AKT-mTOR Signaling-Mediated Rescue of PRKAG2 R302Q Mutant-Induced Familial Hypertrophic Cardiomyopathy by Treatment with β-AR Blocker Metoprolol

PRKAG2 cardiac syndrome, as a common form of metabolic hypertrophic cardiomyopathy (HCM) caused by mutations in PRKAG2 gene, often shows myocardial hypertrophy and abnormal glycogen deposition in cardiomyocytes. However, it remains incurable due to lacking of a management guideline for treatment. Herein, a β1-AR blocker Metoprolol was applied to 5 patients with PRKAG2 cardiac syndrome identified from a PRKAG2 R302Q mutant family, resulting in significantly postponed progression of cardiac hypertrophy. Overexpression of PRKAG2 R302Q in primary cardiomyocytes increased the activity of AMPK, induced cellular hypertrophy and glycogen storage, and promoted the phosphorylation levels of AKT-mTOR signaling. Application of either β1-AR blocker metoprolol or protein kinase A (PKA) inhibitor H89 to the cardiomyocytes rescued the HCM-like phenotypes induced by PRKAG2 R302Q, including suppression of both AKT-mTOR phosphorylation and AMPK activity. In conclusion, the current study not only determined the mechanism regulating HCM induced by PRKAG2 R302Q mutant, but also demonstrated a therapeutic strategy using β1-AR blocker to treat the patients with PRKAG2 cardiac syndrome.

Abstract P491: Myosin-7 E848G Mutation Induces P53-associated Cell Death That Leads To Impaired Tissue Contractility In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy

Introduction: Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and occasional impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood. Hypothesis: We previously identified a novel MYH7 E848G mutation associated with HCM. We hypothesize E848G induces cell death that results in impaired tissue contractility in a dose-dependent manner. Methods: We created MYH7 expressing CMs with WT/WT, E848G/WT, or E848G/E848G alleles by CRISPR/Cas9 gene-editing patient-specific induced pluripotent stem cells (hiPSCs). hiPSC-derived cardiomyocytes were metabolically purified and cocultured with stromal cells on PDMS posts to create 3D engineered heart tissues (EHTs) or cultured as a monolayer. Results: Day 65 monolayer E848G/E848G CMs had 48.5% effective cell number relative to WT/WT. p53 (2.80 ± .11-fold), p21 (7.24 ± .18-fold), and BAX (1.64 ± 0.14-fold) mRNA transcripts were upregulated in day 60 monolayer E848G/E848G relative to WT/WT. E848G/E848G EHTs (n = 12) exhibited lower maximum active twitch force (104.6 ± 18.2 μN) and smaller 2D projected area (5.31 ± 0.22 mm 2 ) at day 14 relative to WT/WT (n = 15; 238.0 ± 20.4 μN; 6.87 ± 0.26 mm 2 ). E848G/WT EHTs (n = 7) had intermediate twitch force (168.7 ± 12.2 μN) and 2D area (6.18 ± 0.36 mm 2 ). Conclusion: These results suggest the MYH7 E848G mutation induces p53-associated cell death that leads to reduced tissue contractility. Ongoing studies will elucidate the molecular mechanism through which E848G activates cell death pathways. Figure: Representative EHTs. L-R: WT/WT, E848G/WT, E848G/E848G.

Abstract P417: Cardiomyopathy-associated Variant In Troponin T Tail Domain Promotes Disruption Of Both Frank-starling Mechanism And Cardiac Myofilament Performance

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) tail region has been linked to familial hypertrophic cardiomyopathy (HCM), arrhythmia, and sudden cardiac death. It has been reported that inotropic stimulation with high extracellular Ca 2+ or isoproterenol led to diastolic dysfunction in both isolated and in vivo HcTnT-I79N mice hearts. Although HcTnT-I79N effects are acknowledged to be dependent on the inotropic state of the cardiac muscle, little is known about how this pathogenic variant affects the Frank-Starling law of the heart. To further investigate the functional and structural consequences of this deadly variant in a stretch-dependent manner, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements. Sarcomere length (SL 1.9, 2.1 or 2.3 μm) was set at pCa 8 using HeNe laser diffraction and then Ca 2+ -dependence of isometric force, sinusoidal stiffness (SS, 0.2% PTP length oscillation) and rate of tension redevelopment ( k TR ) were measured. We observed that HcTnT-I79N tissue exhibited increased myofilament Ca 2+ -sensitivity of force, increased SS, slower k TR at all levels of Ca 2+ -activation, and diminished length-dependent activation (LDA). Small-angle X-ray diffraction revealed that HcTnT-I79N permeabilized cardiac muscles exhibit smaller myofilament lattice spacing at longer SLs (2.1 μm and 2.3 μm) compared to NTg. Using 3% Dextran T500 to osmotically compress the myofilament lattice (SL 2.1 μm), HcTnT-I79N showed no change in myofilament lattice spacing and little change in contractile indices associated with LDA. Interestingly, upon osmotic compression, HcTnT-I79N displayed a decrease in disordered relaxed state (DRX, ON state) of myosin and an increase in super-relaxed state (SRX, OFF state) of myosin. We conclude that altered cardiac myofilament performance, lack of responsiveness to osmotic compression, and reduced LDA observed with HcTnT-I79N are partially due to a combination of smaller myofilament lattice and disturbed ON and OFF states of myosin.

Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.

A deleterious mutation in the ALMS1 gene in a naturally occurring model of hypertrophic cardiomyopathy in the Sphynx cat

Background Familial hypertrophic cardiomyopathy is a common inherited cardiovascular disorder in people. Many causal mutations have been identified, but about 40% of cases do not have a known causative mutation. Mutations in the ALMS1 gene are associated with the development of Alstrom syndrome, a multisystem familial disease that can include cardiomyopathy (dilated, restrictive). Hypertrophic cardiomyopathy has not been described. The ALMS1 gene is a large gene that encodes for a ubiquitously expressed protein. The function of the protein is not well understood although it is believed to be associated with energy metabolism and homeostasis, cell differentiation and cell cycle control. The ALMS1 protein has also been shown to be involved in the regulation of cell cycle proliferation in perinatal cardiomyocytes. Although cardiomyocyte cell division and replication in mammals generally declines soon after birth, inhibition of ALMS1 expression in mice lead to increased cardiomyocyte proliferation, and deficiency of Alstrom protein has been suggested to impair post-natal cardiomyocyte cell cycle arrest. Here we describe the association of familial hypertrophic cardiomyopathy in Sphynx cats with a novel ALMS1 mutation. Results A G/C variant was identified in exon 12 (human exon 13) of the ALMS1 gene in affected cats and was positively associated with the presence of hypertrophic cardiomyopathy in the feline population (p < 0.0001). The variant was predicted to change a highly conserved nonpolar Glycine to a positively charged Arginine. This was predicted to be a deleterious change by three in silico programs. Protein prediction programs indicated that the variant changed the protein structure in this region from a coil to a helix. Light microscopy findings included myofiber disarray with interstitial fibrosis with significantly more nuclear proliferative activity in the affected cats than controls (p < 0.0001). Conclusion This study demonstrates a novel form of cardiomyopathy associated with ALMS1 in the cat. Familial hypertrophic cardiomyopathy is a disease of genetic heterogeneity; many of the known causative genes encoding for sarcomeric proteins. Our findings suggest that variants in genes involved with cardiac development and cell regulation, like the ALMS1 gene, may deserve further consideration for association with familial hypertrophic cardiomyopathy.

ACE Genotype Distributions Differ between Sporadic and Familial Hypertrophic Cardiomyopathy

This study investigated the frequency of ACE genotypes in sporadic hypertrophic cardiomyopathy (HCM) and compared these frequencies to those found in the general population and in familial HCM. Delineation of the genotype of a 287 bp fragment in the ACE gene of 10 patients with confirmed sporadic HCM demonstrated that 2 (20%) were of the DD genotype, 5 (50%) of the ID genotype, and 3 (30%) of the II genotype. These genotype distributions did not differ significantly from controls (p < 0.57). Comparison of the present results with genotype frequencies in familial HCM reported in prior studies revealed a significant difference in genotype distribution between sporadic and familial HCM (p < 0.04). These findings indicate that the frequency of the ACE genotype does not appear to differ between patients with sporadic HCM and the general population. However, the results suggest that, with regard to the ACE polymorphism studied, genetic differences may exist between the sporadic and familial forms of HCM.