A selective inhibitor of mitofusin 1-βIIPKC association improves heart failure outcome in rats

We previously demonstrated that beta II protein kinase C (βIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that βIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. βIIPKC siRNA or a βIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-βIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMβA, a rationally-designed peptide that selectively antagonizes Mfn1-βIIPKC association. SAMβA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMβA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMβA may be a potential treatment for patients with heart failure.


Study design
The study with βII V5-3 treatment in healthy male rats included two groups: sham, n=6; and βII V5-3 -treated, n=6. Four weeks after sham surgery, rats were randomized to drug treatments. Each group was treated with TAT 47-57 -βII V5-3 peptide (global βIIPKC inhibitor, 3mg Kg -1 day -1 ) or equimolar concentration of TAT 47-57 -carrier peptide (negative control), using Alzet osmotic pumps under the skin on the back of the animal for sustained drug delivery, and pumps were replaced every 2 weeks. At the end of the protocol, cardiac function was re-evaluated by an observer blinded to the treatment groups. Forty-eight hours later, all the rats were euthanized by decapitation for other analyses.

Infarct size and apoptosis
Cardiac slices were fixed using 4% buffered formalin and embedded in paraffin for routine histological processing. 5 µm cardiac sections were stained with Masson's trichrome and the quantification of myocardial infarct area, performed in the left ventricle free wall, was done with computer-assisted morphometric system (Leica Quantimet 500, Cambridge). The myocardial infarcted area was expressed as a percentage of the total surface area of the left ventricle. Cell apoptosis in the myocardium was determined by TUNEL staining, according to the manufacturer's instructions (Apoptosis Detection Kit, Trevigem 4812/30-k). TUNEL labeling was performed in 5 µm cardiac sections, visualized using a fluorescence microscope and the data are expressed as the TUNEL-positive cardiomyocytes relative to total nuclei.

Mitochondrial DNA
To quantify the relative amount of mitochondrial DNA (mtDNA) per nucleus DNA (nDNA), we isolated total DNA from cardiac samples using DNeasy Blood & Tissue Kit (Qiagen 69504) according to manufacturer's protocol. DNA was then purified by phenol-chloroform extraction and ethanol precipitation. Quantitative real time polymerase chain (qPCR) reaction were carried out separately and amplifications were performed with an ABI Prism 7500 Sequence Detection System by using Maxima® SYBR Green ROX qPCR Master Mix (Fermentas K0221). Melting point dissociation curves were used to confirm the purity of the amplification products. Results were expressed using the comparative cycle threshold (Ct) method as described by the manufacturer. Calculation of the mtDNA copy number relative do nuclear DNA (nDNA) was performed using the formula: 2 x 2ΔCt, where ΔCt is the nDNA Ct values minus mtDNA Ct values. MTCO1 and ND1 genes were used to quantify mtDNA and Rplp0 gene to nDNA. Data are expressed as the percentage of sham. Primer sequences:  Supplementary Table 1 and Supplementary Table 3. Whenever significant F-values were obtained, Duncan adjustment was used for multiple comparison purposes. GraphPad Prism Statistics was used for the analysis and statistical significance was considered achieved when the value of P was <0.05.

Supplementary Figures
Supplementary Figure 1 βIIPKC inhibition does not alter cardiac mitochondrial content in failing rat hearts. a Representative western blots and protein levels of b mitochondrial electron transport chain subunits (ATP5A, NDUF9 and Ubiquinol), c mitochondrial membrane proteins (TOM70 and VDAC) and d mitophagy markers (p62, NDP52 and OPT); e mitochondrial DNA copy number of MTCO1 and ND1 genes and f cardiac mitochondrial MFN2 immunoprecipitate probed against anti-MFN2 and βIIPKC antibodies; and βIIPKC immunoprecipitate from heart lysate probed against anti-βIIPKC and MFN2 (representative blot of three independent experiments) from heart samples of sham (white bars, n=7), TATtreated heart failure (HF-Ctr, gray bars, n=9) and βII V5-3 -treated heart failure (HF-βII V5-3 , red bars, n=7). Biochemical measurements were performed in the cardiac remote (viable) zone. These measurements were performed at the end of the experimental protocol as described in Figure 1A. Data are means ± SEM. *P<0.05 vs. Sham. One-way analysis of variance (ANOVA) with post-hoc testing by Duncan. LVEDd [left ventricular end-diastolic dimension] measured by echocardiography at the end of the experimental protocol, input: delta of measurements performed before and after treatment; c representative cardiac transmission electron micrographs (scale bar: 2 µm); and d quantification of intermyofibrillar mitochondrial number and area in the transmission electron micrographs in heart samples from sham (white bars, n=10) and βII V5-3 -treated sham animals (βII V5-3 , red bars, n=6). Peptide treatment was continuous (for six weeks) using an Alzet pump, delivering at a rate of 3mg Kg -1 day -1 . All measurements were performed at the end of the experimental protocol as described in Figure 1A. Data are means ± SEM.

Supplementary Figure 3
Inhibition of global βIIPKC (βII V5-3 ) and βIIPKC-Mfn1 proteinprotein interaction (SAMβA) improves mitochondrial function in heart failure in vivo. a Mitochondrial state-dependent oxygen control rates, absolute H 2 O 2 release and H 2 O 2: O 2 in heart samples from sham (white bars, n=10), TAT-treated heart failure (HF-Ctr, gray bars, n=10) and βII V5-3 -treated heart failure (HF-βII V5-3 , red bars, n=10). These measurements were performed at the end of the experimental protocol as described in Figure 1A. b Mitochondrial state-dependent oxygen control rates, absolute H 2 O 2 release and H 2 O 2 :O 2 in heart samples from sham (white bars, n=7), TAT-treated heart failure (HF-Ctr, gray bars, n=8) and SAMβAtreated heart failure (HF-SAMβA, blue bars, n=11). These measurements were performed at the end of the experimental protocol as described in Figure 6A.