| Aims:We aimed to explore the effect of a bioactive peptide (PAP) on angiotensin II (Ang II) stimulation induced cardiac hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models.Methods: PAP (Pak activating peptide) TSNSQKYMSFTDKSA was derived from Pakl autoinhibitory region and was linked to11-amino acid sequence YGRKKRRQRRR derived from HIV-1trans-activating regulatory protein.Neonatal rat cardiomyocytes (NRVMs) were isolated from1-2day old rats using a standard enzymatic method. Isolated NRVMs were plated onto laminin-coated coverslips. To examine the effect of PAP on angiotensin II (Ang II, Sigma-Aldrich) induced hypertrophy in NRVMs, NRVMs were treated with Ang II (500nM) accompanied with or without PAP (20μg/ml) for48h and NRVMs without treatment were taken for control. Thereafter, NRVMs were subjected to immunocytochemistry. Cardiac hypertrophy was induced by administration of Ang II at1mg/kg/day for7days using osmotic mini-pumps (Alzet) implanted subcutaneously in3-month-old male C57BL/6mice. After the treatment, the heart weight (HW) and tibia length (TL) were measured and the HW/TL ratios were calculated to indicate cardiac hypertrophy. Protein extracts (50μg) were subjected to immunoblot analysis.Immunecomplexes were detected by enhanced chemi luminescence with anti-rabbit immunoglobulin G coupled to horseradish peroxidise as the secondary antibody Ventricular tissue was frozen in OCT and10μm sections were collected using a cryostat. Sections were used for Masson’s trichrome stain and Hematoxylin and eosin (HE) Staining. To monitor cardiac rhythms we carried out in vivo ECG analysis on mice anesthetized with isoflurane (2.5%). For the programmed electrical stimulation, Mice were killed by cervical dislocation,and the heart was excised, cannulated and mounted onto a Langendorff system, then perfused To assess propensity to exhibit ventricular arrhythmias, For Ca2+transients recording,Ventricular myocytes isolated from control, Ang II or Ang II plus PAP treated hearts were incubated with fluo-4AM (10μM) for15min,then myocytes were stimulated at1Hz by carbon-fibre electrodes placed at the side of the superfusion bath. Myocytes were imaged with a confocal microscope system that consisted of a Leica TCS NT scanning head coupled to a Leica DMIRB inverted microscope with a100x oil immersion objective lens (1.2NA, Leica) in line scan mode (2.6ms per line). For calcium spark recording, myocytes were incubated and imaged with similar method as above.Results:1. PAP enhances Pakl activity in cardiac myocytes.2. PAP abrogates Ang-II induced hypertrophy in in vitro and in vivo models.3. PAP protects heart from ventricular arrhythmia associated with hypertrophy.4. PAP Attenuates Ang II-induced hypertrophic associated Ca2+sparks, waves and dyssynchronous Ca2+transientsConclusions: PAP antagonizes ventricular arrhythmias associated with Ang II induced cardiac hypertrophy in C57BL/6mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pakl activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias. |
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