A novel αB-crystallin R123W variant drives hypertrophic cardiomyopathy by promoting maladaptive calcium-dependent signal transduction.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in sarcomere genes can account for a substantial proportion of familial cases of HCM, 40%-50% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYABR123W) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYABR123W promotes the HCM phenotype remains unclear. Here, we generated mice carrying the CryabR123W knock-in allele and demonstrated that hearts from these animals exhibit increased maximal elastance at young age but reduced diastolic function with aging. Upon transverse aortic constriction, mice carrying the CryabR123W allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased ejection fraction. Crossing of mice with a Mybpc3 frame-shift model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in the CryabR123W model are independent of the sarcomere. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYABR123W despite its potent effect on driving cellular hypertrophy. Mechanistically, we uncovered an unexpected protein-protein interaction between CRYAB and calcineurin. Whereas CRYAB suppresses maladaptive calcium signaling in response to pressure-overload, the R123W mutation abolished this effect and instead drove pathologic NFAT activation. Thus, our data establish the CryabR123W allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiac pathological hypertrophy.

Deficiency of miR-409-3p improves myocardial neovascularization and function through modulation of DNAJB9/p38 MAPK signaling.

Angiogenesis is critical for tissue repair following myocardial infarction (MI), which is exacerbated under insulin resistance or diabetes. MicroRNAs are regulators of angiogenesis. We examined the metabolic regulation of miR-409-3p in post-infarct angiogenesis. miR-409-3p was increased in patients with acute coronary syndrome (ACS) and in a mouse model of acute MI. In endothelial cells (ECs), miR-409-3p was induced by palmitate, while vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) decreased its expression. Overexpression of miR-409-3p decreased EC proliferation and migration in the presence of palmitate, whereas inhibition had the opposite effects. RNA sequencing (RNA-seq) profiling in ECs identified DNAJ homolog subfamily B member 9 (DNAJB9) as a target of miR-409-3p. Overexpression of miR-409-3p decreased DNAJB9 mRNA and protein expression by 47% and 31% respectively, while enriching DNAJB9 mRNA by 1.9-fold after Argonaute2 microribonucleoprotein immunoprecipitation. These effects were mediated through p38 mitogen-activated protein kinase (MAPK). Ischemia-reperfusion (I/R) injury in EC-specific miR-409-3p knockout (KO) mice (miR-409ECKO) fed a high-fat, high-sucrose diet increased isolectin B4 (53.3%), CD31 (56%), and DNAJB9 (41.5%). The left ventricular ejection fraction (EF) was improved by 28%, and the infarct area was decreased by 33.8% in miR-409ECKO compared with control mice. These findings support an important role of miR-409-3p in the angiogenic EC response to myocardial ischemia.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

Transdermal Delivery of High Molecular Weight Antibiotics to Deep Tissue Infections via Droplette Micromist Technology Device (DMTD).

Wound infection by multidrug-resistant (MDR) bacteria is a major disease burden. Systemic administration of broad-spectrum antibiotics colistin methanesulfonate (CMS) and vancomycin are the last lines of defense against deep wound infections by MDR bacteria. However, systemic administration of CMS and vancomycin are linked to life-threatening vital organ damage. Currently there are no effective topical application strategies to deliver these high molecular weight antibiotics across the stratum corneum. To overcome this difficulty, we tested if high molecular weight antibiotics delivered by Droplette micromist technology device (DMTD), a transdermal delivery device that generates a micromist capable of packaging large molecules, could attenuate deep skin tissue infections. Using green fluorescent protein-tagged E. coli and live tissue imaging, we show that (1) the extent of attenuation of deep-skin E. coli infection was similar when treated with topical DMTD- or systemic IP (intraperitoneal)-delivered CMS; (2) DMTD-delivered micromist did not spread the infection deeper; (3) topical DMTD delivery and IP delivery resulted in similar levels of vancomycin in the skin after a 2 h washout period; and (4) IP-delivered vancomycin was about 1000-fold higher in kidney and plasma than DMTD-delivered vancomycin indicating systemic toxicity. Thus, topical DMTD delivery of these antibiotics is a safe treatment for the difficult-to-treat deep skin tissue infections by MDR bacteria.

MLK3 mediates impact of PKG1α on cardiac function and controls blood pressure through separate mechanisms.

cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation after pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules that mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload and in regulating BP. In a transaortic constriction HF model, PKG activation with sildenafil preserved LV function in MLK3+/+ but not MLK3-/- littermates. MLK3 coimmunoprecipitated with PKG1α. MLK3-PKG1α cointeraction decreased in failing LVs. PKG1α phosphorylated MLK3 on Thr277/Ser281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the soluble guanylate cyclase (sGC) stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserved LV function, whereas MLK3 kinase-independent signaling regulated BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.

Mineralocorticoid Receptor in Smooth Muscle Contributes to Pressure Overload-Induced Heart Failure.

BACKGROUND: Mineralocorticoid receptor (MR) antagonists decrease heart failure (HF) hospitalization and mortality, but the mechanisms are unknown. Preclinical studies reveal that the benefits on cardiac remodeling and dysfunction are not completely explained by inhibition of MR in cardiomyocytes, fibroblasts, or endothelial cells. The role of MR in smooth muscle cells (SMCs) in HF has never been explored. METHODS: Male mice with inducible deletion of MR from SMCs (SMC-MR-knockout) and their MR-intact littermates were exposed to HF induced by 27-gauge transverse aortic constriction versus sham surgery. HF phenotypes and mechanisms were measured 4 weeks later using cardiac ultrasound, intracardiac pressure measurements, exercise testing, histology, cardiac gene expression, and leukocyte flow cytometry. RESULTS: Deletion of MR from SMC attenuated transverse aortic constriction-induced HF with statistically significant improvements in ejection fraction, cardiac stiffness, chamber dimensions, intracardiac pressure, pulmonary edema, and exercise capacity. Mechanistically, SMC-MR-knockout protected from adverse cardiac remodeling as evidenced by decreased cardiomyocyte hypertrophy and fetal gene expression, interstitial and perivascular fibrosis, and inflammatory and fibrotic gene expression. Exposure to pressure overload resulted in a statistically significant decline in cardiac capillary density and coronary flow reserve in MR-intact mice. These vascular parameters were improved in SMC-MR-knockout mice compared with MR-intact littermates exposed to transverse aortic constriction. CONCLUSIONS: These results provide a novel paradigm by which MR inhibition may be beneficial in HF by blocking MR in SMC, thereby improving cardiac blood supply in the setting of pressure overload-induced hypertrophy, which in turn mitigates the adverse cardiac remodeling that contributes to HF progression and symptoms.

CRD-733, a Novel PDE9 (Phosphodiesterase 9) Inhibitor, Reverses Pressure Overload-Induced Heart Failure.

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 Ser273 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.

Sacubitril/Valsartan Improves Left Ventricular Function in Chronic Pressure Overload Independent of Intact Cyclic Guanosine Monophosphate-dependent Protein Kinase I Alpha Signaling.

BACKGROUND: Combined angiotensin receptor/neprilysin inhibition with sacubitril/valsartan (Sac/Val) has emerged as a therapy for heart failure. The presumed mechanism of benefit is through prevention of natriuretic peptide degradation, leading to increased cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) signaling. However, the specific requirement of PKG for Sac/Val effects remains untested. METHODS AND RESULTS: We examined Sac/Val treatment in mice with mutation of the cGMP-dependent protein kinase I (PKGI)α leucine zipper domain, which is required for cGMP-PKGIα antiremodeling actions in vivo. Wild-type (WT) or PKG leucine zipper mutant (LZM) mice were exposed to 56-day left ventricular (LV) pressure overload by moderate (26G) transaortic constriction (TAC). At day 14 after TAC, mice were randomized to vehicle or Sac/Val by oral gavage. TAC induced the same degree of LV pressure overload in WT and LZM mice, which was not affected by Sac/Val. Although LZM mice, but not WT, developed LV dilation after TAC, Sac/Val improved cardiac hypertrophy and LV fractional shortening to the same degree in both the WT and LZM TAC mice. CONCLUSION: These findings indicate the beneficial effects of Sac/Val on LV structure and function in moderate pressure overload. The unexpected finding that PKGIα mutation does not abolish the Sac/Val effects on cardiac hypertrophy and on LV function suggests that signaling other than natriuretic peptide- cGMP-PKG mediates the therapeutic benefits of neprilysin inhibition in heart failure.

Distinct Phenotypes Induced by Three Degrees of Transverse Aortic Constriction in Mice.

Transverse aortic constriction (TAC) is a well-established model of pressure overload-induced cardiac hypertrophy and failure in mice. The degree of constriction "tightness" dictates the TAC severity and is determined by the gauge (G) of needle used. Though many reports use the TAC model, few studies have directly compared the range of resulting phenotypes. In this study adult male mice were randomized to receive TAC surgery with varying degrees of tightness: mild (25G), moderate (26G) or severe (27G) for 4 weeks, alongside sham-operated controls. Weekly echocardiography and terminal haemodynamic measurements determined cardiac remodelling and function. All TAC models induced significant, severity-dependent left ventricular hypertrophy and diastolic dysfunction compared to sham mice. Mice subjected to 26G TAC additionally exhibited mild systolic dysfunction and cardiac fibrosis, whereas mice in the 27G TAC group had more severe systolic and diastolic dysfunction, severe cardiac fibrosis, and were more likely to display features of heart failure, such as elevated plasma BNP. We also observed renal atrophy in 27G TAC mice, in the absence of renal structural, functional or gene expression changes. 25G, 26G and 27G TAC produced different responses in terms of cardiac structure and function. These distinct phenotypes may be useful in different preclinical settings.