Phosphoproteomics of Hypertrophic Cardiomyopathy Patient Myocardium and Novel hiPSC-CM Model Reveal Protein Kinase A as a Modulator of Microtubule Repolymerization

This study integrates phosphoproteomics of human hypertrophic cardiomyopathy (HCM) myocardium with hiPSC-CM modeling to demonstrate that altered microtubule dynamics in HCM are driven not by changes in modifying enzyme levels, but by Protein Kinase A-mediated regulation of microtubule repolymerization in response to beta-adrenergic stimulation.

The Gist

The Big Picture: A Stiff Heart and a Broken Spring

Imagine your heart is a high-performance engine that needs to squeeze (contract) to pump blood and then instantly relax to refill. In a condition called Hypertrophic Cardiomyopathy (HCM), the heart muscle gets too thick and stiff. It squeezes hard, but it struggles to relax, like a door with a rusty hinge that won't swing open all the way. This leads to shortness of breath and fatigue.

Scientists have known for a while that inside these stiff heart cells, there is a "skeleton" made of tiny tubes called microtubules. In healthy hearts, these tubes are flexible. In HCM hearts, they become too stable and rigid, acting like steel rods instead of flexible springs. This rigidity stops the heart muscle from relaxing properly.

This paper asks a simple question: Why do these tubes become so stiff?

Part 1: The "Construction Crew" Theory (What Didn't Happen)

Usually, when you see a building get too stiff, you might think the construction crew (enzymes) is working overtime to add more cement, or the demolition crew is taking a break.

The researchers looked at the "construction crew" responsible for modifying these microtubules:

• The "Glue" Crew (Enzymes that add stability): They checked if there was too much glue being applied.
• The "Demolition" Crew (Enzymes that remove stability): They checked if the demolition crew was missing.

The Surprise: The crew members were actually working at normal levels! In fact, some of the "glue" makers were even lower in HCM patients. This was a shock. It meant the stiffness wasn't caused by the workers building too much; something else was telling the tubes to stay rigid.

Part 2: The "Foreman" Theory (The Real Culprit)

If the workers aren't the problem, maybe the Foreman (a signaling molecule called a kinase) is giving the wrong orders.

The researchers used a high-tech "magnifying glass" (phosphoproteomics) to look at the chemical signals in the heart tissue. They found two major issues with the Foremen:

1. The "Over-Active" Foreman (EGFR/MAPK): This foreman was shouting too loud, telling the cells to grow and thicken.
2. The "Sleeping" Foreman (PKA): This is the most important one. PKA is like the "Relaxation Coach." In a healthy heart, when you exercise or get excited, PKA wakes up and tells the heart, "Okay, squeeze hard, but then relax quickly!"
   • In HCM: The PKA coach was asleep (hypoactive). Because the coach wasn't giving the "relax" orders, the microtubules stayed stiff and didn't repolymerize (reset) correctly.

Part 3: Testing the Theory with "Mini-Hearts"

To prove this, the scientists didn't just look at old heart tissue; they grew mini-hearts (called hiPSC-CMs) in a lab. These were created from stem cells of a patient with a specific HCM mutation (the Dutch founder mutation).

• The Experiment: They watched these mini-hearts grow. They saw that the microtubules in the mutant hearts were indeed trying to "repolymerize" (rebuild themselves) too aggressively, making them stiff.
• The Fix: They woke up the sleeping coach by adding a drug (Isoprenaline) that activates PKA.
• The Result: As soon as PKA was activated, the microtubules stopped being so rigid. They became flexible again.

The Analogy: Imagine the microtubules are a crowd of people holding hands in a circle. In HCM, they are holding on too tight (stiff). The PKA coach is supposed to tell them to loosen their grip. In the disease, the coach is asleep, so they hold on too tight. When the scientists "woke up" the coach, the crowd loosened up and became flexible again.

Part 4: The "Traffic Light" of the Heart

The study also found that specific proteins (like CLASP1, MAST4, and MAP1A) act like traffic lights for these microtubules.

• In a healthy heart, PKA turns the light green for flexibility.
• In HCM, because PKA is asleep, the light stays red, and the traffic (microtubules) gets jammed and stiff.

Why Does This Matter?

1. It's Not Just About Building: We used to think heart stiffness was just about building too much "glue." This paper shows it's actually about communication. The instructions to relax aren't getting through.
2. New Treatments: Since the problem is a "sleeping" PKA coach, maybe we can treat HCM not by trying to break the stiff tubes, but by waking up the PKA coach or fixing the specific traffic lights (proteins) that PKA controls.
3. Personalized Medicine: The researchers found that different genetic mutations in HCM patients might affect these signals differently, suggesting that one day, doctors might choose drugs based on exactly which "Foreman" is broken in a specific patient.

The Bottom Line

The heart in HCM patients is stiff not because the construction workers are overzealous, but because the Relaxation Coach (PKA) has fallen asleep. By waking up this coach, we can potentially make the heart's internal skeleton flexible again, helping the heart relax and pump blood more efficiently. This opens the door to new, smarter ways to treat heart disease.

Technical Summary

1. Problem Statement

Hypertrophic Cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and diastolic dysfunction. A known pathological feature in HCM myocardium is an altered "microtubule (MT) code," specifically elevated levels of $\alpha$-tubulin and its post-translational modifications (PTMs): detyrosination and acetylation. These modifications increase cardiomyocyte stiffness, contributing to impaired relaxation.

However, the upstream mechanisms driving these changes remain unknown. Previous hypotheses suggested that altered expression levels of the enzymes responsible for these PTMs (e.g., $\alpha$TAT1 for acetylation, HDAC6 for deacetylation, VASH/SVBP for detyrosination, and TTL for re-tyrosination) might be the cause. Furthermore, the role of kinase signaling networks in regulating MT dynamics in human HCM has not been comprehensively explored. The study aims to:

1. Determine if altered levels of MT-modifying enzymes explain the HCM MT code.
2. Identify upstream kinase regulators via phosphoproteomics.
3. Validate these findings using a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model.

2. Methodology

The study employed a multi-modal approach combining human tissue analysis, omics, and cellular modeling:

• Human Myocardial Cohort:

  • Samples: Interventricular septal tissue from 24 HCM patients (15 genotype-positive [G+]: MYBPC3 and MYH7 mutations; 9 genotype-negative [G-]) and 8 non-failing (NF) controls.
  • Protein Analysis: Western blotting to quantify protein levels of MT-modifying enzymes ($\alpha$TAT1, HDAC6, VASH1, VASH2, TTL).
  • Phosphoproteomics: Large-scale mass spectrometry to profile serine/threonine (pSer/pThr) and tyrosine (pTyr) phosphorylation sites.
  • Kinome Analysis: Used INKA (Integrated Kinase Activity) analysis to infer kinase activity based on kinase and substrate phosphorylation states.
  • Correlation Analysis: Correlated kinase scores and phosphosites with clinical parameters (e.g., E/e', LVOT gradient, wall thickness).

• hiPSC-CM Model:

  • Generation: Created a homozygous MYBPC3^Arg943X (HOM) hiPSC line using CRISPR/Cas9, derived from a Dutch founder mutation. The line carries a mTag-RFP-T-TUBA1B tag to visualize microtubules.
  • Differentiation: Cells were differentiated into cardiomyocytes and analyzed at 30 and 60 days.
  • Functional Assays:
    • Contractility/Ca$^{2+}$: High-throughput optical measurement of beating frequency, relaxation times, and calcium transients.
    • MT Dynamics: Live-cell imaging using nocodazole to induce depolymerization, followed by washout to measure repolymerization rates.
  • Pharmacological Interventions: Treatment with Isoprenaline (ISO, PKA agonist), EGFR inhibitors, EpoY (detyrosination inhibitor), and Tubacin (HDAC6 inhibitor).

3. Key Contributions

• First Comprehensive Enzyme Profiling: This is the first study to quantify both mRNA and protein levels of key MT-modifying enzymes in a well-characterized HCM cohort, revealing that enzyme abundance does not correlate with the altered MT code.
• Dual-Phosphoproteome Landscape: Provided the first extensive pTyr and pSer/pThr map of HCM myocardium, identifying distinct kinase signaling signatures (hyperactive EGFR/IGF1R-MAPK and hypoactive PKA).
• Novel hiPSC-CM Platform: Developed a MYBPC3^Arg943X hiPSC-CM model with live MT imaging capabilities to dissect the etiological sequence of MT changes.
• Mechanistic Insight: Identified Protein Kinase A (PKA) as a critical regulator of MT repolymerization, linking beta-adrenergic signaling directly to cytoskeletal stability.

4. Key Results

A. MT-Modifying Enzymes Are Not the Primary Drivers

• Protein Levels: Contrary to the hypothesis, $\alpha$TAT1 (acetyltransferase) was decreased, and HDAC6 (deacetylase) was increased in HCM myocardium compared to controls.
• Detyrosination: Levels of VASH1 (detyrosinating enzyme) showed a non-significant trend toward increase, while TTL (re-tyrosinating enzyme) remained unchanged.
• Conclusion: The increased MT PTMs in HCM cannot be explained by the abundance of the modifying enzymes.

B. Phosphoproteomics and Kinase Signaling

• Global Changes: Identified >1900 differentially phosphorylated pSer/pThr sites and 160 pTyr sites.
• Kinase Activity (INKA):
  • Hyperactive: EGFR, IGF1R, INSR, and downstream MAPKs (ERK1/2). This suggests increased EGFR/IGF1R-MAPK signaling.
  • Hypoactive: PKA (Protein Kinase A) signaling was significantly reduced, consistent with previous findings of blunted $\beta$-adrenergic response in HCM.
• Correlations: Specific phosphosites on cytoskeletal proteins (e.g., CLASP1, MAP1A, MAST4) correlated with the MT code and clinical diastolic dysfunction markers.

C. hiPSC-CM Model Findings

• Phenotype: MYBPC3^Arg943X cells exhibited hypercontractility and altered calcium handling (slower release/peak width) at 60 days.
• MT Dynamics: HOM cells showed increased MT repolymerization rates (faster recovery after nocodazole washout) compared to WT, indicating more stable/long-lived MTs.
• PKA Role:
  • Treatment with Isoprenaline (ISO) to activate PKA decreased MT repolymerization in HOM cells, effectively reversing the pathological stabilization.
  • This confirms that low PKA activity in HCM drives MT stabilization.
• Enzyme Inhibitors: Inhibiting detyrosination (EpoY) reduced repolymerization, but inhibiting acetylation (Tubacin) did not, suggesting detyrosination is more critical for the repolymerization defect in this model.

D. Downstream Targets

Phosphoproteomics of ISO-treated hiPSC-CMs identified specific substrates that were hypophosphorylated in HCM but restored by PKA activation. Key candidates include:

• CLASP1 (S646)
• MAP1A (S1776, S1818, S2022)
• MAST4 (S1370, S1467)
  These proteins are potential direct effectors of PKA-mediated MT destabilization.

5. Significance

• Paradigm Shift: The study shifts the focus from "enzyme abundance" to "kinase-mediated regulation" as the driver of the altered MT code in HCM.
• Therapeutic Target: It identifies PKA activation (or downstream modulation of CLASP1/MAP1A/MAST4) as a potential therapeutic strategy to reduce cardiomyocyte stiffness and improve diastolic function.
• Disease Mechanism: It elucidates a mechanism where disease-mediated changes in the MT code exert both direct (stiffness) and indirect (blunted adrenergic response) effects on cardiac function.
• Resource: The study provides a unique phosphoproteomic dataset and a validated hiPSC-CM model for future research into early-stage HCM pathophysiology and drug screening.

In summary, the paper demonstrates that the stiffening of cardiomyocytes in HCM is driven by a hypoactive PKA signaling pathway leading to hyper-stable microtubules, rather than an overabundance of the enzymes that modify them. Restoring PKA activity or targeting its downstream cytoskeletal effectors could ameliorate diastolic dysfunction.