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.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH2-terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3−/−) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3−/− mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3+/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3−/− TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3−/− TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

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.

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Brain mechanisms underpinning attention deficit/hyperactivity disorder (ADHD) are incompletely understood. The adolescent spontaneously hypertensive rat (SHR) is a widely studied preclinical model that expresses several of the key behavioral features associated with ADHD. Yet, little is known about large-scale functional connectivity patterns in the SHR, and their potential similarity to those of humans with ADHD. Using an approach comparable to human studies, magnetic resonance imaging in the awake animal was performed to identify whole-brain intrinsic neural connectivity patterns. An independent components analysis of resting-state functional connectivity demonstrated many common components between the SHR and both Wistar Kyoto and Sprague-Dawley control strains, but there was a divergence in other networks. In the SHR, three functional networks involving the striatum had only weak correlations with networks in the two control strains. Conversely, networks involving the visual cortex that was present in both control strains had only weak correlations with networks in the SHR. The implication is that the patterns of brain activity differ between the SHR and the other strains, suggesting that brain connectivity patterns in this animal model of ADHD may provide insights into the neural basis of ADHD. Brain connectivity patterns might also serve to identify brain circuits that could be targeted for the manipulation and evaluation of potential therapeutic options.

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Introduction: It is unknown how the timing between doses might affect nicotine's impact on neural activity. Our objective was to examine how the interdose interval affects nicotine's impact on resting-state functional connectivity (rsFC). Materials and Methods: Adult male Sprague-Dawley rats were administered nicotine daily (0.4 mg/ kg) over 6 days while control animals received saline vehicle. Functional magnetic resonance imaging was used to measure rsFC before and after a challenge dose of nicotine (0.4 mg/kg) delivered for the first time and 3, 6, 12, or 24 hr after the previous dose. Results: As the interval between nicotine doses increased from 3 to 24 hr, the strength of rsFC increased in some circuits, particularly the nucleus accumbens and prefrontal circuits, and decreased in others, namely the interpeduncular nucleus, hippocampus, caudoputamen, retrosplenial cortex, ventral tegmental, and the insular circuits. Conclusions: These data indicate that the effect that nicotine has on the brain is affected by the amount of time that has passed since the previous dose. The effect on rsFC of cumulative doses is not additive. This may have important implications for the study of nicotine addiction as it implies that the same dose of nicotine might have a different impact on the brain depending on the time elapsed from the previous exposure.