Use of salt has always been a matter of debate for health professionals and medical scientists, particularly with its association with cardiac toxicity. Studies have established that the average salt intake in a large population worldwide exceeds the physiological requirement (Khan et al. 2018). Immense interest of researchers has thus developed in deciphering the role of high salt diet on cardiovascular diseases (CVD) (Lampuré et al. 2020). The modern diet with salt leads to several physiological and pathological changes in organ functions that are independent of blood pressure regulation (Catena et al. 2017). These changes are most commonly associated with endothelial dysfunction(Cheng et al. 2019) and vascular injury (Sanders &Alcaide 2023), that may aid the progression to cardiac toxicity. Hypertension, dyslipidemia, myocardial infarction, obesity, diabetes, alcohol and smoking have been exhaustively discussed for their roles in development cardiac toxicity (Hasebe &Hasebe 2022).
Myocardial tissue is a rich source of CKMB enzyme, one of the essential biomarkers for normal cardiac function, enzyme released in response to a pathological injury to cardiac muscle, e.g., doxorubicin- induced injury or due to altered physiological activity (Aziz et al. 2020). Consequently, we found markedly elevated CKMD levels in all the animals induced with doxorubicin (2 mg/kg). However, treatment with salt-diet reduced CKMB levels in the both, DTS and DSS groups, suggesting the beneficial effect of salts in preventing cardiac toxicity (Fig. 1). The reduction of CKMB may have occurred due to downregulation of genes associated with TGF-β dependent signaling pathways in left ventricle and kidneys(Chen et al. 2022), reported to prevent the cardiac toxicity and improve CVS functions (Methatham et al. 2022).
C- Reactive protein (CRP) is a well reported marker of inflammation underlying tissue injury in atherosclerosis and myocardial infarction. Growing evidences suggest that it is an independent predictor of several cardiovascular events (Dykun et al. 2022, Kones 2010). CRP increases in patients with unstable cardiac functions, and increase many folds quickly at the onset of an acute phase infection or diffuse tissue destruction (Gabay &Kushner 1999). Thus, in this study, doxorubicin – induced cardiac tissue injury resulted in significantly elevated CRP level in DC group. However, controlled increase in CRP level in DTS and DSS group indicated potential of salt in cardiac protection against atherosclerosis and myocardial infarction, thereby preventing cardiac toxicity.
Hyperlipidemia generally progresses to atherosclerosis. Nevertheless, serum lipids start alterations in cardiac morphology independently, before the onset of atherosclerosis. We observed in this study that there was a markedly high level of TC, TG and LDL (p < 0.0001), and low level of HDL (p < 0.01) in doxorubicin- induced DC group as compared to normal healthy rats of NC group. High level of circulating cholesterol blocks coronary arteries, reduces capillary density and reserves the blood flow. This results in apoptosis of capillary endothelium and left ventricular (LV) impairment. LDL and TG have a strong potential to increase the LV mass (Conkar Tunçay et al. 2022). Hypercholesterolemia, therefore, by increasing LV mass, contributes to myocardial toxicity as was observed in our study as well. However, when treated with table and natural sea- salt diet, lipid profile improved in the doxorubicin induced DTS and DSS animals (Fig. 2). Increased LV mass makes a person more prone to the risk of heart attack despite there is no history of coronary heart disease or atherosclerosis induced myocardial infarction. Low level of plasma HDL-C in hypertensive patients is also known to be associated with increase in LV mass (Trimarco et al. 2022). Thus, regulation of lipid levels by table salt and natural sea salt, as found in our study, may play a good role in prevention of cardiac toxicity in patients. Our findings were though contradictory to those of Harsha et al, who reported that sodium intake exerts no significant difference on TC, HDL, LDL or TG (Harsha et al. 2004)
One of the characteristics of the cardiac muscle architecture is a complex network of extracellular matrix proteins containing abundance of fibrillar collagen. Inflammation- induced loss of cardiac myocytes are replaced with collagen- based proteins in ventricular cells (Leonard et al. 2012) However, excessive deposition of these proteins may result in altered cardiac pathology, progressing to fibrosis and biventricular fibrotic modeling(Bacmeister et al. 2019). In our study, DC group presented with increased diameter and decreased flexibility of cardiac muscles, which underlies myocytes metabolic stress. Furthermore, interstitial spaces, distorted intercalated disc, irregular cell arrangement with an increased deposition of fat, and fibrinogen along with irregular and increased distribution of eosinophilic infiltration were seen (Fig. 3D). DTS group illustrated similar morphological alterations with reduced deposition of fat and fibrinogen (Fig. 3E). On the contrary, the pathological characteristics showed similar features to that of NC group, with only a mild alteration (++) in fibrinogen deposits. DC group on the other hand, presented with a significant deposition of fibrinogen (+++) as compared to NC (Fig. 3F).
It is well illustrated that metabolic disorders may interrupt the community richness of gut microbiome and alter the microbial diversity(Le Chatelier et al. 2013). Impact of dysbiosis on renal and cardiac functions has been seen in the manifestations of dyslipidemia, CHF, chronic renal diseases, hypertension and atherosclerosis(Li &Tang 2018). Bile acids, Tri-methyl N-oxide (TMAO), short chain fatty acids (SCFAs) and other bacterial metabolites, along with Vitamin-B, vitamin- K and gut hormones significantly contribute to the pathogenesis of these diseases. Indirectly, dysbiosis also manifests the disease by altering the immune system functions(Levy et al. 2017). A rich gut microbial environment(Lach et al. 2023), on the other hand, is involved in the regulation of cardiac -renal functions and thus maintains homeostasis(Woźniak et al. 2021). Bacterial diversity comprises a measure of microbial richness by the number of observed OTUs, Chao1, Inverse Simpson and Goods Coverage, and is inversely associated with stress- induced cardiac injury a relation well illustrated in several studies(Anwar et al. 2022a). We observed in our study that doxorubicin induction disrupted the microbiota richness in DC group, which was evidenced by a sharp fall in OUT when compared with NC animals. Administration of sea- salt diet in doxorubicin- induced animals (DSS) restored the microbial richness and microflora diversity to normal. Gut microbial diversity was also rejuvenated in animals fed with table-salt diet (DTS group), but to an extent lesser than that of DSS group (Fig. 5) substantiating the results are in accordance to the findings reported in other studies(Kelly et al. 2016, Xu et al. 2020). We established that abundance of distinct microbial taxa is more imperative than their relative frequency in preventing cardiovascular diseases.
The pathogenesis of heart failure, or better rephrased as gut hypothesis of heart failure, is currently a hot- cup of research among the health scientists globally. Our results indicated that the Firmicutes, which comprise of more than 84 species, majorly belong to Clostridium genera. Alteration in this phyla expose to lifetime cardiovascular (Tsai et al. 2021). We observed a significantly high percentage of Firmicutes in DC group as compared to NC. Also, administration of salt diet in DTS and DSS groups decreased the Firmicute levels to about one third of the DC group. Our results are in accordance to the existing studies(Anwar et al. 2022a), showing that alteration in Firmicutes is responsible for cardiac toxicity (Fig. 5) including Lachnospiraceae, Tyzzerella, Coprococcus 2 and Ruminococcus associated with cardiac toxicity and other complications.
Alternatively, the second abundant phylum, Bacteroidetes regulate normal functions of cardiac activity and play an important role in obesity, diabetes – associated cardiovascular complications(Zhang et al. 2022). We found that as compared to NC animals, DC group had a very low percent of Bacteroidetes. It Treatment with table and sea salt increased this percentage to more than twice to that in DC group (Fig. 5). It was interesting to observe that the microbial percentage in NC and DC was almost similar, thus, indicating a direct link cardiovascular disease with the dietary salt of any origin.
Reports are available that demonstrate the alteration of Spirochaetes in a metabolic disease like obesity (Khan et al. 2018) but to our knowledge, there are no reports that have correlated the Spirochetes with cardiac disease. Our results have a significant impact on this bacterial phylum (Fig. 5). There was a significant fall of this phylum in the D.C. group compared to the normal control group. However, the treatment with sea salt and table salt increases the population of this phylum to 36-fold and 9-fold respectively as compared to the D.C. group, justifying the importance of this bacterial phylum in cardiac toxicity.
Proteobacteria phylum constitutes 30% of the total organism following bacterial kingdom in the normal control group. a sharp fall of 7-fold was observed after the administration of doxorubicin. However, normal sea salt and table salt in the doxorubicin treated groups were able to restore this population to double and triple respectively (Fig. 5). These results of varying percentages for Proteobacteria in various groups leave much unanswered questions that whether it’s the diet with salt composition is changing the percentage or it is the disease condition that is playing some hidden role in alteration of the composition of this phylum.
Multivariate analysis of the samples (Fig. 5) demonstrates that there is a high variation in bacterial diversity, supported by Khan et al., where they hence stated the multi-variation of bacterial species in high-fat diet model and similar observations were made in our results of different salt in diet (Khan et al. 2018). This variation may be due to disease condition, or it may be due to variation in electrolytes or lipids profile or it may be due to other factors that need to be deciphered.