Date of Award

5-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

First Advisor

Frank Christopher Howarth

Second Advisor

Sandeep Subramanya

Third Advisor

Saadeh Suleiman

Abstract

Diabetes mellitus is one of the most common endocrine disorders and its global prevalence is increasing at an alarming rate. Cardiovascular disease is the major cause of morbidity and mortality among diabetic patients. In addition to defects in contraction, the diabetic heart also frequently suffers from disturbances to the electrical conduction system. This study investigates the effects of diabetes on the sinoatrial node (SAN) of the rat heart. Previous in vivo and in vitro experiments have demonstrated reduced heart rate, longer SAN action potential duration, prolonged pacemaker cycle length and Sino-atrial conduction time in streptozotocin (STZ) - induced diabetic rat heart. Thus it is of paramount importance to identify the molecular basis of electrical disturbances in SAN of diabetic heart. The hypothesis is that alterations in mRNA encoding various proteins associated with the generation and transmission of electrical signals in the heart may be responsible for the functional disturbances in the SAN of diabetic heart. Experiments were performed in male Wistar rats 10-12 weeks of age after STZ treatment with a single intraperitoneal injection (60 mg/kg body weight). Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of cardiac proteins, involved in electrical transmission, were measured in SAN and right atrial biopsies using real time reverse transcription polymerase reaction technique. Expression of proteins in SAN biopsies was measured using sodium dodecyl sulphate polyacramide gel electrophoresis and Western blotting technique. Ultrastructure of the STZ-SAN was studied using transmission electron microscopy. The heart rate was significantly lower in STZ compared to controls. Of the 85 genes studied, there were some changes of particular interest. These include an increase in the expression of Gja7 (connexin), Cacna1g, Cacna1h, Cacnb3 (calcium channel), Nppa, Nppb (natriuretic peptide) Kcnj5 and Kcnk3 (potassium channel), in STZSAN. There was also decrease in the expression of Scn7a (sodium channel), Kcna2 (potassium channel), a 6-fold decrease in Kcnd2 and a 7-fold decrease in Cacng4. Some of the proteins encoded by these genes showed similar changes in expression including Ryr3, Cav3.1 and Kv4.2 while others like Pro-ANP and BNP did not. The ultrastructure of STZ-SAN had reduced mitochondria. These changes in gene and protein expression may contribute to the electrophysiological disturbances seen in the diabetic SAN. Further studies will be required to investigate whether these changes in mRNA and protein translate into changes in electrophysiological function.

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