Michael E Mendelsohn's research while affiliated with Beverly Hospital, Boston MA and other places

Phosphodiesterase 5 inhibition (PDE5i) activates cGMP‐dependent protein kinase (PKG) and ameliorates heart failure; however, its impact on cardiac mitochondrial regulation has not been fully determined. Here, we investigated the role of the mitochondrial regulator peroxisome proliferator‐activated receptor γ co‐activator‐1α (PGC1α) in the PDE5i‐conferred cardioprotection, utilizing PGC1α null mice. In PGC1α+/+ hearts exposed to 7 weeks of pressure‐overload by transverse aortic constriction, chronic treatment with the PDE5 inhibitor sildenafil improved cardiac function and remodeling, with improved mitochondrial respiration and upregulation of PGC1α mRNA in the myocardium. By contrast, PDE5i‐elicited benefits were abrogated in PGC1α‐/‐ hearts. In cultured cardiomyocytes, PKG overexpression induced PGC1α, while inhibition of the transcription factor CREB abrogated the PGC1α induction. Together, these results suggest that the PKG–PGC1α axis plays a pivotal role in the therapeutic efficacy of PDE5i in heart failure. The phosphodiesterase 5 (PDE5) inhibitor sildenafil replenished myocardial expression of the mitochondrial regulator PGC1α, maintained mitochondrial ATP generating capacity and reduced oxidative stress, leading to the amelioration of maladaptive remodeling in later stages of heart failure during pressure‐overload. Neither the metabolic nor the physiological benefits of sildenafil treatment were observed in hearts lacking PGC1α. Our data provide evidence for the cardioprotection conferred by the cGMP‐PKG‐PGC1α axis upon PDE5 inhibition.

Background Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. Methods Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days ( P <0.001). Mice then received daily treatment with CRD-733 (600 mg/kg per day; n=10) or vehicle (n=17), alongside sham-operated controls (n=10). Results CRD-733 treatment reversed existing LV hypertrophy compared with vehicle ( P <0.001), significantly improved LV ejection fraction ( P =0.009), and attenuated left atrial dilation ( P <0.001), as assessed by serial echocardiography. CRD-733 prevented elevations in LV end diastolic pressures ( P =0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle ( P <0.001), alongside increased phosphorylation of Ser ²⁷³ of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. Conclusions The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

Introduction: Regulator of G protein signaling 2 (RGS2) is a negative regulator of G protein-coupled signaling, and is potentiated by cGMP-PKG. Our prior work using a global deletion model demonstrated the protective role for RGS2 against mal-adaptive cardiac remodeling, which is essential to the cGMP-PDE5 inhibitor efficacy; however, cell type-specific contribution remains to be determined, given the expression of RGS2 in myocytes, fibroblasts and vascular smooth muscle cells. We hypothesized that myocyte RGS2 might serve as a primary contributor to the cardio-protection and mediates cGMP-PKG benefits. Method: We generated cardiomyocyte-specific RGS2 knockout mice (cKO) on a C57BL/6 background. Pressure overload was produced by transverse aortic constriction (TAC) in male RGS2-cKO mice (RGS2cKO) and their littermate controls (WT). To investigate cGMP-PKG enhancing effects, a soluble guanylyl cyclase stimulator (riociguat) was orally administrated daily (3mg/kg/day). Invasive hemodynamics, anatomy, histology and molecular signaling were assessed at 1wk after TAC. Results: RGS2-cKO revealed exacerbated dilative cardiac remodeling (LVDD: cKO 3.57±0.36 vs WT 2.90±0.16) and severely impaired cardiac function (dPdt/IP: cKO 166±3.49 vs WT 195±7.40), while their basal phenotype was undistinguishable from WT. cKO-TAC hearts showed larger cardiomyocyte size and more fibrosis than WT-TAC hearts, associated with aberrant gene expression induction including Myh6, Myh7, Col1a2, and TGFβ . Intriguingly, concomitant riociguat treatment reduced LV chamber size and fibrosis but failed to improve cardiac performance in cKO-TAC hearts, while riociguat ameliorated all these in WT-TAC hearts. Conlusion: These results suggest that RGS2-PKG axis in cardiac myocytes critically contributes to cardiac functional performance and that cGMP-PKG exerts anti-fibrotic effects independently of myocyte conditions.

Background Heart failure is a significant contributor to cardiovascular mortality. cGMP inhibits cardiac remodeling in part via cGMP‐dependent protein kinase G I α (PKGIα). Knock in mice with a mutant PKGIα Leucine Zipper (LZ) have both decreased cardiac function and JNK activity after transaortic constriction (TAC). JNK is regulated by MAPKs including mixed lineage kinase 3 (MLK3) which contains an LZ interacting motif. We hypothesized PKGIα interacts with MLK3 via the PKGIα LZ domain to regulate JNK. Results The MLK3‐PKGIα interaction was observed in mouse heart by co‐immunoprecipitation of MLK3 and PKGIα (n=3). In Cos1 cells MLK3 co‐precipitated with PKGIα but was prevented by mutation of the PKGIα LZ domain (n=3). In cardiomyocytes JNK stimulation by 8‐Br‐cGMP was prevented in MLK3‐depleted cells (siRNA, n=3). The role of MLK3 in the heart was tested by subjecting MLK3 knockout mice (MLK3 ‐/‐ ) to TAC (7 days). Compared to WT littermates MLK3 ‐/‐ hearts had increased hypertrophy and LV end diastolic pressure indicating advanced cardiac dysfunction (n=8‐10) Conclusion These data reveal a novel interaction between PKGIα‐MLK3 that requires the LZ domain. In cardiomyocytes MLK3 is necessary for cGMP dependent JNK activation. Loss of MLK3 in vivo promotes adverse cardiac remodeling. This work was supported by the NIH.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone's involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

We recently reported that mutation of the cGMP-dependent Protein Kinase G I alpha (PKGIα) N-terminal leucine zipper (LZ) domain (in the PKGIα LZ mutant, or LZM, mouse) accelerates LV remodeling and heart failure after TAC, and prevents the anti-remodeling effect of sildenafil. We therefore hypothesized that PKGIα attenuates remodeling by regulating cardiac signaling pathways that are dependent on substrate interactions mediated by its LZ domain. As a first step to identifying cardiac proteins downstream of PKGIα, we screened myocardial lysates for PKGIα LZ domain-interacting proteins. Our previous work revealed a requirement for the PKGIα LZ domain for the activation of anti-remodeling myocardial JNK activity after LV pressure overload. MLK3 is an MAPKKK that contains an LZ domain and activates JNK. We now demonstrate, by immunoprecipitation, that MLK 3 interacts with the PKGIα LZ domain in myocardial lysates. We show further that 8-Br-cGMP induces MLK3 phosphorylation on Threonine 277 and Serine 281 in WT, but not LZM myocardial lysates. And, in 293 cells transfected with FLAG-MLK3, 8Br-cGMP induced PKGIα-MLK3 co-precipitation, and increased phosphorylation of MLK3 on Thr277/Ser281. Co-transfection of MLK3 and PKGIα also induced MLK3 phosphorylation at the same sites. We next examined the cardiovascular effect of MLK3 deletion in vivo. Male 8 week old MLK3 -/- mice display basal bi-ventricular hypertrophy compared with littermate controls (LV/Tibia length 42.8 + 0.6 mg/cm in WT, 52.9 + 1.8 in MLK3 -/-; P <0.01; RV/TL 10.8 + 0.1 mg/cm in WT, 13.3 + 0.3 in MLK3 -/-; P <0.01; n= 7 WT, 5 MLK3 -/-). By 14-16 weeks of age, LVH progressed in the MLK3 -/- mice (LV/TL 47.7 + 1.3 mg/cm in WT, 59.8 + 7.5 in MLK3-/-; n= 6 WT, 9 MLK3-.-; P <0.01). Arterial blood pressure was modestly increased, though still normal, in the MLK3 -/- mice (SBP 93 + 1 in WT, 113 + 1 in MLK3 -/-). And, 14-16 week MLK3 -/- mice have impaired LV diastolic function (tau 3.2 + 0.1 ms WT, 3.7 + 0.1 MLK3-/-; P 0.06). Our studies reveal a previously unknown function of MLK3 as a myocardial PKGIα effector and inhibitor of LVH. Together these results support the strategy of exploring LZ-dependent PKGIα substrates in the myocardium to identify novel therapeutic targets for cardiac remodeling.

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.

Hypertensive heart disease causes significant mortality in older patients, yet there is an incomplete understanding of molecular mechanisms that regulate age-dependent hypertensive left ventricular hypertrophy (LVH). Therefore, we tested the hypothesis that the cGMP-dependent protein kinase G I alpha (PKGIα) attenuates hypertensive LVH by evaluating the cardiac phenotype in mice with selective mutations of the PKGIα leucine zipper domain. These leucine zipper mutant (LZM) mice develop basal hypertension. Compared with wild-type controls, 8-month-old adult LZM mice developed increased left ventricular end-diastolic pressure but without frank LVH. In advanced age (15 months), the LZM mice developed overt pathological LVH. These findings reveal a role of PKGIα in normally attenuating hypertensive LVH. Therefore, mutation of the PKGIα LZ domain produces a clinically relevant model for hypertensive heart disease of aging.