TREATMENT OF CARDIAC REMODELLING
20230173029 · 2023-06-08
Assignee
Inventors
Cpc classification
International classification
Abstract
The present invention provides a polypeptidic compound for use in prevention or treatment of cardiac remodeling in a subject, the polypeptidic compound comprising the amino acid sequence of proANP.sub.31-67, or an amino acid sequence having at least 80% sequence identity thereto.
Claims
1. A method of treating or preventing cardiac remodeling in a subject, comprising administering to said subject a polypeptidic compound comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto.
2. The method of claim 1, wherein said polypeptidic compound is the proANP.sub.31-67 peptide, consisting of the amino acid sequence set forth in SEQ ID NO: 1.
3. The method of claim 1, wherein said cardiac remodeling is ventricular hypertrophy and/or ventricular fibrosis.
4. The method of claim 1, wherein said therapy comprises administering said polypeptidic compound subcutaneously to the subject at a dosage in the range of 25 to 2000 ng/kg/day.
5. The method of claim 4, wherein said dosage is in the range 25-95 ng/kg/day.
6. The method of claim 1, wherein said therapy comprises administering said polypeptidic compound to the subject intravenously at a dosage in the range of 5 to 1000 ng/kg/day.
7. The method of claim 4, wherein said dosage is in the range 6-23 ng/kg/day.
8. The method of claim 1, wherein said therapy comprises administering said polypeptidic compound to the subject intranasally at a dosage in the range of 5 to 1000 ng/kg/day.
9. The method of claim 4, wherein said dosage is in the range 8-30 ng/kg/day.
10. The method of claim 1, wherein the subject does not have heart failure with a symptom severity level of NYHA Class III or Class IV.
11. The method of claim 1, wherein the subject does not have overt symptoms of chronic congestive heart failure (CHF) or acute decompensated congestive heart failure (ADCHF).
12. The method of claim 10, wherein the subject does not have overt symptoms of heart failure and/or a cardiorenal syndrome.
13. The method of claim 12, wherein the subject is at risk of heart failure.
14. The method of claim 12, wherein said subject has hypertension.
15. The method of claim 14, wherein said subject has resistant hypertension.
16. The method of claim 12, wherein said subject has: (i) an endocrine disorder, optionally wherein said endocrine disorder is diabetes; (ii) a heart valve disorder; (iii) myocarditis; (iv) amyloidosis; (v) cardiomyopathy; (vi) an arrhythmia, optionally wherein said arrhythmia is tachycardia or bradycardia; (vii) haemochromatosis; (viii) chronic obstructive pulmonary disease; (ix) sarcoidosis; or (x) has had a heart attack; or wherein said subject is undergoing cardiotoxic therapy, optionally wherein said cardiotoxic therapy is chemotherapy or radiotherapy.
17. The method of claim 10, wherein further cardiac remodeling is prevented.
18. The method of claim 10, wherein diastolic dysfunction is reduced or prevented.
19. The method of claim 17, wherein administration of said peptide to said subject reduces or prevents the development of heart failure.
20. The method of claim 1, wherein said treatment or prevention does not also comprise administration of a diuretic agent to said subject.
21. (canceled)
22. (canceled)
Description
FIGURE LEGENDS
[0105]
[0106] (A) Δ SBP indicates the difference in systolic blood pressure at 6 weeks relative to baseline, within each group. Δ SBP was higher in both groups fed on a high-salt diet (the hypertensive (HT) groups) compared to the group fed on a normal salt diet (the normotensive (NT) group). No difference in Δ SBP was observed between untreated HT rats and those treated with proANP.sub.31-67.
[0107] (B) Cardiac hypertrophy in HT rats is indicated by the percentage increase in HW/BW (relative to NT rats). Untreated HT rats exhibited an increase in HW/BW of more than 15%, whereas proANP.sub.31-67-treated rats exhibited almost no increase in HW/BW.
[0108] (C) The E/A ratio is the ratio of peak velocity blood flow from gravity in early diastole (the E wave) to peak velocity flow in late diastole caused by atrial contraction (the A wave); The decrease in the E/A ratio in untreated HT rats (relative to NT controls) indicated that the hearts of the untreated HT rats had become stiffer, whereas proANP.sub.31-67-treated HT rats had an essentially unchanged E/A ratio, indicating preserved diastolic function.
[0109] (D) LA (%) indicates the percentage left atrium (LA) enlargement (measured by the increase in LA diameter) relative to NT rats, secondary to hypertension. Much greater LA enlargement was seen in the HT untreated group than in the proANP.sub.31-67-treated group.
[0110] (E) Approximately 70% of the untreated HT rats (grey circles) developed concentric hypertrophy (top-right quadrant), whereas about 70% of the proANP.sub.31-67-treated HT rats (white circles) had normal geometry (bottom-left quadrant). All NT rats (black circles) had normal cardiac geometry at 6 weeks (bottom-left quadrant). RWT is relative wall thickness, and LV mass is left ventricle mass.
[0111] (F) The level of Myh7 expression was measured by qPCR. As shown, Myh7 expression is essentially unchanged in treated HT rats relative to NT rats, but significantly higher in untreated HT rats than in NT rats and treated HT rats.
[0112]
[0113] (A) Illustrative images of the interstitial and perivascular left ventricle (LV) areas obtained by Masson's trichrome staining (top), and of LV perivascular collagen cross-linking (CCL) obtained by Picrosirius Red staining (bottom). Scale bars in top panel indicate distance of 40 μm. Images in bottom panel are 10× magnified.
[0114] (B, C) Cardiac collagen deposition was elevated in both interstitial and perivascular LV areas of the untreated HT rats compared to NT rats. ProANP.sub.31-67-treated rats had significantly reduced fibrosis compared to HT untreated rats.
[0115] (D) Untreated HT rats had elevated levels of LV perivascular CCL compared to NT rats. ProANP.sub.31-67-treated rats had significantly lower perivascular CCL compared to untreated HT rats (essentially unchanged relative to NT rats).
[0116] (E) Levels of nuclear phosphorylated SMAD2 were measured by quantitative Western blot. Levels of nuclear pSMAD2 in HT and treated HT rats are shown relative to levels in NT rats. Nuclear pSMAD2 levels were substantially higher in HT rats than NT rats, but slightly lower in treated HT rats than NT rats.
[0117]
[0118] (A) t-tubules were visualized with di-8-ANEPPS in the three Dahl/SS rat groups (upper panels). Distances from all points in the cytosol to the nearest t-tubule or surface sarcolemma were also obtained together with the fraction of transversely-oriented tubules (Trans.) and longitudinally-oriented tubules (Longi.) (bottom panels). Scale bars are shown for the upper 3 rows of panels.
[0119] (B) Cardiomyocyte size was increased in untreated HT rats compared to NT rats, whereas it was unchanged in proANP.sub.31-67-treated HT rats.
[0120] (C) t-tubule density was lower in hypertrophic hearts of untreated HT rats than in NT rats, but was preserved in proANP.sub.31-67-treated rats.
[0121] (D) Consistent with the reduction in t-tubule density, untreated HT rats had a longer distance among t-tubules compared to NT rats, whereas proANP.sub.31-67-treated rats maintained maximum intracellular distances between t-tubules and the sarcolemmal membrane. (n of NT cells=60 from 4 hearts, n of untreated HT cells=60 from 4 hearts, n of proANP.sub.31-67-treated HT cells=53 from 3 hearts.)
[0122]
[0123] Rat neonatal cardiomyocytes were stimulated for 24 h with ANGII to induce hypertrophy in vitro. ProANP.sub.31-67 at 37.5 ng/ml and 150 ng/ml blocked the pathological actions of ANGII.
[0124]
[0125] (A) Δ SBP indicates the difference in systolic blood pressure at 6 weeks relative to baseline, within each group. Δ SBP was higher in both groups fed on a high-salt diet (the hypertensive (HT) groups) compared to the group fed on a normal salt diet (the normotensive (NT) group). No difference in Δ SBP was observed between untreated HT rats and those treated with proANP.sub.31-67.
[0126] (B) HW/BW is presented for three groups of rats (NT, untreated HT and HT treated with low dosage (25 ng/kg/day) proANP.sub.31-67). An increase in HW/BW is indicative of cardiac hypertrophy. As shown in
[0127] (C) As shown in
[0128] (D) The LA diameter was greater in the untreated HT group compared to the NT group, secondary to hypertension. An increase in LA diameter was also seen in the HT group treated with a low dose of proANP.sub.31-67, but this increase was significantly less than in the untreated group.
[0129] (E) As shown in
[0130] (F) The level of Myh7 expression was measured by qPCR. As shown, Myh7 expression is essentially unchanged in treated HT rats relative to NT rats, but significantly higher in untreated HT rats than in NT rats and treated HT rats.
EXAMPLES
Example 1
Effect of proANP.SUB.31-67 .at 50 or 100 ng/kg/day on Hypertensive Rats
Methods
Materials
[0131] ProANP.sub.31-67 was obtained from Madeline Pharmaceuticals Pty Ltd. (Mount Barker, SA, Australia). The drug manufacturer recommends using 200 mM NaCl with 10 mM sodium acetate buffer (pH 5.5) as vehicle for the drug. Teklad high salt (4.0% NaCl) diet was obtained from Envigo (TD.92034; Madison, Wis., USA). Normal salt (0.3% NaCl) diet was from SDS (Special Diets Services; UK).
Experimental Groups
[0132] A total of 29 adult Dahl salt-sensitive (Dahl/SS) male rats with an initial weight of 150 to 200 g were included in the study. Rats were purchased from Charles River Laboratories (USA) and housed in a room with a 12/12-hour light cycle, a temperature of 21° C., and a humidity of 55%. Rats were maintained on a normal salt diet up to seven weeks of age. Twenty-two Dahl/SS were then randomly switched to a high salt diet for 6 weeks to induce hypertension, whilst 7 rats were kept normotensive (NT) on normal salt diet. Drinking water and food was provided ad libitum.
[0133] After 2 weeks of high salt diet, 15 hypertensive (HT) rats were started on treatment with proANP.sub.31-67. Rats treated with proANP.sub.31-67 received one of two dosages: 50 ng/kg/day (n=8) or 100 ng/kg/day (n=7). A total of 14 rats received only vehicle: control rats on a normal salt diet (n=7); and HT rats on a high salt diet (n=7). Rats were randomly assigned to the 3 groups (i.e. control normal salt; high salt untreated; high salt plus treatment). Post-analysis of the treated groups revealed that there was no dose-dependent effects between treatment with 50 or 100 ng/kg/day proANP.sub.31-67, hence treated rats in this example were pooled and analysed as a single group, named “HT+proANP.sub.31-67” (“hypertensive treated”).
[0134] Drug or vehicle was delivered via Alzet osmotic mini-pumps (Model 2004; mean pumping rate 2.28±0.07 μL/hr, mean fill volume 1997.6±18.3 μL) implanted subcutaneously (as instructed by the manufacturer) for 28 days. Animals were treated with buprenorphine (0.05 mg/kg s.c.) as analgesic 30 minutes before and up to 1 day following implantation of pumps.
Study Protocol
[0135] The study conformed to the regulations governing the laboratory animal facility (Comparative Medicine-Ullevål, OUS, Norway). The protocol was approved by The Norwegian Food Safety Authority committee (Mattilsynet) for animal research (FOTS protocol number 12582).
Echocardiography
[0136] Cardiac function was assessed by transthoracic echocardiography using a VEVO 2100 high resolution in vivo imaging system from VisualSonics (Canada). Briefly, animals were maintained under anesthesia (1.5-2% isoflurane mixed with oxygen) on a pre-warmed ECG transducer pad with body temperature and ECG monitored. Measurements were made with an MS250 transducer, frequency set at 24 MHz. M-mode in the parasternal long axis view was performed to assess the function and dimension of the left ventricle and left atrium. LVEF was calculated as 100×((LV Vol;d−LV Vol;s)/LV Vol;d). LV mass was estimated by the formula: 1.053×((LVID;d+LVPW;d+IVS;d)3−LVID;d3). Relative wall thickness (RWT) was calculated as 2×LVPW;d/LVID;d.21 Normal geometry of the heart was defined as the 95.sup.th percentile for both LV Mass and RWT of the NT group. E and A waves in left ventricular filling velocities were assessed via pulsed-wave Doppler in a parasternal long axis view. Echocardiographic analysis were performed by an operator blinded to treatment group.
Blood Pressure Measurement
[0137] Measurement was carried out using the CODA non-invasive blood pressure acquisition system for rats (Kent Scientific Corporation). Animals were kept in restraint tubes and placed over a heating platform (preheated to 33 to 35° C.) and blood pressure measured by a tail-cuff system. Each recording session consisted of 25 acclimation cycles (not used in the analysis), followed by 20 inflation and deflation cycles (the occlusion cuff is inflated to 250 mm Hg and deflated over 20 s). Rats were trained for at least 5 consecutive days before blood pressure measurements were recorded.
Histochemistry
[0138] Hearts were excised, rinsed in PBS, quickly blotted on gauze, and then fixed in 10% formalin for a minimum of 24 hr. The bi-ventricular apex of the heart was embedded in paraffin and cut into 4 μm sections. Sections were stained with Masson's trichrome (Polysciences, Inc., Warrington, Pa., USA) to assess collagen abundance. Stained sections were scanned (20× magnification) with AxioScan Z1 (Carl Zeiss), to obtain whole cross-sections for collagen quantification. Total fibrosis area (%) and perivascular fibrosis (ratio of the area of fibrosis surrounding the vessel wall to the lumen area) were quantified using ZEN2 blue edition (Zeiss, Jena, Germany). In addition, heart sections were stained with Picrosirius Red (Polysciences, Inc.) and visualized under bright-field and polarized light (10× magnification) to assess cross-linked collagen. The degree of cross-linked collagen was calculated as the ratio between cross-linked collagen and total collagen. All histological quantifications were independently performed by three trained researchers blinded to rat groups.
Gene Expression
[0139] Total RNA was extracted from left ventricle tissue using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Cat. #74704). RNA concentration and quality was assessed by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). cDNA synthesis was performed using an iSCRIPT synthesis kit (Bio-Rad). Transcript levels of Mhy7 (Rn00568328_m1) and Rpl32 (Rn00820748_g1) were determined using TaqMan assays (Applied Biosystems) detected on a QuantStudio3 (Thermo Fisher Scientific) and analysed using QuantStudio™ Design (Thermo Fisher Scientific). Mhy7 mRNA levels were calculated by the ΔΔCt method and normalized to Rpl32 transcript value.
Western Blot
[0140] Enriched nuclear protein fraction from left ventricle tissue was isolated using Compartment Protein Extraction Kit (Merck Millipore, Cat. #2145), according to the manufacturer's specifications. Protein concentration was measured by BCA assay kit (Thermo Fisher Scientific, Cat. #23225). The tissue lysates were subjected to (4-15% precast polyacrylamide) SDS-PAGE electrophoresis, and the proteins were transferred to PVDF membranes using the iBlot® system (Thermo Fisher Scientific). Membranes were analysed with rabbit anti-SMAD2/3 (1:1000, Cell Signaling Technology, Cat. #8685) and rabbit anti-phospho-SMAD2 (Ser465/467) (1:500, Cell Signaling Technology, Cat. #3108). The membranes were developed using the ECL system (Pierce Protein Research Products) and ChemiDoc XRS (Bio-Rad), and quantified using Image Lab™ software (version 6.0.1, Bio-Rad).
Cardiomyocyte Isolation and t-Tubule Structure
[0141] A subset of rats was used for analyses of t-tubule structure in isolated left ventricular cardiomyocytes. These animals were anaesthetised as described above and euthanised by removal of the heart. The excised heart was then placed in cool buffer containing 130 mM NaCl, 25 mM HEPES, 5.4 mM KCl, 0.5 mM MgCl2, 0.4 mM NaH.sub.2PO.sub.4, and 5.5 mM D-glucose (pH 7.4), before mounting on a Langendorff setup for retrograde perfusion though the aorta.
[0142] Cardiomyocytes were then isolated as previously described (Frisk et al., American Journal of Physiology—Heart and Circulatory 307: H609-620, 2014). In brief, hearts were perfused with 200 U/mL collagenase type II (Worthington Biochemical, Lakewood, N.J.) at 37° C. for 15 min. Thereafter, the LV was cut out, minced, and triturated with a cut-off pipette before filtering the solution through a 200 μm nylon-mesh. t-tubules were stained with 10 μM di-8-ANEPPS for 20 min prior to imaging using an LSM800 Airyscan confocal microscope (Zeiss) with a 63× oil-immersed objective. Fluorescence was excited at 488 nm and emitted light above 500 nm was measured. t-tubule captures were recorded with 1871×1871-pixel XY images, recorded as z-stacks (spatial resolution: 77×77×500 nm). Image sequences were deconvolved with Huygens Essential software before analysis. Cell area, t-tubule density and distance to nearest t-tubule or sarcolemmal membrane were analysed as previously described (Frisk et al., Cardiovascular Research 112: 445-451, 2016).
Primary Cultures of Neonatal Cardiac Myocytes
[0143] Primary cultures of rat cardiomyocyte cells were prepared as previously described (Larsen et al., Cardiovascular Research 80(1): 47-54, 2008). In brief, hearts from 1-3 day old Wistar rats (Taconic), were isolated by collagen and pancreatin digestion. Cardiomyocytes were separated from noncardiomyocytes by differential attachment to uncoated culture flasks (90151; Techno Plastic Products). Cardiomyocytes were allowed to attach to 6- or 12-well culture plates (Corning International, Corning, N.Y.) coated with gelatine/fibronectin (G-1890/F1141; Sigma, St. Louis, Mo.) overnight in plating medium (2 or 1 ml) at a density of 2.5×10.sup.5 cells/ml medium [DMEM (41965; GIBCO-BRL, Invitrogen) supplemented with penicillin/streptomycin/glutamine (G6784; Sigma), medium 199 (31150; GIBCO-BRL), HEPES (15630; GIBCO-BRL), horse serum (14-703E; Bio-Whittaker, Lonza), and fetal calf serum (14-701E; Bio-Whittaker)]. The cardiomyocytes were maintained in plating medium without serum before being stimulated with 1 μM ANG II (A9525; Sigma), proANP.sub.31-67 37.5 ng/mL or 150 ng/mL, or vehicle for 24 h, and washed twice with DPBS (BE17-512F; BioWhittaker) before being harvested for analysis.
In Vitro Leucine Incorporation in Neonatal Cardiomyocytes
[0144] 5 μCi/ml [3H]leucine (American Radiolabeled Chemicals) were added at the same time as ANG II and the cells were washed six times in 95% EtOH before being harvested in 0.2 M NaOH 24 h after stimulation, as previously described (Halvorsen et al., Journal of Lipid Research 39(4): 901-912, 1998). Serum-stimulated cells served as positive control. [3H]leucine incorporation was quantified by measuring counts per minute in duplicates from each sample with the Wallac Winspectral 1414 liquid scintillation counter (PerkinElmer). Samples were diluted in Pico-Fluor 40 (cat. no. 6013349; PerkinElmer).
Statistics
[0145] Comparisons between 2 groups were made with an unpaired 2-tailed t-test. Comparisons between >2 groups were made with a 1-way ANOVA followed by Holm-Sidak post-hoc test. P values for each comparison are shown in each respective figure, and P<0.05 was considered statistically significant. Values are reported as mean±SEM. All statistical tests were performed with GraphPad Prism 8.0.1 (San Diego, Calif.).
Results
Blood Pressure
[0146] Dahl/SS rats fed a high salt diet showed elevated systolic blood pressure, which was not lowered by proANP.sub.31-67 (Table 1;
Cardiac Structure and Function
[0147] Dahl/SS rats exhibited characteristic signs of adverse cardiac remodeling and function after 6 weeks of high salt diet (Table 1;
[0148] Echocardiographic examination showed preserved systolic function in all groups, as assessed by left ventricular ejection fraction and fractional shortening (Table 1). Cardiac stiffness increased in untreated HT rats, as indicated by a decrease in E/A ratio, but was preserved in proANP.sub.31-67-treated rats (Table 1). Relative fold change to NT controls indicated that the E/A ratio dropped by approximately 50% in untreated HT rats, whereas the E/A ratio was preserved in proANP.sub.31-67-treated rats (
[0149] Cardiac ultrasound revealed that untreated HT animals had increased cardiac wall thickening, which was attenuated in proANP.sub.31-67-treated rats. Both the LV mass and RWT in proANP.sub.31-67-treated rats were similar to in NT rats (Table 1;
[0150] Interstitial and perivascular LV fibrosis increased by 79% and 78%, respectively, in the untreated HT rats compared to NT rats. ProANP.sub.31-67 treatment alleviated the development of both forms of fibrosis with a modest and not significant increase of 14% and 35% for interstitial and perivascular fibrosis, respectively (
Cardiomyocyte Effects
[0151] Cardiomyocyte size was increased in untreated HT rats, whereas it was preserved in proANP.sub.31-67-treated HT animals (
TABLE-US-00001 TABLE 1 Haemodynamic, autoptic and echocardiographic characteristics of the study groups at 6 weeks. NT HT HT + proANP.sub.31-67 (n = 7) (n = 7) (n = 15) SBP (mmHg) 163.9 ± 2.3 177.7 ± 5.6 * 179.3 ± 2.4 ** DBP (mmHg) 115.9 ± 3.6 121.2 ± 7.9 128.2 ± 2.3 MAP (mmHg) 131.6 ± 3.1 139.7 ± 6.8 144.9 ± 2.3 * BW (g) 338.3 ± 1.7 335.6 ± 8.3 348.6 ± 4.4 HW (mg) 1258 ± 48.4 1519 ± 108.1 * 1365 ± 30.1 HW/BW (mg/g) 3.76 ± 0.1 4.62 ± 0.3 **.sup.†† 3.97 ± 0.1 EF (%) 75.9 ± 4.1 82.6 ± 2.1 78.2 ± 1.9 FS (%) 46.5 ± 3.5 53.0 ± 2.3 48.7 ± 1.8 E/A 1.5 ± 0.1 1.1 ± 0.1 .sup.†† 1.6 ± 0.1 LA (mm) 3.8 ± 0.1 4.9 ± 0.2 **.sup.†† 4.2 ± 0.1 * LV Mass (mg) 823.3 ± 67.9 992.0 ± 43.4 * 891.7 ± 12.9 RWT 0.49 ± 0.03 0.65 ± 0.03 **.sup.†† 0.52 ± 0.02 LV Vol; d (μl) 240.8 ± 21.5 232.1 ± 13.8 265.1 ± 12.4 LV Vol; s (μl) 54.6 ± 4.6 41.7 ± 7.4 58.0 ± 6.3 IVS; d (mm) 1.7 ± 0.0 1.9 ± 0.0 *.sup.†† 1.7 ± 0.0 IVS; s (mm) 3.0 ± 0.0 3.3 ± 0.1 3.1 ± 0.0 LVPW; d 1.7 ± 0.1 2.2 ± 0.1 *.sup.† 1.8 ± 0.0 LVPW; s 3.0 ± 0.1 3.4 ± 0.1 *.sup.†† 2.9 ± 0.1 Legend SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; BW = body weight; HW = heart weight; EF = ejection fraction; FS = fractional shortening; E/A = ratio E wave to A wave; LA = left atrium diameter; LV Mass = left ventricular mass; RWT = relative wall thickness; LV Vol; d = left ventricular volume (diastole); LV Vol; s = left ventricular volume (systole); IVS; d = inter-ventricular septum (diastole); IVS; s = inter-ventricular septum (systole); LVPW; d = left ventricular posterior wall (diastole); and LVPW; s = left ventricular posterior wall (systole). * p < 0.05 or ** p < 0.01 for difference from NT .sup.† p < 0.05 or .sup.†† p < 0.01 for difference from HT + proANP.sub.31-67
In Vitro Effects on ANGII-Treated Cardiomyocytes
[0152] Neonatal cardiomyocytes were stimulated for 24 h with the octapeptide ANGII in order to induce hypertrophy as indexed by [3H]-leucine incorporation. At the same time, cells were treated with either vehicle or with proANP.sub.31-67 at two different concentrations: 37.5 ng/ml or 150 ng/ml. As shown in
Discussion
[0153] Administration of proANP.sub.31-67 to HT rats at the dosages used in this example has a cardioprotective effect. The peptide was also shown to have protective effects on renal function and structure (data not shown). Notably, proANP.sub.31-67 at the dosages used in this example did not reduce hypertension, indicating that these actions are independent of haemodynamic changes, indicating direct cardiorenal protective properties. While a direct effect of proANP.sub.31-67 on the kidneys was previously known, a direct effect of the peptide on the heart has not previously been identified.
Example 2
Effect of proANP.SUB.31-67 .at 25 ng/kg/day on Hypertensive Rats
Methods
[0154] Experiments were performed as described above in Example 1, using a group of 8 Dahl/SS rats which, at 7 weeks of age, were switched to a high salt diet as described above. After 2 weeks of high salt diet, this group was started on proANP.sub.31-67 treatment. The rats were administered proANP.sub.31-67 at a renal sub-therapeutic dosage of 25 ng/kg/day.
Results
[0155] As for the higher doses of proANP.sub.31-67 tested in Example 1, the dosage used in this example had no effect on systolic blood pressure (Table 2;
TABLE-US-00002 TABLE 2 Haemodynamic, autoptic and echocardiographic characteristics of the study groups at 6 weeks. NT HT HT + proANP.sub.31-67 (n = 7) (n = 7) (n = 8) SBP (mmHg) Hg) 163.9 ± 2.3 177.7 ± 5.6 * 176.5 ± 3.4 DBP (mmHg) Hg) 115.9 ± 3.6 121.2 ± 7.9 120.1 ± 4.7 MAP (mmHg) 131.6 ± 3.1 139.7 ± 6.8 138.6 ± 4.1 BW (g) 338.3 ± 1.7 335.6 ± 8.3 354.0 ± 8.3 HW (mg) 1258 ± 48.4 1519 ± 108.1 .sup.† 1304 ± 41.4 HW/BW 3.76 ± 0.1 4.62 ± 0.3 * .sup.†† 3.68 ± 0.07 EF (%) 75.9 ± 4.1 82.6 ± 2.1 79.6 ± 1.6 FS (%) 46.5 ± 3.5 53.0 ± 2.3 49.89 ± 1.7 E/A 1.49 ± 0.14 1.11 ± 0.09 .sup.† 1.47 ± 0.08 LA (mm) 3.65 ± 0.19 4.76 ± 0.18 * 4.13 ± 0.17 LV Mass (mg) 823.3 ± 67.9 992.0 ± 43.4 * 872.5 ± 22.5 RWT 0.49 ± 0.03 0.65 ± 0.03 ** .sup.†† 0.46 ± 0.02 LV Vol; d (μl) 240.8 ± 21.5 232.1 ± 13.8 288.5 ± 15.4 LV Vol; s (μl) 54.6 ± 4.6 41.7 ± 7.4 59.44 ± 6.2 IVS; d (mm) 1.73 ± 0.03 1.89 ± 0.04 * .sup.†† 1.67 ± 0.03 IVS; s (mm) 3.05 ± 0.05 3.31 ± 0.13 3.12 ± 0.10 LVPW; d (mm) 1.75 ± 0.10 2.16 ± 0.08 ** .sup.†† 1.67 ± 0.04 LVPW; s (mm) 3.02 ± 0.07 3.39 ± 0.70 * .sup.†† 2.81 ± 0.08 Legend SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; BW = body weight; HW = heart weight; EF = ejection fraction; FS = fractional shortening; E/A = ratio E wave to A wave; LA = left atrium diameter; LV Mass = left ventricular mass; RWT = relative wall thickness; LV Vol; d = left ventricular volume (diastole); LV Vol; s = left ventricular volume (systole); IVS; d = inter-ventricular septum (diastole); IVS; s = inter-ventricular septum (systole); LVPW; d = left ventricular posterior wall (diastole); and LVPW; s = left ventricular posterior wall (systole). * p < 0.05 or ** p < 0.01 for difference from NT .sup.† p < 0.05 or .sup.†† p < 0.01 for difference from HT + proANP.sub.31-67
Discussion
[0156] The lower, 25 ng/kg/day dosage of proANP.sub.31-67 was found not to display the protective effects on renal function seen for the higher dosages used in Example 1. However, the same cardioprotective effects were seen, demonstrating that the cardioprotective effects of proANP.sub.31-67 are independent of its renal activity.