Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor is Facilitated by Dual Conjugation with Myristic Acid and Trans Activator of Transcription to Attenuate Myocardial Ischemia/Reperfusion Injury
Location
Philadelphia, PA
Start Date
11-5-2022 1:00 PM
End Date
11-5-2022 4:00 PM
Description
INTRODUCTION: Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 µM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration.
OBJECTIVES: The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury.
METHODS: Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min). Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis.
RESULTS: Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05).
CONCLUSION: The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts in a porcine myocardial I/R model.
Embargo Period
5-26-2022
Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor is Facilitated by Dual Conjugation with Myristic Acid and Trans Activator of Transcription to Attenuate Myocardial Ischemia/Reperfusion Injury
Philadelphia, PA
INTRODUCTION: Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 µM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration.
OBJECTIVES: The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury.
METHODS: Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min). Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis.
RESULTS: Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05).
CONCLUSION: The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts in a porcine myocardial I/R model.