Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery

Abstract

Proximal spinal muscular atrophy (SMA) is a debilitating neurological disease marked by isolated lower motor neuron death and subsequent atrophy of skeletal muscle. Historically, SMA pathology was thought to be limited to lower motor neurons and the skeletal muscles they control, yet there are several reports describing the coincidence of cardiovascular abnormalities in SMA patients. As new therapies for SMA emerge, it is necessary to determine whether these non-neuromuscular systems need to be targeted. Therefore, we have characterized left ventricular (LV) function of SMA mice (SMN2+/+; SMNΔ7+/+; Smn-/-) and compared it with that of their unaffected littermates at 7 and 14 days of age. Anatomical and physiological measurements made by electrocardiogram and echocardiography show that affected mouse pups have a dramatic decrease in cardiac function. At 14 days of age, SMA mice have bradycardia and develop a marked dilated cardiomyopathy with a concomitant decrease in contractility. Signs of decreased cardiac function are also apparent as early as 7 days of age in SMA animals. Delivery of a survival motor neuron-1 transgene using a self-complementary adeno-associated virus serotype 9 abolished the symptom of bradycardia and significantly decreased the severity of the heart defect. We conclude that severe SMA animals have compromised cardiac function resulting at least partially from early bradycardia, which is likely attributable to aberrant autonomic signaling. Further cardiographic studies of human SMA patients are needed to clarify the clinical relevance of these findings from this SMA mouse.

Figures

Figure 1.

Echocardiographic measurements of (A) LV mass, (B) wall thickness and (C) 2xPWD/LVDD show significant decreases in SMA mice compared with WT and scAAV9-treated animals, suggesting that SMA mice are undergoing eccentric hypertrophy and are at increased risk for heart failure. Although scAAV9-treated mice also have decreased LV mass compared with WT, wall thickness and 2xPWD/LVDD measurements are not significantly changed. Symbols indicate P < 0.05 when comparing SMA with WT (*), AAV9 with WT (#) and AAV9 with SMA (+).

Figure 2.

Echocardiographic measurements of cardiac function in p14 mice. Cardiac function of the SMA mice was significantly lower than that of WT mice when we assessed heart rate (A), SV (B), cardiac output (C) and FS (D). scAAV9-treated animals have heart rates indistinguishable from WT (A), but SV (B) is similar to SMA animals, thereby decreasing the overall cardiac output as well (C). FS, a measure of contractility, is decreased in both SMA and AAV9 mice, though scAAV9-treated mice contract significantly better than untreated SMA mice (D). Tei index is increased in SMA mice, consistent with worse combined systolic and diastolic function, whereas scAAV9-treated mice have similar values to WT, indicating preserved function (E). Symbols indicate P < 0.05 when comparing SMA with WT (*), AAV9 with WT (#) and AAV9 with SMA (+).

Figure 3.

Echocardiographic measurements of cardiac function in p7 mice. As with p14 animals, measures of LV mass (A), wall thickness (B), dilation (C), heart rate (D), FS (E) and Tei index (F) all indicate decreased function in SMA mice, whereas functional measures in scAAV9-treated animals are relatively preserved. Symbols indicate P < 0.05 when comparing SMA with WT (*), AAV9 with WT (#) and AAV9 with SMA (+).

Figure 4.

Cardiac histology of p14 mice. H&E mid-ventricular sections show the typical thinning heart walls and dilated ventricles of SMA mice (B) compared with WT (A) (tiled images, scale = 500 µm). Higher-power images (×40 magnification, scale = 30 µm) of WT (C) and SMA mice (D) both show absence of inflammation or other signs of overt tissue pathology at p14. TEM of WT mice (E) show normal mitochondria (M) and myofibers with well-organized fibrils and well-defined sarcomeres (asterisk, Z-line) (scale = 1 µm). TEM sections of SMA heart tissue (F) show swollen myofibers with disorganized Z-lines (asterisk) and no clearly defined I bands, A bands or H zones (scale = 1 µm). Mitochondria in SMA myocytes are swollen with evidence of crystolysis (mitochondria marked by ‘M’), and in many fields, these organelles occupy most of the sarcoplasm. The inset (F) shows a clearer view of degenerating mitochondria with myelin figure formation (evidence of advanced crystolysis and membrane degradation) (arrowhead) (scale = 0.5 µm).

Figure 5.

Dobutamine stress challenge in p14 WT and SMA mice. Heart rate, as expected, increases upon dobutamine administration in WT and SMA mice (A). FS also increases upon dobutamine administration in both groups, whereas in SMA mice, FS increases to a significantly lesser degree (B). In B, asterisk denotes significance (P < 0.05) compared with WT.

Figure 6.

A schematic overview of the key neuronal structures controlling heart rate (A). The sympathetic structures observed were the PVN, the NTS, the IML and the SG. The parasympathetic structures observed include the MNX, the NA, the NTS and the CG. scAAV9 transduction can be assessed by visualizing the presence of a green fluorescent protein (GFP) transgene (B–I). The MNX (D), NA (E), CG (F) and SG (H) were all highly transduced, whereas we found no evidence of transduction of the PVN (B), NTS (C) and IML (G) neurons. Cardiac myocytes were also found to be mostly positive for the GFP transgene (I). Green, GFP in B–I; Red, tyrosine hydroxylase in B and H, neuronal nuclei (NeuN) in C, choline acetyl-transferase (ChAT) in D, E and G, neurofilament-160 (NF-160) in F and dystrophin in I. Scale = 50µm in B–F, H and I, and 20µm in G.