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Of Mitochondria and Stiff Hearts

Exploring the Mechanisms Behind Hypertrophic Cardiomyopathy

One might consider the heart as a tireless workhorse, pumping blood through our bodies to sustain life. But like any biological system, our hearts are susceptible to myriad diseases, one of which is hypertrophic cardiomyopathy (HCM). Affecting 1 in 500 of the general population, HCM, an inherited disorder, strikes the young with shocking force, making it the leading cause of sudden cardiac death in the 5–15 age bracket.

HCM primarily occurs due to genetic mutations in proteins that make up the sarcomeres, the basic units of muscle contraction. These mutations cause the heart to become stiff and overactive, leading to a whole host of complications. In a recent study, scientists have broken new ground, establishing a link between cardiac stiffness and the disease’s progression, using a novel in vitro model of HCM. They have unraveled a mechanosensing feedback mechanism that might be crucial in understanding the disease and potentially treating it.

Mimicking the Stiff Heart: An In Vitro Model of HCM

To unravel the mysteries of HCM, the researchers ingeniously recreated an in vitro HCM model using hydrogel technology. By adjusting the stiffness of these hydrogels, researchers could simulate the conditions experienced by cardiac myocytes, the heart’s primary contractile cells, in a heart burdened with HCM.

The myocytes cultured on hydrogels mirroring the stiffness of an HCM-affected myocardium shifted to a hypermetabolic mitochondrial state, resembling the behavior of myocytes isolated from a murine model of human HCM (cTnI-G203S). This hypermetabolic shift is believed to be a precursor to the hypertrophic state characteristic of HCM.

Conversely, when these affected myocytes were cultured on hydrogels approximating the softness of a healthy heart, mitochondrial function reverted to normal. This highlights the influence of extracellular matrix (ECM) stiffness on the progression of HCM and points to potential therapeutic pathways.

A Tale of Two Connections: The LTCC and the ECM

The heart’s ability to function efficiently hinges on several complex components, two of which – the L-type calcium channel (LTCC) and the extracellular matrix (ECM) – have been linked to HCM. Both of these components play key roles in modulating mitochondrial function, albeit through different pathways.

The LTCC serves a dual role: it orchestrates the “calcium-induced calcium release,” crucial for maintaining cardiac contraction, and simultaneously regulates mitochondrial function via calcium-dependent and calcium-independent mechanisms, implying a critical link between the LTCC and the mitochondria.

On the other hand, the ECM, a network providing structural and biochemical support to cells, communicates with cardiac myocytes via transmembrane-spanning mechanosensing proteins such as integrins. This ECM-myocyte interaction alters cytoskeletal proteins such as F-actin, which interact directly with mitochondria by binding to outer mitochondrial docking proteins.

Behind the Pathology of HCM: The Role of β1 Integrin and mTOR

Delving deeper into the involvement of the LTCC and β1 integrin in HCM progression, the researchers scrutinized expression levels in heart tissues from pre- and post-cardiomyopathic cTnI-G203S mice using Western blots leveraging Alomone’s anti-CaV1.2 antibody. While no marked difference was observed in LTCC expression, β1 integrin expression demonstrated significant alterations, especially in post-cardiomyopathic hearts (Figure 1). 

 L-type calcium channel expression and analysis

Figure 1. Immunoblot analysis of L-type calcium channel (a, b) and β1 integrin (c, d) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots were probed with L-type calcium channel α1C subunit (CaV1.2, a) or β1 integrin (c) antibody, then GAPDH monoclonal antibody. Densitometry analysis of CaV1.2 (b) or β1 integrin (d) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA (b) or Kruskal-Wallis test (d) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic (e) and nuclear (f) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA (e) or Kruskal-Wallis test (f) determined statistical significance. g Schematic indicating a structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state. Image and legend from Viola, al. Commun Biol 6, 4 (2023).

The investigation revealed that changes in mitochondrial metabolic activity, mediated by LTCC, and a subsequent hypermetabolic state manifest before the onset of cTnI-G203S pathology. Furthermore, the researchers noted a significant elevation in relative mTOR expression, particularly in pre- and post-cardiomyopathic cTnI-G203S nuclear fractions compared to their healthy counterparts. This suggests that mTOR activation could play a part in the early stages of HCM. 

The Stiff Heart’s Miscommunication

In HCM, this delicately balanced system falls apart. The normal, physiological communication between the LTCC and the mitochondria becomes dysregulated, leading to a hypermetabolic mitochondrial state.

This “communication breakdown” is believed to precede the development of HCM. Furthermore, increased stiffness in the ECM, linked to HCM progression, seems to negatively impact this intracellular signaling. By mimicking the conditions of a stiff heart, researchers have established that changes in substrate stiffness can alter the communication between the LTCC and the mitochondria.

Decoding the Stiff Heart: Future Directions

Though the researchers have made significant strides in understanding the complex interplay between cardiac stiffness, the LTCC, and mitochondrial function in HCM, many questions remain. HCM is a complex disease, with over 1,500 different gene mutations known to cause it. It is also possible that each mutation leads to a unique pathophysiology, necessitating further research to confirm the proposed mechanism’s involvement.

From a treatment perspective, this study provides an encouraging platform. By targeting the cellular responses to ECM stiffness, potential therapeutic strategies could be developed. The identified ‘linker’ sites connecting the LTCC and mitochondria or sarcomeric proteins modification may become novel therapeutic targets.

Taken together, the results of this study take a significant step towards understanding the stiff, overworking heart in HCM. By decoding the mechanosensing feedback mechanism and elucidating the role of cardiac stiffness, this research paves the way for the development of preventative therapies. In the struggle against HCM, knowledge truly is power, and each piece of the puzzle brings us closer to treatment.


Pharmacological Tools

Blockers/Antagonists: peptides/peptide toxins

  • Calcicludine (#SPC-650)

    Cat #: SPC-650
    Type: Blockers  | Source: Synthetic peptide | MW: 6979 Da | Target: L-type Ca2+ channels

  • FS-2 (#F-700)

    Cat #: F-700
    Type: Blockers  | Source: Synthetic peptide | MW: 7026 Da | Target: L-type Ca2+ channels

  • Calciseptine (#SPC-500)

    Cat #: SPC-500
    Type: Blockers  | Source: Synthetic peptide | MW: 7036 Da | Target: L-type Ca2+ channels

Blockers/Antagonists: small molecules

  • Amitriptyline hydrochloride (#A-155)

    Cat #: A-155
    Type: Blockers  | Source: Synthetic | MW:  313.86 | Target: L-type Ca2+ channels, α-adrenergic receptors, 5-HT2 serotonin receptors

  • (R)(+)-Bay K8644 (#B-352)

    Cat #: B-352
    Type: Blockers  | Source: Synthetic | MW:  356.3 | Target: L-type Ca2+ channels

  • Diltiazem hydrochloride (#D-135)

    Cat #: D-135
    Type: Blockers  | Source: Synthetic | MW:  451  | Target: L-type Ca2+ channels

  • Isradipine (#I-100)

    Cat #: I-100
    Type: Blockers  | Source: Synthetic | MW:  371.4  | Target: CaV1.2, CaV1.3 Ca2+ channels

  • (±)-Naringenin (#N-110)

    Cat #: N-110
    Type: Blockers  | Source: Synthetic | MW:  272.25  | Target: L-type Ca2+ channels

  • SR 33805 oxalate (#S-105)

    Cat #: S-105
    Type: Blockers  | Source: Synthetic | MW:  654.77  | Target: L-type Ca2+ channels

Activators/Agonists: small molecules

  • (±)-Bay K8644 (#B-350)

    Cat #: B-350
    Type: Activators  | Source: Synthetic | MW:  356.3  | Target: L-type Ca2+ channels

  • FPL 64176 (#F-160)

    Cat #: F-160
    Type: Activators  | Source: Synthetic | MW:  347.4  | Target: L-type Ca2+ channels

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