Novel Calcium Channel Modulator Ameliorates Hypertrophic Cardiomyopathy

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Hypertrophic cardiomyopathy (HCM) is characterized by incomplete penetrance. Therefore, in patients carrying a mutation associated with HCM, a diagnosis is confirmed on presentation of left ventricular hypertrophy assessed by echocardiography. Consequently, current therapeutics including mavacamten are prescribed after hypertrophy is established. Furthermore, mavacamten has not demonstrated efficacy in the ODYSSEY phase 3 clinical trial for nonobstructive HCM. Because one-third of patients with HCM develop a nonobstructive phenotype, there is a significant unmet need to develop therapeutics that are effective in nonobstructive HCM.

We have previously identified a structural-functional link between the L-type calcium channel (LTCC) and the mitochondria that can be targeted to prevent the development of HCM in mice. The β subunit of the LTCC is tethered to the cytoskeleton via the Neuroblast differentiation–associated protein AHNAK, providing a structural-functional link for the LTCC to the mitochondria. The opening of the LTCC transmits a mechanical signal through the cytoskeleton via the β subunit to the mitochondria to alter mitochondrial function independently of Ca^2+1,2 (Figure [A]). Depolymerization of cytoskeletal filaments attenuates the ability of the LTCC to alter mitochondrial function. In HCM, cytoskeletal disarray is present and contributes to the hypermetabolic state.^1,2

Figure.

Treatment of cTnI-G203S mice with AIDP14-TAT or AIDP16-TAT significantly ameliorates the hypertrophic phenotype after 15 weeks of treatment. A, Schematic of the structural-functional link between the L-type calcium channel and mitochondria in cardiomyocytes. B, Normalization of flavoprotein oxidation and JC-1 (5,5’,6,6’ tetrachloro1,1’tetraethylbenzimidazolylcarbocyanine iodide) in cTnI-G203S mice after a 10-week treatment regime. Mean±SEM (left) and representative traces (right). To confirm the signals were mitochondrial in origin, 50 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added in flavoprotein experiments, and 40 mM sodium cyanide (NaCN) was added in JC-1 experiments. C, Some echocardiography and Doppler parameters improved after treatment with 5 µM AIDP14-TAT or 5 µM AIDP16-TAT for 10 weeks. D, All echocardiography and Doppler parameters significantly improved after 15 weeks’ treatment with 10 μM alpha-interaction domain (AID) variant peptides. E, Representative images showing Masson’s trichrome staining of sections of the left ventricle from wt or cTnI-G203S mice after treatment as indicated. Mean±SEM of collagen volume fraction normalized to wt average as indicated. Scale bar=50 µm. To address independent sampling a nonparametric hierarchical randomization test with Benjamini-Hochberg post hoc test was performed for flavoprotein, JC-1 and fibrosis analyses using the hierarch python package.³ For echocardiography studies, parametric data were analyzed with Welch ANOVA and Dunnet T3 post hoc test. Nonparametric data were analyzed with the Kruskal-Wallis test with Dunn post hoc test. AHNAK indicates neuroblast differentiated associated protein; AIDP14-TAT, a variant alpha-interaction domain peptide (RKKRRQRRR-6 amino hexanoic acid-QQEEEDLKGYLDWITQAE-NH₂); AIDP16-TAT, a variant alpha-interaction domain peptide (RKKRRQRRR-6 amino hexanoic acid-QQREEDLKGYLDWITQAE-NH₂); EF, ejection fraction; FS%, fractional shortening; IVSd, interventricular septum diastole; IVSs, interventricular septum systole; LVIDd, left ventricular internal diameter diastole; LVIDs, left ventricular internal diameter systole; LVOT, left ventricular outflow tract; LVPWd, left ventricular posterior wall diastole; LVPWs left ventricular posterior wall systole; maxPG, maximum pressure gradient; MV E/A, ratio early to late ventricular peak filling velocities; N, number of animals; n, number of cells (flavoprotein or JC-1) or number of sections (fibrosis); Rx, treatment; VDAC, voltage-dependent anion channel; and Vmax, maximum velocity. Figure A was created in BioRender. Er, T. (2026) https://BioRender.com/l3bjxwd.

Disruption of the interaction between the β and α1c subunits with peptides derived against the alpha-interaction domain (AID) can normalize the hypermetabolic state in HCM mice and improve contractility by correcting cytoskeletal organization.^4,5 The AID variant peptides do not alter calcium conductance through the channel.^4,5 We have previously reported that the treatment of young cTnI-G203S and αMHC^403/+ mice with the AID variant peptides can prevent the development of HCM.^4,5 In this study, we investigated whether 2 of these variant AID peptides were effective in ameliorating established HCM in αMHC^403/+ and cTnI-G203S mice.

We treated cTnI-G203S and αMHC^403/+ mice with 5 µM AIDP14-TAT (RKKRRQRRR-6 amino hexanoic acid-QQEEEDLKGYLDWITQAE-NH₂) or AIDP16-TAT (RKKRRQRRR-6 amino hexanoic acid-QQREEDLKGYLDWITQAE-NH₂) 3× per week for 10 weeks via intraperitoneal injection. Similar to our previous studies,⁴ the treatment did not alter liver or kidney function, or blood pressure in the mice. Application of the LTCC agonist BayK(−) significantly increased flavoprotein oxidation and ψm (Figure [B]) in myocytes from cTnI-G203S mice treated with AID(S)-TAT (a scrambled inactive peptide) compared with wild-type myocytes, consistent with the hypertrophic phenotype. The in vivo treatment of cTnI-G203S mice with AIDP14-TAT or AIDP16-TAT resulted in normalization of flavoprotein oxidation and ψm (Figure [B]) to wild-type levels in myocytes. The peptides also normalized flavoprotein oxidation and ψm in αMHC^403/+ mice. The normalization of ψm occurs independent of Ca²⁺ (as evidenced in JC-1 experiments performed in Ca²⁺ free solution (Figure [B])). Here, the mechanistic action of these peptides is to normalize the hypermetabolic state in HCM by altering the communication between the LTCC and the mitochondria.

We then assessed the efficacy of these peptides in ameliorating hypertrophy and myocardial fibrosis. Treatment of cTnI-G203S mice for 10 weeks with 5 µM AIDP14-TAT or AIDP16-TAT 3× per week did not significantly alter echocardiography parameters, but diastolic function improved (Figure [C]). There were also significant improvements in fractional shortening, diastolic function (LVOT and mitral valve E/A ratio [MV EA ratio]), and a reduction in myocardial fibrosis in the αMHC^403/+ mice, but there was no change in myocardial fibrosis after treatment of cTnI-G203S mice with AIDP14-TAT or AIDP16-TAT.

Because some beneficial effects were recorded after 10 weeks’ treatment, we assessed the effects of increasing the duration and concentration of the peptides. Treatment of cTnI-G203S mice 3× per week for 15 weeks with 10 µM AIDP14-TAT or 10 µM AIDP16-TAT via subcutaneous injections significantly improved all echocardiography parameters (Figure [D]), Doppler parameters (Figure [D]), significantly ameliorated myocardial fibrosis in the left posterior wall (Figure [E]) and normalized flavoprotein oxidation and ψm to wild-type levels. Our data demonstrate that prolonging the treatment period and increasing the concentration of the peptides were effective in ameliorating established hypertrophy and fibrosis in cTnI-G203S mice.

We have shown previously that progression of HCM occurs due to a maladaptive feedback loop involving the LTCC and cytoskeleton, leading to hypercontractility and altered mitochondrial function.⁶ Treatment with AIDP14-TAT or AIDP16-TAT corrected the cytoskeletal disorganization and normalized mitochondrial energetics improving contractile function. By targeting mitochondrial function, we can disrupt the maladaptive feedback that contributes to HCM and thus ameliorate the hypertrophic phenotype.

Here, we report 2 peptides as first in class safe modulators of LTCC function that are effective at reversing HCM without causing negative inotropic effects or toxicity in translationally relevant HCM models. We propose that the variant peptides will be effective in the treatment of HCM broadly because the mechanism of action involves the modification of mitochondrial function and impaired energy metabolism that is a common characteristic and driver of the pathology in HCM.

Article Information

Author Contributions

L.C. Hool, J.E. Camacho Londoño, and P. Ponnuswamy developed the research plan. L.C. Hool designed the peptide variants. T.S. Er, D. Reinke, H. Cserne Szappanos, C.M. Richworth and A.J. Francis performed JC-1 (5,5’,6,6’ tetrachloro1,1’tetraethylbenzimidazolylcarbocyanine iodide) and flavoprotein fluorescence studies. T.S. Er, D. Reinke, and H. Cserne Szappanos performed echocardiography, Doppler ultrasound and blood pressure measurements. D. Reinke, C.M. Richworth, A.J. Francis, and T.S. Er performed toxicity, fibrosis studies heart weight:tibia length and muscle weight measurements. T.S. Er and L.C. Hool wrote the manuscript. All authors assisted with analysis and edited the manuscript.

Data Availability

Raw data are available on request by contacting the corresponding author.

Disclosures

P. Ponnuswamy and J.E. Camacho Londoño are employees of CSL Innovation GmbH, Germany. The other authors report no conflicts.

Supplemental Materials

Major Resources Table

Supplementary Material

res-139-e329377-s001.pdf ^{(238.8KB, pdf)}

Funding Statement

This work is supported by CSL Ltd Research Acceleration Initiative Award (L.C. Hool); National Health and Medical Research Council of Australia Ideas Grant APP2004282 (L.C. Hool); National Health and Medical Research Council of Australia Senior Research Fellowship APP1117366 (L.C. Hool); Woodside Energy Grant (L.C. Hool); L.C. Hool is the Wesfarmers, The University of Western Australia (UWA)-Victor Chang Cardiac Research Institute (VCCRI) Chair in Cardiovascular Research.