Introduction

Arrhythmias and premature sudden death are tragic sequelae in patients with inherited heart disease that can occur with increased sympathetic activity1,2,3. Familial hypertrophic cardiomyopathy (FHC) is a primary disorder of the myocardium characterized by cardiac hypertrophy in the absence of other loading conditions. It is an autosomal dominant condition caused by defects in many sarcomere protein encoding genes. The majority of disease-causing variants are located in beta myosin heavy chain (MYH7) and myosin binding protein C (MYBPC3)4. It is well recognized that the progression of FHC involves altered energy metabolism, myocyte remodeling, disorganization of cytoskeletal proteins and fibrosis, and results in major adverse cardiac events such as heart failure and sudden cardiac death5,6,7. Intense exercise is thought to promote ventricular tachyarrhythmias, therefore FHC patients are advised to avoid intense physical activity and competitive sport8. Although fibrosis and hypertrophy are recognized substrates for arrhythmias, alterations in the electrical properties of the cardiac myocyte and its response to adrenergic stimulation can also contribute to the genesis of ventricular arrhythmias and sudden cardiac death1,9.

In humans, the R403Q variant in MYH7 causes a severe form of FHC characterized by early-onset and progressive myocardial dysfunction with a high incidence of sudden cardiac death10. Mice expressing the R403Q mutation in MYH6, encode the predominant myosin isoform in the adult mouse heart that is highly homologous in sequence with MYH7, develop hallmark features of hypertrophic cardiomyopathy from 30 weeks of age5. Homozygous mice are viable at birth and look anatomically normal, but die by day 7 with severe dilated cardiomyopathy. Mice heterozygous for the R403Q MYH6 mutation (αMHC403/+) have a normal lifespan, and preserved cardiac function. Young heterozygous mice demonstrate myofibril disorientation, myocyte disarray5, alterations in L-type calcium channel kinetics and altered mitochondrial metabolic activity7 that precede the development of myocyte hypertrophy, myocyte injury and fibrosis. Regardless of the presence of hypertrophy, hearts exhibit impaired diastolic function, myocyte cytoskeletal disarray and altered energetics11.

Similar to FHC patients, αMHC403/+ hypertrophic mice can experience serious arrhythmias with vigorous exercise5,12. The pathophysiology contributing to the development of arrhythmias is unclear. A study using high-resolution optical mapping of αMHC403/+ hypertrophic hearts during ventricular pacing, found no direct correlation between the amount or the pattern of fibrosis and inducibility of arrhythmias13. Arrhythmia formation at the cellular level centers on two key concepts: altered calcium homeostasis and reduced repolarization reserve14,15. In addition to an increase in myofilament calcium sensitivity16, αMHC403/+ mice demonstrate a significant reduction in sarcoplasmic reticulum calcium content7 due to decreased expression of calsequestrin, triadin, junctin and ryanodine receptor 2 (RyR2), the proteins forming the cardiac calcium release unit11. Interestingly, although no differences in diastolic or systolic calcium concentrations were measured in cardiac myocytes isolated from pre-cardiomyopathic αMHC403/+ mice, calcium channel blockers such as diltiazem prevented development of the hypertrophy11,17. In addition an early remodeling of repolarizing K+ currents has been reported prior to the development of hypertrophy in the αMHC403/+ mouse that contributes to alterations in repolarization18. However the effect of sympathetic nervous system stimulation on arrhythmia formation is unclear.

The objective of this study was to investigate the mechanisms for induction of arrhythmias in the αMHC403/+ murine model of FHC with developed hypertrophy in the absence and presence of β-adrenergic receptor stimulation. We performed a suite of in vivo and in vitro studies and found that contrary to effects observed in wt hearts, action potentials in αMHC403/+ myocytes were prolonged and β-adrenergic receptor stimulation shortened the action potential while increasing the frequency of delayed afterdepolarizations and ventricular tachyarrhythmias. This was recapitulated in αMHC403/+ mice following in vivo challenge with isoproterenol. Consistent with the cytoskeletal disarray, CaV1.2-β1 adrenergic receptor colocalization was altered assessed by super-resolution nanoscopy. CaV1.2 was unresponsive to isoproterenol due to increased phosphorylation in mutant hearts and connexin 43 expression was significantly decreased. We conclude that altered ion channel expression, location and function contribute to altered β-adrenergic receptor signaling and increased automaticity. Our data demonstrate for the first time an association between cytoskeletal disarray and arrhythmia formation in the R403Q mutant heart.

Results

Sympathetic stimulation induces sustained arrhythmias in αMHC403/+ mice with a hypertrophic phenotype

First we examined the effect of the β-adrenergic receptor agonist isoproterenol (ISO) on arrhythmia inducibility in vivo. ECGs were recorded in 35–45 week old αMHC403/+ and wt mice before and after intraperitoneal injection of 20 mg/kg ISO. Isoproterenol has a half-life of 2.5 to 5 min19. To monitor arrhythmia inducibility for an extended time period, a second dose of 20 mg/kg ISO was applied 10 min after the first injection (Fig. 1A–D). The dose is well tolerated and does not induce myocardial damage20. Lead II ECGs were recorded using subdermal needle electrodes with no programmed electrical stimulation. Prior to administration of ISO, αMHC403/+ mice exhibited significantly longer QT, QTc intervals and Tpeak Tend duration compared to wt mice (Table 1). In the absence of ISO, single premature ventricular contractions (PVC) were occasionally recorded in αMHC403/+ mice. None of the mice exhibited atrial arrhythmias at baseline or following administration of isoproterenol.

Figure 1
figure 1

Representative electrocardiograms (ECG) before (A–B) during (C–D) and 2 h after (E–F) i. p. injection of 2 × 20 mg/kg ISO in a wt (A, C, E) and a αMHC403/+ mouse (B, D, F). Signal-averaged complexes show distinct effects of ISO on wt (G) and αMHC403/+ mouse (H). Representative ECG recordings in an αMHC403/+ mouse before (I) and after 1 mg/kg atenolol (J) and following ISO (K) treatment. (L) Recovery of the αMHC403/+ mouse 2 h post-ISO. (M–O) R–R intervals plotted against time for ISO treated wt (M), αMHC403/+ mouse (N) and αMHC403/+ mouse treated with atenolol (Ate) 10 min prior to ISO as indicated (O). The irregular RR intervals representing arrhythmias can be seen as dots following ISO.

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Table 1 Electrocardiogram parameters recorded on wt and αMHC403/+ mice.
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Following ISO treatment, both wt and αMHC403/+ mice exhibited a significantly higher heart rate shown as an increase in beats per minute (Table 1). Although QT and QTc intervals, and Tpeak Tend duration were slightly shortened in αMHC403/+ mice following ISO, all remained significantly longer compared to wt mice, while R and T amplitudes became significantly reduced (Table 1, Fig. 1G,H). Arrhythmic events were calculated as number of irregular beats each second of recording and included single PVC’s and ventricular tachycardia (Fig. 1D). One of the eight αMHC403/+ mice suffered cardiac arrest shortly after the ISO injection and significant arrhythmias were recorded in six of the seven surviving αMHC403/+ mice (Table 1). One of the αMHC403/+ mice showed no spontaneous or inducible ventricular arrhythmias. Our data are consistent with reports of arrhythmias and sudden death in αMHC403/+ mice following vigorous swimming5.

A final ECG was taken two hours following administration of ISO. The relative occurrence of arrhythmic events in αMHC403/+ mice was tenfold higher 2 h post-ISO than wt mice challenged with ISO with a higher heart rate (Fig. 1E,F,N vs. M and Table 1). In addition we found that the 6 αMHC403/+ mice that exhibited prolonged exacerbated arrhythmic events demonstrated poor recovery and lethargy 2 h following ISO injection observed as difficulty mobilizing and decreased activity moving around the cage assessed using BAR (bright, alert, responsive) animal monitoring criteria and scoring (see original Monitoring Sheets in Supplementary Information File). This was in contrast to wt mice that remained active and spontaneously groomed and fed following ISO injection.

Consistent with the development of a hypertrophic phenotype, αMHC403/+ mice displayed a significant increase in left ventricular posterior wall thickness and significant decrease in left ventricular internal diameter compared to wt mice (Table 2 and Fig. S1A,B). Stroke volume and diastolic parameters were reduced in mutant mice consistent with previous reports21. In wt mice, ISO treatment induced a significantly greater decrease in left ventricular internal diameter at end systole (LVIDs) and end systolic volume (ESV) compared with mutant hearts. In αMHC403/+ mice treated with ISO the change in ejection fraction (14% increase) was less marked than wt (22%, Table 2). These findings confirm that the αMHC403/+ mouse heart has difficulty complying with the increased contractile demands imposed during sympathetic nervous system stimulation.

Table 2 Echocardiography parameters recorded on wt and αMHC403/+ mice.
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To further investigate the effect of ISO, mice were pre-treated for 10 min with atenolol, a selective β1 AR antagonist. Low dose (1 mg/kg, i.p.) atenolol significantly reduced the heart rate in both wt and αMHC403/+ mice (Fig. 1I,J,O) but the effect was more pronounced in the mutant mice (Table 1). As expected, selective β-blocker pretreatment also relaxed the left ventricles (Table 2, Fig. S1F,H vs. E,G). ISO increased the heart rate in the presence of atenolol in both wt and αMHC403/+ mice (Fig. 1K and Table 1), but importantly heart rate did not remain elevated in the mutant mice treated with atenolol after 2 h’ recovery (Table 1, Fig. 1L,O). The reduction in R amplitude and increase in the relative occurrence of arrhythmic events was less pronounced in the presence of the β1-AR blocker in αMHC403/+ mice (Table 1, Fig. 1O). The recovery from in vivo ISO treatment when atenolol was present was similar to wt mice (Table 1, Figs. 1L,O, S1I,J). αMHC403/+ mice were bright, alert, mobile and active similar to wt mice when pre treated with atenolol followed by ISO. Our data confirm that in addition to facilitating ventricular filling, cardiac selective β1AR blockers can reduce arrhythmogenic activity in αMHC403/+ hypertrophic hearts and decrease lethargy post sympathetic nervous system stimulation.

αMHC403/+ ventricular myocytes exhibit prolonged action potential duration that shortens in the presence of ISO

Next we assessed the AP characteristics of cardiac myocytes isolated from adult hypertrophic αMHC403/+ mice in the absence and presence of acute exposure to ISO (100 nM). Under control conditions, at 1 Hz the resting membrane potential of αMHC403/+ myocytes was slightly depolarized and AP duration significantly prolonged (APD90: 165.1 ± 12.7 vs. 47.2 ± 4.1; n = 62 and n = 63, respectively) (Fig. 2A–C and Table 3). No significant difference in the amplitude of the AP was recorded. Consistent with this the expression level of the cardiac sodium channel protein NaV1.5 was unchanged in αMHC403/+ hearts (Fig. S2). wt cardiac myocytes showed no triggered spontaneous automaticity, while 88.2% of αMHC403/+ cardiac myocytes developed triggered activity, and delayed afterdepolarizations (DADs) at low (1 Hz) stimulation frequency (Table 3). Exposure to ISO caused a prolongation in action potential duration in wt cardiac myocytes (APD50 and APD90, Fig. 2A, Table 3) but shortened αMHC403/+ myocyte APD90 (Fig. 2B,C and Table 3). ISO also significantly increased the probability of DADs in αMHC403/+ myocytes (Fig. 2E,G, Table 3 and Fig. S3).

Figure 2
figure 2

Representative AP recordings from wt (A) and αMHC403/+ ventricular myocytes (B), in the absence (black lines) or presence of 100 nM ISO (red). AP durations at 90% repolarization in the absence (wt n = 62, N = 21; αMHC403/+ n = 63, N = 19), or presence of isoproterenol (wt n = 10, N = 7; αMHC403/+ n = 17, N = 5) (C). Representative AP train recordings (9 Hz) on wt (D and F) and αMHC403/+ cardiac myocytes (E and G), under control conditions (D, E) or the presence of 100 nM ISO (F, G). Zoomed area shows the last 3 s of the 10 s recordings. Representative AP recordings from wt (H) and αMHC403/+ cardiac myocytes (I), in the absence (black) or presence of 10 μM forskolin (blue). Representative AP train recordings in control conditions (J) or the presence of 10 μM forskolin (K) on αMHC403/+ cardiac myocytes. Arrows indicate DADs.

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Table 3 Action potential parameters of wt and αMHC403/+ ventricular myocytes and frequency of delayed afterdepolarizations.
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It is well known that APD depends on heart rate or stimulation frequency15. Pacing αMHC403/+ ventricular myocytes at their baseline heartbeat frequency (9 Hz or 540 beats/min) revealed irregular AP patterns. At the high frequency the cycle lengths of the impulses were shorter than the triggered APD, resulting in ineffective repolarization, early afterdepolarizations (EADs) and consequently depolarized resting membrane potential (Fig. 2E and inset vs. wt control on Fig. 2D). ISO had no effect on the pacing pattern at low stimulation frequency (Fig. S3B), but high frequency stimulus aggravated the effect of ISO in αMHC403/+ cardiac myocytes (Fig. 2G). Furthermore ISO induced DADs in the αMHC403/+ cardiac myocytes, at all stimulation protocols (at 9 Hz in Fig. 2G vs. F and at 3 Hz showed on right panel in Fig. S3A vs. B). A long APD also results in a long refractory period, leading to impaired impulse conduction and reentry in the heart15. Overall, these data indicate that ISO increased the excitability and induction of arrhythmias in αMHC403/+ myocytes.

To further explore the role of the β-adrenergic receptor pathway, we applied the adenylyl cyclase activator forskolin and recorded changes in AP configuration during current clamp. DAD frequency increased in current clamped cardiac myocytes (1.158 ± 0.313 vs. 0.270 ± 0.073, Table 3 and Fig. 2J,K) and altered the AP characteristics compared to ISO (Fig. 2H,I and Table 3). Both forskolin and ISO shortened the AP in αMHC403/+ myocytes, but forskolin also significantly increased the APD50 (Fig. 2I). Isoproterenol did not alter the APD50, but shortened APD90, indicating a predominant effect on repolarization (Phase 3).

To confirm that ISO was increasing susceptibility to arrhythmias in αMHC403/+ ventricular myocytes via protein kinase A, we pre-treated cells for 30 min with a cell-permeable protein kinase A inhibitor PKI (myristoylated PKI 14–22 amide, Tocris). 3 μM PKI attenuated the arrhythmogenic effect of ISO, and DAD frequency in αMHC403/+ myocytes in the presence of 100 nM ISO (0.216 ± 0.116 + PKI vs. 0.525 ± 0.0192 p < 0.05 Table 3). Isoproterenol, in the presence of the protein kinase A inhibitor only slightly shortened the APD (Table 3). Our results indicate that PKA phosphorylation activated by the β-adrenergic signaling cascade is responsible for increased arrhythmic activity in mutant ventricular myocytes.

Cardiac myocytes isolated from αMHC403/+ mice exhibit distinct electrophysiological features in the absence and presence of ISO

Arrhythmia formation in mouse cardiac myocytes can occur as a result of alterations in potassium or calcium currents22. Significant decreases in Ito, IKslow and Isust components of repolarizing potassium currents have been previously reported18 in pre-hypertrophic αMHC403/+ cardiac myocytes. In this study we recorded decreases in Ipeak, Ito, IKslow and Isust components of the repolarizing potassium currents in left ventricular myocytes isolated from hypertrophic hearts (Fig. 3A vs. B,F vs. E). Corresponding with the electrophysiological changes, significantly decreased expression levels of KV4.2, Kir2.1 and Kir6.2 channel proteins were detected in αMHC403/+ versus wt hearts. Localization of the ATP-sensitive K+ channel Kir6.2 was also altered in αMHC403/+ myocytes (Fig. 3G). The expression and localization of other potassium channel proteins: KV1.4, KV1.5, KV2.1, KV11.1 and KV7.1, or their auxiliary subunit proteins TASK1, and KCNE1/MinK were not significantly altered in αMHC403/+ hearts (Fig. S2).

Figure 3
figure 3

Representative potassium current recordings from wt (A, C) and αMHC403/+ (B, D) ventricular myocytes under control conditions (A, B) or the presence of 100 nM ISO (C, D). Whole-cell voltage-gated outward K+ (KV) currents were evoked in response to 500 ms depolarizing voltage steps to test potentials between − 60 and + 40 mV, in 10 mV increments, from a holding potential of − 70 mV (− 70, 0 mV and positive test potentials shown). Inward rectifying K+ currents (IK1) evoked in response to hyperpolarization to − 120 mV. (E–F) Scatter plot with bar graphs show IK density (pA/pF) values for different KV current components as means ± SEM. wt n = 8–15, N = 5 αMHC403/+ n = 10–16 N = 5*p < 0.05 control versus ISO, †p < 0.05 wt versus αMHC403/+ (G) Representative immunofluorescence and corresponding Western blot images for KV4.2, Kir2.1, Kir6.2 potassium channels from wt and αMHC403/+ hearts as indicated. Relative optical density (Rel OD) values were calculated using VDAC (voltage dependent anion channel) as loading control. For further details please see section “Materials and methods”.

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Interestingly αMHC403/+ cardiac myocytes exhibited a significant increase in sensitivity of Ito and IKslow currents to 100 nM ISO (Fig. 3D,F) versus wt myocytes (Fig. 3C,E). Reduced repolarization reserve and increased sensitivity of Ito and IKslow to ISO may contribute to the APD shortening observed in αMHC403/+ cardiac myocytes (Fig. 2B,C). Nevertheless this would only explain in part the increased arrhythmogenicity during sympathetic activation.

It is well recognised that Synapse Associated Protein 97 (SAP97) co-localises with Kir2 and KV channel proteins, anchoring them to the plasma membrane, and aiding correct folding and function23. In addition to AKAP proteins, SAP97 participates in β1-adrenergic receptor localization and PKA phosphorylation24. We measured a significant decrease in SAP97 protein expression in cardiac tissue and myocytes isolated from αMHC403/+ mice versus wt mice (Fig. 4). Furthermore confocal imaging revealed differences in SAP97 protein localization with a relatively preserved surface membrane presentation of the protein, and reduced intracellular SAP97 content. Our data (Figs. 3G, 4) are consistent with changes in cell size and myofilament organization that are characteristic when structural changes such as hypertrophy are present. We measured a profound decrease (~ 40%) in connexin 43 protein expression in αMHC403/+ hearts (Fig. 4). This likely contributes to altered conductivity causing impulse propagation heterogeneity which in combination with decreased repolarization and impaired calcium handling provides a substrate for reentry arrhythmias15,22. Altered calcium handling in the αMHC403/+ model has been reported as a consequence of reduced SR calcium content and calcium accumulation by mutant myofilaments11,16,21. However systolic and diastolic calcium concentrations measured in wt and hypertrophic αMHC403/+ hearts are similar17. Consistent with our previous findings7, in the absence of ISO, quiescent adult αMHC403/+ cardiac myocytes demonstrated a small, but significant decrease in the kinetics of calcium current inactivation (Fig. 5B vs. 5A and E) of the L-type calcium channel (ICa), with no change in peak amplitude (Fig. 5A,B), current density (Fig. 5D), activation or deactivation assessed as the integral of the current (Fig. S4A–C). As expected cell capacitance was increased in αMHC403/+ myocytes consistent with a hypertrophic phenotype (Fig. 5C). Immunoblot studies indicated that there was no significant difference in CaV1.2 protein expression in heart homogenates from αMHC403/+ versus wt mice (Fig. 5F).

Figure 4
figure 4

Representative immunofluorescence images of ventricular myocytes isolated from wt and αMHC403/+ hearts, immunolabelled with connexin 43, caveolin-3 and SAP97. Scale represents 20 μm. Representative Western blot images with relative optical density values are also shown for the same proteins, along with corresponding loading controls. *p < 0.05 wt versus αMHC403/+. For further detail see section “Materials and methods”.

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Figure 5
figure 5

Representative calcium current recordings and IV traces from wt (A) and αMHC403/+ (B) ventricular myocytes, under control conditions (black lines) or the presence of ISO (red). Scatter plot with bar graphs show the cell capacitance (C) used to calculate ICa density (pA/pF) (D); and (E) the rate of inactivation (tau). Number of cells used for the study: wt n = 26, with ISO n = 13; αMHC403/+ n = 42, with ISO n = 22. (F) Representative Western blot images of CaV1.2 protein using total heart homogenates from wt (N = 5) and αMHC403/+ hearts (N = 4) repeated in triplicate. (G) In vitro PKA phosphorylated immunoprecipitated protein samples were used to fluorescently detect total phosphoprotein as well as PKA-specific phosphorylation. (H, I) Representative Fluo-4 calcium transients acquired on current clamped ventricular myocytes isolated from wt (H) and αMHC403/+ mice (I) under control conditions (black traces, n = 23, N = 5; and n = 18, N = 5 respectively) or the presence of 100 nM ISO (red traces, (n = 13, N = 5; and n = 11, N = 5 respectively). Scatter plots with bar graphs show relative amplitude (as F/F0, J), exponential rise time (ms) (K) and exponential decay (tau) (L). (M–N) Representative calcium current recordings from wt (M) and αMHC403/+ (N) cardiac myocytes under control condition (black) or the presence of 10 μM forskolin (blue). Scatter plot with bar graphs show ICa density (pA/pF) (O); and the rate of inactivation (tau) of ICa traces (P) presented as means ± SEM *p < 0.05 control versus ISO, or wt versus αMHC403/+, number of asterisks representing increasing significance in p value. Number of cells used: wt n = 30 ctrl, n = 6 with forskolin and αMHC403/+ n = 45 ctrl, n = 10 with forskolin.

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However, most surprisingly acute ISO exposure (100 nM) had no effect on current density (Fig. 5D), activation or deactivation parameters of ICa in αMHC403/+ (Fig. S4A–C), although it substantially increased ICa in wt cardiac myocytes (Figs. 5D,E, S4A–C). Under paced conditions (1 Hz), no significant difference in calcium transients was recorded in αMHC403/+ versus wt myocytes (Fig. 5H–L), in agreement with previous reports17. But in the presence of 100 nM ISO, the peak amplitude of the calcium transient was increased significantly in wt myocytes only (Fig. 5H, J). We examined the phosphorylation levels of immunoprecipitated CaV1.2 protein and found increased basal phosphorylation in αMHC403/+ hearts. Exposure of the immunoprecipitated CaV1.2 to PKA increased the phosphorylation level of the CaV1.2 channel in wt but not αMHC403/+ hearts (Fig. 5G), suggesting that CaV1.2 protein was already significantly phosphorylated in αMHC403/+ hearts under basal conditions. This was consistent with the lack of response of ICa to ISO in αMHC403 myocytes.

αMHC403/+ myocytes exhibit altered CaV1.2 and β1AR clustering and co-localization

In vitro electrophysiological and ex vivo biochemical studies demonstrated no difference in CaV1.2 (Fig. 5F) and β1-adrenergic receptor expression (Fig. S2), or basal calcium current in αMHC403/+ versus wt hearts under control conditions (Fig. 5A,B,D). Recent studies have demonstrated super-clustering of CaV1.2 promoted by β1-adrenergic receptor stimulation in mouse cardiac myocytes25. To investigate a potential role for altered channel clustering and CaV1.2– β1-AR colocalization, we performed super resolution microscopy experiments.

Pre-treatment with ISO (100 nM isoproterenol, 10 min) induced a significant increase in the formation of superclusters of CaV1.2 in wt but not in αMHC403/+ myocytes (Fig. 6)). While β1-AR cluster area was not significantly different in αMHC403/+ cardiac myocytes compared to wt myocytes under control conditions (Fig. 7A,C,E), ISO treatment significantly altered β1AR cluster area size in αMHC403/+, but not in wt myocytes (Fig. 7B,D,E).

Figure 6
figure 6

ISO-induced super-clustering response of CaV1.2 in ventricular myocytes. (A–B) TIRF images (top row) and super-resolution GSD localization maps (bottom row) of immunostained CaV1.2 channels in fixed adult ventricular myocytes isolated from wt (A) and αMHC403/+ mice (B). Control (left column) and ISO-stimulated (right column) myocytes are displayed side-by-side for comparison. Cluster ROIs indicated by yellow boxes in the GSD images appear magnified below the relevant image (bottom row). Mean CaV1.2 channel cluster areas ± S.E.M. (indicated by red lines and error bars) are summarized for each condition in the aligned dot-plot (C). *p < 0.05 for comparisons as indicated. n = 14–27 ventricular myocytes, from N = 4–7 mice.

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Figure 7
figure 7

Colocalization of β1ΑRs with CaV1.2 is altered in αMHC403/+ mutant myocytes. (A) and (B): TIRF images (top row), super-resolution GSD localization maps (middle row) and binarized images (bottom row) of immunostained CaV1.2 channels and β1ARs in representative fixed adult ventricular myocytes isolated from WT mice under control (A) or ISO-stimulated conditions (B). Merged two-channel image showing relative distributions of CaV1.2 and β1ARs is shown in the third column with the colocalized binarized image. (C, D) Same layout format for myocytes isolated from αMHC403/+ mice. (E) Mean β1ARs cluster areas ± S.E.M. (indicated by red lines and error bars) are summarized for each condition in the aligned dot-plot. (F) % colocalization of CaV1.2 with β1ARs and G: β1ARs and CaV1.2 are summarized. *p < 0.05 for comparisons as indicated. n = 14–27 ventricular myocytes, from N = 4–7 mice.

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Colocalization analysis of αMHC403/+ and wt cardiac myocytes labelled with CaV1.2 and β1AR antibodies revealed a relatively small number of CaV1.2 colocalized with β1ARs in wt cardiac myocytes and a significantly higher proportion of the CaV1.2 colocalized with β1ARs in αMHC403/+ myocytes under control conditions (Fig. 7A–D,F). ISO treatment resulted in more CaV1.2 protein colocalizing with β1ARs in wt cardiac myocytes, but under the same conditions, comparatively less CaV1.2 were found to be localized with β1ARs in αMHC403/+ myocytes (Fig. 7A–D,F). Colocalization of the CaV1.2 with the β1AR may explain the higher phosphorylation level of the channel protein in αMHC403/+ cardiac myocytes (Fig. 5G). There was no change in β1AR co-localizing with CaV1.2 (Fig. 7A–D,G).

Previous studies have demonstrated a role for caveolin-3 in forming macromolecular complexes with β-adrenergic receptors and in β-adrenergic signaling26,27. Although αMHC403/+ myocytes displayed a reduced level of caveolin-3 expression (that was not statistically significant) versus wt myocytes (Fig. 4), no alterations in cluster area or colocalization with CaV1.2 protein were observed (Fig. S5).

Our super resolution study indicated that CaV1.2 protein and β-adrenergic receptor localization are altered in αMHC403/+ myocytes. To further confirm the altered CaV1.2-β1AR co-localization, we examined the effect of forskolin on ICa. In contrast to the lack of response to ISO, the addition of 10 µM forskolin significantly increased the amplitude, activation and inactivation of ICa in αMHC403/+ myocytes similar to that of wt myocytes (Figs. 5M–P, S4D–F). These data indicate that altered ion channel and β1AR localization are responsible for altered calcium handling in αMHC403/+ myocytes.

CaV1.2 is near maximally phosphorylated in αMHC403/+ mice

To further investigate the mechanisms for the poor recovery of αMHC403/+ mice following ISO treatment, hearts were collected following in vivo isoproterenol challenge, and phosphorylation of CaV1.2 assessed. Untreated αMHC403/+ mouse hearts demonstrated phosphorylation of both CaV1.2 and total protein compared to wt, that remained unchanged following ISO treatment (Fig. S6A–C). These data suggest that CaV1.2 is phosphorylated under control conditions in αMHC403/+ mice. Creatine kinase (CK) activity was also significantly increased in hearts from ISO treated αMHC403/+ mice versus wt mice suggesting ongoing myocardial injury (Fig. S6D).

Discussion

Hypertrophic cardiomyopathy is characterized by disorganization of cytoskeletal proteins and myofibrils, myocyte remodeling, fibrosis and altered energy metabolism10. We and others have demonstrated that αMHC403/+ mice develop clinical features similar to those found in human disease at both cellular and whole heart level.7,21. Alterations in the electrical properties of the cardiac myocyte that occur in response to stressors such as increased adrenergic stimulation can contribute to the genesis of ventricular arrhythmias and lead to sudden cardiac death9. The objective of this study was to investigate the mechanisms for increased arrhythmogenic activity resulting from the human FHC disease causing mutation during sympathetic stimulation.

Our data indicate that AP characteristics in αMHC403/+ myocytes were significantly prolonged and contrary to responses in wt cells, the AP shortened under conditions of increased adrenergic stimulation. At normal murine heartbeat-frequency (9 Hz) in the presence of ISO, membrane repolarization was incomplete in αMHC403/+ myocytes, the resting membrane potential became more depolarized, resulting in more early and delayed afterdepolarizations. In addition the AP refractory period was prolonged, which is a recognized substrate for impaired impulse conduction and reentry in the heart15.

The most apparent difference between human and murine AP kinetics is in the plateau phase. In mouse heart depolarizing ICa is less pronounced, while repolarizing Ito and IKur are more prominent, and as a result, murine APs demonstrate a more rapid repolarization. Nevertheless similarities in structure, excitation–contraction coupling, recovery and propagation of excitation can be investigated at the molecular, cellular, tissue, organ, and whole-animal level in the mouse22,28. To clarify any differences in APs between mouse and man, we performed additional experiments and assessed the AP characteristics of hiPSC-CMs (see SI methods) from a hypertrophic cardiomyopathy patient carrying R403Q MYH7 mutation (MYH7 403/+, see family pedigree, Fig. S7C). Action potential measurements were performed with the kinetic imaging cytometry (KIC) platform. Similar to the mouse mutant myocytes, the AP duration of MYH7 403/+ was significantly prolonged (Fig. S7B,E,F) versus the isogenic CRISPR corrected control, MYH7 403/− (Fig. S7A,D,F). Also similar to the αMHC403/+ myocytes, application of 1 µM ISO significantly shortened MYH7 403/+ AP duration while ISO slightly prolonged the isogenic CRISPR corrected control AP. Therefore action potential alterations in hiPSC-CMs are similar to αMHC403/+ myocytes and electrical remodeling at the cardiac myocyte level occurs early in FHC. In support of this, a high incidence of arrhythmias and sudden cardiac death in cardiac troponin T (TnT-I79N) mutant mice have been reported, in the absence of a hypertrophic phenotype. Introducing the TnT-I79N mutation into human induced pluripotent stem cells with CRISPR/Cas9 technique reproduced key features of FHC including myofilament disarray, hypercontractility and diastolic dysfunction, as well as alterations in the ventricular AP29. However the proposed mechanisms often differ between specific mutations. In the TNNT2-R92Q mouse model30, a decrease in NaV1.5 and increase in CaV1.2 expression and late sodium current (INaL) contribute to the phenotype. Isoproterenol further prolongs the AP. In our model, we found no difference in NaV1.5 or CaV1.2 expression and isoproterenol shortened the AP duration.

In addition to a decrease in the expression and function of some potassium channels, we report here an increase in the sensitivity of Ito and IKslow to ISO that appeared to contribute to the APD shortening observed in αMHC403/+ cardiac myocytes. SAP97 protein expression was decreased in mutant hearts consistent with an alteration in the trafficking and localization of K+ channels. SAP97 protein also localized to the β1-adrenergic signaling complex. Importantly we demonstrated a substantial decrease in connexin 43 protein expression that correlates with morphological and histological changes in the hypertrophic myocardium. This can contribute to impaired impulse conduction leading to impulse propagation heterogeneity15,22.

Consistent with previous studies, we found no difference in diastolic or systolic intracellular calcium levels in αMHC403/+ myocytes or in CaV1.2 expression compared with wt hearts7,17. However, despite the small, but significant difference in the inactivation rate of the channel and calcium transients, we could not increase the Cav1.2 current when αMHC403/+ myocytes were exposed to β1-adrenergic stimulation. Consistent with this we revealed an elevated phosphorylation state of the CaV1.2 channel protein extracted from cardiomyopathic αMHC403/+ mice. We cannot rule out the possibility that altered phosphatase activity contributes to the elevated phosphorylation state of the channel.

It is well documented that β1-adrenergic stimulation enhances the cardiac L-type calcium channel activity. To facilitate co-operative gating the CaV1.2 molecules form super-clusters25. Assessed with super-resolution microscopy we demonstrate that in ventricular myocytes isolated from hypertrophic αMHC403/+ hearts CaV1.2 proteins form clusters, similar to wt cells, but isoproterenol treatment only increased the size of these clusters significantly in wt cardiac myocytes. The CaV1.2 molecules not only clustered differently, but there were more CaV1.2 localized together with β1AR’s in mutant myocytes. This demonstrates for the first time a direct interaction between CaV1.2 and β1ARs, which can explain the higher phosphorylation level of the channel protein in αMHC403/+ cardiac myocytes. In vitro ISO treatment stimulated colocalization of CaV1.2 and β1ARs in wt cardiac myocytes, but in αMHC403/+ myocytes it decreased the number of the CaV1.2 localized with β1ARs.

Here we demonstrate that β-adrenergic stimulation alone is sufficient to increase the probability of arrhythmic activity in αMHC403/+ mice. Hypertrophic hearts of the αMHC403/+ mice not only demonstrated ISO-induced arrhythmias, but the mice did not recover well from the ISO challenge resulting in fatigue and tissue damage assessed as a significant increase in creatine kinase activity in the αMHC403/+ hearts but not wt hearts. FHC patients reportedly experience serious cardiovascular events and fatigue following vigorous exercise12.

Our results suggest that cytoskeletal disarray contributes to the alterations in ion channel and β1 adrenergic receptor localization and function in the αMHC403/+ heart. Reduced repolarization reserve and altered conduction velocity are associated with the generation of arrhythmias during β1-adrenergic receptor stimulation in the αMHC403/+ heart. However, pro-arrhythmic mechanisms may vary depending on the underlying gene mutation reinforcing the need to individualize treatment options with the genetic mutation. We find that inhibition of the β1AR with atenolol relaxed ventricular muscle and improved filling, but also significantly reduced the occurrence of arrhythmic events and allowed the mice to recover fully from the adrenergic challenge. Our data indicate that treatment with selective β1AR blockers may be sufficient to manage arrhythmias in patients carrying an R403Q mutation. Furthermore we highlight the significance of cytoskeletal disarray in altering ion channel location and function and β-adrenergic receptor signaling, leading to electrical instability in the FHC heart.

Materials and methods

Mouse model

Male 35–45 wk old heterozygous αMHC403/+ mice expressing the human disease-causing mutation R403Q in MYH6 were used. We used male mice because female mice carrying the αMHCR403Q/+ mutation develop hypertrophic cardiomyopathy less consistently than males. Mice were used to establish a colony received as a gift from C and J Seidman (Department of Genetics, Harvard Medical School, MA). Negatively genotyped male age-matched littermates were used as wild type (wt) controls. A total number of 68 wt and 92 αMHC403/+ mice were used in the study. In the text N indicates number of animals, n indicates number of cells.

All experiments were approved by The Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 8th Edition, 2013; updated 2021) and all methods reported in accordance with the ARRIVE guidelines.

Electrocardiography and echocardiography studies

Mice were anesthetized using methoxyflurane and placed on a warming plate (37 °C). Electrocardiograms (ECGs) were recorded with s.c. bipolar leads (lead II) using a PowerLab data acquisition system with Animal BioAmp for 10 min prior to (control) and following i.p. isoproterenol (ISO, two doses of 20 mg/kg administered 10 min apart) or atenolol (1 mg/kg). Parameters were measured on signal-averaged complexes derived from 10 s of contiguous data. QT interval was corrected for heart rate using the Mitchell method31. The relative occurrence of arrhythmic events was measured as number of irregular beats per second of recording (LabChart ADInstruments).

In parallel experiments, echocardiograms were recorded using i13L probe on a Vivid 7 Dimension (GE Healthcare) as previously described32.

Electrophysiology

Action potential recordings

Left ventricular cardiac myocytes were isolated as described32, for more details please see SI. Cells were stimulated in current clamp mode at 1, 3 or 9 Hz with 0.2 ms suprathreshold stimuli. Glass pipettes (4–5 MΩ) were filled with pipette solution (in mM): 120 K-glutamate, 20 KCl, 10 NaCl, 2 MgCl2, 0.1 EGTA, 5 Hepes, 5 MgATP, 0.03 CaCl2, pH 7.05. Experiments were performed at 37 °C in Tyrode solution (in mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.5 MgCl2, 5.5 Hepes, 11 glucose, pH 7.4 at room temperature. AP duration was measured at 90% (APD90) and 50% (APD50) of repolarization.

Potassium (K+) and calcium (Ca2+) current recordings

For recording of whole-cell K+ currents, pipettes were filled with AP recording pipette solution. Bath solution contained (in mM): 136 NaCl, 4 KCl, 2 MgCl2, 1 CaCl2, 10 Hepes, 10 glucose, 0.02 tetrodotoxin (TTX, Tocris), 0.002 nisoldipine, pH 7.4.. Whole-cell voltage-gated K+ currents were evoked in response to 500 ms depolarizing voltage steps to test potentials of − 120 mV and between − 60 and + 40 mV (10 mV increments) from a holding potential of − 70 mV. Experiments were performed at 37 °C. Peak KV current and IK1 amplitudes were measured as the maximal amplitudes of currents. Ito amplitude was determined from exponential fit to the decay phase of the outward K+ current, Isustained component as the current amplitude at the end of the test pulse and IK,slow was calculated as the difference between Ito and Isustained18.

Ca2+ currents were recorded in bath solution (in mM): 140 NaCl, 5.4 CsCl, 1 CaCl2, 0.5 MgCl2, 5.5 HEPES, 11 glucose, pH 7.4 at 37 °C as previously described32. Pipette solution contained (in mM): 115 CsCl, 1 CaCl2, 20 TEA-Cl, 10 HEPES, 10 EGTA, 5 MgATP, 0.1 Tris-GTP, 10 phosphocreatine, pH7.05. Ca2+ currents were monitored by applying a 100 ms test pulse to 10 mV after a 50 ms prepulse to − 30 mV once every 10 s. Kinetics of calcium current inactivation was analysed by fitting current decay after channel activation with a bi-exponential function (yielding tau 1 and tau 2). Current activation and inactivation were also assessed by calculating the integral or “area under a curve” as the area between the graph of y = f(x) and the x-axis.

Both APs and whole cell currents were recorded using an Axopatch 200B voltage-clamp amplifier (Molecular Devices) and an IBM compatible computer with a Digidata 1400 interface and pClamp10 software (Molecular Devices). Cardiac myocytes isolated from the same animal were used for AP and Ca2+ or K+ current measurements. Data analyses were executed with Clampfit10 and GraphPad Prism8, results are reported as mean ± SEM.

Immunoblotting

Tissue was weighted, homogenised in 1:4 RIPA buffer, consisting of (in mM): 150 NaCl, 50 Tris, 20 Na4P2O7, 2 Na3VO4, 1 NaF, 0.5% Na deoxycholate, 1% Triton X-100, 0.1% SDS, EDTA-free Complete protease inhibitor cocktail (Roche), and Phosphatase inhibitor cocktail, pH7.4. The homogenate was centrifuged at 10,000 g for 5 min at 4 °C. 25 μg of pooled tissue homogenate (N = 3 wt and 3 αMHC403/+ hearts) was loaded into precast 10% Bio-Rad Mini-Protean TGX Stain-FreeTM SDS–polyacrylamide gel, then electrophoretically transferred to 0.2 µm nitrocellulose membrane (Trans-Blot TurboTM Transfer Pack (Bio-Rad) using the Bio-Rad Trans-Blot TurboTM Transfer System. Western blot experiments were run in triplicate, representative images are shown in figures. Antibodies used in the study are listed in SI. Densitometry was performed using ImageJ software. Background subtracted intensity values were normalized to loading controls VDAC or GAPDH signal on the same blot. For more details and for full length blots see SI.

Immunprecipitation and in vitro phosphorylation of CaV1.2 protein

Anti-CaV1.2 antibody pre-incubated with Dynabeads Protein G (Thermo Fisher Scientific) used to pull out the CaV1.2 protein from tissue homogenates. 2 Unit PKA catalytic subunit (Promega) was used per each μg of immunprecipitated protein to perform in vitro phosphorylation as previously described33. All immunoblot experiments were run as triplicate, representative images were shown on figures. For more details and for original blots see SI.

Immunocytochemistry

Precision cover glass (No. 1.5H, Marienfeld Superior) cleaned then coated with poly-L-lysine (0.01%, 20 min) and laminin (20 μg/ml, 45 min; Life Technologies).

For confocal microscopy cells were fixed with 4% formaldehyde, solubilized with 0.5% TritonX-100. Primary antibodies, used for Western blots (1:100 dilution, 5% BSA, 2 h, RT) and fluorescently labelled (Alexa Fluor 488 or Alexa Fluor 555) secondary antibodies (1:1000, 1 h RT, Abcam) were used. After mounting with ProLong™ Glass Antifade Mountant (ThermoFisher), cells were imaged with Nikon C2 confocal microscope coupled with NIS elements software.

For super-resolution studies, some coverslip adherent cells were treated for 10 min with 100 nM isoproterenol prior to fixation in ice-cold methanol for, 5 min. After washing cells were blocked (45 min, RT) in blocking buffer: 20% SEA Block (Thermo Fisher Scientific), 0.05% v/v Triton X‐100 in PBS. Cells were incubated overnight at 4 °C with mouse monoclonal anti‐CaV1.2 (UC Davis/NIH NeuroMab Facility, clone N263/31; 1:100) with rabbit polyclonal anti-caveolin-3 or rabbit polyclonal β1 adrenergic receptor antibody. Secondary antibodies used were Alexa Fluor 647‐conjugated goat anti‐mouse IgG2b or Alexa Fluor 555‐conjugated goat anti‐rabbit (Life Technologies, 1:1000).

Super-resolution nanoscopy

Cells were imaged on a super-resolution Ground State Depletion (GSD) microscope (Leica Microsystems, Wetzlar, courtesy of Dr. F Santana) in TIRF mode with 150 nm penetration depth as previously described34. Fluorescence was detected through a Leica high-power TIRF quad filter cube (QGS HP-T) with emission band-pass filters. The collected frames were reconstructed into super-resolution localization maps using Leica Application Suite (LAS AF) software. Cluster area size was measured from binary masks of the localization maps with a 10 nm pixel size in ImageJ/Fiji as previously described35.

Assessment of changes in intracellular calcium under paced conditions

Myocytes were incubated in Fluo-4-AM (5 μM, Life Technologies, 20 min), then stimulated in current clamp mode with suprathreshold stimuli using an Axopatch 200B voltage-clamp amplifier (Molecular Devices). Signal was recorded using a Zyla 5.5 sCMOS camera and MetaMorph 7.10.3 sotware.

Statistical analysis

Results are reported as means ± SEM. Statistical analysis was performed using Prism GraphPad software. The Shapiro–Wilk normality test was used to assess whether the data were normally distributed. If the data were normally distributed a Brown-Forsyth and Welch ANOVA was used to analyse differences between wt and αMHC403/+ groups. Where data were not normally distributed a Kruskal–Wallis ANOVA was performed. A Dunn’s test was used to correct for multiple comparisons. All chemicals and reagents were purchased from Sigma-Merck unless otherwise specified.