Myocardial Matrix Metalloproteinase‐2 Activation Impairs Amplitude and Frequency of Spontaneous Intracellular Ca <sup>2+</sup> Transients in Doxorubicin Cardiotoxicity
Bibliographic record
Abstract
Anthracyclines, such as doxorubicin (DXR), are effective antineoplastic agents prescribed in many cancer chemotherapeutic regimens. However, 11% of cancer patients treated with DXR develop heart failure. There is no treatment to prevent DXR cardiotoxicity beyond limiting cumulative dosage and hence compromising its therapeutic efficacy. The mechanism of DXR cardiotoxicity is incompletely understood but two key characteristics are elevated oxidative stress and impaired Ca 2+ signaling (notably decreased intracellular Ca 2+ release, a key component of excitation‐contraction coupling). Elevated oxidative stress can activate matrix metalloproteinase‐2 (MMP‐2), a protease which can cleave sarcomeric proteins (e.g. α‐actinin and troponin I) and the Ca 2+ regulatory protein calreticulin. We hypothesize that MMP‐2 plays a role in DXR cardiotoxicity by proteolyzing these proteins upon activation by oxidative stress, impairing Ca 2+ signaling and cardiac contractile function. To test this, neonatal rat ventricular myocytes (NRVM) were treated with 0.5μM DXR ± the MMP inhibitors ARP‐100 or ONO‐4817 (1μM each) for 2–24h. This concentration of DXR is equivalent to that found in the blood of patients undergoing chemotherapy. 24h DXR caused no cell death in NRVM, but, as expected for a chemotherapeutic drug, this treatment protocol caused 15% of human fibrosarcoma HT1080 cells to undergo necrosis. By 12h of DXR treatment, NRVM exhibited increased oxidative stress, as shown by a 20% reduction in aconitase activity, and increased intracellular MMP‐2 activity by threefold relative to control. Intracellular MMP‐2 activation was accompanied by a twofold increase in MMP‐2 protein levels. ARP‐100 or ONO‐4817 attenuated the DXR‐increased MMP‐2 activity by 60%, but not MMP‐2 protein expression. 24h DXR reduced levels of the known MMP‐2 target troponin I by 40%, but this was not prevented by MMP inhibitors, indicating that this effect on troponin I was not due to MMP‐2. The effect of DXR‐induced MMP‐2 activation on intracellular Ca 2+ transients, likely indicative of sarcoplasmic reticulum Ca 2+ release and re‐uptake, was visualized in live NRVM using confocal microscopy and the Ca 2+ ‐binding fluorescent dye Fluo‐8L AM. 24h DXR decreased the amplitude of Ca 2+ transients by 72% and 63% under basal and isoproterenol‐stimulated (100nM) conditions, respectively. DXR also reduced the frequency of both basal and stimulated Ca 2+ transients by 73%. ARP‐100 restored both the DXR‐reduced amplitude and frequency of Ca 2+ transients. To understand how inhibiting MMP‐2 may protect against DXR‐impaired intracellular Ca 2+ transients, we measured the levels of calreticulin in NRVM lysates. However, calreticulin was unaffected by DXR. We hypothesized that SERCA, required for Ca 2+ uptake by the sarcoplasmic reticulum, could be proteolyzed by MMP‐2. MMP‐2 successfully proteolyzed SERCA in vitro, yielding a 50kDa fragment, confirmed by mass spectrometry. Future experiments will investigate the effect of DXR on SERCA levels in membrane fractions. In conclusion, DXR, at a clinically relevant concentration, increases oxidative stress and acutely activates myocardial MMP‐2. MMP‐2 activation appears to impair spontaneous intracellular Ca 2+ release in cardiomyocytes. Thus, the results of this study provide mechanistic insight into the role of MMP‐2 in DXR cardiotoxicity, and support the potential use of MMP inhibitors as an adjuvant therapy for patients receiving DXR chemotherapy. Support or Funding Information Canadian Institutes of Health Research Foundation Scheme Grant (to RS); Women and Children's Health Research Institute and Novartis Pharmaceuticals Canada Inc Graduate Studentships (to BC).
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".