Role of matrix metalloproteinase-2 in sarcoplasmic reticulum dysfunction during myocardial ischemia-reperfusion injury

• Author / Creator

  Roczkowsky, Andrej

• Matrix metalloproteinase-2 (MMP-2) is an intra- and extra-cellular protease which is activated during myocardial ischemia-reperfusion (IR) injury by reactive oxygen and nitrogen species, namely peroxynitrite. Upon its activation, MMP-2 impairs cardiac contractility by cleaving sarcomeric and other intracellular proteins. MMP-2 has been localized to several subcellular sites inside cardiac myocytes, including the sarcoplasmic reticulum (SR); however, its precise role in this organelle is unknown. I investigated two critical SR proteins, which are known to be proteolyzed during IR injury, as putative targets of MMP-2: the Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, and the structural protein junctophilin-2 (JPH-2), which maintains T-tubule structure. I hypothesized that MMP-2 is activated near the SR during cardiac IR injury, resulting in the proteolysis of SERCA2a, to impair its activity, and JPH-2, to impair cardiac contractile function. Isolated rat hearts were perfused in working mode aerobically or subjected to IR injury in the presence and absence of the MMP inhibitor ARP-100. Inhibition of MMP activity significantly improved the recovery of contractile function of hearts following IR injury. I found a putative 70 kDa degradation product of SERCA2a increased two-fold in IR hearts, which was prevented with ARP-100. SERCA activity assessed in SR enriched microsomes prepared from rat hearts was decreased by IR injury; however, this was not prevented with ARP-100. Incubation of purified proteoliposomes containing SERCA2a with MMP-2 in vitro resulted in its proteolysis. Similarly, incubation of SR enriched microsomes isolated from control rat hearts with MMP-2 caused proteolysis of SERCA2a to ~70 kDa products. The presence of MMP-2 in SR enriched microsomes was confirmed by gelatin zymography. JPH-2 was identified as a target of MMP-2 using in silico software. A potential JPH-2 degradation product was increased by IR in an MMP-dependent manner. MMP-2 cleaves JPH-2 in vitro. This is the first study to identify two novel substrates of MMP-2 in the SR, as well as identify MMP-2 activity in SR enriched microsomes. Understanding the diverse repertoire of MMP-2 substrates may aid in the development of selective MMP inhibitors for use in ischemic heart disease to mitigate IR injury.

• Subjects / Keywords

  • MMP-2
  • proteolysis
  • ischemia-reperfusion
  • sarcoplasmic reticulum
  • SERCA2a
  • JPH-2
  • heart

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-f02m-jq12

• License

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