observed in our study, atrial weight increased in the high-salt diet-fed Dahl S rats (Table 3), where EF and FS decreased (Fig. 2) and plasma BNP level increased (Fig. 4A), indicating the presence of HF leading to atrial enlargement in this animal model. The longer AF cycle length observed in this animal model might be associated with atrial enlargement. Drug treatments effectively ameliorated the increment of atrial weight in the high-salt diet-fed Dahl S rats, with the greatest effect observed in the rats receiving cilnidipine and valsartan. Based on these findings, we speculate that the decrease in AF cycle length was associated with anatomical changes in the atrium by cilnidipine and valsartan, as atrial weight was lowest in this combination out of all the treatment groups (Table 3).
Fibrosis is a hallmark of structural remodeling, resulting in a substrate vulnerable to AF, and increased collagen deposition has been reported in patients with AF.20) Gene expression analysis in Table 4 shows increased expression of the fibrosis-related genes, Col1a1 and Ctgf, increased expression of inflammation-related genes, Ccl2 and Osteopontin, and increased expression of the apoptosis-related gene, Caspase-3. This suggests that in Dahl-S rats, hypertension due to salt loading, increased neojugularity from pressure loading, and the formation of an inflammatory state led to increased fibrosis due to renewed secretion of extracellular matrix (ECM). On the contrary, the drug-treated group showed suppression of fibrosis-related gene upregulation, which could have led to suppression of cardiac remodeling and fibrosis. Osteopontin, a type of anti-inflammatory cytokine involved in the regulation of ECM,21) was lower in the CV and AV groups than that in the Val group, suggesting that the combination of ARB and CCB could more strongly suppress ECM production, thereby reducing fibrosis. Cilnidipine is also reported to have suppressive effects on myocardial senescence and heart failure after myocardial infarction via suppression of mitochondrial hyperfission.22) Therefore, the combined effect of valsartan and cilnidipine is presumed due not only to the additive effect on the L- and N-type calcium channel of cilnidipine added on ARB’s effect but also to the multifaceted pharmacological action of cilnidipine and its ability to suppress the side effects of ARB. Nevertheless, in this study, the evaluation was limited to gene expression. Therefore, for a more comprehensive understanding of the mechanism, further analyses, including protein levels, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, and mitochondrial function, should be conducted in future studies.
Utility of the Combination Therapy with Valsartan and CilnidipineCardiac and renal disorders are known as common risk factors for AF, contributing to structural and electrical issues that promote AF.23) In contrast, AF can increase the risk of progression to end-stage renal disease.24) Considering preventive therapy for AF, drugs with cardiorenal protective effects can be effective in suppressing AF. In this study, stronger antihypertensive effects were observed in the CV and AV groups treated with CCB than those in the Val group (Table 1), along with a suppression of organ weight gain. In addition, the duration of AF was lower in the CV group than that in the Vehicle group (Fig. 1B). Furthermore, only in the CV group urinary albumin excretion was significantly decreased (Table 1). These results suggest that the combination of valsartan and CCBs can further reduce pressure and pressure overload, thereby suppressing cardiac fibrosis and persistent AF. Among the CCBs, cilnidipine could be expected to exert not only cardioprotective but also renoprotective effects. A previous clinical study in patients with type II diabetes showed that cilnidipine, in combination with valsartan, reduced albuminuria.25) Similar additive effects on AF may be expected based on the current animal studies.
Furthermore, ARBs have been reported to increase blood angiotensin levels,26) and in this experiment, blood angiotensin levels were higher in the Val group than that in the Vehicle group, although this increase was suppressed by the combination of CCBs, especially cilnidipine (Fig. 4). Taken together, these results suggest that the combination therapy of Valsartan and Cilnidipine is a beneficial regimen. However. As this study was conducted in animal models, the extrapolation of these findings to clinical applications in humans is limited. Therefore, obtaining clinical results in humans is crucial to thoroughly evaluate the effects of such drug combination effects.
In this study using high-salt diet-fed Dahl S rats, the shorter duration of AF induced by atrial burst stimulation, greater left ventricular ejection fraction and left ventricular fractional shortening, and lower urinary albumin excretion were detected in the CV group, which collectively suggests that the combination of cilnidipine with valsartan could be useful in terms of its anti-AF action as well as for improving cardiac and renal functions.
This work was conducted in Ajinomoto Pharmaceuticals Co., Ltd.
Eri Harada and Kazumi Sugino are employees of Ajinomoto Co., Inc. At the time of this experiment, they belonged to Ajinomoto Pharmaceuticals (now EA Pharma Co., Ltd.). Akira Takahara received research funding from Ajinomoto Pharmaceuticals Co., Ltd.
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