Mechanistic dissection of global proteomic changes in rats with heart failure and preserved ejection fraction

Abstract

Introduction: Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, pulmonary congestion and exercise intolerance. Previous preclinical studies show that treatment with cardiosphere-derived cells (CDCs) improves diastolic function, attenuates arrhythmias and prolongs survival in a rat HFpEF model. Here we characterize the myocardial proteome and diastolic function in HFpEF, with and without CDC therapy. As an initial strategy for identifying pathways worthy of further mechanistic dissection, we correlated CDC-responsive proteomic changes with functional improvements. Methods and Results: Dahl salt-sensitive rats fed high-salt diet, with verified diastolic dysfunction, were randomly assigned to intracoronary CDCs or placebo. Dahl rats fed a low salt diet served as controls. Phenotyping was by echocardiography (E/A ratio) and invasive hemodynamic monitoring (time constant of relaxation Tau, and left ventricular end-diastolic pressure [LVEDP]). CDC treatment improved diastolic function as indicated by a normalized E/A ratio, a 33.3% reduction in Tau, and a 47% reduction of LVEDP. Mass spectrometry of left ventricular tissues (n=6/group) revealed changes in transcription and translation pathways in this rat HFpEF model and was also recapitulated in human HFpEF. These pathways were enhanced following CDC treatment in the animal model (205 proteins and 32 phosphorylated residues accounting for 37% and 19% of all changes, respectively). Among all CDC-sensitive pathways, 65% can be linked to at least 1 of 7 upstream regulators, among which several are of potential relevance for regulating protein expression. To probe newly-synthesized proteins AHA labeling was carried out in isolated rat cardiomyocytes obtained from HFpEF groups, with and without CDC therapy. Five of the initial upstream regulators (HNF4A, MTOR, MYC, TGFβ1, and TP53) were linked to proteins expressed exclusively (or increased) with CDC treatment. All 32 phosphorylated residues of proteins involved in transcription/translation altered specifically by CDC treatment had predicted kinases (Protein kinase C (PKC) being the most dominant) and known to be regulated by MYC, TGFβ1 and/or TP53. Western blot analysis of those 5 upstream regulators showed that TGFβ1, TP53, and Myc were significantly decreased in LV from CDC treated animals, whereas MTOR and HNF4A showed a significant increase compared to HFpEF alone. The cellular quantities of several upstream regulator correlated with indices of diastolic function (E/A ratio, Tau and/or LVEDP). Since CDCs act via the secretion of exosomes laden with signaling cargo, it is relevant that all 7 upstream regulators could, in principle, be regulated by proteins or miRNA that are present in CDC-derived exosomes. Conclusion: We identified key cellular regulators of transcription and translation that underlie the therapeutic effects of CDCs in HFpEF, whose levels correlate quantitatively with measures of diastolic function. Among the multifarious proteomic changes associated with rat model of HFpEF which were also observed in human HFpEF samples, we propose that these regulators, and downstream effector kinases, be prioritized for further dedicated mechanistic dissection.

Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure

“…It is of interest if these phosphorylation events driven by PKC correlate with specific cardiac dysfunctions known to HFpEF and if those functional parameters are reversing in correlation with PKC concentrations following CDC treatment. The cellular quantity of each PKC isoforms correlated negatively with E/A ratio suggesting that the increased PKC concentrations are driving, at least in part, the reduced E/A ratio that is observed in HFpEF 31 and is supported by the reduction of PKC isoform concentrations following CDC treatment correlating to an increase in E/A ratio post-CDC treatment. Since LVEDP pressure specifically correlated with PKCβ suggests that concentration changes of this isoform could have the strongest impact on cardiac function in HFpEF and a reduction of PKCβ could be key to reduce diastolic pressure in HFpEF.…”

“…To address the potential importance of particular PKC isoforms to specific heart functions, we correlated the cellular concentration and phosphorylation status of each PKC isoform (based on WB) for each individual animal with the functional data for E/A ratio, Tau, and/or LVEDP obtained for the same corresponding animal (functional data is listed in Soetkamp et al) 31 (Figure 6). Although PKCα, PKCβ, and PKCδ cellular concentrations were each negatively correlating with the E/A ratio (R 2 =−0.233, −0.28, and −0.718, respectively), the PKCδ isoform was highly correlative to the E/A ratio.…”

“…To address the potential importance of particular PKC isoforms to specific heart functions, we correlated the cellular concentration and phosphorylation status of each PKC isoform (based on WB) for each individual animal with the functional data for E/A ratio, Tau, and/ or LVEDP obtained for the same corresponding animal (functional data is listed in Soetkamp et al) 31 (Figure 6). Taken together, ≈30% of the myofilament and myofilament regulating proteins within PKC consensus sequences were affected downstream by PKC manipulations (Table IIIA through IIIC in the Supplemental Material).…”