Calpains as Potential Therapeutic Targets for Myocardial Hypertrophy.

Despite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca2+-dependent cysteine proteases calpains in regulating the hypertrophic process and highlight the feasibility of their long-term inhibition as a pharmacological strategy. In this review, we discuss the existing evidence implicating calpains in the development of cardiac hypertrophy, as well as the latest advances in unraveling the underlying mechanisms. Finally, we provide an updated overview of calpain inhibitors that have been explored in preclinical models of cardiac hypertrophy and the progress made in developing new compounds that may serve for testing the efficacy of calpain inhibition in the treatment of pathological cardiac hypertrophy.

High-fat diet improves tolerance to myocardial ischemia by delaying normalization of intracellular PH at reperfusion.

Reports on the effect of obesity on the myocardial tolerance to ischemia are contradictory. We have described that obesity induced by high-fat diet (HFD) reduces infarct size in B6D2F1 mice submitted to transient coronary occlusion. In this study, we analysed the mechanism by which dietary obesity modifies the susceptibility to myocardial ischemia and the robustness of this effect. B6D2F1 (BDF), C57BL6/J (6J), C57BL6/N (6N) male mice and BDF female mice were fed with a HFD or control diet for 16 weeks. In all three strains, HFD induced obesity with hyperinsulinemia and hypercholesterolemia and without hyperglycemia, hypertension, ventricular remodelling or cardiac dysfunction. In obese mice from all three strains PDK4 was overexpressed and HSQC NMR spectroscopy showed reduced 13C-glutamate and increased 13C-lactate and 13C-alanine, indicating uncoupling of glycolysis from glucose oxidation. In addition, HFD induced mild respiratory uncoupling in mitochondria from BDF and 6N mice in correlation with UCP3 overexpression. In studies performed in isolated perfused hearts submitted to transient ischemia these changes were associated with reduced ATP content and myocardial PCr/ATP ratio at baseline, and delayed pHi recovery (31PNMR) and attenuated hypercontracture at the onset of reperfusion. Finally, in mice subjected to 45 min of coronary occlusion and 24 h of reperfusion, HFD significantly reduced infarct size respect to their respective control diet groups in male BDF (39.4 ±â€¯6.1% vs. 19.9 ±â€¯3.2%, P = 0.018) and 6N mice (38.0 ±â€¯4.1 vs. 24.5 ±â€¯2.6%, P = 0.017), and in female BDF mice (35.3 ±â€¯4.4% vs. 22.3 ±â€¯2.5%, P = 0.029), but not in male 6J mice (40.2 ±â€¯3.4% vs. 34.1 ±â€¯3.8%, P = 0.175). Our results indicate that the protective effect of HFD-induced obesity against myocardial ischemia/reperfusion injury is influenced by genetic background and appears to critically depend on inhibition of glucose oxidation and mild respiratory mitochondrial uncoupling resulting in prolongation of acidosis at the onset of reperfusion.

Calpains mediate isoproterenol-induced hypertrophy through modulation of GRK2.

Inhibition of the Ca2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5 mg/Kg/day isoproterenol intraperitoneally for 1 week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpain-mediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

Studies on the role of apoptosis after transient myocardial ischemia: genetic deletion of the executioner caspases-3 and -7 does not limit infarct size and ventricular remodeling.

Although it is widely accepted that apoptosis may contribute to cell death in myocardial infarction, experimental evidence suggests that adult cardiomyocytes repress the expression of the caspase-dependent apoptotic pathway. The aim of this study was to analyze the contribution of caspase-mediated apoptosis to myocardial ischemia-reperfusion injury. Cardiac-specific caspase-3 deficient/full caspase-7-deficient mice (Casp3/7DKO) and wild type control mice (WT) were subjected to in situ ischemia by left anterior coronary artery ligation for 45 min followed by 24 h or 28 days of reperfusion. Heart function was assessed using M-mode echocardiography. Deletion of caspases did not modify neither infarct size determined by triphenyltetrazolium staining after 24 h of reperfusion (40.0 ± 5.1 % in WT vs. 36.2 ± 3.6 % in Casp3/7DKO), nor the scar area measured by pricosirius red staining after 28 days of reperfusion (41.1 ± 5.4 % in WT vs. 44.6 ± 8.7 % in Casp3/7DKO). Morphometric and echocardiographic studies performed 28 days after the ischemic insult revealed left ventricular dilation and severe cardiac dysfunction without statistically significant differences between WT and Casp3/7DKO groups. These data demonstrate that the executioner caspases-3 and -7 do not significantly contribute to cardiomyocyte death induced by transient coronary occlusion and provide the first evidence obtained in an in vivo model that argues against a relevant role of apoptosis through the canonical caspase pathway in this context.

Implications of Iron Deficiency in STEMI Patients and in a Murine Model of Myocardial Infarction.

In patients with a first anterior ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention, iron deficiency (ID) was associated with larger infarcts, more extensive microvascular obstruction, and higher frequency of adverse left ventricular remodeling as assessed by cardiac magnetic resonance imaging. In mice, an ID diet reduced the activity of the endothelial nitric oxide synthase/soluble guanylate cyclase/protein kinase G pathway in association with oxidative/nitrosative stress and increased infarct size after transient coronary occlusion. Iron supplementation or administration of an sGC activator before ischemia prevented the effects of the ID diet in mice. Not only iron excess, but also ID, may have deleterious effects in the setting of ischemia and reperfusion.

Delayed, oral pharmacological inhibition of calpains attenuates adverse post-infarction remodelling.

Calpains activate during myocardial ischemia-reperfusion and contribute to reperfusion injury. Studies in transgenic animals with altered calpain/calpastatin system subjected to permanent ischemia suggest that calpains are also involved in post-infarction remodelling and heart failure. AIMS: To determine whether delayed oral administration of the calpain inhibitor SNJ-1945 reduces adverse myocardial remodelling and dysfunction following transient coronary occlusion. METHODS AND RESULTS: Male Sprague-Dawley rats were subjected to 30 min of ischemia followed by 21 days of reperfusion and received the calpain inhibitor SNJ-1945 intraperitoneally at the onset of reperfusion (Acute group), orally starting after 24 h of reperfusion and for 14 days (Chronic group), or the combination of both treatments. Calpain-1 and calpain-2 protein content increased and correlated with higher calpain activity in control hearts. Administration of SNJ-1945 attenuated calpain activation, and reduced scar expansion, ventricular dilation and dysfunction in both acute and chronic groups. Acute treatment reduced infarct size in hearts reperfused for 24 h and inflammation measured after 3 days. Delayed, chronic oral administration of SNJ-1945 attenuated inflammation, cardiomyocyte hypertrophy and collagen infiltration in the non-infarcted myocardium at 21 days in correlation with increased levels of IĸB and reduced NF-ĸB activation. In cultured fibroblasts, SNJ-1945 attenuated TGF-ß1-induced fibroblast activation. CONCLUSIONS: Our data demonstrate for the first time that long-term calpain inhibition is possible with delayed oral treatment, attenuates adverse post-infarction remodelling, likely through prevention of NF-ĸB activation, and may be a promising therapeutic intervention to prevent adverse remodelling and heart failure in patients with acute myocardial infarction.

Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception.