The Muscle Ankyrin Repeat Proteins CARP, Ankrd2, and DARP Are Not Essential for Normal Cardiac Development and Function at Basal Conditions and in Response to Pressure Overload

Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload

Nebulette knockout mice have normal cardiac function, but show Z-line widening and up-regulation of cardiac stress markers

Aims: Nebulette is a 109 kDa modular protein localized in the sarcomeric Z-line of the heart. In vitro studies have suggested a role of nebulette in stabilizing the thin filament, and missense mutations in the nebulette gene were recently shown to be causative for dilated cardiomyopathy and endocardial fibroelastosis in human and mice. However, the role of nebulette in vivo has remained elusive. To provide insights into the function of nebulette in vivo, we generated and studied nebulette-deficient (nebl-/-) mice. Methods and results: Nebl-/- mice were generated by replacement of exon 1 by Cre under the control of the endogenous nebulette promoter, allowing for lineage analysis using the ROSA26 Cre reporter strain. This revealed specific expression of nebulette in the heart, consistent with in situ hybridization results. Nebl-/- mice exhibited normal cardiac function both under basal conditions and in response to transaortic constriction as assessed by echocardiography and haemodynamic analyses. Furthermore, histological, IF, and western blot analysis showed no cardiac abnormalities in nebl-/- mice up to 8 months of age. In contrast, transmission electron microscopy showed Z-line widening starting from 5 months of age, suggesting that nebulette is important for the integrity of the Z-line. Furthermore, up-regulation of cardiac stress responsive genes suggests the presence of chronic cardiac stress in nebl-/- mice. Conclusion: Nebulette is dispensable for normal cardiac function, although Z-line widening and up-regulation of cardiac stress markers were found in nebl-/- heart. These results suggest that the nebulette disease causing mutations have dominant gain-of-function effects

Aortic pathology from protein kinase G activation is prevented by an antioxidant vitamin B12 analog.

People heterozygous for an activating mutation in protein kinase G1 (PRKG1, p.Arg177Gln) develop thoracic aortic aneurysms and dissections (TAAD) as young adults. Here we report that mice heterozygous for the mutation have a three-fold increase in basal protein kinase G (PKG) activity, and develop age-dependent aortic dilation. Prkg1R177Q/+ aortas show increased smooth muscle cell apoptosis, elastin fiber breaks, and oxidative stress compared to aortas from wild type littermates. Transverse aortic constriction (TAC)-to increase wall stress in the ascending aorta-induces severe aortic pathology and mortality from aortic rupture in young mutant mice. The free radical-neutralizing vitamin B12-analog cobinamide completely prevents age-related aortic wall degeneration, and the unrelated anti-oxidant N-acetylcysteine ameliorates TAC-induced pathology. Thus, increased basal PKG activity induces oxidative stress in the aorta, raising concern about the widespread clinical use of PKG-activating drugs. Cobinamide could be a treatment for aortic aneurysms where oxidative stress contributes to the disease, including Marfan syndrome

Delta-Sarcoglycan Gene Therapy Halts Progression of Cardiac Dysfunction, Improves Respiratory Failure, and Prolongs Life in Myopathic Hamsters

The BIO14.6 hamster provides a useful model of hereditary cardiomyopathies and muscular dystrophy. Previous delta-sarcoglycan (δSG) gene therapy (GT) studies were limited to neonatal and young adult animals, and prevented the development of cardiac and skeletal muscle dysfunction. GT of a pseudo-phosphorylated mutant of phospholamban (S16EPLN) moderately alleviated the progression of cardiomyopathy

Increased Infarct Wall Thickness by a Bio-Inert Material Is Insufficient to Prevent Negative Left Ventricular Remodeling after Myocardial Infarction

Several injectable materials have been shown to preserve or improve cardiac function as well as prevent or slow left ventricular (LV) remodeling post-myocardial infarction (MI). However, it is unclear as to whether it is the structural support or the bioactivity of these polymers that lead to beneficial effects. Herein, we examine how passive structural enhancement of the LV wall by an increase in wall thickness affects cardiac function post-MI using a bio-inert, non-degradable synthetic polymer in an effort to better understand the mechanisms by which injectable materials affect LV remodeling.Poly(ethylene glycol) (PEG) gels of storage modulus G' = 0.5±0.1 kPa were injected and polymerized in situ one week after total occlusion of the left coronary artery in female Sprague Dawley rats. The animals were imaged using magnetic resonance imaging (MRI) at 7±1 day(s) post-MI as a baseline and again post-injection 49±4 days after MI. Infarct wall thickness was statistically increased in PEG gel injected vs. control animals (p<0.01). However, animals in the polymer and control groups showed decreases in cardiac function in terms of end diastolic volume, end systolic volume and ejection fraction compared to baseline (p<0.01). The cellular response to injection was also similar in both groups.The results of this study demonstrate that passive structural reinforcement alone was insufficient to prevent post-MI remodeling, suggesting that bioactivity and/or cell infiltration due to degradation of injectable materials are likely playing a key role in the preservation of cardiac function, thus providing a deeper understanding of the influencing properties of biomaterials necessary to prevent post-MI negative remodeling

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Dietary linoleic acid supplementation fails to rescue established cardiomyopathy in Barth syndrome

Barth syndrome (BTHS) is a mitochondrial lipid disorder caused by mutations in TAFAZZIN (TAZ), required for cardiolipin (CL) remodeling. Cardiomyopathy is a major clinical feature, with no curative therapy. Linoleic acid (LA) supplementation is proposed to ameliorate BTHS cardiomyopathy by enhancing linoleoyl group incorporation into CL. While the beneficial effect of dietary LA supplementation in delaying the development of BTHS cardiomyopathy has been recently tested, its potential to reverse established BTHS cardiomyopathy remains unclear. Our study revealed that LA supplementation cannot rescue established BTHS cardiomyopathy in mice, highlighting the importance of early initiation of LA supplementation for maximum benefits