Abstract
Background:
This study aimed to show the impairment of autonomic cardiac conduction causing bradycardia and/or electrocardiographic alterations in children affected by spinal muscular atrophy type 1 and 2 (SMA 1 and 2). Methods: We included 25 spinal muscular atrophy patients, admitted from November 2016 to May 2017. All patients underwent an electrocardiographic examination and we studied PR and QRS intervals, P-waves and QRS amplitudes, and heart rate in spinal muscular atrophy patients compared to a control group.
Results:
In all patients, we found longer PRi and QRSi (P < .05), lower P-wave and QRS complex amplitudes (P < .01), and a decreased heart rate (P < .01) with respect to controls. When we divided our patients into SMA1 and SMA2 subgroups, we found that statistical differences were maintained for P-wave and QRS complex amplitudes and heart rate, but not for PRi and QRSi with respect to controls.
Conclusion:
We suggest the hypothesis of SMN expression on cardiac tissue condition and/or autonomic cardiac conduction.
Spinal muscular atrophy (SMA) is a genetic neurologic disease with a mortality of approximately 30% at 2 years, and an incidence of 1/10 000 live births. 1 -13 It is caused by homozygous deletion or mutation in the spinal motor neuron 1 (SMN1) gene on chromosome 5q13 1 -13 (SMN1, Online Mendelian Inheritance in Man [OMIM] #600354). The SMN protein is an essential protein necessary for the efficient assembly of ribonucleoprotein complexes. 1 -8
In 95% of cases, the SMN1 gene is absent, whereas 5% of spinal muscular atrophy patients have small mutations in this gene (eg, small deletions, splice mutants, and missense mutations).1-3 The SMN gene is present as a single copy of the telomere SMN1 gene and a variable number of the centromere SMN2 genes (OMIM 601627); both copies have 9 exons, designated as exon 1, 2a, 2b, and 3-8. 1,2 The presence of the SMN2 gene allows patients to survive during the embryonic and fetal life, but compensates partially for the loss of SMN1 in postnatal life, so a neurodegenerative process occurs with different phenotypes of SMA expression. 2,8,9,11
The centromeric SMN2 gene is present in variable copies. The variation in SMN2 copy numbers explains partly differences in SMN protein levels between patients; this is the most important modifier of spinal muscular atrophy severity. The increases in SMN2 copy number often modify the phenotype, inversely correlating with the severity of spinal muscular atrophy types. The severity spectrum encompasses prenatal spinal muscular atrophy (type 0), infantile-onset severe spinal muscular atrophy (type 1), an intermediate form (spinal muscular atrophy type 2), childhood-onset spinal muscular atrophy (type 3), and adult-onset spinal muscular atrophy (type 4). 4,10,11
The fundamental pathology in spinal muscular atrophy is neurodegeneration; however, more clinical reports suggest that the SMN protein deficiency is also detrimental to other cell types present in the central nervous system and even outside, such as the skeletal muscle, heart, autonomic and enteric nervous systems, metabolic/endocrine system, lymphatic, bone, and reproductive system. 9 -13
In particular, Heier et al studied the cardiac involvement in mouse models with severe spinal muscular atrophy phenotypes. 14 These mice had a decrease in heart rate and severe bradyarrhythmia, before the onset of neuromuscular symptoms. 15 Therefore, the bradyarrhythmia was considered an early and progressive sign of murine spinal muscular atrophy. Moreover, the end stage of their life was characterized by the progression of severe symptomatic bradycardia to cardiac standstill. 15
On the basis of these assumptions, we present a retrospective observational study on cardiac involvement in patients with spinal muscular atrophy, subdivided into SMA1 and SMA2 groups, in order to study the presence of alteration of the intrinsic cardiac conduction associated with alteration of the heart rate. 16 -22
Methods
Patients
In our study, we included 25 patients affected by spinal muscular atrophy, admitted to the General Pediatrics Ward of the Policlinico-Vittorio Emanuele University Hospital, University of Catania, Italy, and the Pediatric Neurology Unit of the Gaslini Hospital in Geneva, Italy, from November 2016 to May 2017.
We included only the patients with spinal muscular atrophy in whom the diagnosis was confirmed by genetic analysis through quantitative real-time polymerase chain reaction of SMN1 and SMN2 copy numbers. 6,23 -26 In our study we included spinal muscular atrophy patients hospitalized in our unit to perform diagnostic examinations of follow-up. No acute illnesses were diagnosed in our patients during the study period.
The following clinical data were collected for each patient: demographic data (including age and sex), neurologic clinical examination, neurologic development assessment, instrumental diagnostic examinations, genetic results, electrocardiogram analysis and echocardiography.
All patients underwent M-mode and 2D echocardiogram at admission and during follow-up. Measurements on the echocardiograms were obtained according to the recommendations of the American Society of Echocardiography. 27 Echocardiographic measurements were ejection fraction (EF >60%), systolic function (FS > 30%), and E/A ratio (E/A = 1). 28
All patients underwent an electrocardiographic examination at admission.
We then compared the value of the studied measures with the same parameters in a control group. The control group was composed of people of matched age and sex without disease, who were hospitalized for other neurologic diseases such as epileptic diseases, without systemic involvement, or cognitive disturbs. At admission, no signs and/or symptoms of acute illness, or any signs or symptoms of any primitive or secondary cardiac involvement, were diagnosed.
The present study was approved by the Ethical Committee of the University of Catania, Italy.
Electrocardiogram Cycle Analysis
The electrocardiographic cycle analysis included the PR and QRS interval that were calculated from the average of 3 nonconsecutive cardiac cycles over a 2-minute recording period.
P wave is the first short upward movement of the ECG tracing. It indicates that the atria are contracting, pumping blood into the ventricles.
The QRS complex, normally beginning with a downward deflection, Q; a larger upwards deflection, a peak (R); and then a downwards S wave. The QRS complex represents ventricular depolarization and contraction.
PR interval was measured from the beginning of the P-wave to the beginning of the QRS complex. The PR segment was calculated from the end of the P-wave to the beginning of the QRS complex. The QRS complex duration was measured from the first deflection of the Q-wave to the end of the S-wave.
The amplitude of a wave is the height or depth of a wave. The amplitude of P-wave was calculated from the isoelectric line to the peak of wave.
The amplitude of QRS was measured vertically from the highest peak (R wave) to the lowest peak (Q wave or S wave).
The heart ratio was calculated from RR intervals, that was measured from the beginning of the R-wave to the beginning of the next R-wave.
Statistical Analysis
For statistical analysis, we used a dedicated software: JMP (produce of SAS Institute Inc, Cary, NC) e GraphPad 5.0 (La Jolla, CA). We analyzed quantitative parameters as mean ± standard deviation. For these variables, normal distribution was checked by the Kolmogorov-Smirnov 1-sample test and statistics for kurtosis and symmetry. A Student t test was used to compare the studied parameters between groups.
P values under .05 were considered statistically significant.
Results
In our study, we included 25 patients, 14 male and 11 female, mean age 9.8 years. Among the included patients, 12 had type 1, 11 had type 2, and 2 had type 3 spinal muscular atrophy.
We studied cardiac function through the echocardiogram and the ECG. Echocardiographic measurements were within standard normal limits in all patients.
We examined a total of 28 electrocardiograms: 22 patients with an electrocardiogram and the remaining 3 patients with 2 electrocardiograms for each one.
When we compared the electrocardiograms of spinal muscular atrophy patients with those of the control group, we found that spinal muscular atrophy patients had longer PRi than controls, with statistical significant results (P < .05). QRSi were not statistically different between the 2 groups. We moreover found that the P-wave and QRS amplitudes (expressed in heights) were lower in the spinal muscular atrophy group than in controls, with a statistical significant result (P < .01 for both measures). Also, the heart rate was lower in spinal muscular atrophy patients than in controls (P < .01) (Table 1). Nevertheless, the statistical analysis was mostly significant for the heart rate and the P-wave and QRS amplitudes (P < .01) than the PRi and QRSi (P < .05).
Statistical Analysis by Student t Test of Electrocardiogram Parameters in SMA Patients Versus Controls.
Abbreviations: ECG, electrocardiogram; M, mean; SD, standard deviation; SMA, spinal muscular atrophy.
We then divided our cohort into 2 subgroups: the SMA1 and SMA2 groups, comparing each group with controls. We found lower P-wave and QRS amplitudes (P < .01 for both measures) and lower heart rates (P < .01) in SMA1 patients than controls, whereas PRi and QRSi did not differ statistically between the 2 groups (Table 2). Similar results were found in the comparison between SMA2 patients and controls (Table 3).
Statistical Analysis by Student t Test of Electrocardiogram Parameters in SMA1 Patients Versus Controls.
Abbreviations: ECG, electrocardiogram; M, mean; NS, not statistically significant; SD, standard deviation; SMA, spinal muscular atrophy.
Statistical Analysis by Student t Test of ECG Parameters in SMA2 Patients Versus Controls.
Abbreviations: ECG, electrocardiogram; M, mean; NS, not statistically significant; SD, standard deviation; SMA, spinal muscular atrophy.
We finally compared the same measures between SMA1 and SMA2 patients (Table 4). We found only a statistically significant difference in PRi between the 2 groups (PRi SMA1 0.10 ± 0.02 vs PRi SMA2 0.13 ± 0.03 [mean ± standard deviation], P < .01), with a longer PRi in SMA2 patients. All the other parameters were not statistically different between groups.
Statistical Analysis by Student t Test of ECG Parameters in SMA1 Versus SMA2 Patients.
Abbreviations: ECG, electrocardiogram; M, mean; NS, not statistically significant; SD, standard deviation; SMA, spinal muscular atrophy.
Discussion
The major results of our study showed significant differences in the electrocardiographic measures between spinal muscular atrophy and non–spinal muscular atrophy children. In particular, in all spinal muscular atrophy patients we found longer PRi and QRSi, lower P-wave and QRS complex amplitudes, and decreased heart rate with respect to controls. Moreover, when we divided our patients into SMA1 and SMA2 subgroups, we found that statistical differences were maintained for P-wave and QRS complex amplitudes and heart rate, but no statistical difference was observed for PRi and QRSi with respect to controls.
The PRi and QRSi also had less statistical significance in spinal muscular atrophy patients versus controls, so in the division in the 2 groups, the number of patients dims and also statistical significance was lost.
Statistically significant differences in PRi were observed when compared between the SMA1 and SMA2 subgroups; in particular, it was longer in SMA2 than SMA1 patients.
In our cohort, SMA2 patients were older than SMA1 (mean age 4.5 years of SMA1 children vs 15.07 years of SMA2) so the disease had been affecting the heart for more years.
In the literature, the number of case reports presenting bradyarrhythmia in spinal muscular atrophy patients is increasing, but the electrocardiographic history of spinal muscular atrophy patients is currently lacking.
To determine whether spinal muscular atrophy patients showed alteration of cardiac conduction, we examined electrocardiograms in patients in which the diagnosis of spinal muscular atrophy is confirmed by genetic analysis.
Electrocardiography is crucial to understand the electrical conduction system of the heart and to provide insight into different aspects of cardiac and neurologic pathophysiology. In particular, 3 retrospective studies 16 -18 and 4 case reports 19 -22 on spinal muscular atrophy patients and 7 studies on spinal muscular atrophy model mice 14,15,29 -32 have been published in literature.
Back et al in a retrospective study of type 1 spinal muscular atrophy patients found that 15 of 63 (23.8%) patients had severe bradycardia, and 4 of these had severe bradyarrhythmia progressing to cardiac standstill. 16 The recurrent episodes of bradycardia were also observed in type 0 spinal muscular atrophy. 17
In addition to these severe patients, also in type 2 and 3 spinal muscular atrophy were observed the heart block progressing to the complete atrioventricular block. 18 -22
Three other studies 18,19,21 that investigated adult patients with spinal muscular atrophy, detected 1 patient with a disease of the conduction system (atrioventricular block) that required the implantation of cardiac pacemaker. These clinical reports indicate clearly a connection between the cardiac defects and spinal muscular atrophy, doubting that this association is a coincidence.
Preclinical studies in animal models of spinal muscular atrophy have confirmed the presence of alteration in the cardiac conduction. 15,16,29 -32
In our study, we investigated the intrinsic cardiac conduction by recording PRi and QRSi both in control and in spinal muscular atrophy patients. The charts suggest a difference between the 2 groups; the PR and QRS intervals especially are increased in spinal muscular atrophy patients compared with controls. Therefore, a higher conduction time between the sinoatrial and atrioventricular node was observed in spinal muscular atrophy patients, as indicated by the elongated PRi compared to the PRi of control. Specifically, it is higher in spinal muscular atrophy type 2 than in type 1.
The ventricular depolarization times are also elongated, with an increase in QRS complex duration in spinal muscular atrophy compared to controls. Very little is known about the main causes of cardiac dysfunction in spinal muscular atrophy. Because the heart rate is regulated by the autonomic nervous system that controls the decelerating and accelerating of spontaneous activity of cardiac pacemaker, we assume that autonomic nerves might be affected.
The work of Heier et al 14,15 on the spinal muscular atrophy mouse model might be clarifying. The authors performed immunostaining on the mice heart with a marker of sympathetic nerves, an antibody to tyrosine hydroxylase, and found that spinal muscular atrophy mouse hearts showed reduced levels of sympathetic innervation resulting in autonomic imbalance.
Stimulation by the sympathetic nervous system results in the following effects on the heart: positive chronotropic effect (increase in heart rate), positive inotropic effect (increase of contractility), positive dromotropic effect (enhancement of conduction of the electrical signal, eg, it increases AV conduction velocity).
By analyzing our data, we may suggest that the cause of the lower heart rate and of the elongation of PR and QRS intervals in spinal muscular atrophy children may be related to a reduction in the activity of the sympathetic nervous system; accordingly, the sinoatrial node is less stimulated and the electric signal is less conducted both in the common and specific myocardium. This also causes a reduction in the myocardial contractility as evidenced by the reduction in the amplitude of the P-wave and the QRS complex.
The cause of the reduction of sympathetic innervation might be the SMN deficiency; in fact, we identified a homozygous deletion of SMN1 in exon 7 in all 25 patients.
Heier et al 15 showed an improvement of the cardiac conduction after the treatment with tricostatina A, a histone deacetylase inhibitor, that had previously been shown to increase SMN transcription and protein both in vitro and in vivo. 33,34 In particular, there are various articles in the literature that report a sympathetic-vagal imbalance in spinal muscular atrophy patients: a decreased blood flow to the extremities leading to necrosis and abnormal skin responses to temperature changes, 35,36 but nothing on the heart, except the work of Heier on the spinal muscular atrophy mouse model. 14,15
Moreover, we have also found a greater statistical significance in P-wave and QRS amplitudes, which suggested that the SMN protein might be deficient in the cardiac muscle, and in the myocardiocytes, with subsequent reduction in atrial and ventricular contraction. If we consider that echocardiography of all patients was normal, these findings may suggest that the electrocardiogram is an early marker of alteration in cardiac contraction in spinal muscular atrophy patients.
Conclusions
Our study underlines that the cardiac aspect should be investigated in spinal muscular atrophy patients. Spinal muscular atrophy is more than a neuromuscular disease; the deficiency of SMN protein may affect cardiac function, especially sympathetic innervation, the cardiac conduction system, and the myocardiocytes. Further studies are needed to define the different phenotypic expression of the disease and the extent of involvement of other cells beyond motor neuron in spinal muscular atrophy.
Considering that spinal muscular atrophy infants and child have significant alterations in PR and QRS intervals, heart rates, and in the amplitudes of P wave and QRS complex, the electrocardiogram might be applied as a clinical biomarker of early cardiac involvement.
Spinal muscular atrophy patients should be part of a well-established follow-up that would include repeat cardiologic visits and electrocardiogram execution, in order to avoid the progression to cardiac standstill as reported in the literature.
Footnotes
Authors Contributions
RF, GV, CR, and MR substantially contributed to the conception or design and to acquisition, analysis, or interpretation of data, and drafted the manuscript. ADC contributed to the conception or design and to acquisition, analysis, or interpretation of data and drafted the manuscript. CF, AP, and SA contributed to the acquisition, analysis, or interpretation of data. All authors critically revised the manuscript for important intellectual content, gave final approval, agreed to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical Approval
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as revised in 2000 37 and was approved by the ethic committee of the University of Catania, Italy.