Investigation of novel molecular targets to counteract myocardial remodeling upon pathophysiological stressors

The heart is a highly adaptable organ that grows through hyperplasia in the embryonic and fetal stages but mainly undergoes hypertrophy in response to increased workload in postnatal and adult life. Hypertrophic remodeling in cardiomyocytes (CMs) is associated with increased protein synthesis (thus requiring tight control over protein quality). In parallel, when CMs enlarge due to hypertrophy, energy expenditure increases to uphold higher protein synthesis while ensuring contractile activity. Thus, an increase in mitochondrial function and number is expected to maintain their energetic homeostasis in the long term. Among different types of hypertrophy, two main forms are conventionally recognised, described as physiological and pathological cardiac hypertrophy. While the former involves long-term potentiation of cardiac function without harmful effects, the latter includes initial adaptive changes followed by molecular and structural changes leading to decreased heart function and eventually heart failure. During my Ph.D., I focused on these two aspects of cardiac hypertrophy (protein quality control and mitochondrial function) to identify possible targetable mechanisms to counteract pathologic myocardial remodeling. In the first project I focused on a muscle-specific ubiquitin ligase, MuRF1, which has been shown to participate in cardiac pathological conditions. MuRF1-/- mice do not show cardiac functional defect in the first 6 months, but only later develop diastolic dysfunction, followed by systolic impairment during ageing, accompanied by cellular hypertrophy and a massive interstitial fibrotic remodeling, characterised by increased levels of Collagen VI already in adult animals. Interestingly, these changes occurred in the absence of common markers of maladaptive remodelling (e.g. cell death, fibroblast proliferation, ANF and b-MHC). Our results suggest that the increased collagen expression in fibroblasts is driven by a paracrine CM-to-fibroblast effect, upon MuRF1 ablation. In the second project, I focused on the Mitochondrial Ca2+ Uniporter (MCU), which was demonstrated to be differentially modulated in patients and mouse models of cardiac hypertrophy. Of note, up- (OE) or down-regulation (KD) of MCU exerted opposite effects in the response of mice to pressure overload. While the KD shows an exacerbated maladaptive phenotype (capillary rarefaction, massive fibrotic deposition), the OE showed a phenotype more similar to a physiological adaptation of the heart (lower fibrosis, preserved vascularisation, lower expression of maladaptive markers) despite a higher hypertrophic growth of CMs. To further assess the mechanism behind the observed cardioprotective role, we exploited an in vitro model, inducing CMs hypertrophy through chronic noradrenaline (NE) treatment. Interestingly, already in basal condition, and further exacerbated after NE stimulation, we observed an increase in the phosphorylation levels of Akt, already linked to beneficial effects on cardiac homeostasis during physiologic hypertrophy of the heart. Our results, obtained both in vitro and in vivo, suggest that upon MCU-OE the increased mitochondrial Ca2+ content impinges on mitochondrial ROS production, which in turn indirectly drives Akt activation. As a side project, I contributed to the optimisation of a culture condition to promote the structural and functional maturation of neonatal CMs in vitro. Exploiting morphological, morphometrical and live imaging analyses, our results show that, upon culture with a Low-Glucose-No-Serum (LGNS) medium, neonatal CMs are morphologically and functionally closer to the adult phenotype. In addition, we also tested this novel condition in CMs and sympathetic neuron co-cultures, obtaining functional connections between these cells, allowing the in vitro study of the dynamics occurring at the neuro-cardiac junction.