Original Paper – PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy: https://advances.sciencemag.org/content/5/5/eaaw5870
Heart disease is the leading cause of death in the United States, and has been for some time. A significant number of these deaths involve heart failure (HF), a clinical condition in which the heart is unable to pump enough blood to keep up with the body’s needs. Heart failure may be categorized into two types according to the heart’s ejection fraction (the percentage of blood out of the total volume of blood in the left ventricle that is pumped out with each contraction): heart failure with reduced ejection fraction (HFrEF), characterized by an ejection fraction of less than 40%, and heart failure with preserved ejection fraction (HFpEF), characterized by normal ejection fraction (50%-70%; 41%-49% considered borderline), with an unhealthy reduction in overall blood volume in the left ventricle. In the United States, about one half of heart failure cases are HFpEF, though the ratio of HFpEF to HFrEF cases has begun to increase, and the former is soon expected to account for the majority of cases. Although significant advancements in treatment for HFrEF have lowered mortality in recent decades, there is still no effective pharmacological treatment for HFpEF, representing a significant unmet medical need.1
Cardiac proteinopathy is a general category of diseases caused by increased misfolded proteins, and as a result increased proteotoxic stress (IPTS), in heart muscle cells. It is the job of the proteasome to degrade these misfolded proteins to maintain proteostasis and prevent proteotoxic stress. Targeted degradation by the proteasome occurs after the misfolded protein is bound by ubiquitin by a series of co-enzymes. Multiple ubiquitin units are subsequently added, and the 26S proteasome detects and degrades the poly-ubiquitinated protein, preventing them from aggregating. Past studies have implicated proteasome functional insufficiency in cardiomyopathy and progression to heart failure, highlighting proteasomal regulation as a potential target for treatment.2,4
A previous study by contributor Alfred Goldberg, it was shown that post-translational modification of the 26S proteasome acts as a means of regulation, specifically through the phosphorylation of various subunits, some of which stimulated increased proteasomal activity. Misfolded protein aggregates act as inhibitors to proteasomal activity, making this a promising find in relation to proteostasis maintenance.5 In a later study, Goldberg et al. specifically identified a pathway for proteasome activation by cAMP, via the phosphorylation of the Rpn6 subunit by Protein Kinase A (PKA).6 Author Xuejun Wang identified a similar pathway for proteasomal stimulation by cGMP/PKG.7 These protein kinases are dependent on their respective cyclic nucleotides to function.
Phosphodiesterase (PDE) is a family of enzymes that break phosphodiester bonds. It is the function of a specific cyclic nucleotide PDE, PDE1, to convert cAMP and cGMP to AMP and GMP respectively, thereby playing an important role in signaling regulation. By breaking down the cyclic nucleotides, PDE1 also effectively inhibits PKA and PKG, and thus their ability to stimulate proteasome activity. Knight and Yan identified PDE1 inhibition as a potential therapy for heart diseases.8 In this current study, the authors demonstrate the effectiveness of PDE1 inhibition in treating HFpEF in mice by upregulating proteasomal activity via increased activation of PKA and PKG.
In this study, the authors demonstrate that PDE1 inhibition exhibits a protective effect against cardiac proteinopathy and IPTS, and show that this is done via enhanced proteasome activity in a PKG and PKA dependent manner. The experiments utilized a transgenic mouse model of cardiac proteinopathy, induced by the expression of CryABR120G (mutant CryAB), a misfolded mutant form of the protein CryAB known to cause human disease.1
The authors first show that PDE1 protein levels were significantly increased in mutant CryAB mice compared to wild type (healthy) CryAB mice and non-transgenic (NTG; no CryAB) mice. They also demonstrate the ability of PDE1 inhibition to stimulate the ubiquitin-proteasome system, using a green fluorescent protein, GFPdgn. Mice treated with IC86430, a PDE1 inhibitor, showed 40% lower GFPdgn levels than control mice. GFPdgn mRNA levels remained comparable between both groups however, suggesting that the reduced protein levels seen in experimental mice is due to post-transcriptional action.
Following this, the authors proceed to show the therapeutic effects of PDE1 inhibition on HFpEF. In this experiment, 134 mutant CryAB mice were randomly assigned to two groups, one receiving PDE1 inhibition via IC86430, and the other receiving unaltered vehicle as a control. As expected of mutant CryAB mice, both groups displayed signs of HFpEF prior to treatment. Following the treatment period however, heart malfunction in PDE1-inhibited mice was significantly reversed, with some functions returning to normal expected NTG levels, while the control group did not show the same improvement. Notably, during the 4-week treatment period as well as the 6 weeks following the end of treatment, none of the PDE1-inhibited mice died, while during that same period, 50% of control group mice did die. These results support the ability of PDE1 inhibition to treat cardiac dysfunction, and also potentially implicates the protective effect of PDE1 inhibition as a long term one.
Experiments were also performed to support the mechanism of the therapeutic effect of PDE1 inhibition, that being the stimulation of proteasome activity by PKG and PKA, following their activation by cGMP and cAMP. After showing that PDE1 inhibition stimulates proteasomal degradation of GFPdgn, a general surrogate substrate of the proteasome, the authors wanted to show that the proteasome would target the disease-causing misfolded mutant CryAB specifically, which they confirmed utilizing a novel protein assay that can distinguish proteasomal degradation of misfolded proteins from other proteins undergoing general turnover. The mutant CryAB mice expectedly showed significantly higher levels of mutant CryAB protein, while NTG mice showed none. PDE1 inhibition significantly lowered mutant CryAB protein levels in mutant mice. Authors also found in this experiment a significant increase in Ser14 phosphorylated Rpn6 subunits in PDE1 inhibited mice, which previous studies have confirmed to occur through PKA. These findings support the proposition that PDE1 inhibition leads to increased levels of PKA, which in turn leads to increased phosphorylation of Rpn6 and thus increased proteasome activity, degrading misfolded proteins in the heart. Finally, the authors demonstrate that this process is dependent on PKA and PKG. Using another green fluorescent protein, GFPu, as the proteasomal substrate, the authors treated mice with a PKG inhibitor (KT5823) and a PKA inhibitor (H89) in addition to the PDE1 inhibitor. Inhibiting PKG and PKA significantly reduced the protective effect of PDE1 inhibition, indicating that this pathway is mediated by PKG and PKA.
The findings of this paper uncover a new, potentially highly effective and longlasting treatment for heart failure, and in particular, HFpEF, a category of heart failure for which there are currently few therapeutic options. Future research can further explore the ability of PDE1 inhibition to treat cardiac proteinopathy by varying the onset and length of treatment; the beginning of treatment in this study was at the onset of the disease, and the authors predict an even greater effect if treatment is started earlier or longer lasting. Because this study only uses IC86430, similar studies might also be held with other PDE1 inhibitors to determine which might display the most efficacy. This study relates to our course in highlighting the extreme importance of the ubiquitin-proteasome system in preserving normal cell function, as well as the importance of protein quality control in disease prevention.
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