Cardiovascular disorders remain the first cause of mortality in US, with myocardial infarction (MI) and subsequent heart failure as the major sequela underlying such lethality. Recent studies have reinforced the concept that cardiac fibroblasts (FBs), which are less investigated than cardiomyocytes, are much more than simple regulators of extracellular matrix turnover; indeed, these cells (and their activated phenotype, myoFBs) seem to be key players in heart failure progression. Although relatively understudied, myocardial fibrosis remains one of the major pathophysiologic features of ischemic cardiac disorders. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular calcium release channels located on the endoplasmic reticulum (ER), the main calcium reservoir within the cell. While the role of IP3Rs in cardiomyocytes has been previously investigated, especially in the context of hypertrophy and arrhythmias, their exact function in the activation of cardiac FBs following an ischemic insult has not been explored hitherto. Human and murine FBs express all three isoforms of IP3Rs and their activity and levels are modulated by several stimuli. We hypothesize that IP3Rs play a key role in the activation of cardiac FBs in the infarcted heart and in the present proposal we will test this hypothesis in vivo, ex vivo, and in vitro. We have preliminary data showing that IP3R expression (all isoforms) is increased in cardiac FBs following MI. Moreover, genome-wide association studies revealed a significant association between IP3- mediated signal transduction pathways and ischemic heart disease. However, definitive functional studies examining the mechanistic role of IP3Rs in cardiac fibrosis in vivo are missing. Using a Cre/lox recombination technique, we generated a novel mouse model (IP3RKO) in which IP3Rs are ablated in activated cardiac myoFBs. This model provides an exquisite tool to evaluate the functional contribution of IP3R to cardiac fibrosis and allows us to overcome the difficulties encountered following the knockdown or KO of a single IP3R gene. Our preliminary studies show that following MI, IP3RKO mice display a significantly reduced fibrosis and attenuated myocardial dysfunction compared with control IP3RCre and IP3Rflox littermates. We will further assess the functional relationships between IP3Rs and cardiac fibrosis in primary isolated cardiac FBs at different time points and in response to established stimuli, in order to assess their proliferative, migratory, secretory, and contractile capacity. Additionally, we will examine IP3R-mediated responses in murine embryonic FBs and human cardiac FBs. To delineate the molecular mechanisms underlying the observed phenotype, we propose to explore the following calcium-mediated pathways: mitochondrial dysfunction, autophagy, ER stress, and inflammation. The proposed studies are highly significant and innovative as they will: (a) provide the first assessment of the functional role of IP3R in post-MI cardiac FBs using a specific Cre/lox KO model; (b) delineate previously unrecognized connections between IP3R and autophagy in the pathophysiology of cardiac fibrosis; (c) identify innovative therapeutic strategies that specifically target excessive post-ischemic cardiac fibrosis.
Cardiovascular disorders remain the number one cause of mortality in United States, with ischemic cardiac disease as the major sequela underlying such lethality. This is the first investigation into the functional role of intracellular calcium fluxes in cardiac fibroblasts using a specific and novel animal model in which all isoforms of a main calcium channel are deleted. Our studies, relevant to public health and to the NIH mission to discover basic mechanisms underlying human disease, suggest an unprecedented role for intracellular calcium release channels in the pathophysiology of cardiac fibrosis following myocardial infarction via pathways that involve the activation and persistence of myofibroblasts after ischemic injury.
