Effect of pharmacological heart failure drugs and gene therapy on Danon's cardiomyopathy.

Danon disease is a rare X-linked genetic disease resulting from LAMP2 mutations leading to defective lysosomal function. Heart failure is the main causes of morbidity and mortality. Mice with an LAMP2-exon-6-deletion (L2Δ6), develop cardiac hypertrophy followed by dilated cardiomyopathy, in association with accumulation of autophagosomes, fibrosis and oxidative stress. We investigated the effect of drugs used to treat heart failure and of LAMP2 gene therapy on the phenotype, molecular markers and ROS in LAMP2 cardiomyopathy. L2Δ6 mice were treated with Angiotensin II, Ramipril, Metoprolol or Spironolactone. Gene therapy was delivered by IP injection of Adeno-associated-virus (AAV9) -LAMP2 vector to neonates ("AAVLAMP2-Prevention"), or at 15 weeks of age ("AAVLAMP2-Treatment"). Angiotensin II markedly aggravated the cardiac phenotype. Ramipril and Spironolactone were effective in attenuating left ventricular hypertrophy and preserving the systolic function. Cardiac protection was associated with decreased autophagosome accumulation, reduced fibrosis and oxidative stress. Gene therapy effectively attenuated autophagosome accumulation and ROS in L2Δ6 hearts, lowering troponin release to nearly normal levels. AAVLAMP2-Prevention protected against systolic dysfunction and decreased hypertrophy. AAVLAMP2-Treatment prevented ventricular dilatation and dysfunction but had no effect on wall thickness. We conclude that RAAS inhibitors are highly effective against cardiomyopathy progression in an experimental mouse model of Danon's and shall be considered in human patients for this purpose until novel therapies become clinically available.

LAMP2 Cardiomyopathy: Consequences of Impaired Autophagy in the Heart.

Background Human mutations in the X-linked lysosome-associated membrane protein-2 (LAMP2) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in-frame LAMP2 gene exon 6 deletion mutation (denoted L2Δ6) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in-frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2Δ6 and wild-type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40-week-old L2Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.

Viral delivered gene therapy to treat catecholaminergic polymorphic ventricular tachycardia (CPVT2) in mouse models.

BACKGROUND: The recessive form of catecholaminergic polymorphic ventricular tachycardia 2 (CPVT2) is caused by mutations in cardiac calsequestrin (CASQ2), leading to protein deficiency. OBJECTIVES: The aims of this study were to develop a viral-delivered gene therapy for CPVT2 and to determine the relationship between CASQ2 expression and antiarrhythmic efficacy in a murine model. METHODS: We used a murine model of CPVT2 caused by the D307H human mutation (CASQ2D307H) or CASQ2 knockout (CASQ2Δ/Δ). Adeno-associated virus (AAV) particles containing the CASQ2 gene (AAVCASQ2) were injected into the heart or intraperitoneally to 12-week-old mice. A telemetry device was implanted, and mice underwent provocation testing 7-8 weeks after gene therapy. RESULTS: CASQ2Δ/Δ mice injected intracardiacally with AAVCASQ2 expressed 40% ± 25% of the normal CASQ2 protein level, which was increased compared to untreated CASQ2Δ/Δ mice (n = 10; P < .05). Intraperitoneal therapy led to a significantly elevated expression of the CASQ2 protein, which was comparable in CASQ2D307H (n = 12) and CASQ2Δ/Δ (n = 4) mice. All control mice with CPVT2 had nonsustained ventricular tachycardia (VT) and 8 of 13 had sustained VT on provocation. Expressing ≥33% of the normal CASQ2 level was needed to protect from nonsustained VT as well as stress-induced premature ventricular contractions. Lower levels of expression prevented sustained VT in AAVCASQ2-treated mice (0 of 26; P < .001 vs controls). CONCLUSION: AAVCASQ2 displays a long-lasting capacity to attenuate and potentially cure CPVT2. Systemic delivery is feasible and convenient, reproducibly providing adequate levels of transgene expression. Antiarrhythmic efficacy depends on the CASQ2 level: ≥33% of the normal CASQ2 level is needed to prevent arrhythmia. However, even lower levels of protein protect from sustained VT, thereby potentially reducing the risk of sudden death.