Partial restoration of cardio-vascular defects in a rescued severe model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death. Loss of a gene called Survival Motor Neuron 1 (SMN1) and, as a result, reduced levels of the Survival Motor Neuron (SMN) protein leads to SMA development. SMA is characterized by the loss of functional motor neurons in the spinal cord. However, accumulating evidence suggests the contribution of other organs to the composite SMA phenotype and disease progression. A growing number of congenital heart defects have been identified in severe SMA patients. Consistent with the clinical cases, we have recently identified developmental and functional heart defects in two SMA mouse models, occurring at embryonic stage in a severe SMA model and shortly after birth in a less severe model (SMN∆7). Our goal was to examine the late stage cardiac abnormalities in untreated SMN∆7 mice and to determine whether gene replacement therapy restores cardiac structure/function in rescued SMN∆7 model. To reveal the extent of the cardiac structural/functional repair in the rescued mice, we analyzed the heart of untreated and treated SMN∆7 model using self-complementary Adeno-associated virus (serotype 9) expressing the full-length SMN cDNA. We examined the characteristics of the heart failure such as remodeling, fibrosis, oxidative stress, and vascular integrity in both groups. Our results clearly indicate that fibrosis, oxidative stress activation, vascular remodeling, and a significant decrease in the number of capillaries exist in the SMA heart. The cardiac structural defects were improved drastically in the rescued animals, however, the level of impairment was still significant compared to the age-matched wildtype littermates. Furthermore, functional analysis by in vivo cardiac magnetic resonance imaging (MRI) revealed that the heart of the treated SMA mice still exhibits functional defects. In conclusion, cardiac abnormalities are only partially rescued in post-birth treated SMA animals and these abnormalities may contribute to the premature death of vector-treated SMA animals with seemingly rescued motor function but an average life span of less than 70 days as reported in several studies.

Introduction

Spinal muscular atrophy (SMA) is the leading genetic cause of infantile death and is characterized by the loss of the α-motor neurons in the spinal cord [1]. The clinical spectrum is broadly categorized based upon disease onset and physical milestones [2]. SMA results from the loss of survival motor neuron-1 (SMN1) [3]. A human-specific copy gene is present on the same chromosome called SMN2 [3], [4]. SMN2 is nearly identical to SMN1 and generates low levels of full-length SMN. SMN2 cannot prevent disease development in the absence of SMN1 since the majority of SMN2-derived transcripts are alternatively spliced and encode an unstable protein [5], [6].

The fundamental pathology in SMA is neurodegeneration, however, there are clinical reports demonstrating the contribution of other tissues to the overall phenotype in the most severe forms (Type 0 and Type I). In severe SMA, several congenital heart defects have been documented including atrial septal defects, dilated right ventricle, ventricular septal defects, and hypoplastic left heart syndrome [[7], [8], [9]]. Sudden death without a clear explanation can occur in severe SMA patients [10]. Central apnea due to bulbar dysfunction and respiratory complications is suggested to be the possible cause of sudden death, even though it also occurs in patients with ventilation support [10]. Therefore, vagal hypertonia induced bradycardia has also been considered [10]. Furthermore, palpitations, ST-segment abnormalities, couplets, diastolic dysfunction, and right ventricular overload have been reported in SMA patients [11], [12]. Distal necrosis has also been documented in severe SMA, occasionally with atrial septal defects and asymmetric left ventricular hypertrophy [13], [14]. Similar pathologies have been observed in SMA mice treated with therapeutic compounds or viral vectors [15], [16]. Life span extension of treated SMA mice has led to increased recognition of distal necrosis in the tail or ear pinna. These results suggest that the dysfunction of the autonomic nervous system may lead to impaired regulation of vascular tone and possibly fetal vasculature defects which cannot be completely restored with post-birth treatments. Recently, our lab and others have identified functional and structural heart defects in SMA animal models [[17], [18], [19]]. Bradycardia is the most common autonomic nervous system phenotype reported in these studies. We have identified cardiac defects occurring during embryonic development in a severe SMA mouse model and shortly after birth in a slightly less severe model (SMNΔ7), suggesting that cardiac defects precede motor neuron degeneration.

Recently, intravenous delivery at postnatal day 1 (P1) of scAAV9 expressing full-length SMN cDNA in SMNΔ7 mice led to a profound phenotypic rescue by restoring motor function/neuromuscular physiology and extended survival up to 250 days [20]. The same group reported complete restoration of the heart rate and partial repair of cardiac function in AAV9-injected animals at P14 [17]. Additional groups reported a significantly extended life span using a similar vector, however, sudden deaths, respiratory complications, and average life spans of only 65–70 days were common [21], [22]. To provide a more detailed analysis of the cardiac defects in these important disease models, we have examined remodeling, fibrosis, oxidative stress, and vascular impairments. In each instance, the SMA mice were profoundly impaired compared to healthy animals and these results served as an important baseline from which we could quantitatively assess cardiac structure/function following SMN gene replacement. To directly compare the functional parameters, we carried out in vivo MRI on the heart of the rescued and age-matched wildtype littermates. Our study demonstrates that the heart of the rescued SMA mice, albeit drastically improved compared to untreated mice, still exhibits structural and functional defects compared to age-matched wildtype mice, suggesting that cardiac defects contribute to the shortened life span of scAAV-rescued mice [21], [22].