Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Karin Przyklenk

Abstract

Remote preconditioning is a promising and robust treatment for myocardial

ischemia/reperfusion injury that evokes cardioprotection through endogenous neural

and/or humoral signaling. A recent study has reported that protective signaling is

mediated by exosomes through the circulation; however this concept is supported by

limited and inconsistent evidence. Despite overwhelming success in preclinical studies,

the efficacy of remote preconditioning in human studies is inconclusive. Importantly, the

majority of remote preconditioning studies use healthy animal models despite growing

evidence that comorbidities, such as type-2 diabetes, may negatively influence

outcomes. Nonetheless, the efficacy of remote preconditioning in the setting of type-2

diabetes has not been investigated.

Using an established model of myocardial ischemia/reperfusion in the Zucker

model of type-2 diabetes and a model of hypoxia/reoxygenation in cultured HL-1

cardiomyocytes we tested four hypotheses:

i. remote preconditioning is ineffective in early-stage type-2 diabetes in vivo;

ii. the traditional ultracentrifugation technique for exosomes isolating is inadequate

to isolate protective factor(s) from remote preconditioning;

iii. enhanced ultracentrifugation technique for exosome isolation sequesters a

protective fraction of serum;

iv. the humoral component of remote preconditioning is defective in type-2 diabetes.

In support of Hypothesis I, we demonstrate that remote preconditioning failed to

reduce infarct size caused by ischemia/reperfusion in the Zucker model of early-stage

type-2 diabetes. Our results illustrate that the loss in efficacy is not the result of

hyperglycemia per se nor sensitization of the myocardium to ischemia/reperfusion.

Subsequently, we sought to isolate a subfraction of serum from remote preconditioned

rats which contained exosomes that could communicate protection and render HL-1

cardiomyocytes resistant to hypoxia/reoxygenation-induced cell death. In agreement

with Hypothesis II, we report that the traditional ultracentrifugation isolation technique

(100,000 xg for 2 hr) did not isolate the protective component with the exosome-rich

pellet from serum, suggesting that the protective component remained in the

supernatant. In accordance with these observations, we enhanced the

ultracentrifugation technique to improve exosome sedimentation and obtain a protective

sub-fraction of serum. In agreement with Hypothesis III, the enhanced

ultracentrifugation technique (300,000 xg for 12 hr) isolated a protective exosome-rich

supernatant fraction from remote preconditioned serum. However, our enhanced

ultracentrifugation technique also yielded an additional, exosome-rich pellet and an

exosome-depleted fraction, neither of which evoked protection. Lastly, in support of

Hypothesis IV, we demonstrate that unfractionated serum and the exosome-rich

supernatant fraction obtained from remote preconditioned diabetic Zucker Fatty rats did

not protect HL-1 cardiomyocytes from hypoxia/reoxygenation. In conclusion, our results

illustrate for the first time that the infarct-sparing efficacy of remote preconditioning is

abolished in the setting of early-stage type-2 diabetes. We demonstrate that exosomes,

although not sufficient for protection, may be requisite in the humoral component of

remote preconditioning. Finally, we report that the humoral component of remote

preconditioning is defective in the setting of type-2 diabetes – a defect that may

contribute to the failure of remote conditioning to limit infarct size in this comorbid

model.

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