The rise of type 2 diabetes (T2DM) increases the risk of diabetic cardiomyopathy. Worsened cardiovascular outcomes have been clinically observed in female diabetic patients; however, there is a lack of sex specific treatments for diabetes associated heart complications. Caveolins are integral membrane proteins expressed in caveolae and mitochondria that act as cellular signaling platforms to regulate stress responses. The cardiac-specific caveolin-3 overexpression (Cav3 OE) has been shown to protect the heart from morphological changes induced by stressors and preserve mitochondrial function, but this has not yet been shown in a diabetic heart. We tested the rationale that Cav3 will protect the heart from diabetes-induced metabolic injury and be especially advantageous for females. Mid-aged male and female Cav3 OE mice and transgene-negative (Tg-Neg) littermates were either injected with streptozotocin (STZ) and fed a 60% kcal high-fat diet (HFD) to induce diabetes or injected with citrate buffer and fed a 4% kcal low-fat diet for controls. STZ/HFD significantly increased weight and impaired glucose tolerance of male and female mice, with worsened glucose tolerance in males. Tg-Neg T2DM mice presented cardiac hypertrophy in males and females, mitochondrial dysfunction in females, diastolic dysfunction in males, and cardiac mitochondrial disarray in males and females. Meanwhile, Cav3 OE prevented these incidences in T2DM mice. Therefore, Cav3 may be a novel target to protect diabetic mice, especially females, from cardiac complications.

As the rate of Type II Diabetes Mellitus (T2DM) develops not only in adults but in youth, many other health related problems are starting to arise in association. A major complication related to T2DM is cardiovascular disease, which female diabetic patients have been found to have a worsened outcome compared to male diabetic patients. To get a better understanding of the sex difference in cardiovascular disease and other complications associated with T2DM, mitochondrial function was investigated to find if it was an underlying cause and if there was a difference between males and females. Through an obesity driven T2DM model, young female and male mice were fed a 45% kcal fat diet (HFD) to induce diabetes and a 10% kcal fat diet (LFD) as control. Males on HFD significantly increased weight with a more profound impairment to glucose tolerance, while females on HFD slowly increased weight, developed ulcerative dermatitis (UD), and did not have as profound impairment to glucose tolerance. There was also a statistically significant three-way interaction between mitochondrial function, gender and diet in the hippocampus and kidney. A two-way interaction between mitochondrial function and diet was found to be statistically significant in the heart, but a statistically significant interaction between mitochondrial function and gender was found in the hippocampus, liver and pancreas.

Heart failure is a leading cause of human morbidity and mortality and has been linked to neurohormonal dysregulation of β-adrenergic receptor (βAR) expression and responsivity that occurs with advancing age. No overarching mechanism for this age-dependent loss in βAR response has been identified. However, βARs localize to caveolae, membrane lipid raft microdomains formed by caveolin (Cav) proteins and expression of caveolin-3 (Cav3), the main Cav in caveolae of cardiac myocytes (CMs) decreases in an age-dependent manner. Therefore, aging-related reduction of Cav3 and caveolae may influence the loss of βAR response, and increased Cav3 expression may alter the βAR responsivity of CMs and preserve βAR function into old age.

The studies presented in this dissertation were designed to understand the influence of Cav3 on the βAR signaling pathway. In chapter 1, we reviewed current knowledge of the biogenesis and degradation of Cavs and caveolae, described their composition and roles in compartmentalization of lipid and protein factors of cellular physiology, and identified unanswered questions informed by the state-of-the-art and human disease.

In chapter 2, we tested the hypothesis that CM-specific Cav3 overexpression (Cav3 OE) would modify βAR responsivity in young and aged hearts. We measured the physiological sequelae of activation of βARs in young and aged hearts with the synthetic catecholamine isoproterenol (Iso). We found significant amplification of Iso dose-induced increases in physiological contractility and relaxation of young Cav3 OE hearts, which also exhibited increased responses into old age.

In chapter 3, we tested the hypothesis that Cav3 OE increases caveolar compartmentation of βARs and/or downstream effectors of the βAR response and that the enhanced Cav3 OE response to Iso is mediated by β1ARs or β2ARs. Cav3 OE hearts did not demonstrate enhancement in targeted adenylyl cyclase- or βAR isoform-specific responses. However, we discovered that βAR-dependent cAMP production with Iso is subject to increased regulation by phosphodiesterase activity in Cav3 OE CMs, implicating Cav3 in differential regulation of βAR signals without significant changes in protein distribution.

Our findings from the studies in this dissertation support the hypothesis that Cav3 OE amplifies βAR function in young hearts and preserves responsivity in aged animals. These results are the first to demonstrate that Cav3 OE is protective against loss of βAR responsivity in aging hearts.

cAMP-dependent protein kinase (PKAc) is a pivotal cell signaling protein in eukaryotes. The catalytic activity of this enzyme is in part controlled by four functionally non-redundant regulatory subunit proteins of PKA (RIα, RIβ, RIIα and RIIβ), which bind PKAc in tetrameric “holoenzyme” complexes to maintain kinase inactivity until stimulation with cyclic-AMP (cAMP). The allosteric activation mechanism triggered by cAMP binding to R-subunits is well understood. However, unique biochemical features and subcellular localizations of RIα and other PKA proteins in the heart may serve to activate PKAc via non-canonical, cAMP-independent mechanisms. The aim of this work was to examine the role of 1) disulfide bond oxidation at the dimerization/docking (D/D) domain of RIα, 2) localization of PKA proteins at mitochondria in the heart, and 3) phosphorylation of RIα by cGMP-dependent protein kinase (PKG) at the linker region. It was hypothesized that these mechanisms result in enhancement of PKAc activity through decreased regulation by RIα.

First, the effect of cardiac ischemia/reperfusion (I/R) injury on RIα protein expression, oxidation, and regulation of PKAc activity was assessed. Results show that RIα is decreased in expression upon extended time periods of reperfusion injury. Analysis in cardiomyocyte cultures showed that oxidant stress alone is sufficient to modify and down-regulate RIα protein, while simultaneously triggering PKAc activity. Furthermore, overexpression of RIα increases apoptosis in oxidant stressed cells. In correlation with these results, PKA proteins are specifically enriched within subsarcolemmal mitochondria (SSM) of the heart, wherein the expression of RIα is specifically decreased with I/R stress. In our study of RIα phosphorylation, we validated that PKG targets Ser101 in RIα under both in vitro and in cell conditions. Phospho-mimetic mutation of Ser101 (S101E) displayed enhanced PKAc activity without cAMP while maintaining holoenzyme complex formation.

Taken together, these findings support the notion that Type I holoenzymes can be alternatively activated by means other than classical cAMP-mediated processes. Future experiments will be aimed at delineating whether these mechanisms can work synergistically within the context of oxidative stress. The implications of this work translate to a better understanding of the role PKA signaling in heart disease.