Depressed β‐adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients’ induced pluripotent stem cell–derived cardiomyocytes

Abstract Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X‐linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+‐handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC‐CMs generated from DMD patients feature blunted positive inotropic response to β‐adrenergic stimulation. To test the hypothesis, [Ca2+]i transients and contractions were recorded from healthy and DMD‐CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD‐CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine‐induced Ca2+ release. In contrast to HC, DMD‐CMs exhibited reduced caffeine‐induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD‐CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA‐seq analyses demonstrated that in DMD CMs the RNA‐expression levels of specific subunits of the L‐type calcium channel, the β1‐adrenergic receptor (ADRβ1) and adenylate cyclase were down‐regulated by 3.5‐, 2.8‐ and 3‐fold, respectively, which collectively contribute to the depressed β‐adrenergic responsiveness.


| INTRODUC TI ON
Duchenne muscular dystrophy (DMD), the most common of 9 types of muscular dystrophy, is an X-linked disease affecting boys and teenagers and rarely adult heterozygous females. The incidence in male newborns is 1:3500, and the prevalence is 6:100 000 in the male population. 1 The disease is caused by mutations in the dystrophin gene encoding the dystrophin protein and constitutes the most severe childhood form of the broader family of muscular dystrophies. [2][3][4] Dystrophin is a key structural/functional protein providing strength and stability to the contracting muscle and is essential for maintaining healthy muscle function; its lack leads to cell damage, impaired Ca 2+ homeostasis, elevated oxidative stress and reduced energy production in muscle cells. Consequently, loss of sarcolemmal dystrophin and dystrophinglycoprotein complex (DGC) promotes muscle fibre damage during muscle contraction. 5 Symptoms onset usually occurs between ages 3 and 5, and includes progressive muscle weakness and wasting. 1,2 By the late teens, the heart and respiratory muscles are also affected, ultimately leading to death due to respiratory and/or cardiac failure. 6,7 Dilated cardiomyopathy (DCM), which is a key pathological feature in DMD patients, affects nearly all patients and is a major cause of morbidity and mortality. 4,7,8 Accordingly, many studies reported depressed left ventricular contractile function in DMD patients [9][10][11][12][13][14][15] which may result from impaired β-adrenergic signalling cascade and/or intracellular and abnormal Ca 2+ handling. [17][18][19] To decipher the mechanism(s) underlying the depressed ventricular function in DMD patients, we tested the hypothesis that DMD patients' iPSC-CMs feature blunted positive inotropic response to β-adrenergic stimulation. Our major findings are: adenylate cyclase are down-regulated in DMD cardiomyocytes by 3.5-, 2.8-and 3-fold, respectively, which may collectively contribute to the depressed β-adrenergic responsiveness. These novel findings provide an explanation for the depressed ventricular function in DMD patients and thus may help to introduce therapeutic support for the patients.

| Western blot
See details in Supplementary Material.

| Statistical analysis
Results are presented as mean ± SEM. See details in Supplementary Material.

| DMD iPSC-CMs exhibit blunted β-adrenergic inotropic responsiveness
All control experiments were performed on healthy male and female (clones fse5m and 24.5) cardiomyocytes. The first step was to determine whether the two healthy clones have similar [Ca 2+ ] ihandling and contractile machineries. As seen Figure S1, the [Ca 2+ ] i transient and contraction parameters are similar in the two healthy clones (fse5m and 24.5), and therefore, one lumped healthy (control) group was used for comparison with adult male and adult female DMD cardiomyocytes. In support of our hypothesis, adult male and adult female DMD cardiomyocytes displayed a blunted positive inotropic response to isoproterenol compared to healthy cells ( Figure 1) As illustrated in Figure  cardiomyocytes was expressed as per cent change from control (Tyrode's, Tyr). The effect of isoproterenol on the contraction parameters of adult male (n = 11), adult female (n = 14) and healthy (n = 7) cardiomyocytes was expressed as per cent change from control (Tyrode's, Tyr). Tyr: Tyrode's solution; *P < 0.05, **P < 0.01, ***P < 0.001 (vs control, Tyrode's). Twoway ANOVA test was performed followed by Holm-Sidak posthoc test smaller than adult male cardiomyocytes ( Figure S2C

| Is the β-adrenergic signalling cascade impaired in DMD cardiomyocytes?
To determine whether the blunted inotropic response was caused by impaired β-adrenergic signalling cascade in DMD cardiomyocytes, we measured the positive chronotropic response to isoproterenol.
As we previously reported 16 and shown here as well (Figure 2A,B), DMD cardiomyocytes exhibit lower (P < 0.001 and P < 0.01 in adult male and adult female, respectively) spontaneous firing rate compared to healthy cardiomyocytes. In support of the functionality of the β-adrenergic signalling cascade, adult male and adult female cardiomyocytes presented a healthy-like positive chronotropic response to isoproterenol. Further, adult male cardiomyocytes exhibited a larger chronotropic response than healthy cells ( Figure 2C), which is in agreement with the increased heart rate in DMD patients. 24 From these experiments we concluded that an impaired β-adrenergic cascade is not responsible for the blunted positive inotropic response in DMD cardiomyocytes.

| Intracellular Ca 2+ handling machinery is altered in DMD cardiomyocytes
Since we excluded the possibility that the blunted inotropic response is due to depressed [Ca 2+ ] i -handling and contractile machineries, we tested the hypothesis that impaired downstream element(s) mediating positive inotropic interventions is depressed in DMD cardiomyocytes.

| SR Ca 2+ handling
Because the inotropic response to isoproterenol and elevated [Ca 2+ ] o was blunted in DMD cardiomyocytes, we hypothesized that this was due to a downstream common element-depleted SR Ca 2+ stores. This hypothesis was tested by investigating: (a) caffeineinduced RyR-mediated SR Ca 2+ release; (b) the inotropic effects of ryanodine and cyclopiazonic acid (CPA), both interfering with SR Ca 2+ -handling. 26,27 3.4.3 | Caffeine-induced RyR-mediated SR Ca 2+ release RyR-mediated SR Ca 2+ release was measured by a brief application of caffeine (10 mmol/L), a RyR2 opener. 28 As illustrated in Figure 4, DMD and healthy cardiomyocytes differed in their response to caffeine. As we previously reported, 21,22,29

| The molecular mechanisms underlying the blunted positive inotropic response in DMD cardiomyocytes
To decipher the molecular mechanisms underlying the blunted positive inotropic response in DMD cardiomyocytes, we carried out a comprehensive RNA-seq analysis, comparing healthy and DMD cardiomyocytes. Altogether, the analysis (see Methods) . iPSC-CMs were stimulated at a frequency 20% higher than the spontaneous rate. (D) The effect of ryanodine on contraction amplitude (L Amp )of adult male (n = 11), adult female (n = 5) and healthy (n = 21) iPSC-CMs was expressed as per cent change from Control (no drug). Tyr, Tyrode's solution; *P < 0.05, **P < 0.01, ***P < 0.001 (vs control, no drug). One-way ANOVA test was performed followed by Posthoc Holm-Sidak test. The effects of cyclopiazonic acid (CPA) (30 mmol/L) on healthy (E), adult male (F) and adult female iPSC-CMs (G). iPSC-CMs were stimulated at a frequency 20% higher than the spontaneous rate. (H), The effect of cyclopiazonic acid (30 mmol/L) on the contraction amplitude (LAmp) of adult male (n = 17), adult female (n = 6) and healthy (n = 14) iPSC-CMs was expressed as per cent change from Control (no drug). *P < 0.05, **P < 0.01, ***P < 0.001 (vs healthy). One-way ANOVA test was performed followed by Holm-Sidak Posthoc test groups. As depicted in Figure 7

| Western blot analysis of ATP2ase2 SERCA2 in DMD iPSC-CMs and healthy iPSC-CMs
To further understand the molecular mechanisms underlying the depressed inotropic response of DMD cardiomyocytes and the depleted SR Ca 2+ stores, we performed Western blot analysis of ATPase 2 SERCA2 in healthy and DMD iPSC-CMs (Figures S3 and   S4). As shown in Figures S3 and S4

| D ISCUSS I ON
To decipher the mechanisms underlying the depressed ventricular function in DMD patients we tested the hypothesis that DMD iPSC-CMs feature blunted positive inotropic response to

| The β-adrenergic positive inotropic response is blunted in DMD cardiomyocytes
Since a clinical hallmark of DMD cardiomyopathy is depressed ventricular function, [9][10][11][12][13][14][15] we attempted to determine the cellular basis of this major pathology. Indeed, we found that male and female DMD cardiomyocytes exhibit a blunted positive inotropic response

| Are SR Ca 2+ -stores depleted in DMD cardiomyocytes?
The findings regarding the blunted inotropic response of DMD cardiomyocytes to isoproterenol and [Ca 2+ ] o lead us to hypothesize that the SR-Ca 2+ -handling capacity is impaired. The first step in testing this hypothesis was to measure the response to caffeine, which induces SR-Ca 2+ release by reducing the activation threshold of RyR2. 29 In agreement with our previous studies, 21,22,29  Overall, the complex alterations in β1-adrenergic mechanisms and the dysfunction of calcium homeostasis may both contribute to the abnormalities of contractile function in mdx myocardium. 48 The comprehensive RNA-seq analysis further show that the AC III and V isoforms are down-regulated by 3-fold in DMD F I G U R E 7 Integrated map consisting of key components of contraction, calcium signalling and adrenergic signalling pathways in cardiomyocytes. Colours are concordant with the waterfall and represent the level of significance in the down-regulated (green) and up-regulated (red) DEGs (differently expressed genes). Dark colours indicate significance at the nominal P-value level, and bright colours indicate significance at the FDR-corrected (false discovery rate) P-value level, P ˂ 0.05 cardiomyocytes. Specifically, AC V which is the major cardiac isoform regulates heart rate and contractility. 35 Therefore, downregulation of AC, especially type V, contributes to reduced cAMP levels and to depressed β-adrenergic responsiveness. Another important element involved in the β-adrenergic inotropic responsiveness is the L-type Ca 2+ channel responsible for Ca 2+ entry during the plateau of the action potential, thereby initiating contraction. 49 The RNA-seq analysis show a 3.5-fold down-regulation of the genes

| The SERCA data
To further decipher putative mechanisms underlying the depleted SR Ca 2+ stores in DMD iPSC-CMs, we found that phosphorylated SERCA2 is overexpressed by 11-fold in adult male and by 2.75 in adult female compared to healthy cardiomyocytes ( Figures S3 and   S4). This finding is consistent with similar findings in mdx mice, which express increased levels of the slow Ca 2+ -pump isoform (SERCA2a) (2-fold) in skeletal muscle. 55 In contrast to the protein expression findings, RNA-seq analysis did not show SERCA overexpression, which may indicate that while the overall transcription levels are not changed, SERCA2 activity is increased, as demonstrated by the overexpression of SERCA2 which probably underwent posttranslational modifications. Increased SERCA activity was previously demonstrated to have a beneficial effect on failing hearts, 52,53,56-58 and therefore, the increase SERCA expression can constitute a compensatory attempt to increase SR Ca 2+ -stores and subsequently DMD iPSC-CMs inotropy.

| SUMMARY AND CON CLUS I ON S
Our results demonstrate that DMD iPSC-CMs exhibit blunted positive inotropic response to isoproterenol and elevated [Ca 2+ ] o compared to healthy cardiomyocytes. In addition, the results from the caffeine, ryanodine and CPA experiments suggest that in DMD cardiomyocytes these depressed responses result from depleted SR Ca 2+ stores. We propose that SERCA2 overexpression in DMD cardiomyocytes constitutes a compensatory mechanism to increase SR Ca 2+ -stores and subsequently DMD iPSC-CMs inotropy. The RNA-seq data demonstrate that down-regulation of ADRβ1 and major AC isoforms contribute to the blunted positive inotropic response to isoproterenol. These results emphasize the involvement of abnormal β-adrenergic cascade and [Ca 2+ ] i -handling in DMD cardiac pathophysiology and may provide the basis for further research and the development of new therapeutic agents targeting these abnormalities. and The US-Israel Binational Science Foundation (BSF) to OB [2019039].

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.