The onset of volume overload (VO) sets in motion a sequence of biochemical and inflammatory events in the cardiac interstitial fluid (ISF) space that interact with cell surface molecules to dictate extracellular matrix (ECM) turnover with subsequent maladaptive left ventricular (LV) remodeling. In response to pressure overload there is an increase ECM production while in the pure VO there is a decrease in ECM. The molecular mechanisms that control ECM response in VO are not understood. Currently, therapy with angiotensin-converting enzyme (ACE) inhibitors, which decreases angiotensin II and increases bradykinin (BK), is ineffective in a pathophysiologic LV VO (1). Therefore, the purpose of the current proposal is to understand the mechanisms of ECM loss in order to prevent progressive LV remodeling and failure in VO. We have shown that the acute VO of aortocaval fistula (ACF) in the rat has increased ISF BK (2). BK through activation of the kallikrein/kinin system and BK2 receptor prevents fibrosis and decreases blood pressure in cardiovascular and renal disease (3). In acute VO, BK2 receptor antagonism prevents ECM loss, mast cell (MC) accumulation and LV dilatation (2, 4). However, there was an increase in mean arterial pressure, which precludes long term therapy with BK2 antagonism in VO. In addition to its antifibrotic effect, BK is chemotactic for inflammatory cells, in particular mast cells (MCs), which are the source of the serine protease chymase. We have shown MCs have BK2 receptors and direct BK interstitial infusion causes MC degranulation and chymase release into the ISF space in wild type mice in vivo. Besides its Ang II-forming capacity, chymase activates kallikrein which can further increase ISF BK (5). Kallikrein and chymase also activate matrix metallo- proteinases (MMPs) (6) and directly degrade fibronectin (7), an important component of the focal adhesion complex that controls cell growth and survival. ISF BK and kallikrein gene expression, as well as increased MCs and chymase activity, are increased at both early and late stages of VO, suggesting cardiac BK formation in response to VO. Taken together, our data suggest a vicious cycle of local production of cardiac kallikrein and BK, which in turn, stimulates release of MC-chymase and activates kallikrein, thus perpetuating the increase in cardiac BK in VO. Thus, we hypothesize that the pure stretch of VO induces cardiac kallikrein/kinin production that produces both a decrease in ECM synthesis and increase MC degranulation. Kallikrein inhibition and downregulation will decrease ISF BK formation and MC chymase release and further prevent MMP activation, resulting in improved LV remodeling and function in rat with the VO of ACF.
Aim 1 Determine whether kallikrein activation mediates ECM loss in the heart through MC activation. Using in vivo microdialysis, we have shown that interstitial infusion of BK in the normal rat is associated with increase in MCs and ECM loss. We will determine whether kallikrein infusion induces BK2 receptor activation and subsequently stimulates MC-chymase release and mediates ECM loss. To define the role of mast cell chymase, we will treat mast cell deficient rats with ISF BK infusion or kallikrein infusion in the normal rat heart and in the rat heart subjected to VO with ACF.
Aim 2 Determine whether cardiomyocytes or fibroblasts are the source of increased kallikrein expression and BK release in response to stretch in vitro and VO in vivo. To isolate the effect of stretch alone, neonatal LV myocytes and adult LV fibroblasts will be stretched to simulate VO to determine their differential ability to produce kallikrein and BK. To dissect out the direct vs. systemic effects of kallikrein inhibition (aprotinin) in VO, we will knockdown kallikrein in the acute ACF in vivo and study effects on ISF BK and chymase activity, MMP/TIMPs, ECM and non ECM proteins.
Aim 3 Determine whether inhibition of kallikrein will improve LV remodeling and function in the VO rat heart. We will study the effects of kallikrein inhibition on in vivo LV function, ECM homeostasis, ISF BK and chymase activity, and mast cell degranulation in response to VO in the early and chronic time points.
Project Narrative In the United States, heart failure affects more than 4 million people, who display 6-year mortality rates of greater than 65% once failure develops. The number of affected individuals is expected to rise significantly in our aging population over the next 30 years. Heart failure has many causes, congenital defects and infections induced rheumatic fever, are among the most common symptoms lead to heart valve disease. Our studies will provide novel mechanistic insights into the differential adaptive responses of the circulating versus tissue specific kallikrein/kinin generating mechanism in the evolution of cardiovascular disease and the impact of targeted bradykinin therapy on such changes. Our current proposal will uncover new targets that lead to improved therapeutic strategies for the management of patients with the volume overload of aortic regurgitation and mitral regurgitation which constitutes a large portion of our Veteran patient population.