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Calpain-mediated Protein Targets in Cardiac Mitochondria following Ischemia-Reperfusion

ABSTRACT: Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC) – reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC-REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of the CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional, cardiomyocyte specific CPNS1 knockout (KO) mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC-REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 KO mice underwent 25 min in vitro global ISC and 30 min REP. LDH activity in coronary effluent was measured to reflect cardiac injury. Mitochondria were isolated from the hearts. Cardiac injury was decreased in CPNS1 KO mice following ISC-REP compared to WT. ISC-REP decreased the rate of oxidative phosphorylation in WT but not in CPNS1 KO mice when glutamate + malate was used as complex I substrate. KO of CPNS1 led to a smaller decrease in CRC (calcium retention capacity) following REP compared to WT. The H2O2 generation was decreased in KO mitochondria following ISC-REP compared to WT. KO of CPNS1 also resulted in less cytochrome c release from mitochondria. Proteomic analysis of the isolated mitochondria showed that KO of CPNS1 increased the abundance of ribosomal proteins and mitochondrial biogenesis proteins. These results show that activation of CPN1/2 increases cardiac injury during ischemia-reperfusion by damaging mitochondria.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Ling Li

LAB HEAD: Qun Chen

PROVIDER: PXD022689 | Pride | 2021-12-21

REPOSITORIES: Pride

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Project description:Aims A kinase interacting protein 1 (AKIP1) stimulates physiological growth in cultured cardiomyocytes and attenuates ischemia / reperfusion (I/R) injury in ex vivo perfused hearts. We aimed to determine whether AKIP1 modulates the cardiac response to acute and chronic cardiac stress in vivo. Methods and results Transgenic mice with cardiac-specific overexpression of AKIP1 (AKIP1-TG) were created. AKIP1-TG mice and their wild type (WT) littermates displayed similar cardiac structure and function. Likewise, cardiac remodeling in response to transverse aortic constriction or permanent coronary artery ligation was identical in AKIP1-TG and WT littermates, as evidenced by serial cardiac magnetic resonance imaging and pressure-volume loop analysis. Histological indices of remodeling, including cardiomyocyte cross-sectional diameter, capillary density and left ventricular fibrosis were also similar in AKIP1-TG mice and WT littermates. When subjected to 45 minutes of ischemia followed by 24 hours of reperfusion, AKIP1-TG mice displayed a significant 2-fold reduction in myocardial infarct size and reductions in cardiac apoptosis. In contrast to previous reports, AKIP1 did not co-immunoprecipitate with or regulate the activity of the signaling molecules NF-κB, protein kinase A or AKT. AKIP1 was, however, enriched in cardiac mitochondria and co-immunoprecipitated with a key component of the mitochondrial permeability transition (MPT) pore, ATP-synthase. Finally, mitochondria isolated from AKIP1-TG hearts displayed markedly reduced calcium induced swelling, indicative of reduced MPT pore formation. Conclusions In contrast to in vitro studies, AKIP1 overexpression does not influence cardiac remodeling in response to chronic cardiac stress. AKIP1 does, however, reduce myocardial I/R injury through stabilization of the MPT pore. These findings suggest that AKIP1 deserves further investigation as a putative treatment target for cardioprotection from I/R injury during acute myocardial infarction.

			Action	DRS
	KO-IR-1.raw	Raw
	KO-IR-2.raw	Raw
	KO-IR-3.raw	Raw
	KO-IR-4.raw	Raw
	KO-TC-1.raw	Raw

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Calpain-mediated Protein Targets in Cardiac Mitochondria following Ischemia-Reperfusion

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