Md. Shenuarin Bhuiyan

Enhanced autophagy ameliorates cardiac proteinopathy

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein. Hearts from these mice had enhanced autophagy, but normal morphology and function. We crossed these mice with CryABR120G mice, a model of DRC in which autophagy is significantly attenuated in the heart, to test the functional significance of autophagy activation in a proteotoxic model of heart failure. Sustained Atg7-induced autophagy in the CryABR120G hearts decreased interstitial fibrosis, ameliorated ventricular dysfunction, decreased cardiac hypertrophy, reduced intracellular aggregates and prolonged survival. To determine whether different methods of autophagy upregulation have additive or even synergistic benefits, we subjected the autophagy-deficient CryABR120G mice and the Atg7-crossed CryABR120G mice to voluntary exercise, which also upregulates autophagy. The entire exercised Atg7-crossed CryABR120G cohort survived to 7 months. These findings suggest that activating autophagy may be a viable therapeutic strategy for improving cardiac performance under proteotoxic conditions.

Activation of HtrA2, a Mitochondrial Serine Protease Mediates Apoptosis: Current Knowledge on HtrA2 Mediated Myocardial Ischemia/Reperfusion Injury

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity, thereby eliciting release of multiple death-promoting factors residing in the mitochondrial intermembrane space into the cytosol. Among the death-promoting factors, a serine protease, high temperature requirement A2 (HtrA2) has drawn attention as a key player in the apoptosis pathways in different pathological conditions including myocardial ischemia/reperfusion injury. Heart ischemia/reperfusion results in HtrA2 translocation from the mitochondria to the cytosol, where it promotes cardiomyocyte apoptosis via a protease activity-dependent and caspase-mediated pathway. Once released, cytosolic HtrA2 causes X-chromosome-linked inhibitor of apoptosis protein (XIAP) degradation, caspase activation, and subsequent apoptosis. Consistent with the hypothesis, inhibition of HtrA2 improved postischemic myocardial contractile functions along with reduction of myocardial infarct size. The precise mechanism underlying HtrA2-induced apoptosis in mammalian cells has been studied through biochemical, structural, and genetic studies, in which HtrA2 promotes proteolytic activation of caspases through multiple pathways in heart ischemia. Therapeutic interventions that inhibit HtrA2 expression, translocation, or protease activity (such as by using the ucf-101 inhibitor) may provide an attractive therapeutics in the treatment of cardiovascular diseases.

Targeting sigma-1 receptor signaling by endogenous ligands for cardioprotection.

Introduction: The sigma receptors, initially described as a subtype of opioid receptors, are now considered to be a unique receptor expressed in neonatal rat cardiomyocytes and in the plasma membrane of adult rat cardiomyocytes. A number of sigma receptor ligands influence cardiovascular function and the heart has binding sites for sigma receptor ligands that alter contractility both in vivo and in vitro. The human sigma-1 receptor gene contains a steroid-binding component and gonadal steroid dehydroepiandrosterone (DHEA) which interacts with the sigma-1 receptor. Areas covered: We recently documented that the pathophysiological role of the sigma-1 receptor in the heart and its modulation using DHEA, was cardioprotective. Moreover, agonist-induced activation of the sigma-1 receptor modulates diverse ion channels and thereby regulates heart function. Novel concepts for understanding the pathophysiological relevance of sigma-1 receptors in the progression of heart failure, and developing clinical therapeutics targeting for the receptor in cardiovascular diseases are discussed. Expert opinion: Future studies should attempt to develop cardiac-specific knockdown of the sigma-1 receptor to observe its downstream signaling. We expect that these observations will lead to a novel therapeutic target for which a new class of antihypertrophic drugs can be designed.

Determination of the critical residues responsible for cardiac myosin binding protein C's interactions

Despite early demonstrations of myosin binding protein Cs (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-Cs ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-Cs N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-Cs C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.

Activation of Endothelial Nitric Oxide Synthase by a Vanadium Compound Ameliorates Pressure Overload-Induced Cardiac Injury in Ovariectomized Rats

We here investigated the effect of bis(1-oxy-2-pyridinethiolato) oxovanadium (IV), [VO(OPT)], against myocardial hypertrophy and cardiac functional recovery in pressure overload-induced hypertrophy in ovariectomized female rats and defined mechanisms underlying its cardioprotective action. Wistar rats subjected to bilateral ovariectomy were further treated with abdominal aortic stenosis. VO(OPT) (containing 1.25 and 2.50 mg of vanadium per kg) was administered orally once a day for 14 days starting from 2 weeks after aortic banding. Treatment with VO(OPT) significantly inhibited pressure overload-induced increase both in the heart weight:body weight ratio and the lung weight:body weight ratio. VO(OPT) also attenuated hypertrophy-induced impaired left ventricular end-diastolic pressure, left ventricular developed pressure, and left ventricular contractility (±dp/dtmax). VO(OPT) treatment significantly restored pressure overload-induced impaired endothelial NO synthase activity with concomitant increased phosphorylation of endothelial NO synthase (Ser1179). Moreover, VO(OPT) treatment significantly restored pressure overload-induced reduced Akt activity, as indicated by increased phosphorylation at Ser473 and at Thr308. Treatment with VO(OPT) also secondarily inhibited calpastatin and dystrophin breakdown and decreased myosin light chain phosphorylation. Finally, VO(OPT) treatment significantly attenuated mortality after repeated isoproterenol administration in pressure overloaded-ovariectomized rats. Taken together, VO(OPT) attenuates cardiac myocytes hypertrophy in vivo in pressure overload-induced hypertrophy in ovariectomized rats and prevents the process from hypertrophy to heart failure. These effects are mediated by inhibition of calpastatin and dystrophin breakdown in addition to increased Akt and endothelial NO synthase activities.

Dehydroepiandrosterone‐Mediated Stimulation of Sigma‐1 Receptor Activates Akt‐eNOS Signaling in the Thoracic Aorta of Ovariectomized Rats with Abdominal Aortic Banding

OBJECTIVE Decreased dehydroepiandrosterone (DHEA) levels are associated with endothelial dysfunction and increased cardiovascular mortality in postmenopausal women. Using ovariectomized rats, we first defined whether expression of sigma-1 receptor (Sig-1R) in the aorta is regulated following pressure overload (PO) and also after DHEA treatment. We also investigated effects of DHEA known as Sig-1R agonist on impaired Akt/endothelial nitric oxide synthase (eNOS) signaling in the thoracic aorta under PO. RESEARCH DESIGN/METHODS Wistar rats subjected to bilateral ovariectomy (OVX) were further treated with abdominal aortic stenosis 2 weeks later. DHEA (15 and 30 mg/kg) was administered orally once a day for 14 days starting from 2 weeks after the aortic banding. RESULTS Time course study indicated that expression of Sig-1R expression and eNOS decreased time dependently in the thoracic aorta from 1 to 4 weeks after PO. DHEA treatment significantly inhibited the decreased Sig-1R expression in the thoracic aorta. The DHEA treatment also significantly restored PO-induced impaired Akt phosphorylation and stimulated eNOS protein expression with concomitant increased Akt-mediated eNOS phosphorylation (Ser1177). We did not find any changes in the phosphorylation of ERK1/2 and PKCα in the aorta following PO and after treatment with DHEA. CONCLUSION We here reported, for the first time, that DHEA treatment induces the upregulation and stimulation of Sig-1R in the thoracic aorta that stimulate Sig-1R-mediated Akt-eNOS signaling pathways in ovariectomized rats under PO.

Targeting sigma-1 receptor with fluvoxamine ameliorates pressure-overload-induced hypertrophy and dysfunctions

Objective: We here investigated the effect of sigma-1 receptor (Sig-1R) stimulation with fluvoxamine on myocardial hypertrophy, cardiac functional recovery and defined mechanisms underlying its cardioprotective action. Methods: Wistar rats subjected to bilateral ovariectomy (OVX) were treated with abdominal aortic banding between the right and left renal arteries. To confirm the cardioprotective role of Sig-1R stimulation, we treated the rats with Sig-1R agonist (fluvoxamine, 0.5 and 1 mg/kg) orally once a day for 4 weeks after the onset of aortic banding. Results: Interestingly, the expression of Sig-1R in the left ventricle (LV) decreased significantly 4 weeks after pressure overload (PO)-induced hypertrophy in OVX rats. The fluvoxamine administration significantly attenuated PO-induced myocardial hypertrophy with concomitant increase in the expression of Sig-1R in LV. Fluvoxamine also attenuated hypertrophy-induced impaired LV functions. The cardioprotective effect of fluvoxamine was nullified by treatment with Sig-1R antagonist (NE-100; 1 mg/kg). Fluvoxamine treatment significantly restored PO-induced impaired eNOS and Akt activity in the LV. Conclusion: We here found, for the first time, the potential role of Sig-1R expression in the heart in attenuating PO-induced hypertrophy in OVX rats. Fluvoxamine treatment protects PO-induced cardiac injury via upregulation of Sig-1R and stimulation of Sig-1R-mediated Akt-eNOS signaling in ovariectomized rats.

Mitochondrial Serine Protease HtrA2/Omi as a Potential Therapeutic Target

Deregulation of apoptosis has been shown to contribute to the development of many diseases, including ischemia/reperfusion injury of organs, different types of cancer formation, as well as neurodegenerative and autoimmune disorders. Recently, the mitochondrial serine protease High temperature requirement A2 (HtrA2)/Omi has drawn attention as it played pivotal role in different pathological conditions. We critically discussed the rationale for therapeutically targeting HtrA2 signaling in pathological conditions and explore the molecular mechanisms of HtrA2 inhibition as a novel therapeutic strategy. The precise mode of action and importance of HtrA2 in mitochondrial quality control as well as in apoptosis in mammalian cells has been recently studied through biochemical, structural and genetic studies. This review introduces HtrA2 [correction of HrtA2] from its molecular origins, discusses its modulation and potential as a novel drug target, and considers future therapeutic perspectives.

Cardioprotective Effect of Vanadyl Sulfate on Ischemia/Reperfusion‐Induced Injury in Rat Heart In vivo Is Mediated by Activation of Protein Kinase B and Induction of FLICE‐Inhibitory Protein

Here we explored the mechanism of cardioprotective action of a tyrosine phosphatase inhibitor vanadyl sulfate on myocardial infarction and cardiac functional recovery in rats subjected to myocardial ischemia/reperfusion (MI/R) in vivo. Male Sprague-Dawley rats underwent 30 min heart ischemia by left coronary artery occlusion followed by 24-h reperfusion. Rats were randomized to receive either vehicle or vanadyl sulfate (1 and 5 mg/kg) intraperitoneally 0 min and 30 min after the start of reperfusion. Posttreatment with vanadyl sulfate significantly reduced the infarct size and significantly decreased the elevated left ventricular end diastolic pressure, improved left ventricular developed pressure, and left ventricular contractility (+/- dP/dt) after 72-h reperfusion in a dose-dependent manner. Moreover, treatment with vanadyl sulfate also significantly inhibited the apoptosis-related Caspase-3 and Caspase-9 processing, thereby elicited the antiapoptotic effect. The cardioprotective effect of vanadyl sulfate was closely associated with restoration of reduced protein kinase B (Akt) activity following MI/R injury. The recovered Akt activity correlated with increased phosphorylation of forkhead transcription factors, FKHR and FKHRL-1, thereby inhibiting apoptotic signaling. Furthermore, treatment with vanadyl sulfate significantly increased FLICE-inhibitory protein (FLIP) expression, and decreased expression of Fas ligand and Bim in cardiomyocytes. Taken together, rescue of cardiomyocytes by posttreatment with vanadyl sulfate from MI/R injury was mediated by increased FLIP expression and decreased Fas ligand and Bim expression via activation of Akt. These results demonstrate that treatment with vanadyl sulfate exerts significant cardioprotective effects along with cardiac functional recovery.

Stimulation of Sigma-1 receptor signaling by dehydroepiandrosterone ameliorates pressure overload-induced hypertrophy and dysfunctions in ovariectomized rats

Objective: Decreased dehydroepiandrosterone (DHEA) levels are associated with endothelial dysfunction and increased cardiovascular mortality in postmenopausal women. We investigated the role of DHEA, also known as sigma-1 receptor (Sig-1R) agonist, in myocardial hypertrophy, cardiac functional recovery and defined mechanisms of cardioprotective action. Methods: Wistar rats subjected to bilateral ovariectomy (OVX) were further treated with abdominal aortic stenosis. DHEA (15 and 30 mg/kg) was administered orally once a day for 14 days starting from 2 weeks after aortic banding. Results: Time course study indicated that left ventricle (LV) weight:body weight (BW) ratio increased time-dependently from 1 to 4 weeks after pressure-overload (PO) with significant inversed regulation of Sig-1R expression. Treatment with the Sig-1R agonist, DHEA, significantly attenuated PO-induced myocardial hypertrophy with increased expression of Sig-1R in the LV. DHEA also attenuated hypertrophy-induced impaired LV end diastolic pressure, LV developed pressure and LV contractility (± dp/dtmax). DHEA treatment significantly restored PO-induced impaired eNOS and Akt activity in the LV. Conclusion: We report, for the first time to our knowledge, the potential role of Sig-1R expression in the heart to attenuate PO-induced hypertrophy in ovariectomized rats. DHEA treatment protects against PO-induced cardiac injury via upregulation of Sig-1R and stimulation of Sig-1R-mediated Akt-eNOS signaling.