SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs

Abstract

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T₅₀ for Ca²⁺ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca²⁺ cycling dysregulation.

Fig. 1

Elevated expression of heart failure markers in SMA model mice. qRT-PCR analysis of a BNP and b ACTA1 mRNA expression. Expression levels (mean ± SEM) in SMN-deficient (SMA) heart tissue are represented relative to unaffected controls at each time point. Statistical analysis: p values shown for one-way ANOVA, Tukey’s post hoc test, n = 7

Fig. 2.

Contraction kinetics are slowed in SMA cardiomyocytes. a Representative recordings of sarcomere length obtained from ventricular myocytes isolated from unaffected or SMA mice (aged P12–13). Cells were field stimulated to contract at 1 Hz. Fractional shortening was not markedly different; however, there was a significant decrease in the diastolic sarcomere length. b Shown are normalized average record of a typical single twitch illustrating the slowed contraction and relaxation kinetics in SMA compared to control cardiomyocytes. Both the time to peak shortening and time to 50% relaxation (T₅₀) were significantly increased in SMA myocytes compared to control Statistical analysis: p values shown for t test, n = 36 and 37 for SMA and control, respectively

Fig. 3

Transcriptome analysis suggests Ca²⁺ handling is altered in SMA heart. a Shown is a “volcano plot” of statistical significance versus transcript fold change in SMA compared to unaffected control hearts. A total of 1178 transcripts were up- or down-regulated in SMA heart. b Heat map illustrating a subset of genes that were significantly changed. Red denotes high expression, blue denotes low expression. Of note, the principal gene regulating Ca²⁺uptake into the sarcoplasmic reticulum, Atp2a2 (SERCA2), was downregulated.

Fig. 4

SERCA2a expression is reduced in SMA hearts. a qRT-PCR analysis of SERCA2 mRNA. Expression levels in SMN-deficient cardiomyocytes are represented relative to unaffected controls (n = 3). b Western blot analysis of SERCA2a protein levels. Quantification of SERCA protein level in SMA cells relative to controls at right. c qRT-PCR analysis of SERCA2a and SERCA2b mRNA. Expression levels in SMN-deficient (SMA) cardiomyocytes are represented relative to unaffected controls. Statistical analysis: p values shown for one-way ANOVA, Tukey’s post hoc test, n = 7

Fig. 5

[Ca²⁺]_i transient kinetics are slowed in SMA cardiomyocytes. a Representative [Ca²⁺]_i transient measured in ventricular myocytes isolated from SMA or unaffected hearts (aged P12–13). There was no significant difference in the estimated diastolic or peak systolic [Ca²⁺]_i (n = 21 for both SMA and control). b Typical normalized [Ca²⁺]_i transients from SMA and control cells are shown illustrating that the time to 50% removal of cytoplasmic Ca²⁺ (T₅₀) c was significantly increased in SMA myocytes compared to control in agreement with the slowed relaxation kinetics. Statistical analysis: p values shown for t test, n = 41 and 39 for SMA and control, respectively

Fig. 6

Defects in [Ca²⁺]_i handling are recapitulated in cardiomyocytes derived from SMA patient iPSCs. a Representative [Ca²⁺]_i transients measured in iPSC-derived cardiomyocytes engineered from unaffected control (NCRM1) and SMA patient (GM23240) iPSCs illustrating the slowed removal of cytoplasmic Ca²⁺. b Expression of SERCA2a expression is increased (western blot, left) and T₅₀ is decreased (right) in SMA patient iPSC-derived cardiomyocytes when SMN protein is restored. Cells were transfected on day 18 of differentiation and recordings were made on day 20. c Similarly, expression of SERCA2a expression is reduced (western blot, left) and T₅₀ is increased (right) in unaffected cotnrol iPSC-derived cardiomyocytes when SMN protein is reduced. Cells were transfected on day 18 of differentiation and recordings were made on day 20. Statistical analysis: p values shown for one-way ANOVA, Tukey’s post hoc test, n = 7