We utilized the SMNΔ7
SMA mouse model (JAX line 5025) developed by Le et al
) for these studies. In our laboratory (20
) and in agreement with previous studies (19
), this model exhibited a phenotype apparent by postnatal day (PND) 5 with an average survival of approximately 2 weeks. Debilitating neuromuscular symptoms were overtly visible by PND8, at which point ambulation was severely affected and mice were grossly undersized. Around PND10, SMA mice began to lose weight and exhibit a decreased level of consciousness (LOC). All ECG values were obtained from resting heart rates recorded using a non-invasive method described previously (22
), with a sample size of ten mice unless otherwise noted.
SMA mice exhibit bradyarrhythmia
To determine whether SMA mice show functional cardiac abnormalities, we examined ECGs recorded from affected mutants and unaffected carrier mice. Readings at PND8, when mutant mice show overt SMA symptoms, revealed that bradyarrhythmia was present in SMA mice relative to unaffected heterozygous littermates (Fig. ). To further examine the electrophysiological nature and developmental time course of cardiac arrhythmia in SMA mice, we characterized the natural history of ECG waveform parameters throughout the development of SMA mice and their control littermates.
Figure 1. Conscious SMA mice exhibit bradycardia. Initial survey of several SMA mice revealed a reduction in heart rate in comparison to control littermates. Tracings here come from representative ECGs obtained in conscious SMA (top) and unaffected heterozygous (more ...)
ECG waveforms established in previous works are equivalent in males and females during neonatal time points (22
). To provide further baseline data in our control genotypes of mice, we recorded ECGs from ages PND2 to PND14 in unaffected mice that were either wild-type at the endogenous Smn
) or heterozygous at the endogenous locus (Smn+/−
). All mice were homozygous for the two transgenes present in this model (SMN2+/+
). No difference in heart rate between genotypes was detected at any time point assayed (Fig. ), nor was any difference observed in PR interval duration, QRS interval duration or in measures of heart rate variability (data not shown). Comparison with the previous data obtained from wild-type FVB/N mice showed that all values and time points were consistent with established ECG values (22
). Together, this demonstrated that there is no effect of transgene insertion or Smn
heterozygosity on heart rate or other ECG waveforms.
Figure 2. Transgenic background has no effect on ECG. ECGs were recorded every 2 days from unaffected 5025 mice that were either homozygous wild-type (Smn+/+) or heterozygous carriers of a knockout allele (Smn+/−) at the Smn locus. All data were obtained (more ...)
Bradyarrhythmia with progressive heart block and impaired conduction
Heart rates were reduced in SMA mice at all time points examined (Fig. A). At PND2, the earliest age we could reliably assay, SMA mice had an average heart rate of 378 ± 50 b.p.m., whereas unaffected littermates had a heart rate of 451 ± 22 b.p.m. (n
= 10 per genotype, P
≤ 0.0005). From PND4 to PND8, SMA heart rates increased at a steady rate of approximately 25 b.p.m. each day, consistent with littermate controls as well as previous studies characterizing the development of neonatal heart rhythms (22
). Throughout this time, SMA mice exhibited heart rates that were approximately 100 b.p.m. lower than unaffected mice. At PND10, the heart rate of SMA mice became further reduced compared with control mice (505 ± 51 versus 642 ± 24 b.p.m., respectively). This deficit progressed at PND12 and PND14 where there was a large drop from previous time points in the heart rate of SMA mice. This resulted in heart rates in SMA mice that were approximately half those in unaffected mice as they approached death endpoints (368 ± 119 versus 657 ± 83 b.p.m.; P
). Together, these results elucidate two points. First, a significant bradyarrhythmia phenotype is present in SMA mice as early as PND2. Second, the end stage of life in SMA mice is characterized by severe bradyarrhythmia progressing to cardiac standstill.
Figure 3. Life history of arrhythmia in SMA mice. Resting ECGs recorded for SMA and unaffected littermates every 2 days reveal significant physiological differences in waveforms. All mice were conscious and unrestrained. (A) Heart rate, (B) PR interval, (C) QRS (more ...)
The most common cause of bradyarrhythmia is heart block, detected through the elongation of PR interval duration. In SMA mice, PR interval was significantly elongated at every time point ECGs were recorded (Fig. B). As with heart rate, PR interval showed two phases during the growth of SMA mice. At PND4, SMA mice possessed a first-degree heart block characterized by elongated PR interval durations compared with unaffected littermates (48.6 ± 8.8 versus 36.1 ± 5.7 ms, P ≤ 0.001); this was maintained as PR interval development paralleled that of unaffected mice until PND10. After this age, there was a sharp increase in the PR interval and breakdown of the cardiac rhythm as mice exhibited progressive heart block as they neared the end stage of survival.
We detected a significant elongation of the time of ventricular depolarization for SMA mice through an increase in QRS interval duration, beginning by PND4 (Fig. C). At this point, SMA mice possessed a QRS interval lasting 15.9 ± 4.9 ms, compared with 11.9 ± 3.0 ms for unaffected littermates (P ≤ 0.05). Ventricular depolarization times were precisely maintained in unaffected mice. SMA mice exhibited larger variations in this parameter as they matured until around PND10, at which point there was a sharp increase as mice began to approach death endpoints.
We found low values of heart rate variability in unaffected neonate mice, as previously observed in neonatal stages of humans and multiple mammalian species (24
). However, as SMA mice neared the end stage of disease, they displayed a sharp spike in measures of heart rate variability (Fig. D). This manifested in ECGs as heart rhythms that were erratic and/or contained skipped beats (Fig. E). Ultimately, SMA mice displayed adjacent RR intervals that were highly inconsistent, resulting in average pNN6 values of ~50–60% for PND12 and PND14 mice. Thus, roughly half of the consecutive heart beats in end stage SMA mice differed in duration by an amount significantly exceeding the threshold set for a regular rhythm.
Functional deficits detected by echocardiography
Echocardiography was performed on PND6 mice to gain further insight into the level of cardiac function in SMA mice. Deficiencies in blood flow out of the right ventricle were detected at the pulmonary valve in SMA mice. Peak velocity was significantly decreased in all SMA mice relative to all littermate controls (489.7 ± 162.2 versus 1617.09 ± 630.2 mm/s; P < 0.005), as was peak gradient (1.1 ± 0.5 versus 11.8 ± 10.5 mmHg; P < 0.05) (Fig. A and B). In both groups, heart rates measured while performing echocardiography were lower than previous measurements due to the mice being sedated. Consistent with earlier findings, however, heart rates were significantly lower in SMA mice than in controls (243 ± 58 versus 351 ± 24 b.p.m.; P < 0.005) (Fig. C). Thus, the SMA heart shows significant reductions in both pumping efficiency and blood flow from the right ventricle to the lungs. Together, these data establish that gross deficits in cardiac function are present in neonate SMA mice.
Figure 4. Echocardiography deficits in SMA mice. Echocardiography was performed on sedated PND6 mice. SMA mice had depressed values in (A) PV peak velocity (P < 0.005) and (B) PV peak gradient (P < 0.05). (C) Heart rate was decreased in sedated (more ...)
Examination of SMA heart morphology and sympathetic innervation
We examined heart to body size ratios to determine whether gross defects in heart morphology were present. Defects such as cardiac hypertrophy or hypotrophy could affect cardiac function and result in hemodynamic insufficiency during periods of normally rapid development in growing SMA neonates. After peaking at PND10, SMA mice typically lost weight each day before dying around 13 days of age. Accordingly, we sacrificed mice at PND10 and PND13 to examine the heart at these two points. General observation of heart morphology and histological sections (data not shown) were entirely consistent with vehicle groups presented later, with SMA mouse hearts appearing smaller and flaccid while lacking defined shape with gross attenuation of walls. Here, SMA mice sacrificed at PND13 lost an average of 21 ± 5% of their body weight, whereas unaffected genotypes gained an average of 26 ± 5% (Fig. A). We found at PND10 that SMA mice displayed a heart to body weight ratio comparable to that of unaffected littermates. At PND13, that ratio was maintained, with values comparable between both groups and ages (Fig. B). Equivalent ratios of heart to body length were also observed between both groups and time points (Fig. C). These data suggest first that heart to body size is precisely maintained in both SMA and heterozygous mice, so the decline in SMA body mass does not appear to result from the developing SMA mouse ‘outgrowing’ its cardiac size nor from relative cardiac hypertrophy. Second, the hearts of SMA mice appear to be losing mass at the end stages of disease, since their mass ratio is precisely maintained even as the mice lose one-fifth of their body mass.
Figure 5. Loss of cardiac mass in end stage of life. (A) Body mass decreased 21 ± 5% in SMA mice from PND10 to PND13, whereas that of unaffected littermates increased by 26 ± 5% (*P < 0.05, n = 7 mice per genotype). (B) Heart:body mass ratios (more ...)
Heart rate in healthy mice is controlled by a balance of the sympathetic and vagal components of the ANS. Sympathetic nerve activity accelerates the heart rate, whereas the vagal system decelerates it. Arrhythmia characterized by bradycardia and progressive heart block as detected here can result from an autonomic imbalance resulting from decreased sympathetic tone relative to vagal tone. To investigate whether such a decrease in sympathetic innervation may be present, we harvested hearts from PND12 mice and performed whole-mount immunostaining with an antibody to tyrosine hydroxylase (TH), a marker of sympathetic nerves (Fig. ). We found distinct differences in the TH staining pattern of SMA mice versus controls in both ventral and dorsal views. Ventral views of unaffected hearts contained prominent neurons not observed in SMA hearts (Fig. A and B). In dorsal views, we observed that TH-positive neurons from SMA mice seemed to have fewer branches while also appearing thinner and less distinct (Fig. C and D), though fine structure was still present upon higher magnification. Quantification of the two most prominent sympathetic neurons visible in the dorsal view of the heart confirmed that there were fewer major neuronal branches detected in SMA hearts (Fig. E; n = 7 mice per group, P ≤ 0.05). These results, in conjunction with the types of arrhythmia we observed, suggest that there is a decrease in sympathetic tone present in SMA mice resulting in autonomic imbalance.
Figure 6. SMA hearts show a decrease in sympathetic staining. Whole-mount immunostaining with an antibody to TH was used to visualize sympathetic innervation. Hearts were harvested from PND12 SMA mice and unaffected littermates. (A) Reduced sympathetic patterning (more ...)
Drug treatment improves SMA motor function throughout extended survival
With SMA mouse models becoming a common system for preclinical drug studies, we next sought to explore the utility of ECG as a non-invasive biomarker. Additionally, we wanted to gain insight into the effects that a drug established to display positive-acting effects in SMA mice has on novel cardiac phenotypes we observed, alongside the effects on motor function and survival. For these studies, we chose to use trichostatin A (TSA). TSA, a histone deacetylase inhibitor, has previously been shown to increase SMN transcription and protein both in vitro
and in vivo
and to provide benefits to motor function and survival of SMA model mice (21
). Here, SMA and unaffected heterozygous littermate mice were injected once daily with either TSA at 10 mg/kg or an equivalent volume of vehicle (DMSO) beginning at PND2. ECG was performed on even numbered days by a reader blinded to genotype, drug identity and phenotyping studies, with a sample size of 10 mice per group. Motor function was assayed by measuring the time to right after the placement of each mouse on its back, which was done on each odd numbered day to prevent the elevation of heart rate by a physical activity.
As expected, injection with TSA successfully improved survival, body weight and motor function of SMA mice (Fig. ). Induction of SMN levels and histone acetylation was also confirmed through molecular analyses (Supplementary Material, Fig. S1
). Survival of SMA mice was increased to a median of 22 days when compared with 15.5 days for those injected with vehicle (P
< 0.0005) (Fig. B). Body weights of SMA and unaffected groups were comparable at PND2 and PND4, with vehicle-injected SMA mice weighing 2.5 ± 0.5 g versus unaffected mice weighing 2.7 ± 0.4 g at PND4 (Fig. C). After this, SMA mice were lower in weight than unaffected littermates (P
< 0.005). TSA produced a weight benefit in SMA mice beginning at PND10, at which point vehicle-injected SMA mice began to lose weight, whereas TSA-treated SMA mice continued to gain/maintain weight until approximately PND20. Motor function benefits were observed in SMA mice from PND6 onwards upon TSA treatment (Fig. D). Interestingly, TSA improved the motor function of SMA mice through righting time towards levels comparable to unaffected controls through PND21, though many reached death endpoints during this time or shortly after. Maintenance of motor function at late time points was further demonstrated using ambulation and negative geotaxis assays (Supplemental Material, Videos S1–S4
). Thus, TSA-treated SMA mice with extended survival were frequently found to die without experiencing a visible decline in motor function.
Figure 7. TSA improves motor function throughout extended survival. TSA was administered to SMA mice and unaffected heterozygous littermates at 10 mg/kg by IP injection once daily beginning at PND2. Control groups received an equivalent volume of vehicle (DMSO). (more ...)
TSA prolongs heart beat maturation until death of SMA mice
Heart rates of SMA mice were significantly elevated at PND10–14 upon TSA treatment, along with concurrent decreases in PR and QRS interval durations (Fig. ). These benefits appeared to be maintained until approximately PND20, after which we observed the progression of bradycardia associated with mice reaching death endpoints. In regard to heart rate, we observed no difference between treated and untreated SMA mice before the age of PND10 (Fig. A). Before this point, both groups displayed slopes of heart rate growth that were parallel to those of unaffected mice. At PND10 and onward, however, the vehicle group was found to decline significantly, whereas drug-injected mice continued to exhibit a rate of increase comparable to unaffected mice for several more time points. In unaffected littermates, TSA-injected mice showed a trend of lower heart rates; however, this difference was not significant and was present between these groups at PND2, before the first injection.
Figure 8. ECG of TSA treatment groups. Extended maturation of ECG waveforms was observed in SMA mice treated with TSA. ECGs were obtained every 2 days from PND2 to PND14, with all animals conscious and unrestrained. (A) Heart rate, (B) PR interval, (C) pNN6ms and (more ...)
First-degree heart block was still present in SMA mice upon TSA treatment, though we did not observe the same progression seen in vehicle or untreated SMA mice (Fig. B). We found a sharp increase in PR interval duration at PND22, but did not observe highly erratic and/or skipped beats as were detected in untreated SMA mice. Consistent with a lack of breakdown in heart rate regularity, we observed no increase in measures of heart rate variability in TSA-treated SMA mice over that found in unaffected littermates of either treatment group at any time point (Fig. C).
Time of ventricular depolarization improved upon drug treatment. Values for QRS interval duration in treated SMA mice were equivalent to those in unaffected littermates from PND10 to PND14 (Fig. D). However, at the end stage of life, this was not maintained, as SMA mice showed an increased QRS interval when nearing a death endpoint over unaffected littermates (15.1 ± 5.4 versus 9.38 ± 0.6; P < 0.05).
TSA treatment increases the size of SMA hearts
We next investigated the cardiac morphology in SMA mice and our treatment groups at PND12, when clear electrocardiographic differences are present. Observations of heart morphology in vehicle-injected mice were consistent with those in mice that received no injections. Upon dissection, hearts from vehicle-injected SMA mice appeared grossly undersized and flaccid (Fig. A). In contrast, TSA-treated SMA mice possessed larger hearts that were closer in size to unaffected mice injected with TSA. In addition, both groups treated with TSA were smaller than unaffected mice injected with vehicle (P < 0.005). Hematoxylin and eosin staining of hearts from vehicle-injected SMA mice revealed gross attenuation of walls and dilated cardiomyopathy (Fig. B and C). Often, this resulted in hearts that appeared misshapen or ‘deflated’ and again was consistent with histological observations from SMA mice receiving no injections (data not shown). Upon TSA treatment, SMA hearts more closely resembled those of unaffected mice injected with TSA.
Figure 9. Cardiac morphology of SMA treatment group hearts. Hearts harvested from SMA mice injected with vehicle were smaller than those of unaffected littermates and typically appeared flaccid or unable to hold their shape. TSA treatment corresponded with an increase (more ...)
Treatment with TSA resulted in hearts that were larger in proportion to body size than in mice injected with vehicle (Fig. D). This was true for the heart:body mass ratio of both SMA and unaffected mice (P < 0.05, n ≥ 5 hearts per group). Drug-treated SMA mice also showed an increase in the ratio of the length of their heart to their body (P < 0.05) (Fig. E). At this time, SMA mice treated with drug were continuing to gain body weight. Thus, SMA mice appear to have hearts that are larger both in weight and in volume when treated with TSA and do not appear to lose cardiac mass at this stage of their life as was found for untreated mice at PND13.